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Sample records for grapevine somatic embryos

  1. Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors.

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    Bouamama-Gzara, Badra; Selmi, Ilhem; Chebil, Samir; Melki, Imene; Mliki, Ahmed; Ghorbel, Abdelwahed; Carra, Angela; Carimi, Francesco; Mahfoudhi, Naima

    2017-12-01

    Prospecting of local grapevine ( Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. 'Hencha' were successfully induced from filament, when cultured on Chée and Pool (1987). based-medium, enriched with 2 mg 1 -1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1 -1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% 'Hencha' somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination.

  2. Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors

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    Badra Bouamama-Gzara

    2017-12-01

    Full Text Available Prospecting of local grapevine (Vitis vinifera L. germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3, Grapevine stem pitting associated virus (GRSPaV and Grapevine virus A (GVA, were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. ‘Hencha’ were successfully induced from filament, when cultured on Chée and Pool (1987. based-medium, enriched with 2 mg 1−1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1−1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% ‘Hencha’ somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination.

  3. SOMATIC EMBRYOGENESIS AND MORPHOANATOMY OF Ocotea porosa SOMATIC EMBRYOS

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    Luciana Luiza Pelegrini

    2013-01-01

    Full Text Available Ocotea porosa seeds have strong tegument dormancy, recalcitrant behavior, low and irregular germination and that makes its natural propagation difficult. The aim of this study was to establish a protocol of regeneration of Ocotea porosa from somatic embryogenesis. Immature embryonic axes were inoculated on WPM culture medium supplemented with 2.4-D (200 μM combined or not with hydrolyzed casein or glutamine (0.5 or 1 g l-1, during 90 days. The repetitive embryogenesis was induced on medium with 2.4-D (22.62 μM combined with 2-iP (2.46 μM followed by transfer to culture medium with hydrolyzed casein or glutamine (1 g l-1 during 90 days. The maturation of somatic embryos was tested in culture medium containing NAA (0.5 μM and 2-iP (5; 10 and 20 μM. The highest percentage of somatic embryos induction (8.3% was observed in WPM culture medium containing 200 μM 2.4-D and 1 g L-1 hydrolyzed casein and the development of somatic embryos occurred indirectly. Repetitive somatic embryogenesis was promoted in WPM medium containing hydrolyzed casein or glutamine. However, the culture medium containing hydrolyzed casein promoted the maintenance of embryogenic capacity for more than two years. During the maturity phase, there was a low progression of globular embryos to cordiform and torpedo stages. The different ontogenetic stages of somatic embryos of Ocotea porosa were characterized by histological studies.

  4. SOMATIC EMBRYOGENESIS AND MORPHOANATOMY OF Ocotea porosa SOMATIC EMBRYOS

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    Luciana Luiza Pelegrini

    2013-12-01

    Full Text Available http://dx.doi.org/10.5902/1980509812343Ocotea porosa seeds have strong tegument dormancy, recalcitrant behavior, low and irregular germinationand that makes its natural propagation difficult. The aim of this study was to establish a protocol ofregeneration of Ocotea porosa from somatic embryogenesis. Immature embryonic axes were inoculatedon WPM culture medium supplemented with 2.4-D (200 μM combined or not with hydrolyzed casein orglutamine (0.5 or 1 g l-1, during 90 days. The repetitive embryogenesis was induced on medium with 2.4-D(22.62 μM combined with 2-iP (2.46 μM followed by transfer to culture medium with hydrolyzed caseinor glutamine (1 g l-1 during 90 days. The maturation of somatic embryos was tested in culture mediumcontaining NAA (0.5 μM and 2-iP (5; 10 and 20 μM. The highest percentage of somatic embryos induction(8.3% was observed in WPM culture medium containing 200 μM 2.4-D and 1 g L-1 hydrolyzed casein andthe development of somatic embryos occurred indirectly. Repetitive somatic embryogenesis was promotedin WPM medium containing hydrolyzed casein or glutamine. However, the culture medium containinghydrolyzed casein promoted the maintenance of embryogenic capacity for more than two years. Duringthe maturity phase, there was a low progression of globular embryos to cordiform and torpedo stages.The different ontogenetic stages of somatic embryos of Ocotea porosa were characterized by histologicalstudies.

  5. Determination of abscisic acid and its glucosyl ester in embryogenic callus cultures of Vitis vinifera in relation to the maturation of somatic embryos using a new liquid chromatography-ELISA analysis method.

    Science.gov (United States)

    Prado, María Jesús; Largo, Asier; Domínguez, Cristina; González, María Victoria; Rey, Manuel; Centeno, María Luz

    2014-06-15

    The levels of abscisic acid (ABA), its conjugate ABA-GE, and IAA were determined in embryogenic calli of Vitis vinifera L. (cv. Mencía) cultured in DM1 differentiation medium, to relate them to the maturation process of somatic embryos. To achieve this goal, we developed an analytical method that included two steps of solid-phase extraction, chromatographic separation by HPLC, ABA-GE hydrolysis, and sensitive ELISA quantification. Because the ABA immunoassay was based on new polyclonal antibodies raised against a C4'-ABA conjugate, the assay was characterized (detection limit, midrange, measure range, and cross-reaction) and validated by a comparison of the ABA data obtained with this ELISA procedure and with a physicochemical method (LC-ESI-MS/MS). Radioactive-labeled internal standards were initially added to callus extracts to correct the losses of plant hormones, and thus assure the accuracy of the measurements. The endogenous concentration of ABA in the embryogenic callus cultured in DM1 medium was doubled at the fifth week of culture, concurring with the maturation process of somatic embryos, as indicated by the accumulation of carbohydrates observed through histological analysis. The ABA-GE content was higher than ABA, decreasing at 21 days of culture in DM1 medium but increasing thereafter. The data suggest the involvement of the synthesis and conjugation of ABA in the final stages of development in grapevine somatic embryos from embryogenic callus. IAA levels were low, suggesting that auxin plays no significant role during the maturation of somatic embryos. In addition, the lower ABA levels in calli cultured in DM differentiation medium with PGRs, a medium presenting high precocious germination and deficiencies in somatic embryo development indicate that an increase in ABA content during the development of somatic embryos in grapevine is necessary for their correct maturation. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. Cryopreservation of Arachis pintoi (leguminosae) somatic embryos.

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    Rey, H Y; Faloci, M; Medina, R; Dolce, N; Engelmann, F; Mroginski, L

    2013-01-01

    In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1 degree C per min from 25 degree C to -30 degree C followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30 degree C water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol, we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid and triploid cytotypes. No morphological abnormalities were observed in any of the plants regenerated from cryopreserved embryos and their genetic stability was confirmed with 10 isozyme systems and nine RAPD profiles.

  7. Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae).

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    Correia, Sandra M; Canhoto, Jorge M

    2010-06-01

    The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.

  8. Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm and testa

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    Traud eWinkelmann

    2015-08-01

    Full Text Available Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified.Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos.

  9. Zygotic and somatic embryo morphogenesis in Pinus pinaster: comparative histological and histochemical study.

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    Tereso, Susana; Zoglauer, Kurt; Milhinhos, Ana; Miguel, Célia; Oliveira, M Margarida

    2007-05-01

    We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.

  10. Somatic Embryos in Catharanthus roseus: A Scanning Electron Microscopic Study

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    Junaid ASLAM

    2014-06-01

    Full Text Available Catharanthus roseus (L. G. Don is an important medicinal plant as it contains several anti-cancerous compounds, like vinblastine and vincristine. Plant tissue culture technology (organogenesis and embryogenesis has currently been used in fast mass propagating raw materials for secondary metabolite synthesis. In this present communication, scanning electron microscopic (SEM study of somatic embryos was conducted and discussed. The embryogenic callus was first induced from hypocotyls of in vitro germinated seeds on which somatic embryos, differentiated in numbers, particularly on 2,4-D (1.0 mg/L Murashige and Skoog (MS was medium. To understand more about the regeneration method and in vitro formed embryos SEM was performed. The SEM study revealed normal somatic embryo origin and development from globular to heart-, torpedo- and then into cotyledonary-stage of embryos. At early stage, the embryos were clustered together in a callus mass and could not easily be detached from the parental tissue. The embryos were often long cylindrical structure with or without typical notch at the tip. Secondary embryos were also formed on primary embryo structure. The advanced cotyledonary embryos showed prominent roots and shoot axis, which germinated into plantlets. The morphology, structure and other details of somatic embryos at various stages were presented.

  11. Selection of Norway spruce somatic embryos by computer vision

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    Hamalainen, Jari J.; Jokinen, Kari J.

    1993-05-01

    A computer vision system was developed for the classification of plant somatic embryos. The embryos are in a Petri dish that is transferred with constant speed and they are recognized as they pass a line scan camera. A classification algorithm needs to be installed for every plant species. This paper describes an algorithm for the recognition of Norway spruce (Picea abies) embryos. A short review of conifer micropropagation by somatic embryogenesis is also given. The recognition algorithm is based on features calculated from the boundary of the object. Only part of the boundary corresponding to the developing cotyledons (2 - 15) and the straight sides of the embryo are used for recognition. An index of the length of the cotyledons describes the developmental stage of the embryo. The testing set for classifier performance consisted of 118 embryos and 478 nonembryos. With the classification tolerances chosen 69% of the objects classified as embryos by a human classifier were selected and 31$% rejected. Less than 1% of the nonembryos were classified as embryos. The basic features developed can probably be easily adapted for the recognition of other conifer somatic embryos.

  12. Water relations in culture media influence maturation of avocado somatic embryos.

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    Márquez-Martín, Belén; Sesmero, Rafael; Quesada, Miguel A; Pliego-Alfaro, Fernando; Sánchez-Romero, Carolina

    2011-11-15

    Application of transformation and other biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos capable of germination at an acceptable rate. In this work, we evaluated the effect of different compounds affecting medium water relations on maturation of avocado somatic embryos. Culture media were characterized with respect to gel strength, water potential and osmotic potential. Improved production of mature somatic embryos was achieved with gelling agent concentrations higher than those considered standard. The osmotic agents such as sorbitol and PEG did not have positive effects on embryo maturation. The number of w-o mature somatic embryos per culture was positively correlated with medium gel strength. Gel strength was significantly affected by gelling agent type as well as by gelling agent and PEG concentration. Medium water potential was influenced by sorbitol concentration; incorporation of PEG to a culture medium did not affect medium water potential. The highest maturation results were achieved on a medium gelled with 10 gl(-1) agar. Moreover, these somatic embryos had improved germination rates. These results corroborate the role of water restriction as a key factor controlling maturation of somatic embryos. Copyright © 2011 Elsevier GmbH. All rights reserved.

  13. The Use of Light Microscopy for Detection of Somatic Embryos

    African Journals Online (AJOL)

    usuario

    2014-02-05

    Feb 5, 2014 ... 2,4-D. After four weeks of culture of explants on the callus induction medium, globular structures were obtained. At the end of 20 days in maturation medium, somatic embryos were observed. Histological analysis showed somatic embryos with caulinar and root apex, protodermal tissue, and the vascular ...

  14. Somatic Embryos in Catharanthus roseus: A Scanning Electron Microscopic Study

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    Junaid ASLAM

    2014-06-01

    Full Text Available Catharanthus roseus (L. G. Don is an important medicinal plant as it contains several anti-cancerous compounds, like vinblastine and vincristine. Plant tissue culture technology (organogenesis and embryogenesis has currently been used in fast mass propagating raw materials for secondary metabolite synthesis. In this present communication, scanning electron microscopic (SEM study of somatic embryos was conducted and discussed. The embryogenic callus was first induced from hypocotyls of in vitro germinated seeds on which somatic embryos, differentiated in numbers, particularly on 2,4-D (1.0 mg/L Murashige and Skoog (MS was medium. To understand more about the regeneration method and in vitro formed embryos SEM was performed. The SEM study revealed normal somatic embryo origin and development from globular to heart-, torpedo- and then into cotyledonary-stage of embryos. At early stage, the embryos were clustered together in a callus mass and could not easily be detached from the parental tissue. The embryos were often long cylindrical structure with or without typical notch at the tip. Secondary embryos were also formed on primary embryo structure. The advanced cotyledonary embryos showed prominent roots and shoot axis, which germinated into plantlets. The morphology, structure and other details of somatic embryos at various stages were presented.

  15. In vitro testing of defense reactions in zygotic and somatic embryos of Abies numidica

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    Jiří Hřib

    2011-01-01

    Full Text Available Defense of desiccated cotyledonary somatic embryos and mature zygotic embryos of Abies numidica was tested in vitro by dual cultures with tester, fungus Phaeolus schweinitzii. Both types of embryos expressed defense reactions manifested by inhibited growth of fungal tester towards the embryos. Mycelial growth was described by logistic sigmoid growth model with a single asymptote. Mutual comparisons of mycelial growth in presence of zygotic and somatic embryos showed significant differences in parameters of mycelium growth curves towards the embryos. Larger defense reactions were observed in zygotic embryos relative to somatic embryos and unlimited control cultivations without embryo. The possible role of auxin in the defense response of plant embryos is discussed.

  16. Induction of somatic embryogenesis in soybean: physicochemical factors influencing the development of somatic embryos

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    Bonacin Gisele Aparecida

    2000-01-01

    Full Text Available The embryogenic capability of five soybean cultivars (Renascença, IAS-5, IAC-17, BR-16 and FT-Cometa was studied at different auxin concentrations (8, 10 and 12 mg/l naphthalene acetic acid, NAA, at different pHs (5.8 and 7.0 and at low (8-12 muEm-2 s-1 and high (27-33 mEm-2 s-1 light intensities. The experimental design was completely randomized with four replications. Immature cotyledons 4-6 mm in length were placed in the six induction mediums evaluated and submitted to two light intensities. Twenty immature cotyledons per cultivar were placed on each Petri dish, which was considered to be one replication. The number of somatic embryos per treatment per replication was counted. The results showed genotype influence on somatic embryogenic capability of each cultivar, with the most embryogenic cultivars being BR-16, FT-Cometa and IAS-5. Auxin concentration and pH value also influenced somatic embryo production, with 10 mg/l NAA being the best auxin concentration and 7.0 the best pH value. The interactions cultivar x auxin, auxin x pH and pH x light were significant, while other double interactions were not. All triple and quadruple interactions were significant, except cultivar x pH x light. No significant differences in somatic embryo production were observed in medium with different pHs or when the Petri dishes containing immature cotyledons were exposed to the two light intensities evaluated. However, a higher number of somatic embryos was produced when the medium pH was adjusted to 7.0.

  17. Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata

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    Rakesh Minocha; Dale R. Smith; Cathie Reeves; Kevin D. Steele; Subhash C. Minocha

    1999-01-01

    Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don. During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no...

  18. A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea.

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    Prabhudesai, V; Bhaskaran, S

    1993-03-01

    An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL(-1) BA and 0.1 mgL(-1) NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL(-1)), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.

  19. Maturation and germination of somatic embryos of Sorghum bicolor (L. Moench cultivar 'CIAP 132R-05'

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    Silvio de J Martínez

    2017-03-01

    Full Text Available In sorghum [Sorghum bicolor (L. Moench], developed protocols for plant regeneration via somatic embryogenesis do not include maturation stage. The present work was carried out with the aim of achieving the maturation and germination of sorghum somatic embryos in cultivar 'CIAP 132R-05'. It were studied four concentrations of sucrose (30, 50, 70 and 90 g l-1, two of abscisic acid (0.25 and 0.5 μM and a control without this growth regulator. Germination initiation (days and number of somatic embryos with complete germination were evaluated in three periods (1 - 7, 8 - 14 and 15 - 21 days of culture. In addition, the effect of 6-BAP (8.9, 17.8 and 26.6 μM on somatic embryo germination was determined. The germination start time (days and after 21 days the number of somatic embryos with complete germination and plants with malformations were determined. The addition of 70 g l-1 sucrose in the culture medium without abscisic acid increased the germination of the somatic embryos to 37.2 plants per embryo group (0.5 g of fresh mass. The highest number of somatic embryos germinated was obtained with 17.78 μM 6-BAP in the germination culture medium. It was demonstrated the need of a maturation stage in the sorghum somatic embryogenesis to increase the germination percentage.   Keywords: somatic embryogenesis, sorghum, sucrose, 6-BAP

  20. Somatic embryogenesis from seeds in a broad range of Vitis vinifera L. varieties: rescue of true-to-type virus-free plants.

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    San Pedro, Tània; Gammoudi, Najet; Peiró, Rosa; Olmos, Antonio; Gisbert, Carmina

    2017-11-29

    Somatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L. However, low frequencies of plant embryo conversion are commonly found. In a previous work we obtained from cut-seeds of a grapevine infected with the Grapevine leafroll associated viruses 1 and 3 (GLRaV-1 and GLRaV-3), high rates of direct regeneration, embryo plant conversion and sanitation. The aim of this study is to evaluate the usefulness of this procedure for regeneration of other grapevine varieties which include some infected with one to three common grapevine viruses (GLRaV-3, Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV)). As grapevine is highly heterozygous, it was necessary to select from among the virus-free plants those that regenerated from mother tissues around the embryo, (true-to-type). Somatic embryogenesis and plant regeneration were achieved in a first experiment, using cut-seeds from the 14 grapevine varieties Airén, Cabernet Franc, Cabernet Sauvignon, Mencía, Merlot, Monastrell, Petit Verdot, Pinot Blanc (infected by GFLV and GFkV), Pinot Gris, Pinot Meunier, Pinot Noir, Syrah, Tempranillo (infected by GFLV), and Verdil. All regenerated plants were confirmed to be free of GFkV whereas at least 68% sanitation was obtained for GFLV. The SSR profiles of the virus-free plants showed, in both varieties, around 10% regeneration from mother tissue (the same genetic make-up as the mother plant). In a second experiment, this procedure was used to sanitize the varieties Cabernet Franc, Godello, Merlot and Valencí Blanc infected by GLRaV-3, GFkV and/or GFLV. Cut-seeds can be used as explants for embryogenesis induction and plant conversion in a broad range of grapevine varieties. The high regeneration rates obtained with this procedure facilitate the posterior selection of true-to-type virus-free plants. A sanitation rate of 100% was obtained for GFkV as this virus is not seed-transmitted. However, the presence of GLRaV-3 and GFLV in

  1. Genetic transformation of olive somatic embryos through ...

    African Journals Online (AJOL)

    Administrator

    2011-06-20

    Jun 20, 2011 ... 2Department of Biochemistry, National Center of Genetic Engineering and Biotechnology, Tehran, Iran. Accepted 9 March, 2011. Transformed olive plants were regenerated from inoculated somatic embryos with Agrobacterium tumefacience strain GV3101, which carries the plasmid pBI-P5CS containing ...

  2. Syntheses of nucleic acid and protein in somatic embryos of Fritillaria ussuriensis maxim in different development stages

    International Nuclear Information System (INIS)

    Wang Shuyu; Tang Wei; Wang Hui

    1993-09-01

    After developing a procedure for somatic embryogenesis in Fritillaria ussuriensis, dynamics on the syntheses of DNA, RNA, and protein during globular, heart-shaped, torpedo-shaped, cotyledonary, and mature somatic embryo stages was demonstrated by both autoradiography and scintillation counting. The rates of syntheses of DNA, RNA, and protein gradually increase between the globular and cotyledonary somatic embryos stages. DNA, RNA, and protein synthesis rates are in peak at the cotyledonary later stage, precotyledonary stage, and cotyledonary stage, respectively. It appears that more DNA, RNA, and protein are synthesized in the cotyledonary somatic embryo stage than in other stages. All these results indicate that an increased syntheses of DNA, RNA, and protein is associated with the differentiation of embryogenic cells and organogenesis in somatic embryos

  3. Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

    Science.gov (United States)

    Smith, D. L.; Krikorian, A. D.

    1989-01-01

    Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and

  4. Efficient somatic embryo production of Limau madu ( Citrus ...

    African Journals Online (AJOL)

    Effects of N6-benzylaminopurine (BAP) concentration, initial cell density and carbon sources and concentrations for producing cell suspension and somatic embryos of Limau madu (Citrus suhuiensis Hort. ex Tanaka) were investigated using cell suspension culture. Cells were first inoculated into Murashige and Skoog (MS) ...

  5. Regulation of somatic embryo development in Norway spruce (Picea abies). A molecular approach to the characterization of specific developmental stages

    Energy Technology Data Exchange (ETDEWEB)

    Sabala, I. [Swedish Univ. of Agricultural Sciences, Uppsala (Sweden). Dept. of Forest Genetics

    1998-12-31

    Embryo development is a complex process involving a set of strictly regulated events. The regulation of these events is poorly understood especially during the early stages of embryo development. Somatic embryos go through the same developmental stages as zygotic embryos making them an ideal model system for studying the regulation of embryo development. We have used embryogenic cultures of Picea abies to study some aspects of the regulation of embryo development in gymnosperms. The bottle neck during somatic embryogenesis is the switch from the proliferation stage to the maturation stage. This switch is initiated by giving somatic embryos a maturation treatment i.e. the embryos are treated with abscisic acid (ABA). Somatic embryos which respond to ABA by forming mature somatic embryos were stimulated to secret a 70 kDa protein, AF70. The af70 gene was isolated and characterised. The expression of the af70 gene was constitutive in embryos but was highly ABA-induced in seedlings. Moreover, expression of this gene was stimulated during cold acclimation of Picea abies seedlings. A full length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins (LTPs), was isolated and characterised. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in nonembryogenic callus and seedlings. In situ hybridization showed that Pa18 gene is expressed in all embryonic cells of proliferating somatic embryos but the expression of the gene in mature somatic and zygotic embryos is restricted to the outer cell layer. Southern blot analysis at different stringencies was consistent with a single gene. An alteration in expression of Pa18 causes disturbance in the formation of the proper outer cell layer in the maturing somatic embryos. In addition to its influence on embryo development the Pa18 gene product also inhibits growth of Agrobacterium tumefaciens 195

  6. Effect of the inoculation density in Coffea arabica L. cv. `Caturra rojo' somatic embryos germination in RITA® Temporary Immersion System

    Directory of Open Access Journals (Sweden)

    Raúl Barbon

    2014-04-01

    Full Text Available The development of somatic embryogenesis of coffee (Coffea spp. in liquid culture medium is a viable alternative for the propagation of these species. The use of liquid culture medium and temporary immersion systems could increase the germination of somatic embryos and improve the quality of plants. The objective of this work was to determine the effect of inoculation density on germination of somatic embryos of Coffea arabica L. cv. `Caturra rojo' in temporary immersion systems RITA®. It were used as inoculum densities 40, 50, 60, 70 and 80 somatic embryos per RITA®. After 90 days of culture the number of somatic embryos germinated, hyperhydricity symptoms, number of true leaves, length and root development was quantified. With inoculum density of 70 somatic embryos per RITA®, it was obtained a highest germination percentage (60% with good leaf development and length of the plants. Key words: hyperhydricity, liquid culture medium, partial germination, total germination, somatic embryogenesis

  7. Histology of somatic embryos of eurycoma longifolia (simaroubaceae): relevance in agrobacterium rhizogenes-mediated transformation

    International Nuclear Information System (INIS)

    Balakrishnan, B.; Rabiah, S.S.; Keng, C.L.

    2014-01-01

    Histological analysis conducted on somatic embryos of Eurycoma longifolia shows the developmental structures that are remarkably similar to seeds found in the wild. The primary components of a growing somatic embryo are its shoot and root apical meristems indicated by dense layers of rapidly growing cells. The increased understanding of In vitro culture systems and anatomical changes provide information into cellular processes that govern genetic transformation of E. longifolia with Agrobacterium rhizogenes. The presence of meristematic regions on cultured somatic embryos suggests that they are suitable for genetic transformation as genetic elements could be transported to these regions where growth and differentiation are centered. This allows the successful integration and expression of transferred DNA in the host organism, leading the way for an efficient A. rhizogenes-mediated transformation protocol. (author)

  8. Development of somatic embryos for genetic transformation in Curcuma longa L. and Curcuma mangga Valeton & Zijp

    Directory of Open Access Journals (Sweden)

    Vachiraporn Pikulthong

    2016-07-01

    Full Text Available Buds from rhizomes of Curcuma longa L. variety ‘Chumphon’ and Curcuma mangga Valeton & Zijp variety ‘Phetchaburi’ were cultured on Murashige and Skoog (MS medium supplemented with 2.0 mg/L N6-benzyladenine (BA for multiple shoot induction. Their shoots were cultured on MS medium supplemented with various concentrations of one of two plant growth regulators or a combination of both—2,4-dichlorophenoxyacetic acid (2,4-D and naphthaleneacetic acid (NAA. Interestingly, the medium containing both auxins (5 mg/L 2,4-D and 5 mg/L NAA was best for somatic embryo induction after culturing for 4 weeks. Somatic embryo formation reached 87.50% for Curcuma longa and 95.83% for Curcuma mangga with a high quality of loose, friable and yellowish characters. The best conditions for the formation of shootlets occurred after transferring the somatic embryo to MS medium supplemented with 3.0 mg/L BA, 0.5 mg/L NAA and 3% maltose. The shootlets were rooted by transferring to MS medium containing 3.0 mg/L NAA. This is the first report of a complete in vitro regeneration system from somatic embryos of C. longa and C. mangga which was further used for gene manipulation in these plants. Diketide CoA synthase (DCS and curcumin synthase (CURS genes, which are the two genes involved in curcuminoid biosynthesis in turmeric, were cloned and transferred to these two species using Agrobacterium-mediated transformation. The presence of both target and marker genes, hpt, in the transformed somatic embryos was confirmed by polymerase chain reaction assay. After culturing, the transformed somatic embryos could survive for 4 weeks.

  9. MORPHOLOGICAL CHANGES DURING THE DEVELOPMENT OF SOMATIC EMBRYOS OF SAGO (Metroxylon sagu Rottb.

    Directory of Open Access Journals (Sweden)

    Pauline D. Kasi

    2016-10-01

    Full Text Available Development of somatic embryos of sago (Metroxylon sagu Rottb. on agar-solidified medium are highly varied producing heterogeneous seedlings. Understanding of this phenomenon may help in improving the cultural procedures and conditions of sagosomatic embryogenesis to obtain uniform seedlings in a large scale. This experiment was conducted at the laboratory for plant cell culture and micropropagation, Indonesian Biotechnology Research Institute for Estate Crops from January to March 2006 to examine morphological changes i.e. color and development stages of sago during their somatic embryo development on an agar-solidified medium. Twenty single globular somatic embryos of sago with specific color (yellowish, greenish, and reddish were cultured in a Petri dish supplemented with a solid medium. The medium was a micronutrients-modified MS (MMS with half strength of macronutrients containing 0.01 mg l-1 ABA, 2 mg l-1 kinetin, 20 g l-1 sucrose, 0.5 g l-1 activated charcoal, and 2 g l-1 gelrite. Parameter observed was the percentage of embryo’s number based on color and developmental stage. The result showed that at the end of 6-week culture passage, most originally greenish (80.8% and reddish (95.8% embryos remained unchanged in their colors, whereas almost half of the originally yellowish embryos turned to greenish and only 30%remained yellowish. At the same time, single globular embryos have changed gradually into the next developmental stages, although not all of the embryos were germinated. The initial color of embryo affected the rate of the developmental stage changes. Yellowish and greenish globular embryos developed more rapidly into cotyledon or germinant stages at 58% and 55% respectively, in 6 weeks than the reddish ones (41%. Therefore, the yellowish and greenish embryos are the best sources of material for in vitro mass propagation and synthetic seed production of sago.

  10. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek

    2016-01-01

    in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early......One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...

  11. Effect of CO2 on somatic embryos development Coffea arabica L. cv. ‘Caturra rojo’ and Clematis tangutica K.

    Directory of Open Access Journals (Sweden)

    Raúl Barbon

    2016-07-01

    Full Text Available Studies to optimize somatic embryogenesis have traditionally focused on the components of the culture medium but little other in vitro environment factors have been analyzed such as the composition of the gaseous atmosphere. The objective of this work was to determine the influence of CO2 on the development of the somatic embryo during the transition from the globular to the torpedo stage. The research was carried out on two model species for somatic embryogenesis that they are developed in different climatic zones: Coffea arabica L. cv. ‘Caturra rojo’ and Clematis tangutica K. Three CO2 concentrations (2.5, 5.0 and 10.0% combined with 21% O2 and two controls (passive exchange and forced ventilation were used. The effect of CO2 on the differentiation of somatic embryos from globular to torpedo stage in coffee and clematis was demonstrated, since in the treatments with passive exchange, where there was accumulation of CO2, the differentiation of somatic embryos was superior to treatments with forced ventilation. With 5.0% CO2 the process of differentiation of the embryos in the globular stage was stimulated, because in the treatment with this concentration of CO2 for coffee and clematis the highest proportion of embryos in torpedo stages and low levels of malformation were obtained.   Keywords: carbon dioxide, differentiation, in vitro environment, somatic embryogenesis

  12. Proteome analysis during pod, zygotic and somatic embryo maturation of Theobroma cacao.

    Science.gov (United States)

    Niemenak, Nicolas; Kaiser, Edward; Maximova, Siela N; Laremore, Tatiana; Guiltinan, Mark J

    2015-05-15

    Two dimensional electrophoresis and nano-LC-MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60 g L(-1)) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high pI. Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses. Copyright © 2015 Elsevier GmbH. All rights reserved.

  13. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  14. Effects of copper and arsenic stress on the development of Norway spruce somatic embryos and their visualization with the environmental scanning electron microscope.

    Science.gov (United States)

    Đorđević, Biljana; Neděla, Vilém; Tihlaříková, Eva; Trojan, Václav; Havel, Ladislav

    2018-05-18

    Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 μM and 10-50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Interactions in the Agrobacterium-soybean system and capability of some Brazilian soybean cultivars to produce somatic embryos

    Directory of Open Access Journals (Sweden)

    Mauro Antonio Orlando Di

    2000-01-01

    Full Text Available Twenty-five Brazilian soybean cultivars were studied for susceptibility to four strains of Agrobacterium tumefaciens (C58, Ach5, Bo542 and A281 and for their ability to produce somatic embryos. Twelve plants of each cultivar were inoculated in a greenhouse at 4-6 weeks of age, using 12 inoculation sites per plant. The number of galls formed on plants were counted 8-10 weeks after inoculation. To study ability to produce somatic embryos, immature cotyledons, 4-6 mm in length, were plated onto N10 medium for induction of somatic embryogenesis, using four Petri dishes with 20 cotyledons for each cultivar. The embryogenic tissues were transferred onto new N10 medium six times at 15-day intervals and the number of somatic embryos per cultivar determined. Significant interaction between soybean cultivars and A. tumefaciens strains was observed; the most virulent strain was A281. The opine type apparently had no effect on strain virulence, and the most embryogenic cultivars were IAS-5, Cristalina, FT-Cometa, IAC-7 and OC-3.

  16. Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos.

    Science.gov (United States)

    Sugimura, S; Narita, K; Yamashiro, H; Sugawara, A; Shoji, T; Terashita, Y; Nishimori, K; Konno, T; Yoshida, M; Sato, E

    2009-09-01

    Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (Ptip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.

  17. Somatic embryogenesis and embryo culture coupled with gamma irradiation for generating avocado (Persea americana Miller) mutants in the Philippines

    Energy Technology Data Exchange (ETDEWEB)

    Avenido, R. A. [Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños (Philippines); Crop Science Cluster, College of Agriculture, University of the Philippines Los Baños (Philippines); Galvez, H. F.; Dimaculangan, J. G.; Welgas, J. N.; Frankie, R. B.; Damasco, O. P. [Crop Science Cluster, College of Agriculture, University of the Philippines Los Baños (Philippines)

    2009-05-15

    Plant regeneration through somatic embryogenesis from immature zygotic embryos and embryo cultures from mature fruits were achieved in select avocado accession ‘Semil’ and other seedling trees in the Philippines. Embryogenic cultures were induced from immature zygotic embryos of eight (8) avocado genotypes using either SE1 medium (MS + 30 g/l sucrose + 5 mg/l 2, 4-D + 0.5 mg/l BAP) or SE2 medium (MS + 30 g/l sucrose + 0.1 mg/l picloram). Embryogenic cultures of 2 genotypes namely ‘Semil’ and ‘Mainit’ developed into somatic embryos after repeated subcultures in SE2, SE3 (MS + 30 g/l sucrose + 0.1 mg/l TDZ + 0.5 mg/l GA{sub 3}) and SE4 (MS + 30 g/l sucrose + 2 mg/l BAP + 1 mg/l IBA) media. Plant/shoot regeneration from ‘Semil’ somatic embryos was recorded in 3 trials at 16.3, 23.0 and 20.7%, and was affected by culture age, light treatment and media used. R4 regeneration medium (B5 macro salts + MS minor salts and vitamins + 60 g/l sucrose + 400 g/l glu + 2 mg/l BAP + 4.5 g/l Phytagel was found to be the best. Gamma irradiation (10 to 30 Gy) of embryogenic cultures of ‘Semil’ resulted in reduced proliferation and formation of cotyledonary stage somatic embryos. However, shoot regeneration from somatic embryos from gamma-irradiated cultures was comparable or even higher (17.8 to 26.9%) as compared to the control (18.3%). Over 200 somatic embryo-derived putative variant/mutant lines from tissue culture and gamma irradiation experiments are being maintained as shoot cultures. Due to slow growth and other related problems, micrografting and in vitro rooting were used to rescue and ensure the greenhouse establishment of putative mutant shoots, and fast-track mutant confirmation by genetic analysis. Preliminary genetic analyses by SSR revealed that (a) the 3 asexually propagated ‘Semil’ mother trees are genetically similar, and (b) mutations marked by the generation of a new allele (band) at the SSR locus was evident among the somatic embryo

  18. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    Science.gov (United States)

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  19. Comparing carbohydrate status during norway spruce seed development and somatic embryo formation

    NARCIS (Netherlands)

    Gösslová, M.; Svobodová, H.; Lipavská, H.; Albrechtová, J.; Vreugdenhil, D.

    2001-01-01

    The carbohydrate status of developing seeds of Picea abies was examined in order to provide a frame of reference for the evaluation of changes in carbohydrate content in maturing somatic embryos of the same species. Samples were taken at weekly intervals from 12 May 1998 (estimated time of

  20. Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification.

    Science.gov (United States)

    Adu-Gyamfi, Raphael; Wetten, Andy

    2012-01-01

    Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enhance security of in situ germplasm collections, 2-3 mm floral-derived secondary somatic embryos were cryopreserved by vitrification. This work demonstrates the most uncomplicated clonal cocoa cryopreservation. Optimal post-cryostorage survival (74.5 percent) was achieved by 5 d preculture of SSEs on 0.5 M sucrose medium followed by 60 min dehydration in cold PVS2. To minimise free radical related cryo-injury, cation sources were removed from the embryo development solution and/or the recovery medium, the former treatment resulting in a significant benefit. After optimisation with cocoa genotype AMAZ 15, the same protocol was effective across all five additional cocoa genotypes tested. For the multiplication of clones, embryos regenerated following cryopreservation were used as explant sources, and vitrification was found to maintain their embryogenic potential.

  1. Development of Somatic Embryo Maturation and Growing Techniques of Norway Spruce Emblings towards Large-Scale Field Testing

    Directory of Open Access Journals (Sweden)

    Mikko Tikkinen

    2018-06-01

    Full Text Available The possibility to utilize non-additive genetic gain in planting stock has increased the interest towards vegetative propagation. In Finland, the increased planting of Norway spruce combined with fluctuant seed yields has resulted in shortages of improved regeneration material. Somatic embryogenesis is an attractive method to rapidly facilitate breeding results, not in the least, because juvenile propagation material can be cryostored for decades. Further development of technology for the somatic embryogenesis of Norway spruce is essential, as the high cost of somatic embryo plants (emblings limits deployment. We examined the effects of maturation media varying in abscisic acid (20, 30 or 60 µM and polyethylene glycol 4000 (PEG concentrations, as well as the effect of cryopreservation cycles on embryo production, and the effects of two growing techniques on embling survival and growth. Embryo production and nursery performance of 712 genotypes from 12 full-sib families were evaluated. Most embryos per gram of fresh embryogenic mass (296 ± 31 were obtained by using 30 µM abscisic acid without PEG in the maturation media. Transplanting the emblings into nursery after one-week in vitro germination resulted in 77% survival and the tallest emblings after the first growing season. Genotypes with good production properties were found in all families.

  2. In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

    Science.gov (United States)

    Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

    2015-01-01

    The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

  3. Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High Resolution Nuclear Magnetic Resonance Analysis of Soybean Seeds, Somatic Embryos and Single Cells

    CERN Document Server

    Baianu, I C; Hofmann, N E; Korban, S S; Lozano, P; You, T; AOCS 94th Meeting, Kansas

    2002-01-01

    Novel methodologies are currently being developed and established for the chemical analysis of soybean seeds, embryos and single cells by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching one micron resolution are presented here. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are presented for oil in soybean seeds and somatic embryos under physiological conditions. FT-NIR spectra of oil and proteins were obtained for volumes as small as two cubic microns. Related, HR-NMR analyses of oil contents in somatic embryos are also presented here with nanoliter precision. Such 400 MHz 1H NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. ~20%) compared to non-mutagenized control embryos. Moreover, developmental changes in single soybean seeds and/or somatic embryos may be monitored by FT-NIR with a precision ...

  4. Influences of somatic donor cell sex on and embryo development following somatic cell nuclear transfer in pigs

    Directory of Open Access Journals (Sweden)

    Jae-Gyu Yoo

    2017-04-01

    Full Text Available Objective The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8 was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT groups (31.4±8.3 to 33.4±11.1. After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05 between sexes. Conclusion The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

  5. Stage-specific regulation of four HD-ZIP III transcription factors during polar pattern formation in Larix leptolepis somatic embryos.

    Science.gov (United States)

    Li, Shui-gen; Li, Wan-feng; Han, Su-ying; Yang, Wen-hua; Qi, Li-wang

    2013-06-15

    Polar auxin transport provides a developmental signal for cell fate specification during somatic embryogenesis. Some members of the HD-ZIP III transcription factors participate in regulation of auxin transport, but little is known about this regulation in somatic embryogenesis. Here, four HD-ZIP III homologues from Larix leptolepis were identified and designated LaHDZ31, 32, 33 and 34. The occurrence of a miR165/166 target sequence in all four cDNA sequences indicated that they might be targets of miR165/166. Identification of the cleavage products of LaHDZ31 and LaHDZ32 in vivo confirmed that they were regulated by miRNA. Their mRNA accumulation patterns during somatic embryogenesis and the effects of 1-N-naphthylphthalamic acid (NPA) on their transcript levels and somatic embryo maturation were investigated. The results showed that the four genes had higher transcript levels at mature stages than at the proliferation stage, and that NPA treatment down-regulated the mRNA abundance of LaHDZ31, 32 and 33 at cotyledonary embryo stages, but had no effect on the mRNA abundance of LaHDZ34. We concluded that these four members of Larix HD-ZIP III family might participate in polar auxin transport and the development of somatic embryos, providing new insights into the regulatory mechanisms of somatic embryogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Influence of the in vitro environment on the germination of somatic embryos of Coffea arabica L. cv. 'Caturra rojo' and Clematis tangutica K.

    Directory of Open Access Journals (Sweden)

    Raúl Barbon

    2017-07-01

    Full Text Available The in vitro environment is a factor that in recent years has begun to investigate, because gases such as oxygen, carbon dioxide and ethylene play an important role in the morphogenesis of somatic embryos and their development in plants. The objective of this work was to determine the effect of the CO2 on the germination of coffee somatic embryos (Coffea arabica L. cv. 'Caturra rojo' and clematis (Clematis tangutica K.. Three gas mixtures composed of CO2 concentrations (2.5, 5.0 and 10.0% combined with 21% O2 and two controls (passive exchange and forced ventilation were used. A positive effect of CO2 on the germination of somatic embryos in the torpedo stage in coffee and clematis was obtained, because in the treatments with passive exchange, where there was CO2 accumulation, germination of the somatic embryos was superior to the treatments with Forced ventilation. With 2.5% and 5.0% CO2, the germination process is stimulated while with 10.0% CO2 there is an inhibition of germination with the appearance of malformations and hyperhydricity.   Keywords: gaseous atmosphere, carbon dioxide, somatic embryogenesis, secondary embryogenesis, hyperhydricity

  7. Study on the presence and influence of phenolic compounds in callogenesis and somatic embryo development of cocoa (Theobroma cacao L..

    Directory of Open Access Journals (Sweden)

    Sulistyani Pancaningtyas

    2015-04-01

    Full Text Available Cocoa (Theobroma cacao L. like most tropical trees is recalcitrant in tissue culture. Somatic embryogenesis is generally efficient micropropagation technique to multiply elite material. However, Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. One of the factors often considered as a component of in vitro recalsitrance is a high phenolic content and oxidation of these compounds. In cocoa tissue culture accumulate large amounts of poliphenolics compounds which probably impair further development. This study was conducted to investigate the composition of phenolic compounds in cocoa flower and leaves, and their changes troughout the somatic embryogenesis process. Calli were induced in cacao floral and leaves explants on a half-strenght Murashige and Skoog medium containing 30 g/L Glucose and combination of 2,4 dichlorophenoxyacetic acid (2,4 D with kinetin (kin. Total polyphenol content was observed on Sulawesi 1 cocoa clone. Embryogenic and non-embryogenic callus were also compared. The percentage of callus production from flower tissue is 85%, percentage of embryogenic callus 40 %, although the percentage of somatic embryo production from embryogenic callus callus is 70%. The conservation of callus into somatic embryos followed by decline in phenol content and an increase in peroxidase. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. So that, phenolic compound can influence the production of calli and an absence the phenolic compound can enhance production of somatic embryo.

  8. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig

    2007-01-01

    The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell...

  9. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    International Nuclear Information System (INIS)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

    2014-01-01

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further

  10. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  11. Study on the presence and influence of phenolic compounds in callogenesis and somatic embryo development of cocoa (Theobroma cacao L..

    Directory of Open Access Journals (Sweden)

    Sulistyani Pancaningtyas

    2015-03-01

    Full Text Available Cocoa (Theobroma cacao L. like most tropical trees is recalcitrant in tissue culture. Somatic embryogenesis is generally efficient micropropagation technique to multiply elite material. However, Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. One of the factors often considered as a component of in vitro recalsitrance is a high phenolic content and oxidation of these compounds. In cocoa tissue culture accumulate large amounts of poliphenolics compounds which probably impair further development. This study was conducted to investigate the composition of phenolic compounds in cocoa flower and leaves, and their changes troughout the somatic embryogenesis process. Calli were induced in cacao floral and leaves explants on a half-strenght Murashige and Skoog medium containing 30 g/L Glucose and combination of 2,4 dichlorophenoxyacetic acid (2,4 D with kinetin (kin. Total polyphenol content was observed on Sulawesi 1 cocoa clone. Embryogenic and non-embryogenic callus were also compared. The percentage of callus production from flower tissue is 85%, percentage of embryogenic callus 40 %, although  the percentage of somatic embryo production from embryogenic callus callus is 70%. The conservation of callus into somatic embryos followed by decline in phenol content and an increase in peroxidase. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. So that, phenolic compound can influence the production of calli and an absence the phenolic compound can enhance production of somatic embryo.Kata kunci: Theobroma cacao L., polifenol, embrio somatik, kalus, flavonoid, katekin, in vitro recalcitance

  12. Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent

    Directory of Open Access Journals (Sweden)

    Kwanyuen Prachuab

    2009-11-01

    Full Text Available Abstract Background In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin. Results When tested against different alternate selection agents our studies show that 0.16 μg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl-L-cysteine (AEC and the acetolactate synthase (ALS inhibitors Exceed® and Synchrony® both at 150 μg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS from E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed. Conclusion Genetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate (Roundup®, AEC and the ALS inhibitors Exceed® and Synchrony® against different tissues of soybean

  13. Direct somatic embryogenesis in Swietenia macrophylla King

    Directory of Open Access Journals (Sweden)

    Raúl Collado

    2006-04-01

    Full Text Available Swietenia macrophylla King is difficult to be propagated by tissue culture and there is not an efficient system via organogenesis, due to problems of microbial contamination, phenolic oxidation and death of tissue in the phase of in vitro establishment of explants. In order to establish a protocol for obtaining somatic embryos, zygotic embryos were used as initial plant material. Three combinations of 2,4-D with kinetin were studied, to obtain the formation of somatic embryos. After six weeks of culture, the number of explants with high and low somatic embryogenesis frequency were determined. So that the somatic embryos in globular stage reach the final stages of torpedo and cotyledonal, these were placed in three treatments with 6-BAP (0.2, 0.4 y 0.6 mg.l-1. The number of somatic embryos that reached the torpedo and cotyledonal stages were evaluated after 30 days of culture. Results demonstrated that direct somatic embryogenesis from immature zygotic embryos is obtained in the culture medium composed by MS salts with 4.0 mg.l-1 of 2,4-D and 1.0 mg.l-1 of kinetin. Higher percentage of somatic embryos in cotiledonal stage (91.7 %, was obtained with 0.4 mg.l-1 of 6-BAP. Key word: forestry, growth regulator, mahogany, somatic embryo, tissue culture

  14. Hemoglobin promotes somatic embryogenesis in peanut cultures.

    Science.gov (United States)

    Jayabalan, N; Anthony, P; Davey, M R; Power, J B; Lowe, K C

    2004-02-01

    Critical parameters influencing somatic embryogenesis include growth regulators and oxygen supply. Consequently, the present investigation has focused on optimization of a somatic embryogenic system for peanut (Arachis hypogaea L.) through media supplementation with the auxin, picloram. The latter at 30 mg L(-1) was optimal for inducing regeneration of somatic embryos from cultured explants of zygotic embryos. In contrast, somatic embryogenesis did not occur in the absence of this growth regulator. An assessment has also been made of the beneficial effect on somatic embryogenesis and plant regeneration of the commercial hemoglobin (Hb) solution, Erythrogen. Hemoglobin at 1:50 and 1:100 (v:v) stimulated increases in mean fresh weight (up to a maximum of 57% over control), mean number of explants producing somatic embryos (15%) and mean number of somatic embryos per explant (29%).

  15. Somatic embryo-like structures of strawberry regenerated in vitro on media supplemented with 2,4-D and BAP.

    Science.gov (United States)

    Omar, Genesia F; Mohamed, Fouad H; Haensch, Klaus-Thomas; Sarg, Sawsan H; Morsey, Mohamed M

    2013-09-01

    Somatic embryo-like structures (SELS) were produced in vitro from leaf disk and petiole explants of two cultivars of strawberry (Fragaria x ananassa Duch) on Murashige and Skoog medium with different concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and sucrose to check the embryonic nature of these structures histologically. A large number of SELS could be regenerated in both cultivars on media with 2-4 mg L(-1) 2,4-D in combination with 0.5 -1 mg L(-1) BAP and 50 g x L(-1) sucrose. Histological examination of SELS revealed the absence of a root pole. Therefore these structures cannot be strictly classified as somatic embryos. The SELS formed under the tested culture conditions represent malformed shoot-like and leaf-like structures. The importance of these results for the propagation of strawberries via somatic embryogenesis is discussed.

  16. Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

    Directory of Open Access Journals (Sweden)

    Lin Yuan

    2014-11-01

    Full Text Available Cloned pigs generated by somatic cell nuclear transfer (SCNT show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP. q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.

  17. Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

    Science.gov (United States)

    Yuan, Lin; Wang, Anfeng; Yao, Chaogang; Huang, Yongye; Duan, Feifei; Lv, Qinyan; Wang, Dongxu; Ouyang, Hongsheng; Li, Zhanjun; Lai, Liangxue

    2014-01-01

    Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos. PMID:25429426

  18. In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

    Science.gov (United States)

    Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

    2010-02-18

    The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

  19. Progress towards initiation of somatic embryogenesis from differentiated tissues of radiata pine (Pinus radiata D. Don) using cotyledonary embryos

    DEFF Research Database (Denmark)

    Find, Jens Iver; Hargreaves, Cathy L.; Reeves, Catherine B.

    2014-01-01

    of dissected embryos and a modified Litvay medium, Glitz, was best. This combination gave the highest rate of initiation, and it was possible to initiate somatic embryogenesis (SE) from differentiated cells in the epicotyledonary region of postcotyledonary zygotic embryos from the two tested families...... with an average initiation rate of approximately 24% and 7% from stage five and six embryos, respectively. This is different from established initiation protocols of embryogenic cultures in radiata pine, which has traditionally been based on embryo rescue and continued proliferation of immature zygotic embryos....... A further implication of initiation of SE from excised post-cotyledonary embryos was that the period of initiation of embryogenic cultures was extended from 4 to 12 wk....

  20. Effect of irradiation and colchicine on callus and somatic embryo formation in cassava (Manihot esculenta Crantz)

    International Nuclear Information System (INIS)

    Dzimega, D. A.

    2012-06-01

    A study was conducted to assess the mutagenic effect of gamma radiation on sprouting and height in four local cassava accessions. The four cassava accessions were assessed for their callus induction and somatic embryo formation ability from leaf lobes from gamma irradiated stakes as well as colchicine treated leaf lobes on different concentrations of plant growth regulators, incorporated into Murashige and skoog, (1962) (MS) basal medium. The cassava accessions were irradiated at 0, 32, 35, 45 and 50 Gy and planted in pots filled with loamy soil. The height of the shoots was measured with rule after sprouting. The leaf lobes were collected from the shoots and cultured on MS medium supplemented with 8 mg/l 2, 4-D and 16 mg/l Picloram. Another set of leaf lobes were treated with 0.0, 0.05, 0.1, 0.2, 0.25 g/l colchicine for one hour and thereafter culture on MS medium supplemented with 8 mg/l 2,4-D and 16 mg/l Picloram as described above. Callus induction from leaf lobes in 45 and 50 Gy were significantly (p≤0.05) affected by the irradiation. On the other hand, Callus induction from leaf lobes in 0.1-0.25 g/l colchicine were significantly (p≤0.05) affected by the mutagenic treatment whereas callus induced from leaf lobes in 0.05 g/l colchicine was not significantly (p≤0.05) affected. Callus induced on 8 mg/l 2, 4-D and 16 mg/l picloram gave the best response in Ankrah and all control tested while Tomfa recorded the least. Colchicine at a concentration of 0.05 g/l and radiation dose of 32 Gy treatments gave the best response of callusing. Callus induction decreased with increasing colchicine concentration and gamma irradiation. Callus derived from irradiated and colchicine leaf lobes appeared soft but friable and tiny, compact, respectively, predominately with creamy to brown colouration. Calli obtained were sub-cultures on embryo regeneration medium consisting of MS supplemented with 0.01mg/1 NAA and o.1 mg/1 BAP. There was no plantlet regeneration. Instead

  1. Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Peura, T.T.; Hartwich, K.M.

    2005-01-01

    The processes of cellular differentiation were studied in somatic cell nuvlear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos...... were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients...

  2. Somatic embryogenesis in cassava: A tool for mutation breeding

    International Nuclear Information System (INIS)

    Lee, K.S.; Duren, M. Van; Morpurgo, R.

    1997-01-01

    Cassava is an important food and livestock feed crop. The effect of gamma radiation on somatic embryogenesis and plant regeneration in cassava clones of African origin was investigated. Explants from young leaves of cassava were cultured on MS medium, supplemented with 18.1 mM 2,4-D and 2 mM CuSO4, solidified with 0.3% Phytagel. Compact and friable calli were observed after 10-15 days of explant culture in dark, which produced somatic embryos in all but one clone. The somatic embryos showed morphological aberrations, such as fused cotyledons, lack of meristematic tip, epicotyl elongation, and had low germination rate; desiccation of embryos increased germination. Histological study showed that the somatic embryos were of multicellular origin. Leaf explants were irradiated with doses between 4 to 38 Gy of gamma rays, and cultured on somatic embryo induction medium. In addition, somatic embryos were irradiated with gamma ray doses from 10 to 18 Gy, and analyzed for germination. LD 50 for embryogenic response of leaf-explants was at around 20 Gy, while that for somatic embryo germination was ca. 10 Gy. (author). 7 refs, 2 tabs

  3. Somatic embryogenesis in cassava: A tool for mutation breeding

    Energy Technology Data Exchange (ETDEWEB)

    Lee, K S; Duren, M Van; Morpurgo, R [Agriculture and Biotechnology Laboratory, International Atomic Energy Agency, Seibersdorf (Austria)

    1997-07-01

    Cassava is an important food and livestock feed crop. The effect of gamma radiation on somatic embryogenesis and plant regeneration in cassava clones of African origin was investigated. Explants from young leaves of cassava were cultured on MS medium, supplemented with 18.1 mM 2,4-D and 2 mM CuSO4, solidified with 0.3% Phytagel. Compact and friable calli were observed after 10-15 days of explant culture in dark, which produced somatic embryos in all but one clone. The somatic embryos showed morphological aberrations, such as fused cotyledons, lack of meristematic tip, epicotyl elongation, and had low germination rate; desiccation of embryos increased germination. Histological study showed that the somatic embryos were of multicellular origin. Leaf explants were irradiated with doses between 4 to 38 Gy of gamma rays, and cultured on somatic embryo induction medium. In addition, somatic embryos were irradiated with gamma ray doses from 10 to 18 Gy, and analyzed for germination. LD{sub 50} for embryogenic response of leaf-explants was at around 20 Gy, while that for somatic embryo germination was ca. 10 Gy. (author). 7 refs, 2 tabs.

  4. Somatic embryogenesis in ferns: a new experimental system.

    Science.gov (United States)

    Mikuła, Anna; Pożoga, Mariusz; Tomiczak, Karolina; Rybczyński, Jan J

    2015-05-01

    Somatic embryogenesis has never been reported in ferns. The study showed that it is much easier to evoke the acquisition and expression of embryogenic competence in ferns than in spermatophytes. We discovered that the tree fern Cyathea delgadii offers an effective model for the reproducible and rapid formation of somatic embryos on hormone-free medium. Our study provides cyto-morphological evidence for the single cell origin and development of somatic embryos. Somatic embryogenesis (SE) in both primary and secondary explants was induced on half-strength micro- and macro-nutrients Murashige and Skoog medium without the application of exogenous plant growth regulators, in darkness. The early stage of SE was characterized by sequential perpendicular cell divisions of an individual epidermal cell of etiolated stipe explant. These resulted in the formation of a linear pro-embryo. Later their development resembled that of the zygotic embryo. We defined three morphogenetic stages of fern somatic embryo development: linear, early and late embryonic leaf stage. The transition from somatic embryo to juvenile sporophyte was quick and proceeded without interruption caused by dormancy. Following 9 weeks of culture the efficiency of somatic embryogenesis reached 12-13 embryos per responding explant. Spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process sevenfold in 10-month-long culture, occurred without subculturing. The tendency for C. delgadii to propagate by SE in vitro makes this species an excellent model for studies relating to asexual embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.

  5. Germination of somatic embryos of Psidium guajava L. cv. Cuban Red Dwarf EEA 18-40 in temporary immersion systems

    Directory of Open Access Journals (Sweden)

    Jorge Vilchez Perozo

    2001-04-01

    Full Text Available Somatic embryo germination of Psidium guajava L. cv. Cuban Red Dwarf EEA 18-40 in temporary immersion systems (TIS, in which somatic embryos were cultured in the heart-torpedo stage in MS mediun at mayor half strength salt and suplemented with: 0.25 mg.l-1 of 6-bencilaminopurine (6-BAP, 10 mg.l-1 of Biobras-6 (analogous of brasinoesteriode and 20 g.l-1 of sucrose. As control was used solid cultivation medium (2.5 g.l-1 Gellan gum, Spectrum® of same composition to the one used in the TIS. The variables germination percentage and fresh weight were evaluated statistically. After ten weeks of cultivation the largest values in germination percentage (91.04% and fresh weight (1.22 g were obtained in the TIS, being statistically different to those obtained in solid medium (9.79% and 1.03 g, respectively. Key words: in vitro plant, guayaba, regeneration, RITA®,somatic embryogenesis

  6. Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree.

    Science.gov (United States)

    Maximova, Siela N; Florez, Sergio; Shen, Xiangling; Niemenak, Nicolas; Zhang, Yufan; Curtis, Wayne; Guiltinan, Mark J

    2014-07-16

    Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray. The expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. Large numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation. These differences in gene

  7. The anti-actin drugs latrunculin and cytochalasin affect the maturation of spruce somatic embryos in different ways

    Czech Academy of Sciences Publication Activity Database

    Vondráková, Zuzana; Eliášová, Kateřina; Vágner, Martin

    2014-01-01

    Roč. 221, MAY 2014 (2014), s. 90-99 ISSN 0168-9452 R&D Projects: GA MŠk 7AMB12FR017 Institutional support: RVO:61389030 Keywords : Somatic embryo genesis * Cytoskeleton * Actin Subject RIV: GK - Forestry Impact factor: 3.607, year: 2014

  8. PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Jin, Long; Zhu, Hai-Ying; Guo, Qing; Li, Xiao-Chen; Zhang, Yu-Chen; Zhang, Guang-Lei; Xing, Xiao-Xu; Xuan, Mei-Fu; Luo, Qi-Rong; Yin, Xi-Jun; Kang, Jin-Dan

    2016-09-01

    To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12. Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed. PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

  9. Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment

    Directory of Open Access Journals (Sweden)

    Yali Liu

    2016-11-01

    Full Text Available Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H2 treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA libraries to identify global transcriptome changes at different time points during H2 treatment of larch pro-embryogenic masses (PEMs. A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H2 during somatic embryogenesis.

  10. High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos

    DEFF Research Database (Denmark)

    Li, J.; Østrup, Olga; Villemoes, Klaus

    2008-01-01

    Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim...... transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development....

  11. In-vitro morphogenesis of corn (Zea mays L.) : I. Differentiation of multiple shoot clumps and somatic embryos from shoot tips.

    Science.gov (United States)

    Zhong, H; Srinivasan, C; Sticklen, M B

    1992-07-01

    In-vitro methods have been developed to regenerate clumps of multiple shoots and somatic embryos at high frequency from shoot tips of aseptically-grown seedlings as well as from shoot apices of precociously-germinated immature zygotic embryos of corn (Zea mays L.). About 500 shoots were produced from a shoot tip after eight weeks of culture (primary culture and one subculture of four weeks) in darkness on Murashige and Skoog basal medium (MS) supplemented with 500 mg/L casein hydrolysate (CH) and 9 μM N(6)-benzyladenine (BA). In this medium, shoots formed in shoot tips as tightly packed "multiple shoot clumps" (MSC), which were composed of some axillary shoots and many adventitious shoots. When the shoot tips were cultured on MS medium containing 500 mg/L CH, 9 μM BA and 2.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D), most of the shoots in the clumps were adventitious in origin. Similar shoot tips cultured on MS medium containing 500 mg/L CH, 4.5 μM BA and 2.25 μM 2,4-D regenerated many somatic embryos within eight weeks of culture. Somatic embryos were produced either directly from the shoot apical meristems or from calli derived from the shoots apices. Both the MSC and the embryos produced normal shoots on MS medium containing 2.25 μM BA and 1.8 μM indole-3-butyric acid (IBA). These shoots were rooted on MS medium containing 3.6 μM IBA, and fertile corn plants were grown in the greenhouse. The sweet-corn genotype, Honey N Pearl, was used for the experiments described above, but shoot-tip cultures from all of 19 other corn genotypes tested also formed MSC on MS medium containing 500 mg/L CH and 9 μM BA.

  12. Somatic Embryogenesis Induction and Plant Regeneration in Strawberry Tree (Arbutus unedo L.).

    Science.gov (United States)

    Martins, João F; Correia, Sandra I; Canhoto, Jorge M

    2016-01-01

    Somatic embryogenesis is a powerful tool both for cloning and studies of genetic transformation and embryo development. Most protocols for somatic embryogenesis induction start from zygotic embryos or embryonic-derived tissues which do not allow the propagation of elite trees. In the present study, a reliable protocol for somatic embryogenesis induction from adult trees of strawberry tree is described. Leaves from in vitro proliferating shoots were used to induce somatic embryo formation on a medium containing an auxin and a cytokinin. Somatic embryos germinated in a plant growth regulator-free medium.

  13. DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome...

  14. DNA methylation in porcine preimplantation embryos developed in-vivo or produced by in-vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome...

  15. Studies on Somatic Embryogenesis in Sweetpotato

    Science.gov (United States)

    Bennett, J. Rasheed; Prakash, C. S.

    1997-01-01

    The purpose of this study was to improve the somatic embryo (SE) system for plant production of sweetpotato Ipomoea batatas L.(Lam)l. Explants isolated from SE-derived sweet potato plants were compared with control (non SE-derived) plants for their competency for SE production. Leaf explants were cultured on Murashige-Skoog (MS) medium with 2,4-dichlorophenoxy acetic acid (0.2 mg/L) and 6-benzylaminopurine (2.5 mg/L) for 2 weeks in darkness and transferred to MS medium with abscisic acid (2.5 Explants isolated from those plants developed through somatic embryo-genesis produced new somatic embryos rapidly and in higher frequency than those isolated from control plants. They also appeared to grow faster in tissue culture than the control plants. Current studies in the laboratory are examining whether plants derived from a cyclical embryogenesis system (five cycles) would have any further positive impact on the rapidity and frequency of somatic embryo development. More detailed studies using electron microscopy are expected to show the point of origin of the embryos and to allow determination of their quality throughout the cyclical process. This study may facilitate improved plant micropropagation, gene transfer and germplasm conservation in sweet potato.

  16. Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses.

    Science.gov (United States)

    Morel, Alexandre; Trontin, Jean-François; Corbineau, Françoise; Lomenech, Anne-Marie; Beaufour, Martine; Reymond, Isabelle; Le Metté, Claire; Ader, Kevin; Harvengt, Luc; Cadene, Martine; Label, Philippe; Teyssier, Caroline; Lelu-Walter, Marie-Anne

    2014-11-01

    Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

  17. Efficiency of porcine somatic cell nuclear transfer – a retrospective study of factors related to embryo recipient and embryos transferred

    Directory of Open Access Journals (Sweden)

    Yongye Huang

    2013-10-01

    The successful generation of pigs via somatic cell nuclear transfer depends on reducing risk factors in several aspects. To provide an overview of some influencing factors related to embryo transfer, the follow-up data related to cloned pig production collected in our laboratory was examined. (i Spring showed a higher full-term pregnancy rate compared with winter (33.6% vs 18.6%, P = 0.006. Furthermore, a regression equation can be drawn between full-term pregnancy numbers and pregnancy numbers in different months (y = 0.692x−3.326. (ii There were no significant differences detected in the number of transferred embryos between surrogate sows exhibiting full-term development compared to those that did not. (iii Non-ovulating surrogate sows presented a higher percentage of full-term pregnancies compared with ovulating sows (32.0% vs 17.5%, P = 0.004; respectively. (iv Abortion was most likely to take place between Day 27 to Day 34. (v Based on Life Table Survival Analysis, delivery in normally fertilized and surrogate sows is expected to be completed before Day 117 or Day 125, respectively. Additionally, the length of pregnancy in surrogate sows was negatively correlated with the average litter size, which was not found for normally fertilized sows. In conclusion, performing embryo transfer in appropriate seasons, improving the quality of embryos transferred, optimizing the timing of embryo transfer, limiting the occurrence of abortion, combined with ameliorating the management of delivery, is expected to result in the harvest of a great number of surviving cloned piglets.

  18. Influence of Growth Regulators on Callogenesis and Somatic Embryo Development in Date Palm (Phoenix dactylifera L. Sahelian Cultivars

    Directory of Open Access Journals (Sweden)

    Djibril Sané

    2012-01-01

    Full Text Available This study provides a physiological analysis of somatic embryogenesis in four elite cultivars of date palms: Ahmar, Amsekhsi, Tijib, and Amaside, from the initial callogenesis to establishment and proliferation of embryogenic suspension cultures. Somatic embryos development and in vitro plants rooting were also studied. For each step, auxins and cytokinins concentrations were optimised. The primary callogenesis from leaf explants of seedlings appeared highly dependent on genotype. Ahmar (80% and Amsekhsi (76% appeared highly callogenic, whereas Tijib (10% and Amaside (2% produced low amounts of calluses. 2,4-Dichlorophenoxyacetic acid appeared favorable to the induction of primary callogenesis and its effect was enhanced by the addition of benzyl adenine or adenine sulfate. Secondary friable calli obtained from chopped granular calli were used to initiate embryogenic cell suspensions in media supplied with 2,4-dichlorophenoxyacetic acid. Suspension cultures showed a growth rate of fourfold after four subcultures in presence of 2,4-dichlorophenoxyacetic acid 2 mg/L. Our results showed that a seven-day transitory treatment with benzyl adenine 0,5 mg/L was necessary to optimize embryos development. Naphthalene acetic acid induced the development of primary orthogravitropic roots during embryos germination. The comparison with cytofluorometry of nuclear DNA amounts showed no significant difference in ploidy level between regenerated plants and seedlings.

  19. Mature seeds for in vitro sanitation of the Grapevine leafroll associated virus (GLRaV-1andGLRaV-3 from grape (Vitis vinifera L.

    Directory of Open Access Journals (Sweden)

    Rosa Peiró

    2015-06-01

    Full Text Available The conservation of old grapevine varieties is important since they are adapted to specific climate conditions and may carry genes interesting to breeders. As virus infection is common in grapevine varieties, the use of virus free materials is of great importance. In this work, we used somatic embryogenesis for the sanitation of GLRaV-1 and GLRaV-3 viruses that were found after analyzing the putative presence of the five most common, economically important grape viruses by real-time multiplex RT-PCR in the old cultivar “Grumet Negre”. Unopened and opened inflorescences, fecundated ovaries, and, also, mature seeds were used as starting explants. Explants were cultured on plates with two embryogenesis induction media (Nitsch & McCown Woody plant medium that contained the growth regulator thidiazuron and differed in their salt and vitamin compositions. One half of each kind of explant was cut prior to being cultured. After five months of culture, embryos had only developed from seeds that were cut previous to sowing. To the best of our knowledge, this is the first time that mature seeds have been used for inducing embryogenesis in grape. A total of 42% of the embryos transferred to tubes for germination regenerated into normal plantlets. The absence of both the GLRaV-1 and GLRaV-3 viruses in all regenerated plants was confirmed by real-time uniplex RT-PCR. So, this protocol can be used for sanitation and also for micropropagation.

  20. Mature seeds for in vitro sanitation of the Grapevine leafroll associated virus (GLRaV-1 and GLRaV-3) from grape (Vitis vinifera L.)

    Energy Technology Data Exchange (ETDEWEB)

    Peiró, R.; Gammoudi, N.; Yuste, A.; Olmos, A.; Gisbert, C.

    2015-07-01

    The conservation of old grapevine varieties is important since they are adapted to specific climate conditions and may carry genes interesting to breeders. As virus infection is common in grapevine varieties, the use of virus free materials is of great importance. In this work, we used somatic embryogenesis for the sanitation of GLRaV-1 and GLRaV-3 viruses that were found after analyzing the putative presence of the five most common, economically important grape viruses by real-time multiplex RT-PCR in the old cultivar “Grumet Negre”. Unopened and opened inflorescences, fecundated ovaries, and, also, mature seeds were used as starting explants. Explants were cultured on plates with two embryogenesis induction media (Nitsch & McCown Woody plant medium) that contained the growth regulator thidiazuron and differed in their salt and vitamin compositions. One half of each kind of explant was cut prior to being cultured. After five months of culture, embryos had only developed from seeds that were cut previous to sowing. To the best of our knowledge, this is the first time that mature seeds have been used for inducing embryogenesis in grape. A total of 42% of the embryos transferred to tubes for germination regenerated into normal plantlets. The absence of both the GLRaV-1 and GLRaV-3 viruses in all regenerated plants was confirmed by real-time uniplex RT-PCR. So, this protocol can be used for sanitation and also for micropropagation. (Author)

  1. Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

    Science.gov (United States)

    Inoue, Kimiko; Ogura, Atsuo

    2013-01-01

    The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (Pcloning efficiency using SCNT. PMID:24146866

  2. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

    2011-01-01

    Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-c...... in sequence-specific interactions between the ooplasm and chromatin of another genus. In conclusion, the results demonstrate a possible reason why the intergeneric SCNT embryos never reached the full term....

  3. Comparative proteomic analysis of off-type and normal phenotype somatic plantlets derived from somatic embryos of Feijoa (Acca sellowiana (O. Berg) Burret).

    Science.gov (United States)

    Fraga, Hugo Pacheco de Freitas; Agapito-Tenfen, Sarah Zanon; Caprestano, Clarissa Alves; Nodari, Rubens Onofre; Guerra, Miguel Pedro

    2013-09-01

    Morphological disorders in a relevant portion of emerged somatic embryos have been a limiting factor in the true-to-type plantlet formation in Acca sellowiana. In this sense, the present study undertook a comparison between normal phenotype and off-type somatic plantlets protein profiles by means of the 2-D DIGE proteomics approach. Off-type and normal phenotype somatic plantlets obtained at 10 and 20 days conversion were evaluated. Results indicated 12 exclusive spots between normal and off-type plantlets at 10 days conversion, and 17 exclusive spots at 20 days conversion. Also at 20 days conversion, 4 spots were differentially expressed, up- or down-regulated. Two proteins related to carbohydrate metabolism were only expressed in off-types at 10 days conversion, suggesting a more active respiratory pathway. A vicilin-like storage protein was only found in off-types at 20 days conversion, indicating that plantlets may present an abnormality in the mobilization of storage compounds, causing reduced vigor in the development of derived plantlets. The presence of heat shock proteins were only observed during formation of normal phenotype somatic plantlets, indicating that these proteins may be involved in normal morphogenesis of plantlets formed. These new findings shed light on possible genetic or epigenetic mechanisms governing A. sellowiana morphogenesis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  4. Intrinsic and extrinsic molecular determinants or modulators for epigenetic remodeling and reprogramming of somatic cell-derived genome in mammalian nuclear-transferred oocytes and resultant embryos.

    Science.gov (United States)

    Samiec, M; Skrzyszowska, M

    2018-03-01

    The efficiency of somatic cell cloning in mammals remains disappointingly low. Incomplete and aberrant reprogramming of epigenetic memory of somatic cell nuclei in preimplantation nuclear- transferred (NT) embryos is one of the most important factors that limit the cloning effectiveness. The extent of epigenetic genome-wide alterations, involving histone or DNA methylation and histone deacetylation, that are mediated by histone-lysine methyltransferases (HMTs) or DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) can be modulated/reversed via exogenous inhibitors of these enzymes throughout in vitro culture of nuclear donor cells, nuclear recipient oocytes and/or cloned embryos. The use of the artificial modifiers of epigenomically-conditioned gene expression leads to inhibition of both chromatin condensation and transcriptional silencing the genomic DNA of somatic cells that provide a source of nuclear donors for reconstruction of enucleated oocytes and generation of cloned embryos. The onset of chromatin decondensation and gene transcriptional activity is evoked both through specific/selective inactivating HMTs by BIX-01294 and through non-specific/non-selective blocking the activity of either DNMTs by 5-aza-2'-deoxycytidine, zebularine, S-adenosylhomocysteine or HDACs by trichostatin A, valproic acid, scriptaid, oxamflatin, sodium butyrate, m-carboxycinnamic acid bishydroxamide, panobinostat, abexinostat, quisinostat, dacinostat, belinostat and psammaplin A. Epigenomic modulation of nuclear donor cells, nuclear recipient cells and/or cloned embryos may facilitate and accelerate the reprogrammability for gene expression of donor cell nuclei that have been transplanted into a host ooplasm and subsequently underwent dedifferentiating and re-establishing the epigenetically dependent status of their transcriptional activity during pre- and postimplantation development of NT embryos. Nevertheless, a comprehensive additional work is necessary to determine

  5. Optimization of somatic embryogenesis induction in Iranian melon ...

    African Journals Online (AJOL)

    Jane

    2011-07-11

    Jul 11, 2011 ... embryo induction and the combination of 0.1 mg/l BA and 5 mg/l 2,4-D had significant effect on somatic ... such as genotype, growth regulator, explant and culture ... different stages of somatic embryos development. (globular ...

  6. Induction of somatic embryogenesis by polyethylene glycol and ...

    African Journals Online (AJOL)

    VIJI

    2012-06-05

    Jun 5, 2012 ... supplemented with 2,4-D (2 µM), abscisic acid (3 µM) and glutamine (0.03 mM). The somatic embryos ... essential amino acids of seed proteins, shortened vegetative ... or somatic embryo maturation in diverse plants such as.

  7. Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

    Science.gov (United States)

    Morel, Alexandre; Teyssier, Caroline; Trontin, Jean-François; Eliášová, Kateřina; Pešek, Bedřich; Beaufour, Martine; Morabito, Domenico; Boizot, Nathalie; Le Metté, Claire; Belal-Bessai, Leila; Reymond, Isabelle; Harvengt, Luc; Cadene, Martine; Corbineau, Françoise; Vágner, Martin; Label, Philippe; Lelu-Walter, Marie-Anne

    2014-09-01

    Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets. © 2014 Scandinavian Plant Physiology Society.

  8. Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.).

    Science.gov (United States)

    Adu-Gyamfi, Raphael; Wetten, Andy; Marcelino Rodríguez López, Carlos

    2016-01-01

    While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation.

  9. DOT1L inhibitor improves early development of porcine somatic cell nuclear transfer embryos

    DEFF Research Database (Denmark)

    Tao, Jia; Zhang, Yu; Zuo, Xiaoyuan

    2017-01-01

    Incomplete epigenetic reprogramming of the genome of donor cells causes poor early and full-term developmental efficiency of somatic cell nuclear transfer (SCNT) embryos. Previous research indicate that inhibition of the histone H3 K79 methyltransferase DOT1L, using a selective pharmacological...... inhibitor EPZ004777 (EPZ), significantly improved reprogramming efficiency during the generation of mouse induced pluripotent stem cells. However, the roles of DOT1L in porcine nuclear transfer-mediated cellular reprogramming are not yet known. Here we showed that DOT1L inhibition via 0.5 nM EPZ treatment...

  10. Multi-instrumental Investigation of Affecting of Early Somatic Embryos of Spruce by Cadmium(II) and Lead(II) Ions

    Czech Academy of Sciences Publication Activity Database

    Šupálková, V.; Petřek, J.; Baloun, J.; Adam, V.; Bartušek, Karel; Trnková, L.; Beklová, M.; Diopan, V.; Havel, L.; Kizek, R.

    2007-01-01

    Roč. 7, 5 (2007), s. 743-759 ISSN 1424-8220 R&D Projects: GA ČR(CZ) GA102/07/0389 Institutional research plan: CEZ:AV0Z20650511 Keywords : early somatic embryos of spruces * glutathione * heavy metals * plant cells * esterase activity * fluorescence detection Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 1.573, year: 2007

  11. Development of direct somatic embryogenesis and regeneration on citrus sinesis

    International Nuclear Information System (INIS)

    Chong Saw Peng; Alvina Lindsay Mijen; Rusli Ibrahim

    2004-01-01

    The plant regeneration processes in Citrus sinensis involves direct somatic embryogenesis. Culture medium used was MS basal supplemented with 50 mg/L sucrose, 0.27% agar and 0.1% vitamin at pH 5.8. Sucrose is the major carbon source for the induction of somatic embryo and also the maturation and germination of somatic embryo. (Author)

  12. Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br.

    Science.gov (United States)

    Wang, Y-H; Bhalla, P L

    2004-01-01

    Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium-from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and alpha-naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.

  13. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    Science.gov (United States)

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  14. Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis.

    Science.gov (United States)

    Marum, Liliana; Loureiro, João; Rodriguez, Eleazar; Santos, Conceição; Oliveira, M Margarida; Miguel, Célia

    2009-09-25

    An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

  15. Effect of light quality on somatic embryogenesis of quince leaves

    International Nuclear Information System (INIS)

    D'Onofrio, C.; Morini, S.; Bellocchi, G.

    1998-01-01

    The effect of light quality on somatic embryogenesis in quince BA 29 was investigated. 2,4-D induced leaves were exposed for 25 days to the following light quality treatments: dark, far-red, far-red+blue, far-red+red, blue, white, red+blue, red. After a further 20 days of white light exposure, somatic embryo production was recorded. Somatic embryogenesis was highest in cultures subjected to red light treatment, and decreased progressively with the transition to red+blue and to white. Overall, embryogenic competence showed a correlation with photoequilibrium. Phytochrome appeared to be inductive although this effect was adversely influenced by the blue absorbing photoreceptor, in particular at low photoequilibrium. Independently of light treatments applied, somatic embryos frequently showed severe morphological abnormalities. Conversion of somatic embryos to plantlets was not observed. (author)

  16. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  17. Numerical Chromosome Errors in Day 7 Somatic Nuclear Transfer Bovine Blastocysts

    DEFF Research Database (Denmark)

    Booth, Paul J.; VIUFF, Dorte; Tan, Shijian

    2002-01-01

    Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulat......Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona...

  18. Effects of chilling and ABA on [3H]gibberellin A4 metabolism in somatic embryos of grape (Vitis vinifera L. x V. rupestris Scheele)

    International Nuclear Information System (INIS)

    Pearce, D.; Pharis, R.P.; Rajasekaran, K.; Mullins, M.G.

    1987-01-01

    Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. x V. rupestris Scheele). The authors have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with [ 3 H]GA 4 (of high specific activity, 4.81 x 10 19 becquerel per millimole) for 48 hours at 26 0 C. Chilling had little effect on the total amount of free [ 3 H]GA-like metabolites formed during incubation at 26 0 C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble [ 3 H] metabolites formed at 26 0 C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogeneous GA precursors (e.g., GA 12 aldehyde-, kaurene, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with [ 3 H]GA 4 treatment at 26 0 C, reduced the uptake of [ 3 H] GA 4 but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26 0 C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water-soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs)

  19. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  20. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-01-01

    Highlights: → Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. → The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. → A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  1. ABA Represses the Expression of Cell Cycle Genes and May Modulate the Development of Endodormancy in Grapevine Buds

    Directory of Open Access Journals (Sweden)

    Ricardo Vergara

    2017-05-01

    Full Text Available Recently, the plant hormone abscisic acid (ABA has been implicated as a key player in the regulation of endodormancy (ED in grapevine buds (Vitis vinifera L. In this study, we show that in the vine, the expression of genes related to the biosynthesis of ABA (VvNCED1; VvNCED2 and the content of ABA are significantly higher in the latent bud than at the shoot apex, while the expression of an ABA catabolic gene (VvA8H3 showed no significant difference between either organ. A negative correlation between the content of ABA and transcript levels of cell cycle genes (CCG was found in both tissues. This result suggested that ABA may negatively regulate the expression of CCG in meristematic tissues of grapevines. To test this proposition, the effect of ABA on the expression of CCG was analyzed in two meristematic tissues of the vine: somatic embryos and shoot apexes. The results indicated that cell cycle progression is repressed by ABA in both organs, since it down-regulated the expression of genes encoding cyclin-dependent kinases (VvCDKB1, VvCDKB2 and genes encoding cyclins of type A (VvCYCA1, VvCYCA2, VvCYCA3, B (VvCYCB, and D (VvCYCD3.2a and up-regulated the expression of VvICK5, a gene encoding an inhibitor of CDKs. During ED, the content of ABA increased, and the expression of CCG decreased. Moreover, the dormancy-breaking compound hydrogen cyanamide (HC reduced the content of ABA and up-regulated the expression of CCG, this last effect was abolished when HC and ABA were co-applied. Taken together, these results suggest that ABA-mediated repression of CCG transcription may be part of the mechanism through which ABA modulates the development of ED in grapevine buds.

  2. Studies for Somatic Embryogenesis in Sweet Potato

    Science.gov (United States)

    Bennett, J. Rasheed; Prakash, C. S.

    1997-01-01

    The purpose of this study was to improve the somatic embryo (SE) system for plant production of sweet potato (Ipomoea batatas L(Lam)). Explants isolated from SE-derived sweet potato plants were compared with control (non SE-derived) plants for their competency for SE production. Leaf explants were cultured on Murashige-Skoog (MS) medium with 2,4-dichlorophenoxy acetic acid (0.2 mg/L) and 6-benzylaminopurine (2.5 mg/L) for 2 weeks in darkness and transferred to MS medium with abscisic acid (2.5 mg/L). Explants isolated from those plants developed through somatic embryogenesis produced new somatic embryos rapidly and in higher frequency than those isolated from control plants They also appeared to grow faster in tissue culture than the control plants. Current studies in the laboratory are examining whether plants derived from a cyclical embryogenesis system (five cycles) would have any further positive impact on the rapidity and frequency of somatic embryo development. More detailed studies using electron microscopy are expected to show the point of origin of the embryos and to allow determination of their quality throughout the cyclical process. This study may facilitate improved plant micropropagation, gene transfer and germplasm conservation in sweet potato.

  3. Pluripotency maintenance in mouse somatic cell nuclear transfer embryos and its improvement by treatment with the histone deacetylase inhibitor TSA.

    Science.gov (United States)

    Hai, Tang; Hao, Jie; Wang, Liu; Jouneau, Alice; Zhou, Qi

    2011-02-01

    Reprogramming of somatic cells to pluripotency can be achieved by nuclear transfer into enucleated oocytes (SCNT). A key event of this process is the demethylation of the Oct4 gene and its temporally and spatially regulated expression. Different studies have shown that it occurs abnormally in some SCNT embryos. TSA is a histone deacetylase inhibitor known to increase the efficiency of development to term of SCNT embryos, but its impact on the developmental features of SCNT embryos is poorly understood. Here, we have followed the fate of the pluripotent cells within SCNT embryos, from the late blastocyst to the early epiblast prior to gastrulation. Our data show a delay in development correlated with a defect in forming and maintaining a correct number of Oct4 expressing ICM and epiblast cells in SCNT embryos. As a consequence, during the outgrowth phase of embryonic stem cell derivation as well as during diapause in vivo, part of the SCNT blastocysts completely lose their ICM cells. Meanwhile, the others display a correctly reprogrammed ICM compatible with the derivation of ES cells and development of the epiblast. Our data also indicate that TSA favors the establishment of pluripotency in SCNT embryos.

  4. Somatic embryogenesis for efficient micropropagation of guava (Psidium guajava L.).

    Science.gov (United States)

    Akhtar, Nasim

    2013-01-01

    Guava (Psidium guajava L.) is well known for edible fruit, environment friendly pharmaceutical and commercial products for both national and international market. The conventional propagation and in vitro organogenesis do not meet the demand for the good quality planting materials. Somatic embryogenesis for efficient micropropagation of guava (P. guajava L.) has been developed to fill up the gap. Somatic embryogenesis and plantlets regeneration are achieved from 10-week post-anthesis zygotic embryo explants by 8-day inductive treatment with different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) on MS agar medium containing 5% sucrose. Subsequent development and maturation of somatic embryos occur after 8 days on MS basal medium supplemented with 5% sucrose without plant growth regulator. The process of somatic embryogenesis shows the highest relative efficiency in 8-day treatment of zygotic embryo explants with 1.0 mg L(-1) 2,4-D. High efficiency germination of somatic embryos and plantlet regeneration takes place on half strength semisolid MS medium amended with 3% sucrose within 2 weeks of subculture. Somatic plantlets are grown for additional 2 weeks by subculturing in MS liquid growth medium containing 3% sucrose. Well-grown plantlets from liquid medium have survived very well following 2-4 week hardening process. The protocol of somatic embryogenesis is optimized for high efficiency micropropagation of guava species.

  5. Cryopreservation of somatic embryogenic cultures of Pinus pinaster: effects on regrowth and embryo maturation.

    Science.gov (United States)

    Álvarez, José M; Cortizo, Millán; Ordás, Ricardo J

    2012-01-01

    Pinus pinaster is one of the most economically important conifers in the world. Somatic embryogenesis is a powerful tool in breeding programmes because it allows the generation of a great number of different clonal lines from seeds of superior genotypes. Unfortunately, embryogenic competence decreases with the age of cultures. Therefore, it is necessary to have a cryopreservation protocol that ensures a continuous supply of juvenile mass while allowing good maturation and conversion rates into vigorously growing plants. In this work we studied the influence of several cryopreservation parameters, such as cryoprotectant solution and pre-cooling temperature, on embryogenic culture regrowth and embryo maturation. Recovery of rewarmed samples after cryopreservation in a -150 degree C freezer depended on the cooling temperature reached prior to plunging the tubes into liquid nitrogen. As a result, we present an optimised cryopreservation protocol that ensures high recovery and embryo maturation rates. The protocol presented is a simple and fast alternative and enabled successful cryopreservation and recovery of 100 percent of the lines tested. Cryopreserved lines presented the same maturation rates as non-cryopreserved controls.

  6. Polyphenols distributions and reserve substances analysis in cacao somatic embryogenesis

    OpenAIRE

    Adriana María Gallego Rúa; Ana María Henao Ramírez; Aura Inés Urrea Trujillo; Lucía Atehortúa Garcés

    2016-01-01

    ABSTRACTIn order to understand the causes of lack of regeneration in cacao somatic embryos, two cacao varieties with different responses to regeneration potential were described based on their capacity to store different compounds. It is well known that seed reserves play a central role in the regenerative capability of somatic embryos; thus, we followed histochemical changes and reserve fluctuations of proteins, polysaccharides and polyphenols during somatic embryogenesis (SE) in the two cac...

  7. POLYPHENOLS DISTRIBUTION AND RESERVE SUBSTANCES ANALYSIS IN CACAO SOMATIC EMBRYOGENESIS

    OpenAIRE

    GALLEGO RÚA, Adriana María; HENAO RAMÍREZ, Ana María; URREA TRUJILLO, Aura Inés; ATEHORTÚA GARCÉS, Lucía

    2016-01-01

    In order to understand the causes of lack of regeneration in cacao somatic embryos, two cacao varieties with different responses to regeneration potential were described based on their capacity to store different compounds. It is well known that seed reserves play a central role in the regenerative capability of somatic embryos; thus, we followed histochemical changes and reserve fluctuations of proteins, polysaccharides and polyphenols during somatic embryogenesis (SE) in the two cacao varie...

  8. Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis.

    Directory of Open Access Journals (Sweden)

    Angelo Schuabb Heringer

    Full Text Available The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E and non-embryogenic (NE callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L(-1 of activated charcoal (AC. Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L(-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project, including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus.

  9. In vitro somatic embryogenesis and plant regeneration of cassava.

    Science.gov (United States)

    Szabados, L; Hoyos, R; Roca, W

    1987-06-01

    An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4-16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA3, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.

  10. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  11. Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Li, Qian; Wang, Yong-Sheng; Wang, Li-Jun; Zhang, Hui; Li, Rui-Zhe; Cui, Chen-Chen; Li, Wen-Zhe; Zhang, Yong; Jin, Ya-Ping

    2014-08-01

    Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, pvitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, pVitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

  12. Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

    Directory of Open Access Journals (Sweden)

    Ryutaro Hirasawa

    Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

  13. Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues.

    Science.gov (United States)

    Tarquini, Giulia; Ermacora, Paolo; Bianchi, Gian Luca; De Amicis, Francesca; Pagliari, Laura; Martini, Marta; Loschi, Alberto; Saldarelli, Pasquale; Loi, Nazia; Musetti, Rita

    2018-05-01

    Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.

  14. Influence of the genotype and density of inoculation on the differentiation of somatic embryos of Coffea arabica L. cv. Red Caturra and Coffea canephora cv. Robusta

    Directory of Open Access Journals (Sweden)

    Raúl Barbón

    2003-07-01

    Full Text Available The conditions were established for the differentiation of somatic embryos from cell suspensions in the genotype Caturra rojo (Coffea arabica and Robusta (Coffea canephora. Cell suspensions with high embryogenic potentials and stable coefficients of multiplication were used. While studying the density of inoculation, for the phase of differentiation for both varieties, differences appeared in the embryogenic capacity among them, being reached a whole of 556 500 ES.l-1 for the variety Caturra rojo and 298 670 SE.l-1 for the variety Robusta. The biggest number of embryos in torpedo state, were obtained with a density of inoculation of 0.5 gFW.l-1 for the variety Caturra rojo and 5.0 gMF.l-1 for the variety Robusta. Key Words: cell suspensions, embryogenic potential, somatic Embryogenesis, embryogenic cells

  15. Anatomy of somatic embryogenesis in Carica papaya L.

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    Juliana A. Fernando

    2001-09-01

    Full Text Available Mature zygotic embryos of Carica papaya L. ‘Sunrise Solo’ were used as explants for embryogenesis induction. The explants were inoculated on Murashige and Skoog culture medium supplemented with 2 mg.L-1 2,4-dichlorophenoxyacetic acid and incubated in darkness at 25+2°C. Histological analysis of callogenesis and somatic embryogenesis indicated occurrence of direct and indirect somatic embryogenesis development. Direct somatic embryo formation was observed from hypocotyledonary epidermic cells only from explant 18 days after inoculation. Somatic embryos formed indirectly were originated from embryogenic superficial cells of pre-embryonic complexes located on peripherical and on internal cell layers of callus 49 days after inoculation. Diverse morphological differences including disformed embryos were observed among the somatic embryos.Embriões zigóticos maduros de Carica papaya L. ‘Sunrise Solo’ foram utilizados como explantes para indução da embriogênese. Estes explantes foram inoculados em meio de cultura de Murashige & Skoog suplementado com 2,0 mg.L-1 de 2,4 ácido diclorofenoxiacético (2,4-D e mantidos no escuro em câmara de crescimento à temperatura de 21°C por período de tempo variável. Através da análise histológica foi possível verificar que os primeiros embriões somáticos formaram-se diretamente a partir de células únicas da epiderme hipocotiledonar do explante após o 18º dia de cultura. Porém, os demais embriões somáticos originaram-se indiretamente a partir de células superficiais de complexos pré-embriônicos presentes nas camadas periféricas e internas do calo após o 49º dia de cultura. Foram detectadas algumas diferenças morfológicas entre os embriões somáticos obtidos.

  16. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

    Science.gov (United States)

    Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

    2016-08-25

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

  17. Sulphur depletion altered somatic embryogenesis in Theobroma ...

    African Journals Online (AJOL)

    Somatic embryogenesis is a useful tool for Theobroma cacao improvement and propagation. Depending on culture medium composition, different morphogenetic structures (including somatic embryo) occur in response to alteration of genes expression patterns and biochemical changes. The effect of SO42- ion deficiency ...

  18. Somatic embryogenesis on Musa AAAB, cv. FHIA-18, using liquids culture mediums

    Directory of Open Access Journals (Sweden)

    Luis A. Barranco

    2002-04-01

    Full Text Available Homogenous cell suspensions were iniciated from somatic embryos in the globular stage and the greatest volume of cell biomass on multiplying the suspensions at a density of 3.0% PCV. From the fifteenth day in culture medium for the formation of embryos, structures consisting of proembryos and somatic embryos in the globular stage started to form. With respect to the densities studied, the best results were obtained with 100 mgFW, where 1 871 SE.l-1 formed with a weight of 248 mgFW.l-1 after 30 days. With an initial density of 0.6 gFW in the culture medium for secondary multiplication, an increase of 42.9-fold the initial amount of fresh weight was obtained; after 60 days of culture 15 985 SE.l-1 were obtained. The greatest percentage of maturation was obtained with 400 mgFW with 70% of mature somatic embryos. The positive effect of Biobras-6 (brassinosteroid analogous was confirmed, with a concentration of 0.01 mg.l-1 the best germination percentages were obtained in liquid and semisolid culture medium. Embryo germination in temporaly inmersion (RITA was achieved with an inoculum density of 0.5 gFW for system with 85% germination. One thousand plants obtained from somatic embryos were taken to ex vitro environment, along with plants derived from conventional micropropagation (shoot tips to carry out studies on the possible presence of somaclonal variation. During the first cycle of production, the plants derived from the two methods in vitro culture showed differences with respect to the plants derived from corms in height, diameter and number of suckers. In the second production cycle, the plants from somatic embryos showed similar characteristics to the plants derived from shoot tip and corms with respect to the morphological parameters evaluated, with only 0.2% of the plants with phenotypic changes. Key Words: Banana, cellular density, germination, somaclonal variability, somatic embryo

  19. Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers.

    Science.gov (United States)

    Moncada, Ximena; Pelsy, Frédérique; Merdinoglu, Didier; Hinrichsen, Patricio

    2006-11-01

    Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.

  20. Numerical chromosome errors in day 7 somatic nuclear blastocysts

    DEFF Research Database (Denmark)

    Booth, Paul J; Viuff, Dorthe; Tan, Shijian J

    2003-01-01

    Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulat......Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona...... families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater......; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 +/- 4.5% and 73.7 +/- 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing or=5N chromosome complements, respectively) between two clone families were...

  1. Somatic embryogenesis of Carica Papaya

    International Nuclear Information System (INIS)

    Alvina Lindsay Mijen; Rusli Ibrahim

    2006-01-01

    This paper describes the somatic embryogenesis of Carica papaya. Culture medium used was1/2 strength MS basal medium supplemented with 6% sucrose, 0.27 % agar, glutamine and various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D). After 8 weeks in culture, the best concentration of 2,4-D to induce somatic embryo is at 45.2 μM. (Author)

  2. Optimization of somatic embryogenesis procedure for commercial ...

    African Journals Online (AJOL)

    The first objective of this study was to assess and optimize somatic embryo production in a genetically diverse range of cacao genotypes. The primary and secondary somatic embryogenesis response of eight promising cacao clones and a positive control was evaluated using modified versions of standard protocols.

  3. Somatic embryogenesis and plant regeneration of Capsicum baccatum L.

    Directory of Open Access Journals (Sweden)

    Peddaboina Venkataiah

    2016-06-01

    Full Text Available A plant regeneration protocol via somatic embryogenesis was achieved in cotyledon and leaf explants of Capsicum baccatum, when cultured on MS medium supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D, 0.5–5.0 mg l−1 in combination with Kinetin (Kn, 0.5 mg l−1 and 3% sucrose. Various stages were observed during the development of somatic embryos, including globular, heart, and torpedo-stages. Torpedo stage embryos were separated from the explants and subcultured on medium supplemented with various concentrations of different plant growth regulators for maturation. Maximum percentage (55% of somatic embryo germination and plantlet formation was found at 1.0 mg l−1 BA. Finally, about 68% of plantlets were successfully established under field conditions. The regenerated plants were morphologically normal, fertile and able to set viable seeds.

  4. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate the devel......Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate...... nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...

  5. Encapsulated Somatic Embryos and Zygotic Embryos for Obtaining Artificial Seeds of Rauli-Beech (Nothofagus alpina (Poepp. & Endl. Oerst. Encapsulado de Embriones Somáticos y Embriones Cigóticos para Obtención de Semillas Artificiales de Raulí (Nothofagus alpina (Poepp. & Endl. Oerst.

    Directory of Open Access Journals (Sweden)

    Priscila Cartes R

    2009-03-01

    Full Text Available Somatic and zygotic embryos from mature seeds of rauli-beech, Nothofagus alpina (Poepp. & Endl. Oerst., were encapsulated in different artificial endosperms in order to generate a cover that fulfills the function of nourishment and protection of the embryos, facilitating their later germination. The content of sodium alginate varied by 4%, 3%, and 2%, as did the immersion time in calcium chloride (CaCl2, which acts as complexing agent. The artificial endosperm components of the Murashige and Skoog medium (MS were added, supplemented with 0.5 mg L-1 indolacetic acid (IAA, 0.5 mg L-1 naphthaleneacetic acid (NAA, 2 mg L-1 6-benzylaminopurine (BAP and 30 g L-1 sucrose. The germinative behaviors of encapsulated somatic and zygotic embryos were evaluated after 4 wk. Comparing the percentages of germination reached by encapsulated somatic and zygotic embryos it was observed that they had similar germinative behavior according to the type of encapsulation applied. However, zygotic embryos substantially exceeded the germination levels reached by somatic embryos, 100% vs. 45% respectively.Embriones somáticos y cigóticos provenientes de semillas maduras de raulí, Nothofagus alpina (Poepp. & Endl. Oerst., se encapsularon en diferentes endospermas sintéticos con el fin de generar una cubierta que cumpla la función de nutrir y proteger al embrión para facilitar su posterior germinación. Se varió el contenido de alginato de sodio al 4%, 3% y 2% y el tiempo de inmersión en cloruro de calcio (CaCl2, el que actúa como agente acomplejante. Además, a la matriz artificial se adicionaron componentes del medio Murashige y Skoog (MS suplementado con: 0,5 mg L-1 de indolacetic acid (IAA, 0,5 mg L-1 de ácido naftalenacético (NAA, 2 mg L-1 de 6-bencilaminopurina (BAP y 30 gL-1 de sacarosa. Al cabo de 4 semanas el porcentaje de germinación de los embriones somáticos y cigóticos encapsulados tuvieron similar comportamiento germinativo según el tipo de

  6. Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars.

    Science.gov (United States)

    Aslam, Junaid; Khan, Saeed Ahmad; Cheruth, Abdul Jaleel; Mujib, Abdul; Sharma, Maheshwar Pershad; Srivastava, Prem Shanker

    2011-10-01

    An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1(-l)). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1(-1)) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N(6)-benzyladenine (BAP, 0.75 mg 1(-l)) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1(-l)) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.

  7. Plant regeneration of Michelia champaca L., through somatic ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-05-03

    May 3, 2010 ... as a basic material for perfume, cosmetic, and medicine. The development of an ... Plant regeneration systems of M. champaca through somatic ... The embryogenic cells proliferated and formed somatic embryos (30%) after four to six .... by using MS excel program and Duncan's new multiple range test.

  8. PECTIMORF and BIOBRAS-16 utilization in the potato somatic embryogenesis

    Directory of Open Access Journals (Sweden)

    Jaime R. Hidrobo Luna

    2002-01-01

    Full Text Available With the application of PECTIMORF and BIOBRAS-16, somatic embryos were obtained in potato (Solanum tuberosum, L c.v. Desirèe, of 40 days old callus obtained from stem micropropagated plants. These were used as possible substitutes for crop regulators used in culture media for the induction of somatic embryos. The culture media was composed for 10ml.l-1 of Murashige and Skoog salt, 0.1mg.l-1 ANA, 0.1mg.l-1 kinetin, 0.5mg.l-1 thiamine, 2.5mg.l-1 cistein, 100mg.l-1 mioinositol, 20g.l-1 sucrose and 2.0g.l-1 agar. Four culture medias were tested in distinct combinations that contained different concentration of PECTIMOR and BIOBRAS-16 as substitute of auxins and cytokinins. After 90 days, the results obtained showed the possibility of substituting the auxins (0.5mg.l-1 ANA and the cytokinins (0.5mg.l-1 kinetin in the culture media, because the application of PECTIMORF at 3.2mg.l-1 and BIOBRAS-16 at 1.0mg.l-1, gave friable callus, high fresh weight (more than 1.4g and a brownish color at the end of the process, moment in which the somatic embryos of different phases, appeared at the surface of the callus. Keywords: brasinoesteroids, callus, oligopectate, somatic embryo

  9. Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

    Science.gov (United States)

    Arias, María E; Ross, Pablo J; Felmer, Ricardo N

    2013-01-01

    Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (Pculture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

  10. The prevalence of embryonic remnants following the recovery of post-hatching bovine embryos produced in vitro or by somatic cell nuclear transfer.

    Science.gov (United States)

    Alexopoulos, Natalie I; French, Andrew J

    2009-08-01

    The reliable collection of peri-implantation embryos in the bovine has important ramifications to post-transfer consequences, particularly in the elucidation of mechanisms associated with post-hatching embryo development and to perturbations in developmental growth following transfer. This study analyzed both in vitro produced (IVP) and somatic cell nuclear transfer (SCNT) embryo-like structures (ELS) recovered at Day (D) 14 and D21. The recovered ELS were subsequently processed for histological examination. At D14 and D21, many of the embryos recovered in the IVP group conformed to the appropriate stage of development. However, a significant number of anomalies were present in the SCNT groups when examined in more detail. Histological examination revealed that irrespective of whether these embryos had undergone trophoblast expansion to an ovoid, tubular or filamentous morphology, many had a degenerated hypoblast layer and a large proportion did not possess an epiblast and therefore could not differentiate into any of the three germ layers as would be expected at the neural groove or somite stage. The prevalence of this developmental pattern was random and did not correlate with treatment (IVP or SCNT) or with types of structures recovered. The rapid embryo elongation period also coincides with the time of greatest embryonic loss and these observations could have important implications for assessing the recovery of embryos post-transfer where incorrect morphological assessment could lead to false implantation and pregnancy determination rates. The implementation of additional methodology is required to adequately characterize the quality of IVP and SCNT-derived embryos collected post-transfer.

  11. In vitro regeneration of some Iranian alfalfa (Medicago sativa L. genotypes via somatic embryogenesis

    Directory of Open Access Journals (Sweden)

    Majid Shokrpour

    2014-12-01

    Full Text Available An effective in vitro regeneration system is one of the prerequisites for genetic manipulation of alfalfa (Medicago sativa L. varieties and genotypes. In this research, somatic embryogenesis of four alfalfa genotypes, 6-18 (synthetic, 4-14 (Kara Yonje- Karakozlu, 3-27 (Kara Yonje Maraghe and y-6 (Regen-SY, were investigated using leaf and petiole explants. Formation of callus and somatic embryogenesis was significantly influenced by the explant type and interaction of genotype and culture medium. Petiole explants of genotype 4-14 produced the highest yield of callus (0.406 gr fresh weight of callus. Percentage of somatic embryogenesis and the number of embryos per callus in petiole explants of genotype 4-14 was higher than those of other genotypes and explants. In genotype 6-18, the highest percentage of somatic embryogenesis was achieved on MS medium containing 5 mg/L 2,4-D and 2 mg/L kinetin. There was no significant differences between genotypes and explants in terms of embryo conversion to plantlet, and on average, 58% of somatic embryos converted to plantlet on MS medium. The petiole explants of genotype 6-18 did not exhibit somatic embryogenesis response in medium containing low ratio of 2,4-D:Kinetin (5 mg/L 2,4-D and 2 mg/L kinetin. While, these explants showed somatic embryogenesis in higher ratio of 2,4-D:Kinetin (5:1. The plantlet conversion efficiency of somatic embryos produced through this study was relatively higher and therefore, the method presented in this study could be used in alfalfa genetic manipulation and molecular studies.

  12. Lethals induced by γ-radiation in drosophila somatic cells

    International Nuclear Information System (INIS)

    Ivanov, A.I.

    1989-01-01

    Exposure of 3-hour drosophila male embryos to γ-radiation during the topographic segregation of the germ anlage nuclei caused recessive sex-linked lethals in somatic cells only. The selectivity of the screening was determined by the ratio of mutation frequencies induced in embryos and adult males. Analysis of lethal mutations shows that a minimal rate of the divergence between germinal and somatic patterns of the cell development is observed in the embryogenesis, the 3d instar larva and prepupa, and maximal in the 1st and 2nd larva and pupa

  13. Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore 'Elsa'.

    Science.gov (United States)

    Shen, Hui-Ju; Chen, Jen-Tsung; Chung, Hsiao-Hang; Chang, Wei-Chin

    2018-01-22

    Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l -1 in darkness. TDZ at 3 mg l -1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95-100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore 'Elsa', was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

  14. Replication of somatic micronuclei in bovine enucleated oocytes

    Directory of Open Access Journals (Sweden)

    Canel Natalia

    2012-11-01

    Full Text Available Abstract Background Microcell-mediated chromosome transfer (MMCT was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes. Methods Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+] or not [Micronucleus- injected (−] to a transgene (50 ng/μl pCX-EGFP during 5 min. Enucleated oocytes [Enucleated (+] and parthenogenetic [Parthenogenetic (+] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (−] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−, Parthenogenetic (− and in vitro fertilized (IVF embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05. Results All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had

  15. Somatic embryogenesis and plant regeneration from leaf explants of ...

    African Journals Online (AJOL)

    An attempt was made to study the somatic embryogenesis and plant regeneration from the in vitro leaf explants of Rumex vesicarius L. a renowned medicinal plant, which belongs to polygonaceae family. Effective in vitro regeneration of R. vesicarius was achieved via young leaf derived somatic embryo cultures.

  16. General properties of grapevine viruses occurring in Hungary

    Directory of Open Access Journals (Sweden)

    Eszter Cseh

    2012-03-01

    Full Text Available The past fifty years important advances have been made in the field of grapevine virus research, including characterization of pathogens and control measurements. Still the occurrence of Grapevine fanleaf virus (GFLV, Arabis mosaic virus (ArMV, Tomato black ring virus (TBRV, Grapevine chrome mosaic virus (GCMV, Alfalfa mosaic virus (AMV, Grapevine Bulgarian latent virus (GBLV, Grapevine fleck virus (GFkV, Grapevine leafroll- associated viruses (GLRaV1-4, Grapevine virus A (GVA, Grapevine virus B (GVB and Grapevine rupestris stem pitting- associated virus (GRSPaV have been reported in Hungary and characterized by conventional methods as woody indexing, herbaceous indexing and serological methods. Among grapevine viruses the Grapevine line pattern virus (GLPV seems to be uncial; because it was reported only in Hungary. Causal agents of several grapevine diseases, like enation, vein necrosis and vein mosaic remained undiscovered. These virus-like diseases occurred only sporadically, without economic importance.

  17. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    Science.gov (United States)

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos. (c) 2009 Wiley-Liss, Inc.

  18. Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT embryos

    Directory of Open Access Journals (Sweden)

    María E Arias

    2013-01-01

    Full Text Available Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively. No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01 in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28% compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively. Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA. Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

  19. General properties of grapevine viruses occurring in Hungary

    OpenAIRE

    Eszter Cseh; András Takács; László Kocsis; Richard Gáborjányi

    2012-01-01

    The past fifty years important advances have been made in the field of grapevine virus research, including characterization of pathogens and control measurements. Still the occurrence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tomato black ring virus (TBRV), Grapevine chrome mosaic virus (GCMV), Alfalfa mosaic virus (AMV), Grapevine Bulgarian latent virus (GBLV), Grapevine fleck virus (GFkV), Grapevine leafroll- associated viruses (GLRaV1-4), Grapevine virus A (GVA), Grape...

  20. Somatic embryogenesis in plantain cultivar 'FHIA - 25' (AAB from meristem tips

    Directory of Open Access Journals (Sweden)

    Dayana Rodríguez González

    2015-07-01

    Full Text Available Plantain cultivar 'FHIA – 25' (AAB shows high yielding qualities and high resistance to Black Sigatoka disease, but its sugar content in the fruit is low, so a regeneration method at cell level is necessary, such as somatic embryogenesis supported by biotechnological tools to improve fruit quality. This work was performed with the aim of establishing a plant regeneration method via somatic embryogenesis using initial explants of shoot apices from axillary buds in liquid culture medium. Homogenous embryogenic cell suspensions were obtained from mentioned explants. The highest cellular multiplication rates were achieved at 3,0% density. The incubation of somatic embryos during 30 days in the maturation culture medium permitted to increase germination. During the acclimatization stage, plants regenerated from somatic embryos, as well as plants from organogenesis, showed a high survival percentage (98 and 97 respectively, without somaclonal variation.

  1. Bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Ross, Pablo J; Cibelli, Jose B

    2010-01-01

    Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

  2. In vitropropagation in Temporary Immersion System of sugarcane plants variety `RB 872552' derived from somatic embryos

    Directory of Open Access Journals (Sweden)

    Marina Medeiros de Araújo Silva

    2015-07-01

    Full Text Available In this study, we used a temporary immersion system (TIS to multiply sugarcane (Saccharum spp. plants obtained by somatic embryogenesis (SE. SE was induced from immature leaf segments that were grown in culture medium supplemented with 2,4-D and BAP. Embryo formation occurred in 81% of the inoculated explants and 254 plants were regenerated. Ninety plants were transferred to TIS and cultured in medium supplemented with BAP. After three subcultures, 60 000 plantlets were obtained and transferred to rooting media. After 30 days of acclimatization period plantlets were well developed and exhibited a 96% survival. The results demonstrate the feasibility of the combined use of two important techniques of in vitro culture (SE and shoot multiplication in TIS to sugarcane in vitro propagation. Key words: acclimatization, 6-benzylaminopurine, Saccharum spp.

  3. Forest management guidelines for controlling wild grapevines

    Science.gov (United States)

    H. Clay Smith

    1984-01-01

    Grapevines (Vitis spp.) are becoming a major problem to forest managers in the Appalachians, especially when clearcutting is done on highly productive hardwood sites. Where present, grapevines can reduce tree quality and growth, and eventually kill the tree. Silvical characteristics of grapevines are discussed as background for grapevine control....

  4. Plant regeneration from immature embryos of Kenyan maize inbred ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... their respective single cross hybrids were evaluated for their ability form callus, somatic embryos and .... Callus was induced from embryos excised from ears at. 10, 15, 18, 21 and ..... Plant Cell Tissue Organ Cult., 18: 143-151.

  5. In Vitro Selection of Peanut Somatic Embryos on Medium Containing Culture Filtrate of Sclerotium rolfsii and Plantlet Regeneration

    Directory of Open Access Journals (Sweden)

    YUSNITA

    2005-06-01

    Full Text Available Attempts to identify somaclonal variants of peanut with resistance to Sclerotium stem rot disease due to infection of S. rolfsii were conducted. The objectives of this study were to develop in vitro selection method using culture filtrates of S. rolfsii, identify culture filtrate-insensitive somatic embryo (SE of peanut after in vitro selection and regenerate peanut R0 lines originated from culture filtrate-insensitive SE. To achieve these objectives, peanut embryogenic tissues were cultured on selective medium containing various concentrations of S. rolfsii culture filtrates and sublethal concentration of the filtrates. Medium containing sublethal level of S. rolfsii culture filtrates was used to identify culture filtrate-insensitive SE of peanut. Subsequently, the selected SEs were germinated, plantlets were regenerated and preliminary tested against S. rolfsii. Results of the experiments showed that addition of S. rolfsii culture filtrates into medium for inducing peanut somatic embryos drastically reduced their growth and proliferation. S. rolfsii culture filtrates at 10% concentration has significantly reduced the number of proliferated SE per explant. However, sublethal level was achieved at 30% of culture filtrates concentration. Responses of five peanut cultivars against 30% of culture filtrates were similar, indicating they were similar in their susceptibility against S. rolfsii. A number of culture filtrate-insensitive SE were identified after culturing 1500 clumps of embryogenic tissue of peanut cv. Kelinci for three consecutive passages on medium containing 30% of culture filtrates. Germination of selected SE and regeneration of plantlet from culture filtrate-insensitive SE resulted in 50 peanut R0 lines. These lines have been grown in the plastic house and produced normal seeds for further evaluation. Results of S. rolfsii inoculation indicated the existence of chimera for insensitivity against S. rolfsii.

  6. Genetic stability evaluation of quercus suber l. somatic embryogenesis by rapd analysis

    International Nuclear Information System (INIS)

    Fernandes, P.; Costa, A.; Rocha, A.C.C.; Santos, C.

    2011-01-01

    A reliable protocol for adult Quercus suber L. somatic embryogenesis (SE) was developed recently. To evaluate the potential use of this protocol in cork oak forest breeding programs, it is essential to guarantee somatic embryos/emblings genetic stability. Random Amplification of Polymorphic DNA (RAPD) is currently used to assess somaclonal variation providing information on genetic variability of the micropropagation process. In this work, SE was induced from adult trees by growing leaf explants on MS medium supplemented with 2,4-D and zeatin. Embling conversion took place on MS medium without growth regulators. DNA from donor tree, somatic embryos and emblings was used to assess genetic variability by RAPD fingerprinting. Fourteen primers produced 165 genetic loci with high quality and reproducibility. Despite somatic embryos originated some poor quality PCR-profiles, replicable and excellent fingerprints were obtained for both donor plant and embling. Results presented no differences among regenerated emblings and donor plant. Hence, the SE protocol used did not induce, up to moment, any genetic variability, confirming data previously obtained with other molecular/genetic techniques, supporting that this protocol may be used to provide true-to-type plants from important forestry species. (author)

  7. Targeted heavy-ion microbeam irradiation of the embryo but not yolk in the diapause-terminated egg of the silkworm, bombyx mori, induces the somatic mutation

    International Nuclear Information System (INIS)

    Furusawa, Toshiharu; Fukamoto, Kana; Sakashita, Tetsuya; Funayama, Tomoo; Kobayashi, Yasuhiko; Kakizaki, Takehiko; Wada, Seiichi; Hamada, Nobuyuki; Suzuki, Hiromi; Ishioka, Noriaki; Nagaoka, Shunji

    2009-01-01

    Using heavy-ion microbeam, we report target irradiation of selected compartments within the diapause-terminated egg and its mutational consequences in the silkworm, Bombyx mori. On one hand, carbon-ion exposure of embryo to 0.5-6 Gy increased the somatic mutation frequency, suggesting targeted radiation effects. On the other, such increases were not observed when yolk was targeted, suggesting a lack of nontargeted bystander effect. (author)

  8. Characterization of somatic embryogenesis initiated from the Arabidopsis shoot apex.

    Science.gov (United States)

    Kadokura, Satoshi; Sugimoto, Kaoru; Tarr, Paul; Suzuki, Takamasa; Matsunaga, Sachihiro

    2018-04-28

    Somatic embryogenesis is one of the best examples of the remarkable developmental plasticity of plants, in which committed somatic cells can dedifferentiate and acquire the ability to form an embryo and regenerate an entire plant. In Arabidopsis thaliana, the shoot apices of young seedlings have been reported as an alternative tissue source for somatic embryos (SEs) besides the widely studied zygotic embryos taken from siliques. Although SE induction from shoots demonstrates the plasticity of plants more clearly than the embryo-to-embryo induction system, the underlying developmental and molecular mechanisms involved are unknown. Here we characterized SE formation from shoot apex explants by establishing a system for time-lapse observation of explants during SE induction. We also established a method to distinguish SE-forming and non-SE-forming explants prior to anatomical SE formation, enabling us to identify distinct transcriptome profiles of these two explants at SE initiation. We show that embryonic fate commitment takes place at day 3 of SE induction and the SE arises directly, not through callus formation, from the base of leaf primordia just beside the shoot apical meristem (SAM), where auxin accumulates and shoot-root polarity is formed. The expression domain of a couple of key developmental genes for the SAM transiently expands at this stage. Our data demonstrate that SE-forming and non-SE-forming explants share mostly the same transcripts except for a limited number of embryonic genes and root genes that might trigger the SE-initiation program. Thus, SE-forming explants possess a mixed identity (SAM, root and embryo) at the time of SE specification. Copyright © 2018. Published by Elsevier Inc.

  9. Wild grapevine management

    Science.gov (United States)

    H. Clay Smith

    1989-01-01

    Wild grapevines are a problem for forest managers in many areas of the central hardwood forests. The vines grow on a wide range of soil and site conditions but usually are more concentrated on good sites (northern red oak site index 70 and above), on the faster growing more valuable timber. Presently there is more interest and concern in controlling grapevine for the...

  10. The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus).

    Science.gov (United States)

    Du, Fuliang; Shen, Perng-Chih; Xu, Jie; Sung, Li-Ying; Jeong, B-Seon; Lucky Nedambale, Tshimangadzo; Riesen, John; Cindy Tian, X; Cheng, Winston T K; Lee, Shan-Nan; Yang, Xiangzhong

    2006-02-01

    One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

  11. Grapevine (Vitis vinifera L.).

    Science.gov (United States)

    Torregrosa, Laurent; Vialet, Sandrine; Adivèze, Angélique; Iocco-Corena, Pat; Thomas, Mark R

    2015-01-01

    Grapevine (Vitis) is considered to be one of the major fruit crops in the world based on hectares cultivated and economic value. Grapes are used not only for wine but also for fresh fruit, dried fruit, and juice production. Wine is by far the major product of grapes, and the focus of this chapter is on wine grape cultivars. Grapevine cultivars of Vitis vinifera L. have a reputation for producing premium quality wines. These premium quality wines are produced from a small number of cultivars that enjoy a high level of consumer acceptance and are firmly entrenched in the market place because of varietal name branding and the association of certain wine styles and regions with specific cultivars. In light of this situation, grapevine improvement by a transgenic approach is attractive when compared to a classical breeding approach. The transfer of individual traits as single genes with a minimum disruption to the original genome would leave the traditional characteristics of the cultivar intact. However, a reliable transformation system is required for a successful transgenic approach to grapevine improvement. There are three criteria for achieving an efficient Agrobacterium-mediated transformation system: (1) the production of highly regenerative transformable tissue, (2) optimal cocultivation conditions for both grapevine tissue and Agrobacterium, and (3) an efficient selection regime for transgenic plant regeneration. In this chapter, we describe a grapevine transformation system that meets these criteria. We also describe a protocol for the production of transformed roots suitable for functional gene studies and for the production of semi-transgenic grafted plants.

  12. Developmental localization and the role of hydroxyproline rich glycoproteins during somatic embryogenesis of banana (Musa spp. AAA

    Directory of Open Access Journals (Sweden)

    Menzel Diedrik

    2011-02-01

    Full Text Available Abstract Background Hydroxyproline rich glycoproteins (HRGPs are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somatic embryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis, and by immunomodulation with the JIM11 antibody. Results Immunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs, mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs, proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM. This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP

  13. Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance.

    Science.gov (United States)

    Galambos, A; Zok, A; Kuczmog, A; Oláh, R; Putnoky, P; Ream, W; Szegedi, E

    2013-11-01

    Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance. Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. 'Richter 110' rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58.

  14. Somatic embryogenesis in banana and plantain (Musa spp. from male immature flowers

    Directory of Open Access Journals (Sweden)

    Rafael Gómez-Kosky

    2001-01-01

    Full Text Available In the development of this work, aspects related to the achievement of somatic embryos and regeneration of plants in some cultivars of Musa sp. group AAA, ABB and AABB were studied. Different experiments were carried out to determine the optimum culture conditions; the solidifying agent resulted to be phytagel (SIGMA Co., at a concentration of 2.0 g.l-1, were 68.6% of the explants formed yellow nodular calli. With, respect to the incubation conditions, darkness had a better influence achieving 73.8% of the explants forming yellow nodular calli. The best dosage of 2,4-D could have been determined for the induction of the yellow nodular calli in each one of the cultivars studied: Grande Naine (AAA, Parecido al Rey (AAA and FHIA-03 (AABB 4.0 mg.l-1 and for Bluggoe (ABB 2.0 mg.l-1. Studying the ranges of the hands, it was found that for all the cultivars, the ranges from 5-9 formed more callus with somatic embryogenesis of high frequency than the ranges from 10-14. Once the somatic embryos were transferred to the germination culture medium, a rate of 48.0% was obtained for Grande Naine and 39.0% for Parecido al Rey. Key words: banana, callus, male flowers, somatic embryo

  15. Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

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    Maria Jesús Cánepa

    Full Text Available Reproductive biotechnologies such as in vitro fertilization (IVF and somatic cell nuclear transfer (SCNT enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70, endoplasmic reticulum (ER stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5 and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3 in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART.

  16. TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos.

    Science.gov (United States)

    Cao, Zubing; Hong, Renyun; Ding, Biao; Zuo, Xiaoyuan; Li, Hui; Ding, Jianping; Li, Yunsheng; Huang, Weiping; Zhang, Yunhai

    2017-01-01

    The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells' chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

  17. Direct and Indirect Somatic Embryogenesis from Petiole and Leaf Explants of Purple Fan Flower (Scaevola aemula R. Br. cv. 'Purple Fanfare')

    OpenAIRE

    Shyama Ranjani Weerakoon

    2010-01-01

    Direct and indirect somatic embryogenesis (SE) from petiole and leaf explants of Scaevola aemula R. Br. cv. 'Purple Fanfare' was achieved. High frequency of somatic embryos was obtained directly from petiole and leaf explants using an inductive plant growth regulator signal thidiazuron (TDZ). Petiole explants were more responsive to SE than leaves. Plants derived from somatic embryos of petiole explants germinated more readily into plants. SE occurred more efficiently in ...

  18. Identification and characterization of bZIP-type transcription factors involved in carrot (Daucus carota L.) somatic embryogenesis.

    Science.gov (United States)

    Guan, Yucheng; Ren, Haibo; Xie, He; Ma, Zeyang; Chen, Fan

    2009-10-01

    Seed dormancy is an important adaptive trait that enables seeds of many species to remain quiescent until conditions become favorable for germination. Abscisic acid (ABA) plays an important role in these developmental processes. Like dormancy and germination, the elongation of carrot somatic embryo radicles is retarded by sucrose concentrations at or above 6%, and normal growth resumes at sucrose concentrations below 3%. Using a yeast one-hybrid screening system, we isolated two bZIP-type transcription factors, CAREB1 and CAREB2, from a cDNA library prepared from carrot somatic embryos cultured in a high-sucrose medium. Both CAREB1 and CAREB2 were localized to the nucleus, and specifically bound to the ABA response element (ABRE) in the Dc3 promoter. Expression of CAREB2 was induced in seedlings by drought and exogenous ABA application; whereas expression of CAREB1 increased during late embryogenesis, and reduced dramatically when somatic embryos were treated with fluridone, an inhibitor of ABA synthesis. Overexpression of CAREB1 caused somatic embryos to develop slowly when cultured in low-sucrose medium, and retarded the elongation of the radicles. These results indicate that CAREB1 and CAREB2 have similar DNA-binding activities, but play different roles during carrot development. Our results indicate that CAREB1 functions as an important trans-acting factor in the ABA signal transduction pathway during carrot somatic embryogenesis.

  19. Embryogenic calli induced in interspecific (Elaeis guineensis x E. oleifera hybrid zygotic embryos

    Directory of Open Access Journals (Sweden)

    Paula Cristina da Silva Angelo

    2009-01-01

    Full Text Available The hybridization between oil palm (Elaeis guineensis and caiaué (E. oleifera plants is directed to obtainprogenies presenting high yields like oil palm but with reduced shoot height and resistance to lethal yellowing like caiaué.Cloning F1, BC1 and BC2 progenies can make the replication of selection trials easier. The objective of this work was to inducesomatic embryogenesis in interspecific zygotic embryos collected 100 days after pollination. Three progenies were cultivatedin an induction medium developed for Tenera (E. guineensis tp. dura x pisifera embryos. The number of embryos bearing calliand germinating was recorded and submitted to the Z test. Calli were weighted and submitted to histological analysis.Progenies differed in the number of embryos presenting plumules and calli simultaneously. By the ninth month, the apices ofincompletely developed somatic embryos were observed protruding from the surfaces of nodular calli. Highly embryogenicand friable secondary calli producing globular somatic embryos were not observed.

  20. Monitoring Genetic Stability in Quercus serrata Thunb. Somatic Embryogenesis Using RAPD Markers

    OpenAIRE

    Ramesh C., Thakur; Susumu, Goto; Katsuaki, Ishii; S. Mohan, Jain; Forestry and Forest Products Research Institute; Fukuoka Prefecture Forest Research and Extension Center; Forestry and Forest Products Research Institute; University of Helsinki

    1999-01-01

    Genetic stability of propagules regenerated via somatic embryogenesis is of paramount importance for its application to clonal forestry. Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability in somatic embryogenesis of Quercus serrata Thunb. (Japanese white oak). Forty samples from an embryogenic line, consisting of regenerated plantlets, somatic embryos, and embryogenic calli, were examined using 54 decanucleotide primers. A total of 6520 clear reproduc...

  1. Effect of the time interval between fusion and activation on epigenetic reprogramming and development of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Liu, Jun; Wang, Yongsheng; Su, Jianmin; Wang, Lijun; Li, Ruizhe; Li, Qian; Wu, Yongyan; Hua, Song; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2013-04-01

    Previous studies have shown that the time interval between fusion and activation (FA interval) play an important role in nuclear remodeling and in vitro development of somatic cell nuclear transfer (SCNT) embryos. However, the effects of FA interval on the epigenetic reprogramming and in vivo developmental competence of SCNT embryos remain unknown. In the present study, the effects of different FA intervals (0 h, 2 h, and 4 h) on the epigenetic reprogramming and developmental competence of bovine SCNT embryos were assessed. The results demonstrated that H3 lysine 9 (H3K9ac) levels decreased rapidly after fusion in all three groups. H3K9ac was practically undetectable 2 h after fusion in the 2-h and 4-h FA interval groups. However, H3K9ac was still evidently detectable in the 0-h FA interval group. The H3K9ac levels increased 10 h after fusion in all three groups, but were higher in the 2-h and 4-h FA interval groups than that in the 0-h FA interval group. The methylation levels of the satellite I region in day-7 blastocysts derived from the 2-h or 4-h FA interval groups was similar to that of in vitro fertilization blastocysts and is significantly lower than that of the 0-h FA interval group. SCNT embryos derived from 2-h FA interval group showed higher developmental competence than those from the 0-h and 4-h FA interval groups in terms of cleavage rate, blastocyst formation rate, apoptosis index, and pregnancy and calving rates. Hence, the FA interval is an important factor influencing the epigenetic reprogramming and developmental competence of bovine SCNT embryos.

  2. Perkembangan Praimplantasi Embrio Mencit dengan Materi Genetik yang Berasal dari Parental, Maternal, dan Inti Sel Somatik (PRE-IMPLANTATION DEVELOPMENT OF MOUSE EMBRYO WITH GENETIC MATERIAL DERIVED FROM PARENTAL, MATERNAL AND SOMATIC CELL NUCLEUS

    Directory of Open Access Journals (Sweden)

    Harry Murti

    2014-05-01

    Full Text Available Cloned embryo and parthenogenetic embryo are a potential source of stem cells for regenerativemedicine. Stem cells derived from those embryos are expected to overcome the ethical issues to the use offertilization embryos for therapeutic purposes. The pre-implantation development is a critical step fordeveloping embryos reach the blastocyst stage. The objectives in vivo of this research are to produce mousecloned embryo, parthenogenetic embryo, and fertilized embryo and to study stages of  in vitro pre-implantation development culture. In vivo fertilized embryos, mouse oocytes, and cumulus cells were usedin this study. Treatment was performed on female mice superovulated with PMSG and hCG injections.Two-cell stage of in vivo fertilized embryos were collected on the second day post hCG injection. Clonedembryos were produced through Somatic Cell Nuclear Transfer (SCNT, which included enucleation, nucleartransfer and artificial activation. Parthenogenetic embryos were produced with artificial activationtechnique. The result of the research indicated that SCNT application was able to produce cloned embryos which could develop to blastocyst stage (3,2%. In addition, artificial activation of oocytes could produceparthenogenetic embryos which were able to develop up to the blastocyst stage (8,6%. In conclusion,efficiency level of parthenogenetic embryos that is able to reach the blastocyst stage was higher than in thecloned embryos. Fertilized embryos shows a better development and more efficient compared to in vitrocloned embryos and parthenogenetic embryos cultures.

  3. Recent advancements in cloning by somatic cell nuclear transfer.

    Science.gov (United States)

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-05

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

  4. Recent advancements in cloning by somatic cell nuclear transfer

    Science.gov (United States)

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-01

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model. PMID:23166393

  5. DIRECT AND INDIRECT SOMATIC EMBRYOGENESIS ON ARABICA COFFEE (Coffea arabica

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    Meynarti Sari Dewi Ibrahim

    2013-10-01

    Full Text Available Propagation of Coffea arabica L. through direct and indirect somatic embryogenesis technique is promising for producing large number of coffee seedlings. The objectives of the research were to evaluate methods for direct and indirect somatic embryo-genesis induction of C. arabica var. Kartika. The explants were the youngest fully expanded leaves of arabica coffee. The evalu-ated medium was modified Murashige and Skoog (MS medium supplemented with a combination of 2.26 µM 2,4-D + 4.54 or 9.08 µM thidiazuron; 4.52 µM 2,4-D + 4.54 or 9.08 µM thidiazuron; or 9.04 µM 2,4-D + 9.08 µM thidiazuron. Both calli (100 mg and pre-embryos developed from the edge of leaf explants were subcultured into regeneration medium (half strength MS with modified vitamin, supplemented with kinetine 9.30 µM and adenine sulfate 40 mg L-1. The results showed coffee leaf explant cultured on medium containing 2.26 µM 2,4-D + 4.54 or 9.08 µM thidiazuron to induce direct somatic embriogenesis from explant, while that of 4.52 or 9.04 µM 2,4-D + 9.08 µM thidiazuron to induced indirect somatic embrio-genesis. The medium for calli induction from coffee by explants was medium supplemented with 4.52 or 9.04 µM 2,4-D in combination with 9.08 µM thidiazuron. On the other hand, the best medium for activation of induction of somatic embryos was MS medium supplemented with 9.04 µM 2,4-D + 9.08 µM thidiazuron. Based on this results, the first step for developing micropropagation for coffee has been resolved. The subsequent studies will be directed to evaluate agronomic performance of the derived planting materials.

  6. High frequency induction of somatic embryos and plantlet ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-16

    Nov 16, 2009 ... plant, Hygrophila spinosa through direct somatic embryogenesis from nodal explants excised from 4 week old ... medicinal purposes further curbs propagation via seed. Plant tissue ... buted a great deal of information for the genetic, morpho- ..... analysis of peroxidase in cultured lettuce (Lactuca sativa L.).

  7. Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem.

    Science.gov (United States)

    Oh, H J; Kim, M K; Jang, G; Kim, H J; Hong, S G; Park, J E; Park, K; Park, C; Sohn, S H; Kim, D Y; Shin, N S; Lee, B C

    2008-09-01

    The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.

  8. Somatic Embryogenesis in Juniperus Procera using Juniperus ...

    African Journals Online (AJOL)

    The aim for this particular research was initially an adaptation of optimum half strength lithium chloride-sodium propionate (LP) medium protocol for growth and proliferation of embryogenic ... Additional study on the effect of seed extraction to the growing embryogenic culture showed no effect on mature somatic embryos.

  9. Developmental Localization and Methylesterification of Pectin Epitopes during Somatic Embryogenesis of Banana (Musa spp. AAA)

    Science.gov (United States)

    Xu, Chunxiang; Zhao, Lu; Pan, Xiao; Šamaj, Jozef

    2011-01-01

    Background The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development. Methodology/Principal Findings Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment. Conclusions/Significance These data suggest that both low

  10. Arthropods vector grapevine trunk disease pathogens.

    Science.gov (United States)

    Moyo, P; Allsopp, E; Roets, F; Mostert, L; Halleen, F

    2014-10-01

    Arthropod-mediated dispersal of pathogens is known in many cropping systems but has never been demonstrated for grapevine trunk disease pathogens. Arthropods from vineyards were screened for the presence of pathogens associated with Petri disease and esca using cultural and molecular techniques. The ability of the most abundant pathogen-carrying species to inoculate healthy grapevine vascular tissues was also determined. Millipedes and ants were allowed to associate with a DsRed- Express-transformed Phaeomoniella chlamydospora, after which they were exposed to freshly pruned healthy grapevines under controlled conditions and wounds were monitored for subsequent infection. In addition, the possibility of millipede excreta, commonly found on pruning wounds in the field, to act as inoculum source was determined. A diverse arthropod fauna was associated with declining grapevines and many of these carried trunk disease pathogens. However, spiders, the ant Crematogaster peringueyi, and the millipede Ommattoiulus moreleti were the most abundant pathogen carriers. The ant and millipede species fed on pruning wound sap and effectively transmitted trunk disease pathogens. Millipede excreta contained viable spores of Phaeomoniella chlamydospora and may serve as an inoculum source. Numerous arthropods, including beneficial predators, are potential vectors of grapevine trunk disease pathogens. Our results highlight the need for an integrated approach, including targeted management of ants and millipedes at the time of pruning, to limit the spread of grapevine trunk diseases.

  11. Somatic Embryogenesis in Yam (Dioscorea rotundata

    Directory of Open Access Journals (Sweden)

    Isidro Elías Suárez Padrón

    2011-12-01

    Full Text Available Embryogenic yam (Dioscorea rotundata cultures were induced from petioles of leaves of in vitro grown plants on medium supplemented with different 2.4-D concentrations. Cultures were maintained either on semisolid or in liquid MS medium supplemented with 4.52 µM 2.4-D. The effect of sucrose concentration on somatic embryo development was also evaluated and the effects of different BAP concentrations on somatic embryo conversion were determined. Treatments were distributed using a complete randomized design. The highest rate of induction occurred with 4.52 µM 2.4-D. Sucrose at 131.46 mM significantly enhanced somatic embryo development. The conversion rate was not affected by BAP.Cultivos embriogénicos de ñame (Dioscorea rotundata fueron inducidos a partir de explantes consistentes de hojas con peciolos, aisladas de plantas establecidas en condiciones in vitro, en presencia de diferentes concentraciones de 2,4-D. Los cultivos inducidos fueron mantenidos en medio MS líquido o semisólido suplido con 4,52 µM 2,4-D. El efecto de las concentraciones de sacarosa sobre el desarrollo de embriones somáticos y el efecto de varias concentraciones de BAP sobre la tasa de conversión de embriones somáticos en plantas también fueron evaluados. Todos los tratamientos fueron distribuidos usando un diseño completamente al azar. El mayor porcentaje de inducción de tejidos embriogénicos ocurrió con 4,52 µM de 2,4-D. La adición de 131,46 mM de sacarosa incrementó significativamente el desarrollo de embriones somáticos. La tasa de conversión de embriones somáticos en plantas no fue afectada por las concentraciones de BAP.

  12. [Changes in polyamine levels in Citrus sinensis Osb. cv. Valencia callus during somatic embryogenesis].

    Science.gov (United States)

    Liu, Hua-Ying; Xiao, Lang-Tao; Lu, Xu-Dong; Hu, Jia-Jin; Wu, Shun; He, Chang-Zheng; Deng, Xiu-Xin

    2005-06-01

    Somatic embryogenetic capability and changes in polyamine level and their relationship were analyzed using the long-term (8 years) subcultured calli of Citrus sinensis Osb. cv. Valencia as materials. The results showed that endogenous polyamine contents in embryogenic calli were higher than those in non-embryogenic calli, and the embryogenetic capability was positively correlated to the levels of endogenous polyamines. When the calli were transferred to a differentiation medium, the putrescine content rapidly increased and reached a peak, then fell gradually. Applying exogenous putrescine raised the embryogenesis frequency and endogenous putrescine level. It indicated that increase in putrescine content at early stage of differentiation promoted embryogenesis. With the development of somatic embryo, spermidine content reached its the highest level at globular embryo stage, spermine content rose and reached a peak at a later stage of globular embryo development. Furthermore, changes of the putrescine, spermidine and spermine contents during somatic embryogenesis were similar in Valencia calli which had different ploidy levels, but their contents decreased following the increasing of ploidy level. Changes in arginine decarboxylase activity were positively correlated to the polyamine levels, which suggest that the later is a key factor in regulating the polyamine levels during somatic embryogenesis in citrus plants.

  13. Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

    Science.gov (United States)

    Ono, Yukiko; Kono, Tomohiro

    2006-08-01

    Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as

  14. Rat primary embryo fibroblast cells suppress transformation by the E6 and E7 genes of human papillomavirus type 16 in somatic hybrid cells.

    OpenAIRE

    Miyasaka, M; Takami, Y; Inoue, H; Hakura, A

    1991-01-01

    The E6 and E7 genes of human papillomavirus type 16 (HPV-16) transform established lines of rat cells but not rat cells in primary culture irrespective of the expression of the two genes. The reason for this difference between the susceptibilities of cell lines and primary cells was examined by using hybrid cells obtained by somatic cell fusion of rat cell lines transformed by the E6 and E7 genes of HPV-16 and freshly isolated rat embryo fibroblast cells. In these hybrid cells, transformed ph...

  15. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  16. Somatic Embryogenesis in Two Orchid Genera (Cymbidium, Dendrobium).

    Science.gov (United States)

    da Silva, Jaime A Teixeira; Winarto, Budi

    2016-01-01

    The protocorm-like body (PLB) is the de facto somatic embryo in orchids. Here we describe detailed protocols for two orchid genera (hybrid Cymbidium Twilight Moon 'Day Light' and Dendrobium 'Jayakarta', D. 'Gradita 31', and D. 'Zahra FR 62') for generating PLBs. These protocols will most likely have to be tweaked for different cultivars as the response of orchids in vitro tends to be dependent on genotype. In addition to primary somatic embryogenesis, secondary (or repetitive) somatic embryogenesis is also described for both genera. The use of thin cell layers as a sensitive tissue assay is outlined for hybrid Cymbidium while the protocol outlined is suitable for bioreactor culture of D. 'Zahra FR 62'.

  17. THE ORIGIN OF GRAPEVINE CULTIVATION IN ITALY: THE ARCHAEOBOTANICAL EVIDENCE

    Directory of Open Access Journals (Sweden)

    S. Marvelli

    2013-04-01

    Full Text Available Grapevine remains show that this plant has been important for humans since ancient times. This paper presents a synthesis of archaeobotanical studies on grapevine remains (pollen, wood, charcoal, seed/fruit and other botanical remains from Epigravettian to Bronze Age sites. Carpological remains are the most important ones, because they often allow to distinguish cultivated and wild grapevines. Grapevine findings are rare in Mesolithic sites, they increase during Neolithic period and become frequent in Bronze Age. Archaeobotanical data show that during Neolithic and in the Early Bronze Age a good level of knowledge concerning grapevine utilization was already acquired; during Middle and Late Bronze Age the grapevine diffusion increases. Based on archaeobotanical data, the wild grapevine evolution by indigenous people was probably accompanied by an input of allochtonous vines from Mycenaean world, and then from Hellenic world. Therefore, the critical period of grapevine domestication can be placed between Bronze Age and Early Iron Age.

  18. Complete nucleotide sequence of the RNA-2 of grapevine deformation and Grapevine Anatolian ringspot viruses.

    Science.gov (United States)

    Ghanem-Sabanadzovic, Nina Abou; Sabanadzovic, Sead; Digiaro, Michele; Martelli, Giovanni P

    2005-05-01

    The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71-73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62-64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5' non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively.

  19. Regeneration of Algerian germplasm by stigma/style somatic ...

    African Journals Online (AJOL)

    ... days in most of the cultured genotypes. Formed embryos were cultured in a single tube before in vivo acclimatization. After sanitary assays, regenerated plants were shown to be free from the agents detected in the mother trees. Key words: Algeria, citrus germplasm, plant regeneration, sanitation, somatic embryogenesis.

  20. Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

    Science.gov (United States)

    Shi, Li-Hong; Miao, Yi-Liang; Ouyang, Ying-Chun; Huang, Jun-Cheng; Lei, Zi-Li; Yang, Ji-Wen; Han, Zhi-Ming; Song, Xiang-Fen; Sun, Qing-Yuan; Chen, Da-Yuan

    2008-03-01

    The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development. (c) 2008 Wiley-Liss, Inc.

  1. Influence of plant growth regulators on somatic embryos induction ...

    African Journals Online (AJOL)

    TANOH

    2013-04-17

    Theobroma cacao L.) using Thidiazuron. In vitro Cell Dev. Biol. 34:293-299. Michaux-Ferrière N, Carron MP (1989). Histology of early somatic embryogenesis in Hevea brasiliensis. The importance of timing of subculturing. Plant Cell Tiss ...

  2. The status of Botryosphaeriaceae species infecting grapevines

    Directory of Open Access Journals (Sweden)

    Jose Ramón URBEZ-TORRES

    2011-12-01

    Full Text Available Normal 0 MicrosoftInternetExplorer4 Normal 0 MicrosoftInternetExplorer4 Species in the Botryosphaeriaceae have a cosmopolitan distribution, and occur on a wide range of annual and perennial hosts including grapevines. To date, morphological and taxonomic studies, as well as analyses of nucleotide sequences of multiple genes, have allowed the identification of at least 21 different species in the Botryosphaeriaceae occurring in grapevines worldwide. Grapevine disease symptoms caused by members of this family include leaf spots, fruit rots, shoot dieback, bud necrosis, vascular discoloration of the wood, and perennial cankers, and their current status as pathogens is reviewed. Additionally, the disease name Botryosphaeria dieback is proposed here to describe the different grapevine trunk disease symptoms caused by species of Botryosphaeriaceae. Much has been written during the last decade about the association between species in the Botryosphaeriaceae and grapevine trunk diseases, which has contributed to a better understanding of the role that these fungal taxa play in grapevine diseases. Although virulence has been shown to vary between species and isolates of the same species in different countries, these fungi have become well-recognized as important grapevine pathogens worldwide. Latest and novel findings from studies conducted in different countries, on disease etiology and species distribution, epidemiology and biology are discussed. Much progress has been achieved in the development and implementation of novel diagnostic and detection techniques.Vineyard sanitation techniques, as well as chemical, biological, and cultural control strategies available at the present time to reduce the infection caused by botryosphaeriaceous fungi, are presented in this review. 

  3. Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

    Directory of Open Access Journals (Sweden)

    Yukari Terashita

    Full Text Available Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA, an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2 could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

  4. Imaging of Norway spruce early somatic embryos with the ESEM, Cryo-SEM and laser scanning microscope.

    Science.gov (United States)

    Neděla, Vilém; Hřib, Jiří; Havel, Ladislav; Hudec, Jiří; Runštuk, Jiří

    2016-05-01

    This article describes the surface structure of Norway spruce early somatic embryos (ESEs) as a typical culture with asynchronous development. The microstructure of extracellular matrix covering ESEs were observed using the environmental scanning electron microscope as a primary tool and using the scanning electron microscope with cryo attachment and laser electron microscope as a complementary tool allowing our results to be proven independently. The fresh samples were observed in conditions of the air environment of the environmental scanning electron microscope (ESEM) with the pressure from 550Pa to 690Pa and the low temperature of the sample from -18°C to -22°C. The samples were studied using two different types of detector to allow studying either the thin surface structure or material composition. The scanning electron microscope with cryo attachment was used for imaging frozen extracellular matrix microstructure with higher resolution. The combination of both electron microscopy methods was suitable for observation of "native" plant samples, allowing correct evaluation of our results, free of error and artifacts. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Bottlenecks in bog pine multiplication by somatic embryogenesis and their visualization with the environmental scanning electron microscope.

    Science.gov (United States)

    Vlašínová, Helena; Neděla, Vilem; Đorđević, Biljana; Havel, Ladislav

    2017-07-01

    Somatic embryogenesis (SE) is an important biotechnological technique used for the propagation of many pine species in vitro. However, in bog pine, one of the most endangered tree species in the Czech Republic, limitations were observed, which negatively influenced the development and further germination of somatic embryos. Although initiation frequency was very low-0.95 %, all obtained cell lines were subjected to maturation. The best responding cell line (BC1) was used and subjected to six different variants of the maturation media. The media on which the highest number of early-precotyledonary/cotyledonary somatic embryos was formed was supplemented with 121 μM abscisic acid (ABA) and with 6 % maltose. In the end of maturation experiments, different abnormalities in formation of somatic embryos were observed. For visualization and identification of abnormalities in meristem development during proliferation and maturation processes, the environmental scanning electron microscope was used. In comparison to the classical light microscope, the non-commercial environmental scanning electron microscope AQUASEM II has been found as a very useful tool for the quick recognition of apical meristem disruption and abnormal development. To our knowledge, this is the first report discussing somatic embryogenesis in bog pine. Based on this observation, the cultivation procedure could be enhanced and the method for SE of bog pine optimized.

  6. Somatic embryogenesis in Agave sisalana Perrine: induction, characterization and anatomical regeneration

    Directory of Open Access Journals (Sweden)

    Fernando dos Santos Carneiro

    2014-09-01

    Full Text Available Sisal (Agave sisalana Perrine is used for the extraction of fibers present in their leaves, being considered the main hard fiber produced in the world. However, a decline has been observed for this crop, due to a disease caused by the soil fungus Aspergillus niger. Biotechnology, particularly tissue culture techniques, represents a viable alternative for propagating this species. Among these techniques, the somatic embryogenesis can be pointed out. Thus, this study aimed to characterize the in vitro morphogenesis, during somatic embryogenesis, in A. sisalana Perrine. In all experiments, bulblets were used as explants and also ½ MS medium plus sucrose (30.0 g L-1, solidified with agar (7.0 g L-1 supplemented with different concentrations of auxin and cytokinin. The explants formed callus with compact and semi-compact aspects, pointing out the embryogenic cells converted into somatic embryos with the use of 2,4-D and BAP. The anatomic analyzes showed embryos in globular and torpedo stages and no connection with the mother callus.

  7. Field evaluation of regenerated plants by somatic embryogenesis from shoots apexes of axillary buds in ´Navolean’ (Musa spp., AAB.

    Directory of Open Access Journals (Sweden)

    Jorge López

    2005-04-01

    Full Text Available The use of shoots apexes from axilary buds for callus induction with embryogenic structures in plantain ‘Navolean’ (Group AAB permitted to develop a plant regeneration method through out somatic embryogenesis. In order to know the phenotypic variants that may be produced with the previously mentioned method , 1000 plants were planted in field conditions in comparison to those coming from somatic embryos obtained from multibuds as initial explants and organogenesis-derived plants (shoot tipsand conventionally derived plants (corms, during two growing cycles. The main morphological characters and yield components were evaluated. The total frequency of somaclonal variation during the first growing cycle in plants coming from somatic embryos obtained from shoots apexes from axilary buds as initial explants were 1.1%, and 8,6% in regenerated plants from somatic embryos obtained from multi-buds as initial explants. Later, in this same growing cycle, plants regenerated from somatic embryos (both sources showed a similar performance between them and they were significantly superior in all evaluated variants in comparison to corm-derived plants. In the second growing cycle, significant differences were not observed in yield components of suckers from evaluated plants, in spite of the propagation method used. With regard to somaclonal variation, the best performance was obtained with shoots apexes from axilary buds as explants. Finally, the feasibility of using the new method was shown. Key words: embryogenic cell suspensions, somaclonal variation

  8. Latrunculin A Treatment Prevents Abnormal Chromosome Segregation for Successful Development of Cloned Embryos

    Science.gov (United States)

    Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

    2013-01-01

    Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene—essential for normal development but never before expressed in cloned embryos—was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning. PMID:24205216

  9. Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Gómez, Martha C; Pope, C Earle

    2015-01-01

    In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

  10. Micropropagation of Codiaeum variegatum (L.) Blume and regeneration induction via adventitious buds and somatic embryogenesis.

    Science.gov (United States)

    Radice, Silvia

    2010-01-01

    Codiaeum variegatum (L) Blume cv. "Corazon de oro" and cv. "Norma" are successfully micropropagated when culture are initiated with explants taken from newly sprouted shoots. The establishment and multiplication steps are possible when 1 mg/L BA or 1 mg/L IAA and 3 mg/L 2iP are added to MS medium, according to the cultivar respectively selected.Adventive organogenesis and somatic embryogenesis are induced from leaf explants taken from in vitro buds of croton. On leaf-sectioned of "Corazon de oro" cultured in vitro, 1 mg/L BA stimulates continuous somatic embryos development and induces some shoots too. Replacing BA with 1 mg/L TDZ induces up to 100% bud regeneration in the same explants. On the other hand, leaf-sectioned of C. variegatum cv. Norma does not start somatic embryo differentiation if 1 mg/L TDZ is not added to the MS basal medium. Incipient callus is observed after 30 days of culture, and then, subculture to MS with 1 mg/L BA allows the same process to show on the "Corazon de oro" cultivar. Somatic embryos show growth arrest that is partially overcome by transfer to hormone-free basal medium with activated charcoal. Root induction is possible on basal medium plus 1 mg/L IBA. Plantlets in the greenhouse have variegated leaves true-to-type.

  11. Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae).

    Science.gov (United States)

    Chen, J -T.; Chang, W -C.

    2000-12-07

    An efficient method was established for high frequency somatic embryogenesis and plant regeneration from callus cultures of a hybrid of sympodial orchid (Oncidium 'Gower Ramsey'). Compact and yellow-white embryogenic calli formed from root tips and cut ends of stem and leaf segments on 1/2 MS [11] basal medium supplemented with 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ, 0.1-3 mg/l), 2,4-dichlorophenoxyacetic acid (2,4-D, 3-10 mg/l) and peptone (1 g/l) for 4-7 weeks. Embryogenic callus was maintained by subculture on the same medium for callus induction and proliferated 2-4 times (fresh weight) in 1 month. Initiation of somatic embryogenesis and development up to the protocorm-like-bodies (PLBs) from callus cultures was achieved on hormone-free basal medium. Regenerants were recovered from somatic embryos (SEs) after transfer to the same medium and showed normal development. The optimized protocol required about 12-14 weeks from the initiation of callus to the plantlet formation. Generally, the frequency of embryo formation of root-derived callus was higher than stem- and leaf-derived calli. Combinations of naphthaleneacetic acid (NAA) and TDZ significantly promoted embryo formation from callus cultures. The high-frequency (93.8%) somatic embryogenesis and an average of 29.1 SEs per callus (3x3 mm(2)) was found in root-derived callus on a basal medium supplemented with 0.1 mg/l NAA and 3 mg/l TDZ. Almost all the SEs converted and the plantlets grew well with an almost 100% survival rate when potted in sphagnum moss and acclimatized in the greenhouse.

  12. Grapevine leafroll-associated virus 3

    Directory of Open Access Journals (Sweden)

    Hans J. Maree

    2013-04-01

    Full Text Available Grapevine leafroll disease (GLD is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3, the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant-pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

  13. Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

    Science.gov (United States)

    de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

    2013-05-01

    Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

  14. Somatic embryogenesis from zygotic embryos of Euterpe oleracea Mart. Embriogênese somática em embriões zigóticos de Euterpe oleracea Mart.

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    Ana da Silva Ledo

    2002-12-01

    Full Text Available The aim of this work was to study the morphogenetic responses of zygotic embryos of açai palm (Euterpe oleracea Mart. submitted to several conditions of in vitro culture. Several research experiments were conducted, in laboratory, using vegetable material collected from açai palm plants at Embrapa Amazon Oriental, Belém-PA, Brazil. It was possible to verify the expression of a direct, repetitive and no-synchronized model of somatic embryogenesis in mature zygotic embryos cultivated in primary MS medium supplemented with 2,4-D (339.36 muM and transferred to a secondary MS medium in the presence of NAA (0.537 muM and 2iP (12.30 muM. The conversion of somatic embryos into seedlings was reached after 210 days with the transfer of the cultures to a third medium with sucrose and mineral salts concentrations reduced to a half, without growth regulators.O objetivo do presente trabalho foi estudar as diferentes respostas morfogenéticas de embriões zigóticos de açaizeiro (Euterpe oleracea Mart. submetidos a várias condições de cultura in vitro. Os experimentos foram conduzidos em laboratório, com material vegetal coletado de plantas de açaí da Embrapa Amazônia Oriental, Belém-PA, Brasil. Foi possível verificar a expressão de um modelo de embriogênese somática direto, repetitivo e assincronizado em embriões zigóticos maduros cultivados em meio primário MS, suplementado com 339,36 miM de 2,4-diclorofenoxiacético (2,4-D, e transferidos para meio secundário MS na presença de 0,537 miM de ácido 1-naftalenoacético (ANA e 12,30 miM de 2-isopenteniladenina (2iP. A conversão de embriões somáticos em plântulas foi alcançada aos 210 dias da inoculação com a transferência das culturas para um terceiro meio com a concentração de sais e sacarose reduzida pela metade e ausência de reguladores de crescimento.

  15. The role of chromatin modifications in somatic embryogenesis in plants

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    Clelia eDe-la-Peña

    2015-08-01

    Full Text Available Somatic embryogenesis (SE is a powerful tool for plant genetic improvement, when used in combination with agricultural traditional techniques, and it is being used to understand the different processes that occur during the development of plant embryogenesis. SE onset depends on a complex network of interactions among plant growth regulators, mainly auxins and cytokinins, during the proembryogenic early stages, and ethylene, gibberellic and abscisic acids later in the development of the somatic embryos. These growth regulators control spatial and temporal regulation of multiple genes in order to initiate the change in the genetic program of the somatic cells, as well as the transition among embryo developmental stages. In recent years, epigenetic mechanisms have emerged as critical factors during SE. Some early reports indicate that auxins modify the levels of DNA methylation in embryogenic cells. The changes in DNA methylation patterns are associated with the regulation of several genes involved in SE, such as WUS, BBM1, LEC, and several others. In this review, we highlight the more recent discoveries in the role of epigenetic regulation of SE. In addition, we include a survey of novel approaches to the study of SE, and new opportunities to focus SE studies.

  16. In vitro multiplication and somatic embryogenesis of Perezia pinnatifida (Asteraceae medicinal Andean plant

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    Percy Olivera-Gonzales

    2017-10-01

    Full Text Available This work inform on in vitro propagation of the "valeriana" Perezia pinnatifida (Humb. & Bonpl. Wedd. Shoot multiplication and indirect somatic embryogenesis methodologies were performed. The basal culture medium for all stages was Murashige and Skoog, middle of salts supplemented with 2.0% sucrose, 0.3% phytagel and pH 5.67; the treatments were prepared with or without phytohormones. The hormonal supplements for the shoot multiplication were: BAP 1.0 mg.L-1 + ANA 0.01 mg.L-1, and BAP 1.0 mg.L-1; for embryogenic callus induction were: ANA or 2,4-D (1.0 mg.L-1 and 2.0 mg.L-1; and for the embryo germination were: BAP (0.5 and 1.0 mg.L-1 or BAP 0.5mg.L-1 + ANA 0.05mg.L-1. BAP 1.0 mg.L-1 produced the higher number buds. For somatic embryogenesis, ANA 1.0 mg.L-1 induced a greater area of embryogenic callus, and BAP 0.5 mg.L-1 allowed major germination of the somatic embryos.

  17. Somatic embryogenesis of East Kalimantan local upland rice varieties

    Science.gov (United States)

    Nurhasanah; Ramitha; Supriyanto, B.; Sunaryo, W.

    2018-04-01

    Somatic embryogenesis is the formation, growth and development of embryos from somatic cells. Somatic embryo induction is one of the in vitro plant propagation techniques that is very important for plant developmental purposes. Four local upland rice varieties of East Kalimantan, Mayas Pancing, Gedagai, Siam and Serai, were used in this study. A total of 200 explants (mature rice grains) for each varieties were inoculated on MS solid medium supplemented with 1 mg L-1 2,4 Dichlorophenoxy acetic acid (2,4-D) and 0.5 mg L-1 6-Benzylaminopurine (BAP). The results showed that response of each variety differed to embryosomatic induction, indicated by callus induction rate and callus quality, in terms of callus color and structure. The fastest callus formation was sobserved in Gedagai variety (8 days) while Mayas Pancing (13 days) was the latest one. The rate of callus induction varied from 60 to 98.5 %, and Serai variety has the highest callus induction rate. The highest friable callus structure was found in Siam variety (89.1%) and the lowest was in Gedagai (62.5%). Callus color was dominated by the yellowish-white (transparent) on all varieties tested. Most of the callus was potential as embryogenic callus characterized from the nodular and globular of friable callus structure and its yellowish-white color.

  18. Efficient somatic embryogenesis and molecular marker based analysis as effective tools for conservation of red-listed plant Commiphora wightii

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    ASHOK KUMAR PARMAR

    2014-08-01

    Full Text Available A refined and high efficiency protocol for in vitro regeneration of Commiphora wightii, a red-listed medicinal plant of medicinal importance, has been developed through optimized somatic embryogenesis pathway. Cultures from immature fruits were induced and proliferated on B5 medium supplemented with 2.26 µM 2,4-D. Embryogenic calli were obtained and then maintained for extended periods by alternately subculturing on modified MS medium supplemented with 1.11 µM BAP, 0.57 µM IBA and with 0.5% activated charcoal or without PGR every 3-4 weeks. Cyclic embryogenesis was obtained. Late torpedo and early cotyledonary stages somatic embryos were regularly harvested from PGR-free modified MS medium. It was found that percent moisture available in culture containers play a critical role in maturation and subsequent germination of somatic embryos of C. wighti. Maximum germination of more than 80% was achieved through media recycling. Somatic embryo derived plants (emblings were acclimatized. After 5 months, acclimatized plants were out-planted in experimental field. These morphologically normal plants have been surviving for over 3 years. Molecular polymorphism was clearly evident when these plants were tested using RAPD primers, making the plants suitable for use in its species restoration program.

  19. Ultrastructural changes and the distribution of arabinogalactan proteins during somatic embryogenesis of banana (Musa spp. AAA cv. 'Yueyoukang 1').

    Science.gov (United States)

    Pan, Xiao; Yang, Xiao; Lin, Guimei; Zou, Ru; Chen, Houbin; Samaj, Jozef; Xu, Chunxiang

    2011-08-01

    A better understanding of somatic embryogenesis in banana (Musa spp.) may provide a practical way to improve regeneration of banana plants. In this study, we applied scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to visualize the ultrastructural changes during somatic embryogenesis of banana (Musa AAA cv. 'Yueyoukang 1'). We also used histological and immunohistochemical techniques with 16 monoclonal antibodies to study the spatial distribution and cellular/subcellular localization of different arabinogalactan protein (AGP) components of the cell wall during somatic embryogenesis. Histological study with periodic acid-Schiff staining documented diverse embryogenic stages from embryogenic cells (ECs) to the late embryos. SEM revealed a mesh-like structure on the surface of proembryos which represented an early structural marker of somatic embryogenesis. TEM showed that ECs were rich in juvenile mitochondria, endoplasmic reticulum and Golgi stacks. Cells in proembryos and early globular embryos resembled ECs, but they were more vacuolated, showed more regular nuclei and slightly more developed organelles. Immunocytochemical study revealed that the signal of most AGP epitopes was stronger in starch-rich cells when compared with typical ECs. The main AGP component in the extracellular matrix surface network of banana proembryos was the MAC204 epitope. Later, AGP immunolabelling patterns varied with the developmental stages of the embryos. These results about developmental regulation of AGP epitopes along with developmental changes in the ultrastructure of cells are providing new insights into the somatic embryogenesis of banana. Copyright © Physiologia Plantarum 2011.

  20. Modeling deployment of Pierce’s disease resistant grapevines

    Science.gov (United States)

    Deployment of Pierce’s disease resistant grapevines is a key solution to mitigating economic losses caused by Xylella fastidiosa. While Pierce’s disease resistant grapevines under development display mild symptoms and have lower bacterial populations than susceptible varieties, all appear to remain ...

  1. Effects of embryo-derived exosomes on the development of bovine cloned embryos.

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    Pengxiang Qu

    Full Text Available The developmental competence of in vitro cultured (IVC embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE, as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development, but also following growth to term (in vivo development. Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.

  2. In vitro manipulation techniques of porcine embryos

    DEFF Research Database (Denmark)

    Liu, Ying; Li, Juan; Løvendahl, Peter

    2015-01-01

    During the last 17 years, considerable advancements have been achieved in the production of pigs, transgenic and non-transgenic, by methods of somatic cell nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection, microinjection and sperm-mediated gene transfer by artificial...... insemination. Therefore, a review of the overall efficiency for the developmental competence of embryos produced by these in vitro methods would be useful in order to obtain a more thorough overview of this growing area with respect to its development and present status. In this review a meta-analysis was used...... to analyse data collected from all published articles with a focus on zygotes and embryos for transfer, pregnancy, full-term development and piglets born. It was generally concluded that an increasing level of in vitro manipulation of porcine embryos decreased the overall efficiency for production of piglets...

  3. [TSA improve transgenic porcine cloned embryo development and transgene expression].

    Science.gov (United States)

    Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

    2011-07-01

    Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

  4. Detection and Molecular Characterization of Grapevine Virus A in Jordan

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    G. Anfoka

    2004-12-01

    Full Text Available In a study on grapevines in Jordan conducted between 2002 and 2003, grapevine virus A (GVA was detected in all areas where grapevines were planted. DAS-ELISA analysis of samples from symptomatic trees found that 16.1% of samples were infected with GVA. Using a GVA- specific primer pair (H587/C995, a portion of the coat protein gene of the virus was amplified by IC-RT-PCR and RT-PCR, using leaf extracts and RNA extracted from infected grapevines respectively. After cloning and sequencing the coat protein gene of the Jordanian isolate of GVA (GVA-Jo, the sequence of the amplified product was compared with sequences of other GVA isolates from different countries.

  5. Respecting the Grapevine.

    Science.gov (United States)

    Carroll, David J.

    2001-01-01

    Administrators can create word-of-mouth communication that dispels negative attitudes and build good school reputations by discovering what parents and students are saying, targeting employee satisfaction and retention, providing excellent customer service, actively seeking and handling complaints, nurturing champions, and integrating "grapevine"…

  6. Histology study of the somatic embryogenesis in Agave fourcroydes Lem.

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    Silvia Alemán García

    2002-01-01

    Full Text Available A histological study of the structures embryogenic formed from meristem of Agave fourcroydes, with the objective of observing the behavior of the somatic embryogenesis from the induction until the formation of the embryo was carried out. The meristem cultivated in vitro was subculture in modified MS medium the induction. The samples to be included intheresinJB–4weretakenperiodically.Thehistologicalscutsof5-10µmwerecarriedoutwithrotatorymicrotome and strained in 2% toluidine blue. The sections were observed and photographed in light microscopy Axioplan Zeiis with increases of 20X. The results show to the 60 days of culture the presence of cells meristematic pre-embrygenic. Embryos in different phases of formation globular and scutellum were observed in all the mass of the callus in the following subculture. Key words: Agavaceae, in vitro culture, embryo, histology

  7. Morpho-anatomical characterization of embryogenic calluses from immature zygotic embryo of peach palm during somatic embryogenesis =

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    Simone de Alencar Maciel

    2010-04-01

    Full Text Available The objective of this study was to morpho-anatomically characterizenodular embryogenic calluses from zygotic embryos of peach palm during the induction of somatic embryogenesis. Immature zygotic embryos were pre-treated in MS medium added to Picloram and 2,4-D (25 μM and BAP (0, 5, 10 μM. After three months, primary calluses were transferred to MS induction medium added to Picloram and 2,4-D (450 μM. After six months, the embryogenic calluses were then histologically analyzed and cultivated in the maturation medium. The competent tissues of the zygotic embryos differentiated embryogenic calluses under action of both Picloram and 2,4-D auxins (450 μM, where the presence of multi-granular structures were observed. Histological observations showed that in the nodular embryogenic calluses, the outlying parenchymal cells exhibit cellular characteristics of high mitotic activity. Differentiation of tracheal elements exists in embryogenic calluses connecting the callus to the explant. The evaluated cytokinin/auxin interaction influences the development of embryogenic calluses and globular structures.O objetivo deste trabalho foi caracterizar morfoanatomicamente calos nodulares embriogênicos originados de embriões zigóticos de pupunheira durante a indução da embriogênese somática. Embriões zigóticos imaturos de pupunha foram inicialmente pré-tratados em meio de cultura MS, solidificado com 2,5 g L-1 de phytagel® e suplementado com Picloram e 2,4-D na concentração de 25 μM e BAP (0, 5, 10 μM. Após três meses, os calos primários foram transferidos para meio de indução, com Picloram e 2,4-D (450 μM. Após seis meses, os calosnodulares embriogênicos formados foram então analisados histologicamente e repicados para o meio de maturação para a progressão das estruturas multigranulares embriogênicas. Verificou-seque os tecidos competentes dos embriões zigóticos imaturos diferenciaram nódulos embriogênicos pela ação de ambas

  8. Clonación y micropropagación de curuba (Passiflora mollissima Bailey a partir de embriones somáticos provenientes de hojas | Cloning and micropropagation of banana passionfruit (Passiflora mollissima Bailey from leaf somatic embryos

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    María Del Pilar Acosta-Zambrano

    2017-11-01

    Full Text Available The banana passionfruit (Passiflora mollissima Bailey is a fruit from the Andean region and is used as raw material for the preparation of various food products. Tests were carried out for its in vitro multiplication using somatic embryos obtained from its leaves. A disinfection using sodium hypochlorite for 5 minutes and Murashige and Skoog (MS growth medium protocols with 1 mg/L of gibberellins was designed in order to induce germination. Vitro explants were selected for the multiplication in the MS medium supplemented with 2 mg/L of benzyl aminopurine and 1 mg/L of naphthenic acid. The stronger leaves were selected and inoculated in the MS medium and Woody Plant (WPM. The formation of embryos was observed since the third week. The formed plants were inoculated in a WPM medium with 1 mg/L of benzyl aminopurine. A 2-ip WPM 1 mg/L medium was used for the rooting period. Finally, they were acclimatized in peat with earth or pearlite, and then planted in pots with earth for further studies. Seed disinfection using sodium hypochlorite presented 20% contamination and 80% of plants germination, proving to be the best disinfectant (p < 0.05. From this last procedure, somatic embryos of leaves in a 2-ip WPM with 1 mg/L were obtained. The ideal acclimatization occurred in the medium with peat and earth, in which case the survival level obtained was 100% by comparison with earth and pearlite, in which no plant growth was observed. Micropropagation represents an economic and effective technique in the breeding of pathogen-free plants.

  9. Expression of a gymnosperm PIN homologous gene correlates with auxin immunolocalization pattern at cotyledon formation and in demarcation of the procambium during Picea abies somatic embryo development and in seedling tissues.

    Science.gov (United States)

    Palovaara, Joakim; Hallberg, Henrik; Stasolla, Claudio; Luit, Bert; Hakman, Inger

    2010-04-01

    In seed plants, the body organization is established during embryogenesis and is uniform across gymnosperms and angiosperms, despite differences during early embryogeny. Evidence from angiosperms implicates the plant hormone auxin and its polar transport, mainly established by the PIN family of auxin efflux transporters, in the patterning of embryos. Here, PaPIN1 from Norway spruce (Picea abies [L.] Karst.), a gene widely expressed in conifer tissues and organs, was characterized and its expression and localization patterns were determined with reverse transcription polymerase chain reaction and in situ hybridization during somatic embryo development and in seedlings. PaPIN1 shares the predicted structure of other PIN proteins, but its central hydrophilic loop is longer than most PINs. In phylogenetic analyses, PaPIN1 clusters with Arabidopsis thaliana (L.) Heynh. PIN3, PIN4 and PIN7, but its expression pattern also suggests similarity to PIN1. The PaPIN1 expression signal was high in the protoderm of pre-cotyledonary embryos, but not if embryos were pre-treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This, together with a high auxin immunolocalization signal in this cell layer, suggests a role of PaPIN1 during cotyledon formation. At later stages, high PaPIN1 expression was observed in differentiating procambium, running from the tip of incipient cotyledons down through the embryo axis and to the root apical meristem (RAM), although the mode of RAM specification in conifer embryos differs from that of most angiosperms. Also, the PaPIN1 in situ signal was high in seedling root tips including root cap columella cells. The results thus suggest that PaPIN1 provides an ancient function associated with auxin transport and embryo pattern formation prior to the separation of angiosperms and gymnosperms, in spite of some morphological differences.

  10. Novel technologies using radiation and somatic embryogenesis for Kenaf improvement

    International Nuclear Information System (INIS)

    Rusli Ibrahim; Siti Mariam Mohd Nahar; Siti Hajar Mohd Nahar; Abdul Rahim Harun; Azhar Mohamad; Sobri Hussein

    2010-01-01

    Full text: Kenaf (Hibiscus cannabinus L.) is a plant in the Malvaceae family, similar to roselle (Hibiscus sabdariffa), cotton (Gossypium hirsutum L.) and okra (Abelmoschus esculentus), holds a promising potential in the Malaysian bio composite industry. Its long fibres are suitable in the process of making a number of products such as pulp and paper, fibre and particle boards, as well as fibre reinforced plastic components and chemical absorbent. Most varieties of kenaf are photo period sensitive and vegetative growth increases until the daylight period becomes less than 12 h 30 min. flowering is then initiated and the vegetative growth rate declines. At present, most of the varieties planted by the farmers produced very low yield, between 3-5 tons/ha. The aim of this research proposal is to study the potential of using nuclear technique with the use radiation in combination with biotechnology to induce genetic variability in kenaf using somatic embryogenesis. Since mutation is a single cell event, irradiation of cell cultures such as somatic embryos will induce high rate of mutation for selection of desired traits. One of the main objectives of the project was to establish an efficient and productive regeneration system for intact plants from somatic embryos obtained from the original mother plant varieties: G4, V36 dan G393. Once regeneration protocol has been optimized, somatic embryos were irradiated using both acute (high dose rate) and chronic (lower dose rate) gamma irradiation with effective doses (2-3 doses). It takes between 4-5 months to reach maximum height of 4-6 meters from seed propagated plants before they can be harvested. With the use of in vitro mutagenesis, screening and selection of new mutant lines with traits of interest can be achieved within a short period of time (3-5 years). Field evaluations were carried out in collaboration with National Kenaf and Tobacco Board (NKTB) and Kelantan Biotech Corporation Sdn. Bhd. targeted for desired

  11. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  12. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

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    LiBing Ma

    Full Text Available It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT. Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1 cultured to 70-80% confluency (control group, (2 cultured to full confluency, (3 starved in low serum medium for 4 d, or (4 cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in

  13. Levels of polonium-210 in the grapevine leaves in Alasehir district in Turkey

    International Nuclear Information System (INIS)

    Gurboga, G.; Aytas Oelmez, S.

    2001-01-01

    The objective of present work is the estimation of Po-210 (polonium) content in the edible grapevine leaves (Vitis viniferae, L.cv Sultana syn.) collected from Gediz plain in Western Turkey. Alasehir District in Gediz plain is one of the most important wine culture region of Turkey. Grapevine leaves are important food material for Dolma in Turkish cuisine. Dolma is a name applied to such vegetables as grapevine leaves, cabbage leaves and green peppers stuffed ground meat or spiked rice. Levels of Po-210 in the grapevine leaves had not been analyzed before in Turkey. In this study, after wet ashing of grapevine leaves, Po-210 was spontaneously plated onto a copper disc from dilute hydrochloric acid medium and deposited activity was measured. The results for Po-210 in the grapevine leaves are compared with the other foodstuff values in the literature

  14. Effects of in ovo injection of carbohydrates on somatic characteristics and liver nutrient profiles of broiler embryos and hatchlings.

    Science.gov (United States)

    Zhai, W; Bennett, L W; Gerard, P D; Pulikanti, R; Peebles, E D

    2011-12-01

    Effects of the in ovo injection of commercial diluent supplemented with dextrin or with dextrin in combination with various other carbohydrates on the somatic characteristics and liver nutrient profiles of Ross × Ross 708 broiler embryos and chicks were investigated. Results include information concerning the gluconeogenic energy status of the liver before and after hatch. Eggs containing live embryos were injected in the amnion on d 18 of incubation using an automated multiple-egg injector for the delivery of the following carbohydrates dissolved in 0.4 mL of commercial diluent: 1) 6.25% glucose and 18.75% dextrin; 2) 6.25% sucrose and 18.75% dextrin; 3) 6.25% maltose and 18.75% dextrin; and 4) 25% dextrin. Also, a noninjected control and a 0.4-mL diluent-injected control were included. Body weight relative to set egg weight on d 19 of incubation (E19) was increased by the injection of all carbohydrate solutions, and on the day of hatch was increased by the injection of diluent, sucrose and dextrin, and maltose and dextrin solutions. Hatchability of the fertilized eggs, residual yolk sac weight, and liver weight were not affected by any injection treatment; however, as compared with the 0.4 mL diluent-injected group, all of the supplementary carbohydrates, except for the glucose and dextrin combination group, increased liver glycogen and glucose concentrations on E19. Furthermore, all carbohydrates, except for the 25% dextrin treatment, decreased liver fat concentration on E19. From E19 to the day of hatch, liver glycogen concentrations dropped dramatically from an average of 3.2 to 0.6%. Despite treatment differences observed on E19 for liver glycogen, glucose, and fat concentrations, these differences were lost by the day of hatch. Nevertheless, liver glycogen and glucose concentrations were positively correlated on the day of hatch. In conclusion, the in ovo injection of various supplemental carbohydrates dissolved in 0.4 mL of commercial diluent altered the

  15. Additional mitochondrial DNA influences the interactions between the nuclear and mitochondrial genomes in a bovine embryo model of nuclear transfer.

    Science.gov (United States)

    Srirattana, Kanokwan; St John, Justin C

    2018-05-08

    We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.

  16. Stem cells from residual IVF-embryos - Continuation of life justifies isolation.

    NARCIS (Netherlands)

    Bongaerts, G.P.A.; Severijnen, R.S.V.M.

    2007-01-01

    Embryonic stem cells are undifferentiated pluripotent cells that can indefinitely grow in vitro. They are derived from the inner mass of early embryos. Because of their ability to differentiate into all three embryonic germ layers, and finally into specialized somatic cell types, human embryonic

  17. Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures.

    Science.gov (United States)

    Ganesan, M; Jayabalan, N

    2005-10-01

    Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of maltose, glucose, sucrose and fructose. Maltose (30 g/l) is the best carbon source for embryogenic callus induction and glucose (30 g/l) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/l), thiamine HCI (0.3 mg/l), picloram (0.3 mg/l), Kin (0.1 mg/l) and maltose (30 g/l). During embryo induction and maturation, accelerated growth was observed in liquid medium containing NH3NO4 (1 g/l), picloram (2.0 mg/l), 2 ip (0.2 mg/l), Kin (0.1 mg/l) and glucose (30 g/l). Before embryoid induction, large clumps of embryogenic tissue were formed. These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50 mg/l), thiamine HCl (0.2 mg/l), GA3 (0.2 mg/l), BA (1.0 mg/l) and glucose (30 g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 g/l) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.

  18. PLEADING FOR THE GRAPEVINE CULTURE

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    Ionela Cătălina Guţă

    2012-04-01

    Full Text Available The grapevine is cultivated with good results on hilly terrain, on sand and sandy soils, thus ensuring high recovery of these categories of agricultural land considered unsuitable for other crops. Vineyards, so related to people existence everywhere, became something more than just places of economic interest. What makes the viticulture to be so important is that it refers to the food value, therapeutic, recreational of grapes, must and wine, wine derived products and residues from wine, the great extent of the area occupied by vineyards, to good natural conditions (pedo-climatic existing in our country and also to the aesthetic value of the land planted with vines worldwide. The fitting of the gardens of the house, both in the countryside and in urban areas, includes in most cases the presence of grapevine plants cut in different art forms, their care being an exciting job. In general, by the presence of vines are valued the spaces next to existing buildings (house, yard, various outbuildings, along fences and roads. Grapevines location, cutting types chosen, besides beautifying the yard, must make a harmonious aspect of the whole surrounding. Chosen forms of management (arches, halfarches can protect the strong sunlight places. By the kiosks or other artistic realized forms are created spaces for rest, shade.

  19. Endogenous Quantification of Abscisic Acid and Indole-3-Acetic Acid in Somatic and Zigotic Embryos of Nothofagus alpina (Poepp. & Endl. Oerst Cuantificación Endógena de Ácido Abscísico y Ácido Indol-3 Acético en Embriones Somáticos y Cigóticos de Nothofagus alpina (Poepp. & Endl. Oerst

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    Pricila Cartes Riquelme

    2011-12-01

    Full Text Available Abscisic acid (ABA and indole-3-acetic acid (IAA participate in the propagation of plants by somatic embryogenesis, causing polar structural differentiation of the embryo. The goal of the assay was to compare endogenous levels of ABA and IAA between somatic embryos (SE and zygotic embryos (ZE of Nothofagus alpina (Poepp. & Endl. Oerst. In this study, a somatic embryo maturation assay involving the addition of varying concentrations of exogenous ABA was performed on cotyledonary-stage of N. alpina. Furthermore, the endogenous levels of ABA and IAA were quantified in the immature ZE, the mature ZE, and the embryonic axis of a mature embryo of N. alpina. The current study utilized high performance liquid chromatography (HPLC for quantification. The maturation treatments performed did not present significant differences in the endogenous ABA levels in SE. However, significant differences did exist in levels of ABA and IAA between SE submitted to the different maturation treatments and mature ZE of N. alpina. The application of exogenous ABA to the culture medium increased endogenous ABA levels, therefore, increasing the number of germinated somatic embryos. Thus, the plant conversion process was also successfully completed in somatic embryos of N. alpina.El ácido abscísico (ABA y el ácido indol 3 acético (IAA participan en el proceso de propagación de plantas mediante embriogénesis somática, ya que permiten la diferenciación de la estructura polar del embrión, órganos y regiones meristemáticas de éste. En este estudio se llevó a cabo un ensayo de maduración de embriones somáticos en estado cotiledonar con la adición de diferentes concentraciones de ABA exógeno, además se determinaron niveles endógenos entre ZE inmaduro, ZE maduro, y eje embrionario aislado desde el embrión maduro para luego comparar niveles endógenos de ABA e IAA en embriones somáticos (SE y cigóticos (ZE de raulí, Nothofagus alpina (Poepp. & Endl. Oerst. La

  20. Neural network classification of sweet potato embryos

    Science.gov (United States)

    Molto, Enrique; Harrell, Roy C.

    1993-05-01

    Somatic embryogenesis is a process that allows for the in vitro propagation of thousands of plants in sub-liter size vessels and has been successfully applied to many significant species. The heterogeneity of maturity and quality of embryos produced with this technique requires sorting to obtain a uniform product. An automated harvester is being developed at the University of Florida to sort embryos in vitro at different stages of maturation in a suspension culture. The system utilizes machine vision to characterize embryo morphology and a fluidic based separation device to isolate embryos associated with a pre-defined, targeted morphology. Two different backpropagation neural networks (BNN) were used to classify embryos based on information extracted from the vision system. One network utilized geometric features such as embryo area, length, and symmetry as inputs. The alternative network utilized polar coordinates of an embryo's perimeter with respect to its centroid as inputs. The performances of both techniques were compared with each other and with an embryo classification method based on linear discriminant analysis (LDA). Similar results were obtained with all three techniques. Classification efficiency was improved by reducing the dimension of the feature vector trough a forward stepwise analysis by LDA. In order to enhance the purity of the sample selected as harvestable, a reject to classify option was introduced in the model and analyzed. The best classifier performances (76% overall correct classifications, 75% harvestable objects properly classified, homogeneity improvement ratio 1.5) were obtained using 8 features in a BNN.

  1. Plant regeneration from protoplasts ofVicia narbonensis via somatic embryogenesis and shoot organogenesis.

    Science.gov (United States)

    Tegeder, M; Kohn, H; Nibbe, M; Schieder, O; Pickardt, T

    1996-11-01

    Protoplasts ofVicia narbonensis isolated from epicotyls and shoot tips of etiolated seedlings were embedded in 1.4% sodium-alginate at a final density of 2.5×10(5) protoplasts/ml and cultivated in Kao and Michayluk-medium containing 0.5 mg/I of each of 2,4- dichlorophenoxyacetic acid, naphthylacetic acid and 6 -benzylaminopurine. A division frequency of 36% and a plating efficiency of 0.40-0.5% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred onto gelrite-solidified Murashige and Skoog-media containing various growth regulators. Regeneration of plants was achieved via two morphologically distinguishable pathways. A two step protocol (initially on medium with a high auxin concentration followed by a culture phase with lowered auxin amount) was used to regenerate somatic embryos, whereas cultivation on medium containing thidiazuron and naphthylacetic acid resulted in shoot morphogenesis. Mature plants were recovered from both somatic embryos as well as from thidiazuron-induced shoots.

  2. ‘Bois noir’: new phytoplasma disease of grapevine in Iran

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    Mirchenari Seyed Mehdi

    2015-01-01

    Full Text Available Recently, grapevines showing symptoms suggesting the ‘bois noir’ phytoplasma disease were observed in vineyards located in several central provinces of Iran. Polymerase chain reaction assays using phytoplasma universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR, confirmed the association of phytoplasmas with symptomatic grapevines. The results of RFLP analyses using HpaII, HinfI, MseI, RsaI, and TaqI restriction enzymes, indicated that grapevine phytoplasma isolates in these regions could be related to the 16SrXII group. Sequence analyses of the partial 16S rRNA gene confirmed that Iranian grapevine phytoplasmas are associated with ‘Candidatus Phytoplasma solani’. This is the first report of the ‘bois noir’ disease outbreak in Iran

  3. Control of grapevine wood fungi in commercial nurseries

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    C. Rego

    2009-05-01

    Full Text Available Previous surveys conducted in commercial nurseries found that different wood fungi, namely Cylindrocarpon spp., Botryosphaeriaceae, Phomopsis viticola and Phaeomoniella chlamydospora infect grapevine cuttings. Two field trials were carried out to evaluate the effectiveness of cyprodinil + fludioxonil, pyraclostrobin + metiram, fludioxonil and cyprodinil to prevent or reduce natural infections caused by such fungi. Rootstock and scion cuttings were soaked in fungicidal suspensions for 50 min prior to grafting. After callusing, the grafted cuttings were planted in two commercial field nurseries with and without a previous history of grapevine cultivation. After nine months in the nursery, the plants were uprooted and analysed for the incidence and severity of the wood fungi. Plants uprooted from the field without a previous history of grapevine cultivation were generally less strongly infected by wood fungi. Under this condition, only the mixture cyprodinil + fludioxonil simultaneously reduced the incidence of Cylindrocarpon and Botryosphaeriaceae fungi, as well as the severity of Cylindrocarpon infections. Treatments did not produce significant differences in the incidence and severity of P. viticola, and Pa. chlamydospora. For plants grown in the field with a grapevine history, all fungicides except cyprodinil significantly reduced the incidence and severity of Cylindrocarpon fungi. Also, the incidence and severity of Botryosphaeriaceae pathogens were significantly decreased both by cyprodinil + fludioxonil and by cyprodinil. No significant differences were noticed for P. viticola incidence and severity, and Pa. chlamydospora was not detected again. These results suggest that the practice of soaking grapevine cuttings in selected fungicides prior to grafting significantly reduces Cylindrocarpon spp. and Botryosphaeriaceae infections, thus improving the quality of planting material.

  4. Follow-up of fluorine pollution effect on grapevine

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    Ferjani Ben Abdallah

    2004-12-01

    By another way, our results seem to show that full mature grapevine leaves may constitute an efficient tool to assess fluorine pollution impact. Berries contamination seems to be affected directly by the factory smoke, there is no endogenous supply. Likewise, by its characteristic necrosis in the leaf boundaries, grapevine may be considered as a bioindicator variety of fluorine pollution which can be used in mapping polluted areas.

  5. Developmental control of hypoxia during bud burst in grapevine.

    Science.gov (United States)

    Meitha, Karlia; Agudelo-Romero, Patricia; Signorelli, Santiago; Gibbs, Daniel J; Considine, John A; Foyer, Christine H; Considine, Michael J

    2018-05-01

    Dormant or quiescent buds of woody perennials are often dense and in the case of grapevine (Vitis vinifera L.) have a low tissue oxygen status. The precise timing of the decision to resume growth is difficult to predict, but once committed, the increase in tissue oxygen status is rapid and developmentally regulated. Here, we show that more than a third of the grapevine homologues of widely conserved hypoxia-responsive genes and nearly a fifth of all grapevine genes possessing a plant hypoxia-responsive promoter element were differentially regulated during bud burst, in apparent harmony with resumption of meristem identity and cell-cycle gene regulation. We then investigated the molecular and biochemical properties of the grapevine ERF-VII homologues, which in other species are oxygen labile and function in transcriptional regulation of hypoxia-responsive genes. Each of the 3 VvERF-VIIs were substrates for oxygen-dependent proteolysis in vitro, as a function of the N-terminal cysteine. Collectively, these data support an important developmental function of oxygen-dependent signalling in determining the timing and effective coordination bud burst in grapevine. In addition, novel regulators, including GASA-, TCP-, MYB3R-, PLT-, and WUS-like transcription factors, were identified as hallmarks of the orderly and functional resumption of growth following quiescence in buds. © 2018 John Wiley & Sons Ltd.

  6. Characterization of Cadophora luteo-olivacea and C. melinii isolates obtained from grapevines and environmental samples from grapevine nurseries in Spain

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    David GRAMAJE

    2011-12-01

    Full Text Available Fifty-eight Cadophora luteo-olivacea and three C. melinii isolates were recovered from grapevines showing black vascular streaking and decline symptoms characteristic of Petri disease, and from different stages of the grapevine nursery process in Spain. The isolates were studied by means of phenotypical characterization, DNA analysis and pathogenicity tests. The morphological characters studied included conidiophore, phialide and conidial morphology. Colony characters and pigment production on MEA, PDA and OA were also examined. Phenotypical data were subjected to cluster analysis, which clearly separated C.luteo-olivacea isolates into four groups. Mating tests were performed on all possible combinations for each Cadophora species but no sexual fruiting bodies were produced. Partial sequences of the nuclear ribosomal internal transcribed spacer (ITS, beta-tubulin (BT and the elongation factor 1a (EF were analysed, but no genetic variation occurred within the C. luteo-olivacea isolates or within the C. melinii isolates in any of the regions studied. Pathogenicity tests were conducted on 1-year-old grapevine cuttings of four different rootstocksusing four C. luteo-olivacea isolates and one isolate of C. melinii. All Cadophora isolates except the C.melinii isolate caused significantly longer lesions in the xylem of grapevine rootstocks than in the controls.

  7. Identification of Eutypa lata, a Grapevine Parasite

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    Goran Delibašić

    2006-01-01

    Full Text Available The phytopathogenic fungus Eutypa lata (Pers.: Fr. Tul. and C. Tul., the causing agent of eutypa dieback, has been increasingly often identified in recent times as a cause of grapevine disease. It was first discovered and identified in Australian vineyards (Carter,1973, where it represented one of the most dangerous fungus pathogens of this plant. A few years later it was discovered in European vineyards as well. This polyfagous fungus,known originally as E. armeniaca (Honsf. & Carter, was first discovered on apricot, on which it caused the “gummosis disease”.In Serbia, Eutypa lata has not been determined officially. However, bearing in mind the form of its spreading (anemochory, as well as the fact that our country is a major producer of grape and fruit, we need to pay special attention to this dangerous pathogen since thereare indications that it is already present in our vineyards.During the period between 2003 and 2005, an inspection of a great number of vineyards in the areas of Vršac, Fruška Gora and Kruševac, was conducted. Many of them had grapevines with typical eutypa dieback symptoms. The aim of the inspection was to find grapevines with this disease, to mark them and take samples for laboratory analysis. Marking suspicious grapevines enabled us to monitor the volume of symptoms, as well as other changes on grapevines. Different colours were used for markings, according to the principle “same colour – same year” The procedure revealed that the average periodbetween early and mild disease symptoms and extreme changes, including withering of entire vines, was 2 to 3 years.The signs of eutypa dieback on diseased grapevines are manifested: on leaves in the form of chlorosis, twisting, necrosis of the edges, drying out and falling off; on shoots,where the shortening of internodia is noticable, as well as colour change and “zig-zag” distribution of internodes; on blossoms and clusters, where absence of flowering, partial

  8. Root-associated bacteria promote grapevine growth: from the laboratory to the field

    KAUST Repository

    Rolli, Eleonora

    2016-08-18

    Background and Aims: Laboratory and greenhouse experiments have shown that root-associated bacteria have beneficial effects on grapevine growth; however, these effects have not been tested in the field. Here, we aimed to demonstrate whether bacteria of different geographical origins derived from different crop plants can colonize grapevine to gain a beneficial outcome for the plant leading to promote growth at the field scale. Methods: To link the ecological functions of bacteria to the promotion of plant growth, we sorted fifteen bacterial strains from a larger isolate collection to study in vitro Plant Growth Promoting (PGP) traits. We analysed the ability of these strains to colonise the root tissues of grapevine and Arabidopsis using green-fluorescent-protein-labelled strain derivatives and a cultivation independent approach. We assessed the ability of two subsets randomly chosen from the 15 selected strains to promote grapevine growth in two field-scale experiments in north and central Italy over two years. Parameters of plant vigour were measured during the vegetative season in de novo grafted vine cuttings and adult productive plants inoculated with the bacterial strains. Results: Beneficial bacteria rapidly and intimately colonized the rhizoplane and the root system of grapevine. In the field, plants inoculated with bacteria isolated from grapevine roots out-performed untreated plants. In both the tested vineyards, bacteria-promotion effects largely rely in the formation of an extended epigeal system endowed of longer shoots with larger diameters and more nodes than non-inoculated plants. Conclusions: PGP bacteria isolated in the laboratory can be successfully used to promote growth of grapevines in the field. The resulting larger canopy potentially increased the photosynthetic surface of the grapevine, promoting growth.

  9. Anatomical Study of Somatic Embryogenesis in Glycine max (L. Merrill

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    Juliana Aparecida Fernando

    2002-09-01

    Full Text Available A comparative anatomical analysis of somatic embryogenesis in two soybean (Glycine max (L. Merrill genotypes was carried out. The somatic embryos were originated from cotyledonary explants obtained from immature zygotic embryos. The medium used for somatic embryogenesis induction was Murashige and Skoog, 1962, salts and Gamborg et al., 1968, vitamins (MSB supplemented with 0.8 mg.L-1 of 2,4-D for genotype PI 123439 and 40 mg.L-1 of 2,4-D for ‘Williams 82’. Globular structures, constituted by meristematic cells, originated from subepidermal cell divisions of the cotyledonary mesophyll. In PI 123439, the globular structures presented tracheary differentiation among meristematic cells and they could follow distinct morphogenetic process depending on their location along the explant. For ‘Williams 82’ it was observed globular structures along the cotyledonary explant surface. They gave rise to somatic embryos. These embryos showed different morphologies and they were classified based on their shape and number of cotyledons. The ability of these morphological types to convert to plantlets was discussed.Realizou-se uma análise anatômica comparativa da embriogênese somática em dois genótipos de soja (Glycine max (L. Merrill. Os embriões somáticos foram obtidos a partir de explantes cotiledonares excisados de embriões zigóticos imaturos do genótipo PI 123439, adaptado às condições tropicais, e ‘Williams 82’. O meio utilizado para indução da embriogênese somática constituiu-se de sais de Murashige e Skoog,1962, e vitaminas de Gamborg et al., 1968 (MSB suplementado com 0,8 mg.L-1 de 2,4-D (PI 123439 e 40 mg.L-1 (‘Williams 82’. Estruturas globulares originaram-se a partir de divisões celulares nas camadas subepidérmicas do mesofilo cotiledonar e foram constituídas por células meristemáticas. No genótipo PI 123439, as estruturas globulares apresentaram diferenciação traqueal entre as células meristemáticas e

  10. The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation.

    Science.gov (United States)

    Zhang, Yufan; Clemens, Adam; Maximova, Siela N; Guiltinan, Mark J

    2014-04-24

    The Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network. An ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction. Our study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm.

  11. THE EFFECT OF PICLORAM AND LIGHT ON SOMATIC EMBRYOGENESIS REGENERATION OF PINEAPPLE

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    Ika Roostika

    2012-10-01

    Full Text Available Smooth Cayenne is the largest pineapple type cultivated in Indonesia, but its vegetative planting materials for mass propagation are limited. Somatic embryogenesis is a potential method to be applied. The aim of this study was to investigate the somatic embryogenesis regeneration under the effect of picloram and light. Callus formation was induced by picloram (21, 41 and 62 μM added with 9 μM thidiazuron. The calli were transferred onto MS or Bac medium  enriched with N-organic compounds with or without addition of 21 μM picloram under dark or light condition. The compact calli were subcultured onto MS medium supplemented with 4.65 μM kinetin, while the friable calli were  transferred onto BIG medium (modified MS + 1.1 μM benzyl adenine + 0.9 μM indole butyric acid + 0.09 μM giberelic acid or B medium (MS + 0.018 mM benzyl adenine. The results showed that the events of somatic embryogenesis were started from cell polarization, asymmetrical division, proembryo formation as  embryogenic tissues and friable embryogenic tissues, and embryo development. The best treatment for callus induction was 21 μM picloram. The addition of 21 μM picloram on N-organic enriched medium and the use of light condition  proliferated embryogenic calli. The N-organic enriched Bac medium and light condition yielded the highest number of mature somatic embryos (17 embryos perexplant in 2 months. The B medium was better than BIG medium to develop  somatic embryos from friable embryogenic tissues. The somatic embryogenesis method presented is potential for pineapple mass propagation and artificial seedproduction.Abstrak Bahasa IndonesiaSmooth Cayenne merupakan kultivar nenas yang banyak dibudidayakan di  Indonesia, namun ketersediaan benih untuk perbanyakan massal masih terbatas. Embriogenesis somatikadalah metode yang potensial untuk produksi bibit secara massal. Tujuan penelitian adalah untuk mempelajari pengaruh pikloram dan pencahayaan terhadap regenerasi

  12. BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

    Science.gov (United States)

    Huang, Jiaojiao; Zhang, Hongyong; Yao, Jing; Qin, Guosong; Wang, Feng; Wang, Xianlong; Luo, Ailing; Zheng, Qiantao; Cao, Chunwei; Zhao, Jianguo

    2016-01-01

    Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, Pcloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression. © 2016 Society for Reproduction and Fertility.

  13. Effects of GhWUS from upland cotton (Gossypium hirsutum L.) on somatic embryogenesis and shoot regeneration.

    Science.gov (United States)

    Xiao, Yanqing; Chen, Yanli; Ding, Yanpeng; Wu, Jie; Wang, Peng; Yu, Ya; Wei, Xi; Wang, Ye; Zhang, Chaojun; Li, Fuguang; Ge, Xiaoyang

    2018-05-01

    The WUSCHEL (WUS) gene encodes a plant-specific homeodomain-containing transcriptional regulator, which plays important roles during embryogenesis, as well as in the formation of shoot and flower meristems. Here, we isolated two homologues of Arabidopsis thaliana WUS (AtWUS), GhWUS1a_At and GhWUS1b_At, from upland cotton (Gossypium hirsutum). Domain analysis suggested that the two putative GhWUS proteins contained a highly conserved DNA-binding HOX domain and a WUS-box. Expression profile analysis showed that GhWUSs were predominantly expressed during the embryoid stage. Ectopic expression of GhWUSs in Arabidopsis could induce somatic embryo and shoot formation from seedling root tips. Furthermore, in the absence of exogenous hormone, overexpression of GhWUSs in Arabidopsis could promote shoot regeneration from excised roots, and in the presence of exogenous auxin, excised roots expressing GhWUS could be induced to produce somatic embryo. In addition, expression of the chimeric GhWUS repressor in cotton callus inhibited embryogenic callus formation. Our results show that GhWUS is an important regulator of somatic embryogenesis and shoot regeneration. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Somatic Embryogenesis in Parana Pine (Araucaria angustifolia (Bert. O. Kuntze

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    Santos André Luis Wendt dos

    2002-01-01

    Full Text Available Embryogenic cultures of Araucaria angustifolia were induced from dominant and non-dominant zygotic embryos excised from immature seeds proceeding from three different genotypes and five harvest dates. Zygotic embryos were inoculated in inductive culture medium LP and BM supplemented with or without plant growth regulators 2,4-D (5 µM, BA (2 µM and Kin (2 µM. The genotype of the mother tree and the developmental explant stage affected the induction frequency. In the maintenance phase, embryogenic cultures were maintained at continuous repetitive cell cycles every 20 days in semi-solid or liquid medium. In the maturation phase the culture medium was supplemented with different types and levels of growth regulators, osmotic agents, carbohydrates and derived. Embryogenic cultures inoculated in culture medium supplemented with PEG 3350 (6 and 9%, maltose (6 and 9%, plus BA and Kin (1 µM each resulted in the progression of somatic embryos to globular and torpedo developmental stages.

  15. Recurrent somatic embryogenesis in long-term cultures of Gentiana lutea L. as a source for synthetic seed production for medium-term preservation

    Directory of Open Access Journals (Sweden)

    Holobiuc Irina

    2012-01-01

    Full Text Available Our aim was to establish an efficient and reproducible system for producing synthetic seeds from recurrent somatic embryogenesis in long-term cultures of Gentiana lutea L. This species is a vulnerable medicinal plant, protected both at the national and international levels, and is included in different Red Lists and Books. In vitro culture, as an alternative to classical methods of preservation, allows for the cyclic multiplication of plant material and short-, medium- and long-term preservation of tissue collections. Biotechnological approaches allow for maintenance of the plant material in a confined space and protection against biotic and abiotic factors. Somatic embryogenesis (SE is the most efficient way to regenerate plants, ensuring material for preservation and fundamental research. In our experiment, recurrent somatic embryogenesis was developed in long-term cultures in the presence of sugar alcohols (mannitol, sorbitol and in the absence of growth factors. This process proceeded at a high rate, with adventive somatic embryos being generated in a continuous process, followed by maturation, germination and development into plants. To follow the somatic embryogenesis process, histological samples were made. We used these embryogenic cultures for synthetic seed production and medium-term conservation. The viability of somatic embryos after moderate osmotic stress treatment was tested using TTC. Our methodology relied on the induction of somatic embryogenesis in the presence of auxins in the first cycle of in vitro cultures, long-term high embryogenic culture maintenance in the presence of sugar alcohols and synthetic seed production.

  16. Genomics technologies to study structural variations in the grapevine genome

    Directory of Open Access Journals (Sweden)

    Cardone Maria Francesca

    2016-01-01

    Full Text Available Grapevine is one of the most important crop plants in the world. Recently there was great expansion of genomics resources about grapevine genome, thus providing increasing efforts for molecular breeding. Current cultivars display a great level of inter-specific differentiation that needs to be investigated to reach a comprehensive understanding of the genetic basis of phenotypic differences, and to find responsible genes selected by cross breeding programs. While there have been significant advances in resolving the pattern and nature of single nucleotide polymorphisms (SNPs on plant genomes, few data are available on copy number variation (CNV. Furthermore association between structural variations and phenotypes has been described in only a few cases. We combined high throughput biotechnologies and bioinformatics tools, to reveal the first inter-varietal atlas of structural variation (SV for the grapevine genome. We sequenced and compared four table grape cultivars with the Pinot noir inbred line PN40024 genome as the reference. We detected roughly 8% of the grapevine genome affected by genomic variations. Taken into account phenotypic differences existing among the studied varieties we performed comparison of SVs among them and the reference and next we performed an in-depth analysis of gene content of polymorphic regions. This allowed us to identify genes showing differences in copy number as putative functional candidates for important traits in grapevine cultivation.

  17. Genetic characterization of some Romanian red wine grapevine varieties

    Science.gov (United States)

    Ghetea, Ligia Gabriela; Motoc, Rozalia Magda; Niculescu, Ana-Maria; Litescu, Simona Carmen; Duma, Virgil-Florin; Popescu, Carmen Florentina

    2008-04-01

    In our study we have considered three of the most valuable Romanian red wine grapevine cultivars: Feteasca neagra, Feteasca alba and Novac. We have chosen to study grapevine because grapes and wine are an important part of a healthy diet, and because red grapes have the highest content of proanthocyanidins, that act as antioxidants (free radical scavengers) in the human body. Proanthocyanidins possess anti-mutagenic, anti-tumor, anti-viral activities and they present many other confirmed or potential benefits. Genotyping method was applied in order to asses the genetic profile at 14 microsatellite loci, for two cultivars: Feteasca neagra and Feteasca alba. In order to achieve this, the HPLC-DAD method was used. The content of anthocyans in grape skin from two cultivars - Feteasca neagra and Novac - was measured. Microsatellite markers have been certified as powerful tools for assessing genetic identities and genetic relationships between grapevine gene pools. Genetic characterization of grapevine cultivars can certify their authenticity and purity, two features that have a direct effect on the quality and value of the finished product, the wine. In our country, this is the first attempt in order to establish a genetic profile for valuable Romanian origin grapevine varieties. In some of the 14 microsatellitic loci, Feteasca neagra and Feteasca alba cultivars presented allele size variants different from the values cited in the literature, proving that these cultivars belong to a geographical distinct gene pool. The content of anthocyans in Feteasca neagra grape skin was significantly higher than in Novac.

  18. Influence of embryo handling and transfer method on pig cloning efficiency.

    Science.gov (United States)

    Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong

    2015-03-01

    The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Genetic chimerism of Vitis vinifera cv. Chardonnay 96 is maintained through organogenesis but not somatic embryogenesis

    Directory of Open Access Journals (Sweden)

    Butterlin Gisèle

    2005-09-01

    Full Text Available Abstract Background Grapevine can be a periclinal chimera plant which is composed at least of two distinct cell layers (L1, L2. When the cell layers of this plant are separated by passage through somatic embryogenesis, regenerated plants could show distinct DNA profiles and a novel phenotype which proved different from that of the parent plant. Results Genetically Chardonnay clone 96 is a periclinal chimera plant in which is L1 and L2 cell layers are distinct. Plants obtained via organogenesis through meristematic bulks are shown to be composed of both cell layers. However, plants regenerated through somatic embryogenesis starting from anthers or nodal explants are composed only of L1 cells. These somaclones do not show phenotypic differences to the parental clone up to three years after regeneration. Interestingly, the only somaclone showing an atypical phenotype (asymmetric leave shows a genotypic modification. Conclusion These results suggest that the phenotype of Chardonnay 96 does not result from an interaction between the two distinct cell layers L1 and L2. If phenotype conformity is further confirmed, somatic embryogenesis will result in true-to-type somaclones of Chardonnay 96 and would be well suitable for gene transfer.

  20. Physiological and agronomical responses of Syrah grapevine under protected cultivation

    Directory of Open Access Journals (Sweden)

    Claudia Rita de Souza

    2015-01-01

    Full Text Available The performance of Syrah grapevine under protected cultivation with different plastic films was evaluated during 2012 and 2013 seasons in South of Minas Gerais State. Agronomical and physiological measurements were done on eight years old grapevines, grafted onto ‘1103 Paulsen’ rootstock cultivated under uncovered conditions, covered with transparent and with diffuse plastic films. Both plastic covers induced the highest shoot growth rate and specific leaf area. The diffuse plastic induced greater differences on leaf area, pruning weight and leaf chlorophyll content as compared to uncovered vines. Grapevines under diffuse plastic also had the lowest rates of photosynthesis, stomatal conductance and transpiration. Leaf starch, glucose and fructose contents were not affected by treatment, but leaf sucrose was reduced by transparent plastic. The leaf and stem water potential were higher under diffuse plastic. In 2013, grapevines under diffuse plastic showed the highest yields mainly due to decreased rot incidence and increased cluster weight. Furthermore, berries under diffuse plastic showed the highest anthocyanins concentration. The use of diffuse plastic induces more agronomical benefits to produce Syrah grape under protected cultivation.

  1. VARYING DEGREE OF GRAFTING COMPATIBILITY BETWEEN CV. CHARDONNAY, MERLOT AND DIFFERENT GRAPEVINE ROOTSTOCKS

    Directory of Open Access Journals (Sweden)

    Slavica TODIĆ

    2006-02-01

    Full Text Available Level of affi nity between grapevine rootstock and Vitis vinifera as scion, quality of reproductive materials and technological actions in grapevine rootstock production process determine success in grapevine rootstock production in large extent. Practical training showed that difference in level of compatibility between grapevine rootstock and grafted Vitis vinifera cultivars are existing. Direct effects of these differences are unequal yield of fi rst class grafted grapevine rootlings. In this paper, level of compatibility in nursery between clones of cv. Chardonnay BCL 75, VCR4 and cv. Merlot R18, MCL 519 and grapevine rootstocks Kober 5BB (Vitis berlandieri x V. riparia, SO4 (V. berlandieri x V. riparia and 41B (Chasselas x V.berlandieri were investigated. The trial was conducted in commercial grapevine nursery located in Velika Drenova, Serbia. As an index of compatibility, grade of high quality grapevine grafted rootlings, dry matter in mature shoots and root system development were used. Grafting was done by `tongue grafting` indoor technique. Stratifi cation was done in sand, on temperature of the stratifi cation material of 26-28oC, and humidity of around 90%. Grafted cuttings were waxed twice: before stratifi cation, and before planting in the nursery. Grafted rootlings were classed in two classes according to regulations of quality, (Yugoslav Offi cial Register, 26/79. Grafted rootlings that did not satisfi ed standard criteria were discarded. Both clones of cv. Chardonnay gave the highest percentage of I class grafted rootlings on grapevine rootstock 41B: clone BCL 75 – 60% and clone VCR4 – 61%. In the same combination, those grapevine grafted rootlings had the highest weight of the root system. Lower percentage of obtained I class grafted rootlings was established on rootstock Kober 5BB, while statistically signifi cantly lower yields were obtained on grapevine rootstock SO4: clone BCL75 – 43% and clone VCR4 – 48%. Dry

  2. Whole genome amplification and microsatellite genotyping of herbarium DNA revealed the identity of an ancient grapevine cultivar

    Science.gov (United States)

    Malenica, Nenad; Šimon, Silvio; Besendorfer, Višnja; Maletić, Edi; Karoglan Kontić, Jasminka; Pejić, Ivan

    2011-09-01

    Reconstruction of the grapevine cultivation history has advanced tremendously during the last decade. Identification of grapevine cultivars by using microsatellite DNA markers has mostly become a routine. The parentage of several renowned grapevine cultivars, like Cabernet Sauvignon and Chardonnay, has been elucidated. However, the assembly of a complete grapevine genealogy is not yet possible because missing links might no longer be in cultivation or are even extinct. This problem could be overcome by analyzing ancient DNA from grapevine herbarium specimens and other historical remnants of once cultivated varieties. Here, we present the first successful genotyping of a grapevine herbarium specimen and the identification of the corresponding grapevine cultivar. Using a set of nine grapevine microsatellite markers, in combination with a whole genome amplification procedure, we found the 90-year-old Tribidrag herbarium specimen to display the same microsatellite profile as the popular American cultivar Zinfandel. This work, together with information from several historical documents, provides a new clue of Zinfandel cultivation in Croatia as early as the beginning of fifteenth century, under the native name Tribidrag. Moreover, it emphasizes substantial information potential of existing grapevine and other herbarium collections worldwide.

  3. An Epigenetic Modifier Results in Improved In Vitro Blastocyst production after Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Zhang, Yunhai; Li, Juan; Villemoes, Klaus

    2007-01-01

    The present study was designed to examine the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on development of porcine cloned embryos. Our results showed that treatment of cloned embryos derived from sow oocytes with 50 nM TSA for up to 24 h after the onset of activation cou...... were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production...

  4. Changes in the synthesis of DNA, RNA and protein during somatic embryogenesis in wheat (triticum aestivum L.)

    International Nuclear Information System (INIS)

    Cui Kairong; Wang Xiaozhe; Chen Xiong; Wang Yafu

    1997-01-01

    Embryogenic and non-embryogenic callus formed from immature embryo of wheat (Triticum aestivum L.) in N 6 B 5 MS medium I supplemented with 2,4-D 2 mg/L, KT 0.5 mg/L, LH300 mg/L, sucrose 3% were sub-cultured and transferred respectively to N 6 B 5 MS medium II (2,4-D was decreased to 0.5 mg/L and 4 mol/L proline was added). Somatic embryos obtained from embryogenic callus, and plantlet formed from non-embryogenic callus through organogenesis respectively. By incorporation of 3 H-thymidine, 3 H-uridine and 3 H-leucine into DNA, RNA and protein respectively, the rate of synthesis of DNA, RNA and protein during somatic embryogenesis were measured. A large amount of RNA and protein synthesized during the early somatic embryogenesis. The activities of RNA and protein synthesis reached the peak on the 4th and the 8th day respectively, then decreased a little, but kept a high level. The synthesis of DNA increased apparently during the early stage. No apparent change occurred when the embryogenic cell masses formed. The synthesis rate of RNA and protein in non-embryogenic callus were much less than that in embryogenic callus. Actinomycin and cycloheximide inhibited not only the synthesis of nucleic acid and protein, but also the growth of embryogenic callus and somatic embryogenesis. The earlier the inhibitors were added, the greater the influence was caused. The results indicate that the active expression of corresponding genes of wheat is the molecular base of somatic embryogenesis

  5. Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

    International Nuclear Information System (INIS)

    Lee, Eugine; Lee, So Hyun; Kim, Sue

    2006-01-01

    Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P < 0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones

  6. RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

    Science.gov (United States)

    Zhu, Hai-Ying; Jin, Long; Guo, Qing; Luo, Zhao-Bo; Li, Xiao-Chen; Zhang, Yu-Chen; Xing, Xiao-Xu; Xuan, Mei-Fu; Zhang, Guang-Lei; Luo, Qi-Rong; Wang, Jun-Xia; Cui, Cheng-Du; Li, Wen-Xue; Cui, Zheng-Yun; Yin, Xi-Jun; Kang, Jin-Dan

    2017-05-01

    To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p  0.05), this was not significant. RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

  7. Survival and ultrastructural features of peach palm (Bactris gasipaes, Kunth) somatic embryos submitted to cryopreservation through vitrification.

    Science.gov (United States)

    Heringer, Angelo Schuabb; Steinmacher, Douglas André; Schmidt, Éder Carlos; Bouzon, Zenilda Laurita; Guerra, Miguel Pedro

    2013-10-01

    Bactris gasipaes (Arecaceae), also known as peach palm, was domesticated by Amazonian Indians and is cultivated for its fruit and heart-of-palm, a vegetable grown in the tree's inner core. Currently, the conservation of this species relies on in situ conditions and field gene banks. Complementary conservation strategies, such as those based on in vitro techniques, are indicated in such cases. To establish an appropriate cryopreservation protocol, this study aimed to evaluate the ultrastructural features of B. gasipaes embryogenic cultures submitted to vitrification and subsequent cryogenic temperatures. Accordingly, somatic embryo clusters were submitted to Plant Vitrification Solution 3 (PVS3). In general, cells submitted to PVS3 had viable cell characteristics associated with apparently many mitochondria, prominent nucleus, and preserved cell walls. Cells not incubated in PVS3 did not survive after the cryogenic process in liquid nitrogen. The best incubation time for the vitrification technique was 240 min, resulting in a survival rate of 37 %. In these cases, several features were indicative of quite active cell metabolism, including intact nuclei and preserved cell walls, an apparently many of mitochondria and lipid bodies, and the presence of many starch granules and condensed chromatin. Moreover, ultrastructure analysis revealed that overall cellular structures had been preserved after cryogenic treatment, thus validating the use of vitrification in conjunction with cryopreservation of peach palm elite genotypes, as well as wild genotypes, which carry a rich pool of genes that must be conserved.

  8. [Effect of TSA and VPA treatment on long-tailed macaque (Macaca fascicularis)-pig interspecies somatic cell nuclear transfer].

    Science.gov (United States)

    Qin, Zu-Xing; Huang, Gao-Bo; Luo, Jun; Ning, Shu-Fang; Lu, Sheng-Sheng; Lu, Ke-Huan

    2012-03-01

    Long-tailed macaque-pig interspecies somatic cell nuclear transfer (iSCNT) is beneficial to yield embryonic stem cells from iSCNT embryos with similar genetic background as human, which can be used as materials for medical and basic research. The primary objective of this study was to investigate the effects of concentrations and treatment duration of two histone deacetylase inhibitors-Trichostatin A (TSA) and Valproic acid (VPA) and two different embryo culture media (PZM-3 and HECM-10) on the in vitro development of iSCNT embryos. The results suggested that when PZM-3 was used as the embryo culture medium, the blastocyst rate of 10 nmol/L TSA treatment for 48 h was significantly higher than the control group (22.78% vs 9.86%, PTSA treatment could enhance the in vitro developmental potential of long-tailed macaque-pig iSCNT embryos.

  9. The grapevine kinome: annotation, classification and expression patterns in developmental processes and stress responses.

    Science.gov (United States)

    Zhu, Kaikai; Wang, Xiaolong; Liu, Jinyi; Tang, Jun; Cheng, Qunkang; Chen, Jin-Gui; Cheng, Zong-Ming Max

    2018-01-01

    Protein kinases (PKs) have evolved as the largest family of molecular switches that regulate protein activities associated with almost all essential cellular functions. Only a fraction of plant PKs, however, have been functionally characterized even in model plant species. In the present study, the entire grapevine kinome was identified and annotated using the most recent version of the grapevine genome. A total of 1168 PK-encoding genes were identified and classified into 20 groups and 121 families, with the RLK-Pelle group being the largest, with 872 members. The 1168 kinase genes were unevenly distributed over all 19 chromosomes, and both tandem and segmental duplications contributed to the expansion of the grapevine kinome, especially of the RLK-Pelle group. Ka/Ks values indicated that most of the tandem and segmental duplication events were under purifying selection. The grapevine kinome families exhibited different expression patterns during plant development and in response to various stress treatments, with many being coexpressed. The comprehensive annotation of grapevine kinase genes, their patterns of expression and coexpression, and the related information facilitate a more complete understanding of the roles of various grapevine kinases in growth and development, responses to abiotic stress, and evolutionary history.

  10. Somatic Embryogenesis in Olive (Olea europaea L. subsp. europaea var. sativa and var. sylvestris).

    Science.gov (United States)

    Rugini, Eddo; Silvestri, Cristian

    2016-01-01

    Protocols for olive somatic embryogenesis from zygotic embryos and mature tissues have been described for both Olea europaea sub. europaea var. sativa and var. sylvestris. Immature zygotic embryos (no more than 75 days old), used after fruit collection or stored at 12-14 °C for 2-3 months, are the best responsive explants and very slightly genotype dependent, and one single protocol can be effective for a wide range of genotypes. On the contrary, protocols for mature zygotic embryos and for mature tissue of cultivars are often genotype specific, so that they may require many adjustments according to genotypes. The use of thidiazuron and cefotaxime seems to be an important trigger for induction phase particularly for tissues derived from cultivars. Up to now, however, the application of this technique for large-scale propagation is hampered also by the low rate of embryo germination; it proves nonetheless very useful for genetic improvement.

  11. Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

    Science.gov (United States)

    Gómez, M. C.; Biancardi, M.N.; Jenkins, J.A.; Dumas, C.; Galiguis, J.; Wang, G.; Earle Pope, C.

    2012-01-01

    Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

  12. Genome-wide analysis of the expansin gene superfamily reveals grapevine-specific structural and functional characteristics.

    Directory of Open Access Journals (Sweden)

    Silvia Dal Santo

    Full Text Available BACKGROUND: Expansins are proteins that loosen plant cell walls in a pH-dependent manner, probably by increasing the relative movement among polymers thus causing irreversible expansion. The expansin superfamily (EXP comprises four distinct families: expansin A (EXPA, expansin B (EXPB, expansin-like A (EXLA and expansin-like B (EXLB. There is experimental evidence that EXPA and EXPB proteins are required for cell expansion and developmental processes involving cell wall modification, whereas the exact functions of EXLA and EXLB remain unclear. The complete grapevine (Vitis vinifera genome sequence has allowed the characterization of many gene families, but an exhaustive genome-wide analysis of expansin gene expression has not been attempted thus far. METHODOLOGY/PRINCIPAL FINDINGS: We identified 29 EXP superfamily genes in the grapevine genome, representing all four EXP families. Members of the same EXP family shared the same exon-intron structure, and phylogenetic analysis confirmed a closer relationship between EXP genes from woody species, i.e. grapevine and poplar (Populus trichocarpa, compared to those from Arabidopsis thaliana and rice (Oryza sativa. We also identified grapevine-specific duplication events involving the EXLB family. Global gene expression analysis confirmed a strong correlation among EXP genes expressed in mature and green/vegetative samples, respectively, as reported for other gene families in the recently-published grapevine gene expression atlas. We also observed the specific co-expression of EXLB genes in woody organs, and the involvement of certain grapevine EXP genes in berry development and post-harvest withering. CONCLUSION: Our comprehensive analysis of the grapevine EXP superfamily confirmed and extended current knowledge about the structural and functional characteristics of this gene family, and also identified properties that are currently unique to grapevine expansin genes. Our data provide a model for the

  13. Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

    Science.gov (United States)

    Roper, M Caroline; Greve, L Carl; Warren, Jeremy G; Labavitch, John M; Kirkpatrick, Bruce C

    2007-04-01

    Xylella fastidiosa is the causal agent of Pierce's disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

  14. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

    Directory of Open Access Journals (Sweden)

    Xuemei Zhang

    2016-10-01

    Full Text Available Myostatin (MSTN can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause “double-muscling” trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

  15. Species of Diatrypaceae associated with grapevine trunk diseases in Eastern Spain

    Directory of Open Access Journals (Sweden)

    Jordi LUQUE

    2013-01-01

    Full Text Available The presence and diversity of Diatrypaceae species occurring on grapevines in Eastern Spain were investigated. Several species were identified on the basis of morphological characters and phylogenetic analyses of the complete sequence of the internal transcribed spacers of the ribosomal DNA and part of the β-tubulin gene. Five species of Diatrypaceae isolated from the wood of diseased grapevines, pruning debris and/or perithecia were identified, including Anthostoma decipiens, Cryptovalsa ampelina, Eutypa lata, Eutypella citricola and Eutypella microtheca. Additionally, four taxa could not be identified to the species level but were closely related to Eutypa tetragona based on phylogenetic analyses. Eutypa lata was the most prevalent species and showed the greatest degree of genetic diversity. Cryptovalsa ampelina and E. microtheca ranked second in the frequency of isolations, while all the remaining species were less frequently isolated. Eutypella citricola and E. microtheca are reported for the first time as occurring on grapevine in Spain and this is the first report of A. decipiens occurring on grapevine.

  16. Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

    Czech Academy of Sciences Publication Activity Database

    Petřek, J.; Zítka, O.; Adam, V.; Bartušek, Karel; Anjum, N. A.; Pereira, E.; Havel, L.; Kizek, R.

    2015-01-01

    Roč. 10, č. 12 (2015), e0144093:1-16 E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) LO1212 Institutional support: RVO:68081731 Keywords : somatic embryogenesis * biochemical parameters Subject RIV: BH - Optics, Masers, Lasers Impact factor: 3.057, year: 2015

  17. Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.).

    Science.gov (United States)

    Mihaljević, Snježana; Radić, Sandra; Bauer, Nataša; Garić, Rade; Mihaljević, Branka; Horvat, Gordana; Leljak-Levanić, Dunja; Jelaska, Sibila

    2011-11-01

    Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin. Copyright © 2011 Elsevier GmbH. All rights reserved.

  18. Negative regulation of P element excision by the somatic product and terminal sequences of P in drosophila melanogaster

    Science.gov (United States)

    A transient in vivo P element excision assay was used to test the regulatory properties of putative repressor-encoding plasmids in Drosophila melanogaster embryos. The somatic expression of an unmodified transposase transcription unit under the control of a heat shock gene promoter (phsn) effectivel...

  19. Endangered wolves cloned from adult somatic cells.

    Science.gov (United States)

    Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hwang, Woo Suk; Hossein, Mohammad Shamim; Kim, Joung Joo; Shin, Nam Shik; Kang, Sung Keun; Lee, Byeong Chun

    2007-01-01

    Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.

  20. SOYBEAN AND CASEIN HYDROLYSATES INDUCE GRAPEVINE IMMUNE RESPONSES AND RESISTANCE AGAINST PLASMOPARA VITICOLA

    Directory of Open Access Journals (Sweden)

    Nihed eLachhab

    2014-12-01

    Full Text Available Plasmopara viticola, the causal agent of grapevine downy mildew, is one of the most devastating grape pathogen in Europe and North America. Although phytochemicals are used to control pathogen infections, the appearance of resistant strains and the concern for possible adverse effects on environment and human health are increasing the search for alternative strategies. In the present investigation, we successfully tested two protein hydrolysates from soybean (soy and casein (cas to trigger grapevine resistance against P. viticola. On Vitis vinifera cv. Marselan plants, the application of soy and cas reduced the infected leaf surface by 76 and 63%, as compared to the control, respectively. Since both hydrolysates might trigger the plant immunity, we investigated their ability to elicit grapevine defence responses. On grapevine cell suspensions, a different free cytosolic calcium signature was recorded for each hydrolysate, whereas a similar transient phosphorylation of two MAP kinases of 45 and 49 kDa was observed. These signalling events were followed by transcriptome reprogramming, including the up-regulation of defence genes encoding pathogenesis-related (PR proteins and the stilbene synthase enzyme responsible for the biosynthesis of resveratrol, the main grapevine phytoalexin. Liquid chromatography analyses confirmed the production of resveratrol and its dimer metabolites, δ- and ε-viniferins. Overall, soy effects were more pronounced as compared to the cas one. Both hydrolysates proved to act as elicitors to enhance grapevine immunity against pathogen attack.

  1. Arabinogalactan-proteins stimulate somatic embryogenesis and plant propagation of Pelargonium sidoides.

    Science.gov (United States)

    Duchow, Stefanie; Dahlke, Renate I; Geske, Thomas; Blaschek, Wolfgang; Classen, Birgit

    2016-11-05

    Root extracts of the medicinal plant Pelargonium sidoides, native to South Africa, are used globally for the treatment of common cold and cough. Due to an increasing economic commercialization of P. sidoides remedies, wild collections of root material should be accompanied by effective methods for plant propagation like somatic embryogenesis. Based on this, the influence of arabinogalactan-proteins (AGPs) on somatic embryogenesis and plant propagation of P. sidoides has been investigated. High-molecular weight AGPs have been isolated from dried roots as well as from cell cultures of P. sidoides with yields between 0.1% and 0.9%, respectively. AGPs are characterized by a 1,3-linked Galp backbone, branched at C6 to 1,6-linked Galp side chains terminated by Araf and to a minor extent by GlcpA, Galp or Rhap. Treatment of explants of P. sidoides with AGPs from roots or suspension culture over 5.5 weeks resulted in effective stimulation of somatic embryo development and plant regeneration. Copyright © 2016. Published by Elsevier Ltd.

  2. The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

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    Hitomi Suzuki

    Full Text Available BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo.

  3. Effectiveness of some microorganisms in the limitation of grapevine cuttings infection by Phomopsis viticola Sacc.

    Directory of Open Access Journals (Sweden)

    Ewa Król

    2013-12-01

    Full Text Available The possibilities of using antagonistic fungi and bacteria in the limitation of grapevine stems infection by Phomopsis viticola Sacc. were studied. Trichodema koningii Oud., T.viride Persoon ex S.F.,T.harzianum Rifai, Gliocladium catenulatum Gilman and Abbott, G.fimbriatum Gilman and Abbott, Bacillus sp., Pseudomonas fluorescens and five unidentified isolates of bacteria i.e.: 22a, 35, 40, 45, 66 were estimated. It was appeared what Trichoderma spp. were the most effective in protection of grapevine stems against the infection by P.viticola. After these antagonistic fungi were used on protected grapevine canes not numerous necrosis were observed and few cultures of pathogen were reisolated from them. Moreover, Trichoderma spp. survived on the grapevine stems during the period of experiment. The abilities of other microorganisms tested to protect grapevine cuttings against P.viticola infection and to exist on the stems were less than Trichoderma spp.

  4. Restricted mobility of Dnmt1 in preimplantation embryos: implications for epigenetic reprogramming

    Science.gov (United States)

    Grohmann, Maik; Spada, Fabio; Schermelleh, Lothar; Alenina, Natalia; Bader, Michael; Cardoso, M Cristina; Leonhardt, Heinrich

    2005-01-01

    Background Mouse preimplantation development is characterized by both active and passive genomic demethylation. A short isoform of the prevalent maintenance DNA methyltransferase (Dnmt1S) is found in the cytoplasm of preimplantation embryos and transiently enters the nucleus only at the 8-cell stage. Results Using GFP fusions we show that both the long and short isoforms of Dnmt1 localize to the nucleus of somatic cells and the cytoplasm of preimplantation embryos and that these subcellular localization properties are independent of phosphorylation. Importantly, photobleaching techniques and salt extraction revealed that Dnmt1S has a very restricted mobility in the cytoplasm, while it is highly mobile in the nucleus of preimplantation embryos. Conclusion The restricted mobility of Dnmt1S limits its access to DNA and likely contributes to passive demethylation and epigenetic reprogramming during preimplantationdevelopment. PMID:16120212

  5. HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

    International Nuclear Information System (INIS)

    Wang, Yingying; Hai, Tang; Liu, Zichuan; Zhou, Shuya; Lv, Zhuo; Ding, Chenhui; Liu, Lei; Niu, Yuyu; Zhao, Xiaoyang; Tong, Man; Wang, Liu; Jouneau, Alice; Zhang, Xun; Ji, Weizhi; Zhou, Qi

    2010-01-01

    Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo.

  6. Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

    Science.gov (United States)

    Wakayama, Teruhiko

    2007-02-01

    Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

  7. Use of somatic cell banks in the conservation of wild felids.

    Science.gov (United States)

    Praxedes, Érika A; Borges, Alana A; Santos, Maria V O; Pereira, Alexsandra F

    2018-05-03

    The conservation of biological resources is an interesting strategy for the maintenance of biodiversity, especially for wild felids who are constantly threatened with extinction. For this purpose, cryopreservation techniques have been used for the long-term storage of gametes, embryos, gonadal tissues, and somatic cells and tissues. The establishment of these banks has been suggested as a practical approach to the preservation of species and, when done in tandem with assisted reproductive techniques, could provide the means for reproducing endangered species. Somatic cell banks have been shown remarkable for the conservation of genetic material of felids; by merely obtaining skin samples, it is possible to sample a large group of individuals without being limited by factors such as gender or age. Thus, techniques for somatic tissue recovery, cryopreservation, and in vitro culture of different wild felids have been developed, resulting in a viable method for the conservation of species. One of the most notable conservation programs for wild felines using somatic samples was the one carried out for the Iberian lynx, the most endangered feline in the world. Other wild felids have also been studied in other continents, such as the jaguar in South America. This review aims to present the technical progress achieved in the conservation of somatic cells and tissues in different wild felids, as well address the progress that has been achieved in a few species. © 2018 Wiley Periodicals, Inc.

  8. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  9. In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii interspecific cloned embryos

    Directory of Open Access Journals (Sweden)

    Guanghua SU,Lei CHENG,Yu GAO,Kun LIU,Zhuying WEI,Chunling BAI,Fengxia YIN,Li GAO,Guangpeng LI,Shorgan BOU

    2014-02-01

    Full Text Available The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

  10. Correction of β-thalassemia mutant by base editor in human embryos

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    Puping Liang

    2017-09-01

    Full Text Available Abstract β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

  11. Histological and biochemical response of Norway spruce somatic embryos to UV-B irradiation

    Czech Academy of Sciences Publication Activity Database

    Eliášová, Kateřina; Vondráková, Zuzana; Malbeck, Jiří; Trávníčková, Alena; Pešek, Bedřich; Vágner, Martin; Cvikrová, Milena

    2017-01-01

    Roč. 31, č. 4 (2017), s. 1279-1293 ISSN 0931-1890 R&D Projects: GA MŠk(CZ) LD13050; GA MŠk(CZ) LD13051 Institutional support: RVO:61389030 Keywords : Oxidative stress * Phenolic acids * Phenylpropanoids * Picea abies (L.) Karst * Polyamines * Somatic embryogenesis Subject RIV: ED - Physiology OBOR OECD: Forestry Impact factor: 1.842, year: 2016

  12. Differential expression of genes of Xylella fastidiosa in xylem fluid of citrus and grapevine.

    Science.gov (United States)

    Shi, Xiangyang; Bi, Jianlong; Morse, Joseph G; Toscano, Nick C; Cooksey, Donald A

    2010-03-01

    Xylella fastidiosa causes a serious Pierce's disease (PD) in grapevine. Xylella fastidiosa cells from a PD strain were grown in a pure xylem fluid of a susceptible grapevine cultivar vs. xylem fluid from citrus, which is not a host for this strain of X. fastidiosa. When grown in grapevine xylem fluid, cells of the PD strain formed clumps and biofilm formed to a greater extent than in citrus xylem fluid, although the PD strain did grow in xylem fluid of three citrus varieties. The differential expression of selected genes of a PD X. fastidiosa strain cultured in the two xylem fluids was analyzed using a DNA macroarray. Compared with citrus xylem fluid, grapevine xylem fluid stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as gacA, algU, xrvA, and hsq, and also genes involved in the biogenesis of pili and twitching motility, such as fimT, pilI, pilU, and pilY1. Increased gene expression likely contributes to PD expression in grapevine, whereas citrus xylem fluid did not support or possibly suppressed the expression of these virulence genes.

  13. Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer

    Science.gov (United States)

    Yamazaki, Yukiko; Makino, Hatsune; Hamaguchi-Hamada, Kayoko; Hamada, Shun; Sugino, Hidehiko; Kawase, Eihachiro; Miyata, Takaki; Ogawa, Masaharu; Yanagimachi, Ryuzo; Yagi, Takeshi

    2001-01-01

    When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5–17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells. PMID:11698647

  14. Exogenous putrescine affects endogenous polyamine levels and the development of Picea abies somatic embryos

    Czech Academy of Sciences Publication Activity Database

    Vondráková, Zuzana; Eliášová, Kateřina; Vágner, Martin; Martincová, Olga; Cvikrová, Milena

    2015-01-01

    Roč. 75, č. 2 (2015), s. 405-414 ISSN 0167-6903 R&D Projects: GA MŠk(CZ) LD13050 Institutional support: RVO:61389030 Keywords : Exogenous putrescine * Somatic embryogenesis * Picea abies Subject RIV: ED - Physiology Impact factor: 2.333, year: 2015

  15. The study of antioxidants in grapevine seeds

    Directory of Open Access Journals (Sweden)

    Lenka Tomášková

    2017-01-01

    Full Text Available Grapevine seeds contain a large amount of antioxidant components, and are therefore recommended in the prevention and treatment of many diseases. For this research, we studied the antioxidant properties of grapevine seeds from the Marlen variety, as evidence suggests that these types have higher resistance against fungal diseases. Through high-performance liquid chromatography with UV/VIS detection, a total of 10 antioxidant components were selected for further investigation, specifically: catechin, epicatechin, rutin, quercitrin, quercetin, caftaric acid, caffeic acid, p-coumaric acid, ferulic acid, and gallic acid. The antioxidant activity was determinated spectrophotometrically through the adoption of three fundamentally different methods (the DPPH assay, the ABTS method, and the FRAP method. Using the Folin-Ciocalteu method, it was possible to determine the content of all the polyphenolic compounds. The results of the assessment antioxidant activity and the content of polyphenolic compounds were recalculated to gallic acid equivalents (GAE. The values of the antioxidant activity as determinated by the DPPH test were 6643 (±154 mg of GAE; 1984 (±88 mg of GAE when using the FRAP method; and 812 (±31 mg of GAE when the ABTS method was utilised. The content of the total polyphenolic compounds came to 6982 (±221 mg of GAE. The most abundant antioxidant was catechin, with a content of 115 mg.L-1, whilst the least represented compound was ferulic acid (0.139 mg.L-1. Overall, this study showed a high antioxidant potential of grapevine seeds. 

  16. Use of Endophytic and Rhizosphere Actinobacteria from Grapevine Plants To Reduce Nursery Fungal Graft Infections That Lead to Young Grapevine Decline.

    Science.gov (United States)

    Álvarez-Pérez, José Manuel; González-García, Sandra; Cobos, Rebeca; Olego, Miguel Ángel; Ibañez, Ana; Díez-Galán, Alba; Garzón-Jimeno, Enrique; Coque, Juan José R

    2017-12-15

    Endophytic and rhizosphere actinobacteria isolated from the root system of 1-year-old grafted Vitis vinifera plants were evaluated for their activities against fungi that cause grapevine trunk diseases. A total of 58 endophytic and 94 rhizosphere isolates were tested. Based on an in vitro bioassay, 15.5% of the endophytic isolates and 30.8% of the rhizosphere isolates exhibited antifungal activity against the fungal pathogen Diplodia seriata , whereas 13.8% of the endophytic isolates and 16.0% of the rhizosphere isolates showed antifungal activity against Dactylonectria macrodidyma (formerly Ilyonectria macrodidyma ). The strains which showed the greatest in vitro efficacy against both pathogens were further analyzed for their ability to inhibit the growth of Phaeomoniella chlamydospora and Phaeoacremonium minimum (formerly Phaeoacremonium aleophilum ). Based on their antifungal activity, three rhizosphere isolates and three endophytic isolates were applied on grafts in an open-root field nursery in a 3-year trial. The field trial led to the identification of one endophytic strain, Streptomyces sp. VV/E1, and two rhizosphere isolates, Streptomyces sp. VV/R1 and Streptomyces sp. VV/R4, which significantly reduced the infection rates produced by the fungal pathogens Dactylonectria sp., Ilyonectria sp., P. chlamydospora , and P. minimum , all of which cause young grapevine decline. The VV/R1 and VV/R4 isolates also significantly reduced the mortality level of grafted plants in the nursery. This study shows that certain actinobacteria could represent a promising new tool for controlling fungal trunk pathogens that infect grapevine plants through the root system in nurseries. IMPORTANCE Grapevine trunk diseases are a major threat to the wine and grape industry worldwide. They cause a significant reduction in yields as well as in grape quality, and they can even cause plant death. Trunk diseases are caused by fungal pathogens that enter through pruning wounds and/or the

  17. Somatic embryogenesis in Malaysian cultivars of sweet potato (Ipomoea batatas L.).

    OpenAIRE

    De Silva, Angela Ee

    2017-01-01

    Three Malaysian sweet potato cultivars, namely Ipomoea batatas (L.) cv. Gendut, Jalomas and Telong, were investigated for their abilities to produce somatic embryos. Shoot meristems were used as the starting materials and cultured on Murashige and Skoog basal media (Murashige and Skoog, 1962) in the presence of auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Auxin 2,4,5-T at 5 µM was most effective for the initiation of primary embryogenic formati...

  18. The p66(Shc adaptor protein controls oxidative stress response in early bovine embryos.

    Directory of Open Access Journals (Sweden)

    Dean H Betts

    Full Text Available The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2-4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2-4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.

  19. Grapevine fruiting cuttings: validation of an experimental system to study grapevine physiology. I. Main vegetative characteristics

    Directory of Open Access Journals (Sweden)

    Nathalie Ollat

    1998-03-01

    Full Text Available A method for producing fruiting cuttings of grapevine cv. Cabernet Sauvignon is described. The main developmental features of these cuttings are presented. The clusters reach maturity after 5 months and the period of cluster development is not shortened. Before flowering, a careful control of stem and leaf growth improves the partitioning of stored carbon towards the roots and the cluster. Vegetative growth occurs mainly between fruit set and veraison. During ripening, the leaf to fruit ratio is between 20 and 30 cm2 of leaf area per gram of fruit. In the cuttings as well as in grapevines from the vineyard, free polyamine content in the leafblade does not change during development. Conjugate polyamine content does not evolve in the same way. It peaks at veraison for vineyard leaves but decreases continuously for cuttings. A proper nutrient solution provides a minerai composition in accordance with viticultural requirements. The cuttings also behave like vineyard plants in response to different levels of potassium in the nutrient solution.

  20. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

    International Nuclear Information System (INIS)

    Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer; Campbell, Keith H.S.

    2005-01-01

    The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells

  1. Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

    Directory of Open Access Journals (Sweden)

    Li-Ying Sung

    2014-12-01

    Full Text Available Summary: Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells. : Sung et al. demonstrate in a mouse model that telomeres of telomerase haplo-insufficient cells can be elongated by somatic cell nuclear transfer. Moreover, ntESCs derived from Terc+/− cells exhibit pluripotency evidenced by generation of Terc+/−ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency.

  2. Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2).

    Science.gov (United States)

    Ganesan, M; Jayabalan, N

    2004-10-01

    Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH4NO3 and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6-7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.

  3. Pyrosequencing detects human and animal pathogenic taxa in the grapevine endosphere.

    Science.gov (United States)

    Yousaf, Sohail; Bulgari, Daniela; Bergna, Alessandro; Pancher, Michael; Quaglino, Fabio; Casati, Paola; Campisano, Andrea

    2014-01-01

    Generally, plants are not considered as hosts for human and animal pathogens (HAP). The recent produce-associated outbreaks of food-borne diseases have drawn attention toward significant deficiencies in our understanding of the ecology of HAP, and their potential for interkingdom transfer. To examine the association of microorganisms classified as HAP with plants, we surveyed the presence and distribution of HAP bacterial taxa (henceforth HAPT, for brevity's sake) in the endosphere of grapevine (Vitis vinifera L.) both in the plant stems and leaves. An enrichment protocol was used on leaves to detect taxa with very low abundance in undisturbed tissues. We used pyrosequencing and phylogenetic analyses of the 16S rDNA gene. We identified several HAPT, and focused on four genera (Propionibacterium, Staphylococcus, Clostridium, and Burkholderia). The majority of the bacterial sequences in the genus Propionibacterium, from grapevine leaf and stem, were identified as P. acnes. Clostridia were detected in leaves and stems, but their number was much higher in leaves after enrichment. HAPT were indentified both in leaves and wood of grapevines. This depicts the ability of these taxa to be internalized within plant tissues and maintain their population levels in a variety of environments. Our analysis highlighted the presence of HAPT in the grapevine endosphere and unexpected occurrence of these bacterial taxa in this atypical environment.

  4. Developmental Competence and Epigenetic Profile of Porcine Embryos Produced by Two Different Cloning Methods.

    Science.gov (United States)

    Liu, Ying; Lucas-Hahn, Andrea; Petersen, Bjoern; Li, Rong; Hermann, Doris; Hassel, Petra; Ziegler, Maren; Larsen, Knud; Niemann, Heiner; Callesen, Henrik

    2017-06-01

    The "Dolly" based cloning (classical nuclear transfer, [CNT]) and the handmade cloning (HMC) are methods that are nowadays routinely used for somatic cloning of large domestic species. Both cloning protocols share several similarities, but differ with regard to the required in vitro culture, which in turn results in different time intervals until embryo transfer. It is not yet known whether the differences between cloned embryos from the two protocols are due to the cloning methods themselves or the in vitro culture, as some studies have shown detrimental effects of in vitro culture on conventionally produced embryos. The goal of this study was to unravel putative differences between two cloning methods, with regard to developmental competence, expression profile of a panel of developmentally important genes and epigenetic profile of porcine cloned embryos produced by either CNT or HMC, either with (D5 or D6) or without (D0) in vitro culture. Embryos cloned by these two methods had a similar morphological appearance on D0, but displayed different cleavage rates and different quality of blastocysts, with HMC embryos showing higher blastocyst rates (HMC vs. CNT: 35% vs. 10%, p cloned embryos were similar on D0, but differed on D6. In conclusion, both cloning methods and the in vitro culture may affect porcine embryo development and epigenetic profile. The two cloning methods essentially produce embryos of similar quality on D0 and after 5 days in vitro culture, but thereafter both histone acetylation and gene expression differ between the two types of cloned embryos.

  5. ADVANCES IN PROPAGATION OF GRAPEVINE IN THE WORLD

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    DANIEL SANTOS GROHS

    2017-12-01

    Full Text Available ABSTRACT Grapevine production by classical grafting methods and in commercial scale emerged over 130 years. This system remained handmade until the mid-1950s, when the first international certification programs aimed at obtaining mother plants with high viral sanity emerged. The necessity to increase the scale of production on industrial model and plant material production based on minimum morphological standards appeared at the end of the 1960s. Along the 1970s, research unlocked knowledge on semi-automated grafting, process hygiene, use of plant growth regulators and understanding of physiological events of rootstock-scion compatibility, callus formation and rooting. So, until the mid-2000s, certification schemes and propagation processes advanced little in technical standard. However, grapevine growing areas were expanded and demands for plant material increased, and new diseases emerged from contaminated nurseries. These new diseases (new viral complexes, phytoplasmas, bacteria and grapevine trunk diseases were discovered by high-sensitivity diagnostic methods. Today, there is a new discussion on the nursery segment worldwide. The propagation techniques have been reviewed from the perspective of reducing the incidence of new diseases and minimum physiological damage of nursery plants during the production stages. Therefore, technological innovations regarding equipment, practices and production inputs have been incorporated in new certification schemes. However, despite these advantages, these schemes have become more complex and multidisciplinary than previous ones, bringing difficulties in adaptation of nurserymen.

  6. Modification of mitochondrial function, cytoplasmic lipid content and cryosensitivity of bovine embryos by resveratrol.

    Science.gov (United States)

    Abe, Takahito; Kawahara-Miki, Ryouka; Hara, Tomotaka; Noguchi, Tatsuo; Hayashi, Takeshi; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2017-10-18

    Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor "EX527", and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.

  7. Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage

    Directory of Open Access Journals (Sweden)

    F.D. Oliveira

    2015-12-01

    Full Text Available In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1 of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

  8. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    International Nuclear Information System (INIS)

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin; Sun, Xiaofang

    2009-01-01

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  9. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China); Sun, Xiaofang, E-mail: xiaofangsun@hotmail.com [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China)

    2009-04-24

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  10. Identification and Prevalence of Grapevine fanleaf virus in Khorasan-Razavi Vineyards

    Directory of Open Access Journals (Sweden)

    Z. Gholampour

    2016-02-01

    Full Text Available Introduction Grapevine fanleaf virus (GFLV is one of the devastating viruses of grapevine cause severe crop loss in vineyards. GFLV is a member of the genus Nepovirus in the family Secoviridae. The GFLV genome consists of two positive-sense single-stranded RNAs. The genome has a poly (A tail at the 3´ terminus and a covalently linked VPg protein at the 5´ terminus. Each genomic RNA encodes a polyprotein from which functional proteins are released by proteolytic processing by the virus-specific protease. GFLV isolates differing in the type of leaf symptoms, ranging from fanleaf, yellow mosaic, vein banding and mottle in different grapevine varieties. GFLV is specifically transmitted from grapevine to grapevine by the ectoparasitic nematode species Xiphinema index. It is also transmitted by grafting, vegetative propagation or mechanical inoculation onto herbaceous plants. GFLV has restricted natural host range, grapevine is the dominant natural host of GFLV and, however, it has been reported on several weeds in Iran. It is thought that the old Persia, especially the region located between the Caspian Sea and the Black Sea, might be the origin of GFLV. Grapevine wide cultures in Khorasan-Razavi province, northeast of Iran, but little information is available for the incidence of GFLV in this region. In the present work, we are interested in the study of the Prevalence of the Grapevine fanleaf virus in Khorasan-Razavi province. Materials and Methods: To identify the distribution of GFLV in Khorasan-Razavi, 280 leaf samples were randomly collected during the growing season of 2011-2012. GFLV was detected in leaf samples by enzyme-linked immunosorbent assay (ELISA using specific antibodies raised against Iranian isolate of the virus (Zakiaghl and Izadpanah 2003. Chenopodium quinoa plants were used as systemic herbaceous host for the propagation of GFLV. The carborundum dusted seedlings were inoculated by extracts of ELISA positive samples in

  11. Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B Produção de anti-soros policlonais a partir de proteínas capsidiais recombinantes de Grapevine leafroll-associated virus 2 e Grapevine virus B

    Directory of Open Access Journals (Sweden)

    Paula Radaelli

    2008-10-01

    Full Text Available The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2 and Grapevine virus B (GVB, developed from expressed coat proteins (CPs in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405 of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.O objetivo deste trabalho foi produzir e caracterizar anti-soros específicos contra isolados brasileiros do Vírus do enrolamento-da-folha da videira 2 (GLRaV-2 e do Vírus B da videira (GVB, desenvolvidos a partir das proteínas capsidiais expressas em Escherichia coli, e testar seu possível uso para a detecção destes dois vírus em videiras infectadas. Os genes da proteína capsidial (CP foram amplificados via RT-PCR, clonados e seqüenciados. Foram, subseqüentemente, subclonados, e os plasmídeos recombinantes foram empregados na transformação das células de E. coli e na expressão das proteínas capsidiais. As proteínas capsidiais recombinantes foram purificadas, e suas identidades foram confirmadas em SDS-PAGE e "Western blot" e utilizadas para imunizar coelhos. Os anti

  12. Somatic embryogenesis in cell cultures of Glycine species.

    Science.gov (United States)

    Gamborg, O L; Davis, B P; Stahlhut, R W

    1983-08-01

    This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.

  13. Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine.

    Science.gov (United States)

    Farace, Giovanni; Fernandez, Olivier; Jacquens, Lucile; Coutte, François; Krier, François; Jacques, Philippe; Clément, Christophe; Barka, Essaid Ait; Jacquard, Cédric; Dorey, Stéphan

    2015-02-01

    Non-self-recognition of microorganisms partly relies on the perception of microbe-associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA-regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long-lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP-non-producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  14. VTCdb: a gene co-expression database for the crop species Vitis vinifera (grapevine).

    Science.gov (United States)

    Wong, Darren C J; Sweetman, Crystal; Drew, Damian P; Ford, Christopher M

    2013-12-16

    Gene expression datasets in model plants such as Arabidopsis have contributed to our understanding of gene function and how a single underlying biological process can be governed by a diverse network of genes. The accumulation of publicly available microarray data encompassing a wide range of biological and environmental conditions has enabled the development of additional capabilities including gene co-expression analysis (GCA). GCA is based on the understanding that genes encoding proteins involved in similar and/or related biological processes may exhibit comparable expression patterns over a range of experimental conditions, developmental stages and tissues. We present an open access database for the investigation of gene co-expression networks within the cultivated grapevine, Vitis vinifera. The new gene co-expression database, VTCdb (http://vtcdb.adelaide.edu.au/Home.aspx), offers an online platform for transcriptional regulatory inference in the cultivated grapevine. Using condition-independent and condition-dependent approaches, grapevine co-expression networks were constructed using the latest publicly available microarray datasets from diverse experimental series, utilising the Affymetrix Vitis vinifera GeneChip (16 K) and the NimbleGen Grape Whole-genome microarray chip (29 K), thus making it possible to profile approximately 29,000 genes (95% of the predicted grapevine transcriptome). Applications available with the online platform include the use of gene names, probesets, modules or biological processes to query the co-expression networks, with the option to choose between Affymetrix or Nimblegen datasets and between multiple co-expression measures. Alternatively, the user can browse existing network modules using interactive network visualisation and analysis via CytoscapeWeb. To demonstrate the utility of the database, we present examples from three fundamental biological processes (berry development, photosynthesis and flavonoid biosynthesis

  15. Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

    Science.gov (United States)

    Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

    2006-11-01

    Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

  16. Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

    Science.gov (United States)

    Jin, Jun-Xue; Li, Suo; Gao, Qing-Shan; Hong, Yu; Jin, Long; Zhu, Hai-Ying; Yan, Chang-Guo; Kang, Jin-Dan; Yin, Xi-Jun

    2013-10-01

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  17. Analysis of high-identity segmental duplications in the grapevine genome

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    Carelli Francesco N

    2011-08-01

    Full Text Available Abstract Background Segmental duplications (SDs are blocks of genomic sequence of 1-200 kb that map to different loci in a genome and share a sequence identity > 90%. SDs show at the sequence level the same characteristics as other regions of the human genome: they contain both high-copy repeats and gene sequences. SDs play an important role in genome plasticity by creating new genes and modeling genome structure. Although data is plentiful for mammals, not much was known about the representation of SDs in plant genomes. In this regard, we performed a genome-wide analysis of high-identity SDs on the sequenced grapevine (Vitis vinifera genome (PN40024. Results We demonstrate that recent SDs (> 94% identity and >= 10 kb in size are a relevant component of the grapevine genome (85 Mb, 17% of the genome sequence. We detected mitochondrial and plastid DNA and genes (10% of gene annotation in segmentally duplicated regions of the nuclear genome. In particular, the nine highest copy number genes have a copy in either or both organelle genomes. Further we showed that several duplicated genes take part in the biosynthesis of compounds involved in plant response to environmental stress. Conclusions These data show the great influence of SDs and organelle DNA transfers in modeling the Vitis vinifera nuclear DNA structure as well as the impact of SDs in contributing to the adaptive capacity of grapevine and the nutritional content of grape products through genome variation. This study represents a step forward in the full characterization of duplicated genes important for grapevine cultural needs and human health.

  18. Atmospheric circulation patterns and phenological anomalies of grapevine in Italy

    Science.gov (United States)

    Cola, Gabriele; Alilla, Roberta; Dal Monte, Giovanni; Epifani, Chiara; Mariani, Luigi; Parisi, Simone Gabriele

    2014-05-01

    Grapevine (Vitis vinifera L.) is a fundamental crop for Italian agriculture as testified by the first place of Italy in the world producers ranking. This justify the importance of quantitative analyses referred to this crucial crop and aimed to quantify meteorological resources and limitations to development and production. Phenological rhythms of grapevine are strongly affected by surface fields of air temperature which in their turn are affected by synoptic circulation. This evidence highlights the importance of an approach based on dynamic climatology in order to detect and explain phenological anomalies that can have relevant effects on quantity and quality of grapevine production. In this context, this research is aimed to study the existing relation among the 850 hPa circulation patterns over the Euro-Mediterranean area from NOAA Ncep dataset and grapevine phenological fields for Italy over the period 2006-2013, highlighting the main phenological anomalies and analyzing synoptic determinants. This work is based on phenological fields with a standard pixel of 2 km routinely produced from 2006 by the Iphen project (Italian Phenological network) on the base of phenological observations spatialized by means of a specific algorithm based on cumulated thermal resources expressed as Normal Heat Hours (NHH). Anomalies have been evaluated with reference to phenological normal fields defined for the Italian area on the base of phenological observations and Iphen model. Results show that relevant phenological anomalies observed over the reference period are primarily associated with long lasting blocking systems driving cold air masses (Arctic or Polar-Continental) or hot ones (Sub-Tropical) towards the Italian area. Specific cases are presented for some years like 2007 and 2011.

  19. Influence of water stress on Botryosphaeriaceae disease expression in grapevines

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    Jan VAN NIEKERK

    2011-12-01

    Full Text Available Several species in Botryosphaeriaceae have been associated with grapevine trunk diseases. To evaluate the effect of water stress on infection of grapevines by Botryosphaeriaceae spp., 1-year-old Shiraz/101-14 Mgt nursery grapevine plants were planted in plastic potting bags and placed outdoors under shade netting. Five weeks after planting, vines were pruned and the pruning wounds inoculated with spore suspensions of Neofusicoccum australe, Neofusicoccum parvum, Lasiodiplodia theobromae or Diplodia seriata. Control treatments consisted of applications of sterile water or a Trichoderma harzianum spore suspension. Stem inoculations were done by inserting a colonised or uncolonised agar plug into a wound made in each stem. Four different irrigation regimes were introduced 12 weeks after planting to simulate varying degrees of water stress. Measurements of stomatal conductance, photosynthetic rate and leaf spectrometry were made to monitor physiological stress. Eight months after inoculation, vines were uprooted and the root, shoot and plant mass of each vine determined. Lesions observed in the inoculated pruning wounds and stems were also measured. Vines subjected to the lowest irrigation regime were significantly smaller than optimally irrigated vines. Water stressed vines also had significantly lower photosynthetic rates and levels of stomatal conductance compared with vines receiving optimal irrigation, indicating that these plants experienced significantly higher levels of physiological stress. The mean lesion length was significantly longer in the pruning wounds and stems of plants subjected to the lowest irrigation regime, with lesion length declining linearly with increasing irrigation volume. These results clearly indicate that when a grapevine is exposed to water stress, colonisation and disease expression by Botryosphaeriaceae spp. are much more severe.

  20. Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster).

    Science.gov (United States)

    Marum, Liliana; Rocheta, Margarida; Maroco, João; Oliveira, M Margarida; Miguel, Célia

    2009-04-01

    Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.

  1. Somatic Embryogenesis in Peach-Palm (Bactris gasipaes) Using Different Explant Sources.

    Science.gov (United States)

    Steinmacher, Douglas A; Heringer, Angelo Schuabb; Jiménez, Víctor M; Quoirin, Marguerite G G; Guerra, Miguel P

    2016-01-01

    Peach palm (Bactris gasipaes Kunth) is a member of the family Arecaceae and is a multipurpose but underutilized species. Nowadays, fruit production for subsistence and local markets, and heart-of-palm production for local, national, and international markets are the most important uses of this plant. Conventional breeding programs in peach palm are long-term efforts due to the prolonged generation time, large plant size, difficulties with controlled pollination and other factors. Although it is a caespitose palm, its propagation is currently based on seeds, as off-shoots are difficult to root. Hence, tissue culture techniques are considered to be the most likely strategy for efficient clonal plantlet regeneration of this species. Among various techniques, somatic embryogenesis offers the advantages of potential automated large-scale production and putative genetic stability of the regenerated plantlets. The induction of somatic embryogenesis in peach palm can be achieved by using different explant sources including zygotic embryos, immature inflorescences and thin cell layers from the young leaves and shoot meristems. The choice of a particular explant depends on whether clonal propagation is desired or not, as well as on the plant conditions and availability of explants. Protocols to induce and express somatic embryogenesis from different peach palm explants, up to acclimatization of plantlets, are described in this chapter.

  2. Gene expression profiling in susceptible interaction of grapevine with its fungal pathogen Eutypa lata: Extending MapMan ontology for grapevine

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    Usadel Björn

    2009-08-01

    Full Text Available Abstract Background Whole genome transcriptomics analysis is a very powerful approach because it gives an overview of the activity of genes in certain cells or tissue types. However, biological interpretation of such results can be rather tedious. MapMan is a software tool that displays large datasets (e.g. gene expression data onto diagrams of metabolic pathways or other processes and thus enables easier interpretation of results. The grapevine (Vitis vinifera genome sequence has recently become available bringing a new dimension into associated research. Two microarray platforms were designed based on the TIGR Gene Index database and used in several physiological studies. Results To enable easy and effective visualization of those and further experiments, annotation of Vitis vinifera Gene Index (VvGI version 5 to MapMan ontology was set up. Due to specificities of grape physiology, we have created new pictorial representations focusing on three selected pathways: carotenoid pathway, terpenoid pathway and phenylpropanoid pathway, the products of these pathways being important for wine aroma, flavour and colour, as well as plant defence against pathogens. This new tool was validated on Affymetrix microarrays data obtained during berry ripening and it allowed the discovery of new aspects in process regulation. We here also present results on transcriptional profiling of grape plantlets after exposal to the fungal pathogen Eutypa lata using Operon microarrays including visualization of results with MapMan. The data show that the genes induced in infected plants, encode pathogenesis related proteins and enzymes of the flavonoid metabolism, which are well known as being responsive to fungal infection. Conclusion The extension of MapMan ontology to grapevine together with the newly constructed pictorial representations for carotenoid, terpenoid and phenylpropanoid metabolism provide an alternative approach to the analysis of grapevine gene expression

  3. Genetic characterization of autochthonous grapevine cultivars from Eastern Turkey by simple sequence repeats (SSRs

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    Sadiye Peral Eyduran

    2016-01-01

    Full Text Available In this research, two well-recognized standard grape cultivars, Cabernet Sauvignon and Merlot, together with eight historical autochthonous grapevine cultivars from Eastern Anatolia in Turkey, were genetically characterized by using 12 pairs of simple sequence repeat (SSR primers in order to evaluate their genetic diversity and relatedness. All of the used SSR primers produced successful amplifications and revealed DNA polymorphisms, which were subsequently utilized to evaluate the genetic relatedness of the grapevine cultivars. Allele richness was implied by the identification of 69 alleles in 8 autochthonous cultivars with a mean value of 5.75 alleles per locus. The average expected heterozygosity and observed heterozygosity were found to be 0.749 and 0.739, respectively. Taking into account the generated alleles, the highest number was recorded in VVC2C3 and VVS2 loci (nine and eight alleles per locus, respectively, whereas the lowest number was recorded in VrZAG83 (three alleles per locus. Two main clusters were produced by using the unweighted pair-group method with arithmetic mean dendrogram constructed on the basis of the SSR data. Only Cabernet Sauvignon and Merlot cultivars were included in the first cluster. The second cluster involved the rest of the autochthonous cultivars. The results obtained during the study illustrated clearly that SSR markers have verified to be an effective tool for fingerprinting grapevine cultivars and carrying out grapevine biodiversity studies. The obtained data are also meaningful references for grapevine domestication.

  4. Regional somatic embryogenesis from in vitro apple leaf and artificial seed of apple

    International Nuclear Information System (INIS)

    Da Kedong; Li Yazhi; Shu Huairui; Wang Bin

    1998-01-01

    First three open leaves of in vitro apple (Malus domastica Borkh.) shoots were used as explants. Each leaf was divided into three regions (upper, middle and lower) from tip to base and each region was composed of two sub-regions (leaf and right) along the main vain. Every explant was pricked with a dissector at the center of one sub-region before inoculated on MS + BA 1 mg/L + NAA 4 mg/L + 2,4-D 0.5 mg/L medium and incubated in darkness for 7 days, then transferred to MS + BA 1 mg/L medium. 85% of the explants regenerated somatic embryos directly around the prick after 40 days. The embryos were encapsulated in 4% sodium alginate and 2% CaCl 2 as artificial seeds, which could germinate and grow into plantlets under aseptic condition

  5. Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

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    Domenico Iuso

    Full Text Available The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT. Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

  6. Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

    Science.gov (United States)

    Iuso, Domenico; Czernik, Marta; Di Egidio, Fiorella; Sampino, Silvestre; Zacchini, Federica; Bochenek, Michal; Smorag, Zdzislaw; Modlinski, Jacek A; Ptak, Grazyna; Loi, Pasqualino

    2013-01-01

    The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

  7. Aberrant epigenetic reprogramming of imprinted microRNA-127 and Rtl1 in cloned mouse embryos

    International Nuclear Information System (INIS)

    Cui Xiangshun; Zhang Dingxiao; Ko, Yoeung-Gyu; Kim, Nam-Hyung

    2009-01-01

    The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent (∼10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63-70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT).

  8. Effect of ploughing-down of grapevine chips on soil structure when using special agricultural machinery

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    Barbora Badalíková

    2012-01-01

    Full Text Available Within the period of 2008–2011, changes in soil structure were studied in two selected localities: one of them was situated in vineyards of the University Training Farm of Mendel University in Žabčice near Brno, the other was in vineyards situated in the cadastre of wine-growing municipality Velké Bílovice. Established were altogether three variants of experiments with application of crushed grapevine wood (chips: Variant 1 – control; Variant 2 – crushed grapevine wood ploughed down to the depth of 0.10 m; Variant 3 – crushed grapevine wood + grass spread on the soil surface as a mulch. Grapevine canes were crushed to chips using a special agricultural machinery while the soil in inter-rows was processed using conventional tilling machines. The obtained results showed that the best coefficient of structurality (expressing the degree of destruction of soil structure was recorded in Variants 2 in both localities. Considering values of this coefficient it could be concluded that just this variant showed a positive effect on soil structure. This variant reduced the compaction of soil caused by the movement of agricultural machines in vineyard inter-rows Crushed grapevine waste wood can therefore compensate losses of organic matter in soil. Better values of structurality coefficient were recorded in the locality Žabčice.

  9. Grapevine dynamics after manual tending of juvenile stands on the Hoosier National Forest, Indiana

    Science.gov (United States)

    Robert C. Morrissey; Martin-Michel Gauthier; John A., Jr. Kershaw; Douglass F. Jacobs; Burnell C. Fischer; John R. Siefert

    2008-01-01

    Large woody vines, most notably grapevines, are a source of great concern for forest and wildlife managers in many parts of the Central Hardwood Forest Region of the United States. We examined grapevine dynamics in stands aged 21 - 35 years. The plots, located in regenerated clearcuts in the Hoosier National Forest (HNF), were evaluated for vine control, site, and tree...

  10. Effects of mutagens on somatic embryogenesis and plant regeneration in groundnut

    International Nuclear Information System (INIS)

    Muthusamy, A.; Vasanth, K.; Sivasankari, D.; Chandrasekar, B.R.; Jayabalan, N.

    2007-01-01

    The embryogenic calli (EC) were obtained from hypocotyl explants of groundnut (Arachis hypogaea L.) cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 0.5 mg dm −3 6-benzylaminopurine (BAP). The EC were exposed to γ-radiation (10–50 Gy) or treated with 1–5 mM of ethyl methane sulphonate (EMS) or sodium azide (SA). The mutated EC were subcultured on embryo induction medium containing 20 mg dm −3 2,4-D. Somatic embryos (SE) developed from these calli were transferred to MS medium supplemented with BAP (2.0 mg dm −3 ) and 0.5 mg dm −3 2,4-D for maturation. The well-developed embryos were cultured on germination medium consisting of MS salts with 2.0 mg dm −3 BAP and 0.25 mg dm −3 naphthaleneacetic acid (NAA). Well-developed plantlets were transferred for hardening and hardened plants produced normal flowers and set viable seeds. The fresh mass of the EC, mean number of SE per explant and regeneration percentage were higher at lower concentrations of mutagens (up to 30 Gy/3 mM). Some abnormalities in regenerated plants were observed, especially variations in leaf shape. (author)

  11. Expression and Function of Cell Wall-Bound Cationic Peroxidase in Asparagus Somatic Embryogenesis

    Science.gov (United States)

    Takeda, Hiroyuki; Kotake, Toshihisa; Nakagawa, Naoki; Sakurai, Naoki; Nevins, Donald J.

    2003-01-01

    Cultured asparagus (Asparagus officinalis L. cv Y6) cells induced to regenerate into whole plants through somatic embryogenesis secreted a 38-kD protein into cell walls. The full-length cDNA sequence of this protein (Asparagus officinalis peroxidase 1 [AoPOX1]) determined by reverse transcriptase-polymerase chain reaction showed similarity with plant peroxidases. AoPOX1 transcripts were particularly abundant during early somatic embryogenesis. To evaluate the in vivo function of AoPOX1 protein, purified recombinant AoPOX1 protein was reacted with a series of phenolic substrates. The AoPOX1 protein was effective in the metabolism of feruloyl (o-methoxyphenol)-substituted substrates, including coniferyl alcohol. The reaction product of coniferyl alcohol was fractionated and subjected to gas chromatography-mass spectrometry analysis and 1H-nuclear magnetic resonance analysis, indicating that the oxidation product of coniferyl alcohol in the presence of AoPOX1 was dehydrodiconiferyl alcohol. The concentration of dehydrodiconiferyl alcohol in the cultured medium of the somatic embryos was in the range of 10−8 m. Functions of the AoPOX1 protein in the cell differentiation are discussed. PMID:12692335

  12. Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines

    NARCIS (Netherlands)

    Cabral, A.; Rego, C.; Nascimento, T.; Oliveira, H.; Groenewald, J.Z.; Crous, P.W.

    2012-01-01

    Black foot is an important disease of grapevines, which has in recent years been recorded with increased incidence and severity throughout the world, affecting grapevines both in nurseries and young vineyards. In the past the disease has been associated with infections by Ilyonectria macrodidyma,

  13. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants.

    Science.gov (United States)

    Chen, M H; Wang, P J; Maeda, E

    1987-10-01

    The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions.

  14. Genetic and somatic effects in animals maintained on tritiated water

    International Nuclear Information System (INIS)

    Carsten, A.L.; Commerford, S.L.; Cronkite, E.P.; Brooks, A.

    1982-01-01

    Somatic and genetic effects of the continuous ingestion of tritiated water (HTO) at concentrations of 0.3, 1.0 and 3.0 μCi/ml were investigated in mice of the Hale-Stoner-Brookhaven strain. At these levels, there was no measurable somatic effect. Although genetic effects as measured by dominant lethal mutation (DLM) assay indicated a significant effect (P>0.01) on the number of viable embryos and early deaths in the 3.0 μCi/ml HTO group and on the number of viable embryos in the 1.0 μCi/ml HTO group, no genetic effects were significantly noted in the 0.3 μCi/ml HTO group. Liver cytogenetic studies showed a significant increase in the number of abnormal cells in the 3.0 μCi/ml HTO group. A reduction in bone marrow stem cells, without an attendant reduction in total marrow cellularity, was noted in the 3.0 and 1.0 μCi/ml HTO groups. There was no significant difference in any of the DLM parameters between animals maintained on 3.0 μCi/ml of HTO and animals exposed to the equivalent 137 Cs gamma dose (22 hours/day exposure). Consideration of the relative amounts and biological half lives of tritium present in the nucleus as water, DNA and histone suggests that after transient exposure to tritiated water, nearly all significant radiation damage can be attributed to tritium present in the nucleus as water. These data suggest that hazards from tritium attendant with normal reactor operation should not at this time be considered as a deterrent to the further development of fission and/or fusion reactor technology. (Namekawa, K.)

  15. Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species.

    Science.gov (United States)

    Lagutina, Irina; Zakhartchenko, Valeri; Fulka, Helena; Colleoni, Silvia; Wolf, Eckhard; Fulka, Josef; Lazzari, Giovanna; Galli, Cesare

    2011-04-01

    The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

  16. Cortisol and somatization.

    Science.gov (United States)

    Rief, W; Auer, C

    2000-05-01

    Somatization symptoms are frequently associated with depression, anxiety, and feelings of distress. These features interact with the activity of the HPA-axis. Therefore we investigated relationships between somatization symptoms and cortisol. Seventy-seven participants were classified into three groups: somatization syndrome (at least eight physical symptoms from the DSM-IV somatization disorder list), somatization syndrome combined with major depression, and healthy controls. The following data were collected: salivary cortisol at three time points (morning, afternoon, evening), nighttime urinary cortisol, serum cortisol after the dexamethasone suppression test (DST), and psychological variables such as depression, anxiety, somatization, and hypochondriasis. Salivary cortisol showed typical diurnal variations. However, the groups did not differ on any of the cortisol variables. A possible explanation may be counteracting effects of somatization and depression. Exploratory correlational analyses revealed that associations between cortisol and psychopathological variables were time-dependent. DST results correlated with psychological aspects of somatization, but not with the number of somatoform symptoms per se.

  17. Somatic embryogenesis and plant regeneration in elite clones of Theobroma cacao Embriogênese somática e regeneração in vitro de clones elite de Theobroma cacao

    Directory of Open Access Journals (Sweden)

    Thiago Édson Ribeiro da Silva

    2008-10-01

    Full Text Available The objective of this work was to evaluated a procedure for somatic embryogenesis and regeneration of cacao (Theobroma cacao L. elite clones. Petal explants from cacao clones TSH 565 and TSH 1188 were cultured on PCG and SCG-2 media, for calli growth. Somatic embryos were formed on the surface of embryogenic calli after transfer to embryo development (ED medium. Clone TSH 565 showed a higher embryogenic potential than TSH 1188. The best combination of carbon source for embryo induction in ED medium was genotype-specific. Embryogenic callus formations increased in micropore tape-sealed Petri dishes, irrespective of cacao genotype. Mature somatic embryos were successfully converted into plantlets.O objetivo deste trabalho foi avaliar um procedimento para embriogênese somática e regeneração de clones elite de cacau. Pétalas dos clones de cacau TSH 565 e TSH 1188 foram cultivadas em meios PCG e SCG-2 para o crescimento de calos. Embriões somáticos desenvolveram-se na superfície dos calos embriogênicos, após a transferência para o meio ED. O clone TSH 565 apresentou maior potencial embriogênico do que o TSH 1188. A melhor combinação de fonte de carbono quanto à indução de embriões em meio ED foi específica do genótipo. A formação de calos embriogênicos foi superior em placas de Petri seladas com fita hipoalergênica, independentemente do genótipo. Embriões maduros de ambos os genótipos foram convertidos em plântulas.

  18. Histologia da embriogênese somática induzida em embriões de sementes maduras de Urochloa brizantha apomítica Histology of somatic embryogenesis induced in embryos of mature seeds of the apomictic Urochloa brizantha

    Directory of Open Access Journals (Sweden)

    Sandra Janeth Lenis-Manzano

    2010-05-01

    Full Text Available O objetivo deste trabalho foi descrever o processo de embriogênese somática em Urochloa brizantha cv. Marandu (Syn. Brachiaria brizantha cv. Marandu e fornecer subsídios para o aprimoramento dos métodos de cultura de tecidos e transformação genética. Calos embriogênicos foram obtidos por indução em embriões isolados de sementes maduras, e cultivados in vitro, em meio de cultura que continha ácido 2,4-diclorofenoxiacético, 6-benzilaminopurina e caseína hidrolisada. Plântulas foram regeneradas a partir dos calos embriogênicos, na presença de ácido naftalenoacético e cinetina. Esse processo foi descrito morfologicamente por observações em microscopia de luz de secções seriadas semifinas de tecidos fixados, ao longo do processo de regeneração, em FAA [formaldeído (40%: ácido acético glacial: etanol (50%, a 5:5:90 v/v/v]. Os embriões das sementes de U. brizantha cv. Marandu não têm epiblasto e são classificados como do tipo panicoide. Nas condições estabelecidas de cultura in vitro, calos embriogênicos e embriões somáticos de U. brizantha cv. Marandu, desenvolvem-se a partir de células meristemáticas do escutelo.The objective of this work was to describe the process of somatic embryogenesis in Urochloa brizantha cv. Marandu (Syn. Brachiaria brizantha cv. Marandu and to provide support for the improvement of tissue culture and genetic transformation methods. Embryogenic calli were obtained by induction in embryos isolated from mature seeds, and cultivated in vitro in culture medium containing 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine and hydrolyzed casein. Plantlets were regenerated from the embryogenic calli in the presence of naphthaleneacetic acid and kinetin. This process was described by morphological observations of serial semithin sections of tissues fixed along the regeneration process in FAA (40% formaldehyde: acetic acid: 50% ethanol, at 5:5:90 v/v/v, using light microscopy. Seed embryos of U

  19. Grapevine responses to terroir: a global approach

    Directory of Open Access Journals (Sweden)

    Alain Deloire

    2005-12-01

    Full Text Available This paper deals with the technical and/or practical treatment of terroir as a study concept, together with related functional aspects. Functioning of the terroir relies on the relation between climate, soil and vine. In addition to this interaction, a comprehensive study concept for terroir requires the consideration of viticultural and enological sciences and techniques necessary to ensure the assurance of wine quality, together with spatial aspects of the grapevine response to environmental factors, as required for vineyard management. In order to comprehend the quality of the harvest, it is necessary to understand the relationship « whole plant - berry ». An easy field and laboratory method to study the relationship between the whole plant and berry and the consequences thereof for wine quality is proposed. Knowledge of grapevine water status and the biochemical evolution within the grape berry from berry set onwards are important issues for the understanding of the role that terroir plays with respect to the quality of the harvest and the wine style or « typicality ».

  20. Weed control and cover crop management affect mycorrhizal colonization of grapevine roots and arbuscular mycorrhizal fungal spore populations in a California vineyard.

    Science.gov (United States)

    Baumgartner, Kendra; Smith, Richard F; Bettiga, Larry

    2005-03-01

    Arbuscular mycorrhizal (AM) fungi naturally colonize grapevines in California vineyards. Weed control and cover cropping may affect AM fungi directly, through destruction of extraradical hyphae by soil disruption, or indirectly, through effects on populations of mycorrhizal weeds and cover crops. We examined the effects of weed control (cultivation, post-emergence herbicides, pre-emergence herbicides) and cover crops (Secale cereale cv. Merced rye, x Triticosecale cv.Trios 102) on AM fungi in a Central Coast vineyard. Seasonal changes in grapevine mycorrhizal colonization differed among weed control treatments, but did not correspond with seasonal changes in total weed frequency. Differences in grapevine colonization among weed control treatments may be due to differences in mycorrhizal status and/or AM fungal species composition among dominant weed species. Cover crops had no effect on grapevine mycorrhizal colonization, despite higher spring spore populations in cover cropped middles compared to bare middles. Cover crops were mycorrhizal and shared four AM fungal species (Glomus aggregatum, G. etunicatum, G. mosseae, G. scintillans) in common with grapevines. Lack of contact between grapevine roots and cover crop roots may have prevented grapevines from accessing higher spore populations in the middles.

  1. Phakopsora euvitis Causes Unusual Damage to Leaves and Modifies Carbohydrate Metabolism in Grapevine

    Directory of Open Access Journals (Sweden)

    Antonio F. Nogueira Júnior

    2017-09-01

    Full Text Available Asian grapevine rust (Phakopsora euvitis is a serious disease, which causes severe leaf necrosis and early plant defoliation. These symptoms are unusual for a strict biotrophic pathogen. This work was performed to quantify the effects of P. euvitis on photosynthesis, carbohydrates, and biomass accumulation of grapevine. The reduction in photosynthetic efficiency of the green leaf tissue surrounding the lesions was quantified using the virtual lesion concept (β parameter. Gas exchange and responses of CO2 assimilation to increasing intercellular CO2 concentration were analyzed. Histopathological analyses and quantification of starch were also performed on diseased leaves. Biomass and carbohydrate accumulation were quantified in different organs of diseased and healthy plants. Rust reduced the photosynthetic rate, and β was estimated at 5.78, indicating a large virtual lesion. Mesophyll conductance, maximum rubisco carboxylation rate, and regeneration of ribulose-1,5-bisphosphate dependent on electron transport rate were reduced, causing diffusive and biochemical limitations to photosynthesis. Hypertrophy, chloroplast degeneration of mesophyll cells, and starch accumulation in cells close to lesions were observed. Root carbohydrate concentration was reduced, even at low rust severity. Asian grapevine rust dramatically reduced photosynthesis and altered the dynamics of production and accumulation of carbohydrates, unlike strict biotrophic pathogens. The reduction in carbohydrate reserves in roots would support polyetic damage on grapevine, caused by a polycyclic disease.

  2. The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

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    Byungkuk Min

    2016-05-01

    Full Text Available Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts.

  3. Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures.

    Science.gov (United States)

    Kitiyanant, Y; Saikhun, J; Chaisalee, B; White, K L; Pavasuthipaisit, K

    2001-01-01

    Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.

  4. Human cloning and embryo research: the 2003 John J. Conley Lecture on medical ethics.

    Science.gov (United States)

    George, Robert P

    2004-01-01

    The author, a member of the U.S. President's Council on Bioethics, discusses ethical issues raised by human cloning, whether for purposes of bringing babies to birth or for research purposes. He first argues that every cloned human embryo is a new, distinct, and enduring organism, belonging to the species Homo sapiens, and directing its own development toward maturity. He then distinguishes between two types of capacities belonging to individual organisms belonging to this species, an immediately exerciseable capacity and a basic natural capacity that develops over time. He argues that it is the second type of capacity that is the ground for full moral respect, and that this capacity (and its concomitant degree of respect) belongs to cloned human embryos no less than to adult human beings. He then considers and rejects counter-arguments to his position, including the suggestion that the capacity of embryos is equivalent to the capacity of somatic cells, that full human rights are afforded only to human organisms with functioning brains, that the possibility of twinning diminishes the moral status of embryos, that the fact that people do not typically mourn the loss of early embryos implies that they have a diminished moral status, that the fact that early spontaneous abortions occur frequently diminishes the moral status of embryos, and that his arguments depend upon a concept of ensoulment. He concludes that if the moral status of cloned human embryos is equivalent to that of adults, then public policy should be based upon this assumption.

  5. Somatic Expression of Psychological Problems (Somatization: Examination with Structural Equation Model

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    Tugba Seda Çolak

    2014-09-01

    Full Text Available The main purpose of the research is to define which psychological symptoms (somatization, depression, obsessive ‐ compulsive, hostility, interpersonal sensitivity, anxiety, phobic anxiety, paranoid ideation and psychoticism cause somatic reactions at most. Total effect of these psychological symptoms on somatic symptoms had been investigated. Study was carried out with structural equation model to research the relation between the psychological symptoms and somatization. The main material of the research is formed by the data obtained from 492 people. SCL‐90‐R scale was used in order to obtain the data. As a result of the structural equation analysis, it has been found that 1Psychoticism, phobic anxiety, and paranoid ideation do not predict somatic symptoms.2There is a negative relation between interpersonal sensitivity level mand somatic reactions.3Anxiety symptoms had been found as causative to occur the highest level of somatic reactions.

  6. Protective, curative and eradicative activities of fungicides against grapevine rust

    Directory of Open Access Journals (Sweden)

    Francislene Angelotti

    2014-01-01

    Full Text Available The protective, eradicative and curative activities of the fungicides azoxystrobin, tebuconazole, pyraclostrobin+metiram, and ciproconazole against grapevine rust, were determined in greenhouse. To evaluate the protective activity, leaves of potted ´Niagara´ (Vitis labrusca vines were artificially inoculated with an urediniospore suspension of Phakopsora euvitis four, eight or forteen days after fungicidal spray; and to evaluate the curative and eradicative activities, leaves were sprayed with fungicides two, four or eight days after inoculation. Disease severity was assessed 14 days after each inoculation. All tested fungicides present excellent preventive activity against grapevine rust; however, tebuconazole and ciproconazole provide better curative activity than azoxystrobin and pyraclostrobin+metiram. It was observed also that all tested fungicides significantly reduced the germination of urediniospore produced on sprayed leaves.

  7. Current perspectives in proteomic analysis of abiotic stress in Grapevines

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    Iniga Seraphina George

    2014-12-01

    Full Text Available Grapes are an important crop plant which forms the basis of a globally important industry. Grape and wine production is particularly vulnerable to environmental and climatic fluctuations, which makes it essential for us to develop a greater understanding of the molecular level responses of grape plants to various abiotic stresses. The completion of the initial grape genome sequence in 2007 has led to a significant increase in research on grapes using proteomics approaches. In this article, we discuss some of the current research on abiotic stress in grapevines, in the context of abiotic stress research in other plant species. We also highlight some of the current limitations in grapevine proteomics and identify areas with promising scope for potential future research.

  8. Interaction of Ulocladium atrum, a Potential Biological Control Agent, with Botrytis cinerea and Grapevine Plantlets

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    Sébastien Ronseaux

    2013-09-01

    Full Text Available The effectiveness of biological control agent, Ulocladium atrum (isolates U13 and U16 in protecting Vitis vinifera L. cv. Chardonnay against gray mold disease caused by Botrytis cinerea, and simulation of the foliar defense responses was investigated. A degraded mycelium structure during cultural assay on potato dextrose agar revealed that U. atrum isolates U13 and U16 were both antagonistic to B. cinerea, mainly when isolates were inoculated two days before Botrytis. Under in vitro conditions, foliar application of U. atrum protected grapevine leaves against gray mold disease. An increase in chitinase activity was induced by the presence of U. atrum isolates indicating that the biological control agents triggered plant defense mechanisms. Moreover, U13 has the potential to colonize the grapevine plantlets and to improve their growth. The ability of U. atrum isolates to exhibit an antagonistic effect against B. cinerea in addition to their aptitude to induce plant resistance and to promote grapevine growth may explain a part of their biological activity. Hence, this study suggests that U. atrum provides a suitable biocontrol agent against gray mold in grapevines.

  9. Transcriptome analyses of the Dof-like gene family in grapevine reveal its involvement in berry, flower and seed development.

    Science.gov (United States)

    da Silva, Danielle Costenaro; da Silveira Falavigna, Vítor; Fasoli, Marianna; Buffon, Vanessa; Porto, Diogo Denardi; Pappas, Georgios Joannis; Pezzotti, Mario; Pasquali, Giancarlo; Revers, Luís Fernando

    2016-01-01

    The Dof (DNA-binding with one finger) protein family spans a group of plant transcription factors involved in the regulation of several functions, such as plant responses to stress, hormones and light, phytochrome signaling and seed germination. Here we describe the Dof-like gene family in grapevine (Vitis vinifera L.), which consists of 25 genes coding for Dof. An extensive in silico characterization of the VviDofL gene family was performed. Additionally, the expression of the entire gene family was assessed in 54 grapevine tissues and organs using an integrated approach with microarray (cv Corvina) and real-time PCR (cv Pinot Noir) analyses. The phylogenetic analysis comparing grapevine sequences with those of Arabidopsis, tomato, poplar and already described Dof genes in other species allowed us to identify several duplicated genes. The diversification of grapevine DofL genes during evolution likely resulted in a broader range of biological roles. Furthermore, distinct expression patterns were identified between samples analyzed, corroborating such hypothesis. Our expression results indicate that several VviDofL genes perform their functional roles mainly during flower, berry and seed development, highlighting their importance for grapevine growth and production. The identification of similar expression profiles between both approaches strongly suggests that these genes have important regulatory roles that are evolutionally conserved between grapevine cvs Corvina and Pinot Noir.

  10. Embryo splitting

    Directory of Open Access Journals (Sweden)

    Karl Illmensee

    2010-04-01

    Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

  11. The sulfated laminarin triggers a stress transcriptome before priming the SA- and ROS-dependent defenses during grapevine's induced resistance against Plasmopara viticola.

    Directory of Open Access Journals (Sweden)

    Adrien Gauthier

    Full Text Available Grapevine (Vitis vinifera is susceptible to many pathogens which cause significant losses to viticulture worldwide. Chemical control is available, but agro-ecological concerns have raised interest in alternative methods, especially in triggering plant immunity by elicitor treatments. The β-glucan laminarin (Lam and its sulfated derivative (PS3 have been previously demonstrated to induce resistance in grapevine against downy mildew (Plasmopara viticola. However, if Lam elicits classical grapevine defenses such as oxidative burst, pathogenesis-related (PR-proteins and phytoalexin production, PS3 triggered grapevine resistance via a poorly understood priming phenomenon. The aim of this study was to identify the molecular mechanisms of the PS3-induced resistance. For this purpose we studied i the signaling events and transcriptome reprogramming triggered by PS3 treatment on uninfected grapevine, ii grapevine immune responses primed by PS3 during P. viticola infection. Our results showed that i PS3 was unable to elicit reactive oxygen species (ROS production, cytosolic Ca(2+ concentration variations, mitogen-activated protein kinase (MAPK activation but triggered a long lasting plasma membrane depolarization in grapevine cells, ii PS3 and Lam shared a common stress-responsive transcriptome profile that partly overlapped the salicylate- (SA and jasmonate-(JA-dependent ones. After P. viticola inoculation, PS3 specifically primed the SA- and ROS-dependent defense pathways leading to grapevine induced resistance against this biotroph. Interestingly pharmacological approaches suggested that the plasma membrane depolarization and the downstream ROS production are key events of the PS3-induced resistance.

  12. Embriogenesis somatik anggrek bulan Phalaenopsis amabilis (L. Bl: struktur dan pola perkembangan

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    Edy Setiti Wida Utami

    2012-02-01

    Full Text Available Research of the structure and development pattern of somatic embryos from callus of leaf explants moon orchid Phalaenopsis amabilis (L Bl had been done. One year old of plantlets were used as explants sources. Basal leaf of these explants were cultured in Somatic Embryo Induction Medium (SEIM e.i.: NP(New Phalaenopsis medium added with 2 mg/L NAA, 1 mg/L BA, 10 g/L sucrose, and 2 g/L gellan gum. Then somatic embryos were transferred to EMM (Embryo Maturation Medium e.i.: NP medium added with 1 mg/L NAA, 1 mg/L BA, 10 g/L sucrose, and 2 g/L gellan gum. Finally, mature somatic embryo were transferred to NP medium without plant growth regulator as Embryo Germination Medium (EGM. The origin of somatic embryos initially from single cell at the pheriphery of embryogenic callus. These cells then devided in mitotic repeatedly formed globular proembryo, elongation embryo, and completed embryo. The structure and development pattern of somatic embryos as the same as with zygotic embryo.

  13. From 'Fire Esca' to 'Esca of Grapevine'

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    A. Graniti

    2006-04-01

    Full Text Available The use of tinder (‘esca’ or ‘amadou’ prepared from basidiocarps of some bracket fungi, e.g. Fomes fomentarius and Phellinus igniarius as an easy-to-burn matter goes back to the man’s conquest of fire. Archaeological finds, such as fragments of tinder, flint-stones and traces of pyrite carried by the ‘Ice man’ on his way across the Alps more than 5,000 years ago, bear evidence of the use of tinder in the Neolithic age. In 1926, on the assumption that P. igniarius was one of the pathogens of the so-called ‘apoplexy’ of grapevine, the name ‘esca’ was given to the disease. For long time, esca was thought to affect old vines only. In the last decades, however, various forms of the disease have been found to be widespread and to cause losses even to young vines. Aetiological studies have shown that esca of grapevine is a complex disease, incited by wilt-inducing ascomycetes (Togninia, Phaeoacremonium, Phaeomoniella and/or the wood-decaying basidiomycete Fomitiporia mediterranea. Since the latter is not a tinder fungus, the advisability of retaining the name ‘esca’ for the disease is discussed.

  14. Ectopic expression of Arabidopsis broad-spectrum resistance gene RPW8.2 improves the resistance to powdery mildew in grapevine (Vitis vinifera).

    Science.gov (United States)

    Hu, Yang; Li, Yajuan; Hou, Fengjuan; Wan, Dongyan; Cheng, Yuan; Han, Yongtao; Gao, Yurong; Liu, Jie; Guo, Ye; Xiao, Shunyuan; Wang, Yuejin; Wen, Ying-Qiang

    2018-02-01

    Powdery mildew is the most economically important disease of cultivated grapevines worldwide. Here, we report that the Arabidopsis broad-spectrum disease resistance gene RPW8.2 could improve resistance to powdery mildew in Vitis vinifera cv. Thompson Seedless. The RPW8.2-YFP fusion gene was stably expressed in grapevines from either the constitutive 35S promoter or the native promoter (NP) of RPW8.2. The grapevine shoots and plantlets transgenic for 35S::RPW8.2-YFP showed reduced rooting and reduced growth at later development stages in the absence of any pathogens. Infection tests with an adapted grapevine powdery mildew isolate En NAFU1 showed that hyphal growth and sporulation were significantly restricted in transgenic grapevines expressing either of the two constructs. The resistance appeared to be attributable to the ectopic expression of RPW8.2, and associated with the enhanced encasement of the haustorial complex (EHC) and onsite accumulation of H 2 O 2 . In addition, the RPW8.2-YFP fusion protein showed focal accumulation around the fungal penetration sites. Transcriptome analysis revealed that ectopic expression of RPW8.2 in grapevines not only significantly enhanced salicylic acid-dependent defense signaling, but also altered expression of other phytohormone-associated genes. Taken together, our results indicate that RPW8.2 could be utilized as a transgene for improving resistance against powdery mildew in grapevines. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Simplification of Bovine Somatic Cell Nuclear Transfer by Application of a Zona-Free Manipulation Technique

    DEFF Research Database (Denmark)

    Booth, Paul J; Tan, Shijian; Reipurth, Rikke

    2001-01-01

    Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The techni......Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods....... The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development......-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20...

  16. Induksi dan Proliferasi Embriogenesis Somatik In Vitro pada Lima Genotipe Kedelai

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    Adam Saepudin

    2017-01-01

    Full Text Available ABSTRACTSomatic embryo induction medium was reported to be genotype dependent for soybean. This study was aimed to obtain the optimum medium for embryo somatic induction and proliferation, and to regenerate somatic embryo of five soybean genotypes. Five soybean genotypes (Tanggamus, Anjasmoro, Yellow Biloxi, CG-22-10, and SP-10-4 were used in this study. The research was divided into four steps: (1 embryogenic callus induction of  five soybean genotypes, (2 embryogenic callus proliferation of five soybean genotypes, (3 optimation of embryo somatic induction on five soybean genotypes and (4 embryo somatic regeneration of five soybean genotypes. The induction experiment showed that based on number of embryogenic callus, the best somatic embryo-induction medium was 3% sucrose+ NAA 5 mg L-1+2,4-D 5 mg L-1+ Vitamin B5. Embryogenic callus number for each genotype tested was increased on proliferation media of 3% sukrosa + 2,4-D 5 mg L-1 + NAA 5 mg L-1+ Vit B5, and Yellow Biloxi gave the highest number of proliferated somatic embryos compared to other genotypes. Increasing number of globular somatic embryo of all genotypes was obtained from the optimation of somatic embryo induction media being used, and Tanggamus genotype gave the highest number of globular somatic embryo which followed by Yellow Biloxi genotype. Tanggamus and Yellow Biloxi genotypes were also successfully formed the four steps of somatic embryos (globular, heart, torpedo, and cotyledonary stages, but in regeneration medium of MS0 and media MS + sukrosa 10 g L-1 + GA3 2 mg L-1 + BAP 4 mg L-1 + Vit B5 only Tanggamus genotype was regenerated into plantlet.  Keywords: 2,4-D, NAA, somatic embryos, induction, proliferation

  17. Spatio-temporal effects of soil and bedrock variability on grapevine water status in hillslope vineyards.

    Science.gov (United States)

    Brillante, Luca; Bois, Benjamin; Mathieu, Olivier; Leveque, Jean

    2014-05-01

    Hillslope vineyards show various and complex water dynamics between soil and plants, and in order to gain further insight into this phenomenon, 8 grapevine plots were monitored during three vintages, from 2010 to 2013, on Corton Hill, Burgundy, France. Plots were distributed along a topolithosequence from 330 to 270 metres a.s.l. Grapevine water status was monitored weekly by surveying water potential, and, at the end of the season, by the use of the δ13C analysis of grape juice. Soil profile of each plot was described and analysed (soil texture, gravel content, organic carbon, total nitrogen, pH, CEC). Soil volumetric humidity was measured weekly, using TDR probes. A pedotransfer function was developed to transform Electrical Resistivity Imaging (ERI) into soil volume wetness and therefore to spatialise and observe variation in the Fraction of Transpirable Soil Water (FTSW). During the three years of monitoring, grapevines experienced great variation in water status, which ranged from low to considerable water deficit (as expressed by pre-dawn leaf water potential and δ13C analysis of grape juice). With ERI imaging, it was possible to observe differences in water absorption pattern by roots, in different soils, and at different depth. In addition, significant differences were observed in grapevine water status in relation to variations in the physical characteristics of the terroir along the hillslope (i.e. the geo-pedological context, the elevation etc.). Grapevine water behaviour and plant-soil water relationships on the hillslope of Corton Hill have been extensively characterised in this study by ultimate technologies, allowing to present this terroir as a very interesting example for future generalisation and modelling of the hillslope vineyard water dynamics.

  18. PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Jun-Xue; Kang, Jin-Dan; Li, Suo; Jin, Long; Zhu, Hai-Ying; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2015-01-02

    Highlights: • First explored that the effects of PXD101 on the development of SCNT embryos in vitro. • 0.5 μM PXD101 treated for 24 h improved the development of porcine SCNT embryos. • Level of AcH3K9 was significantly higher than control group at early stages. - Abstract: In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24 h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P < 0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24 h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P < 0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.

  19. Isolation and pathogenicity of Xylella fastidiosa from grapevine and almond in Iran

    Directory of Open Access Journals (Sweden)

    Naser AMANIFAR

    2014-09-01

    Full Text Available Symptoms similar to those of Pierce’s disease (PD of grapevine and leaf scorch of almond were observed in vineyards and almond orchards in several provinces of Iran. Grafting of scions from symptomatic almond trees onto seedlings of a local almond (cv. Mamaee under greenhouse conditions resulted in the transmission of the leaf scorch agent. A number of symptomatic samples from orchard and greenhouse plants were positive for presence of Xylella fastidiosa when tested by DAS-ELISA and PCR with X. fastidiosa specific antibodies and primers. A Gram-negative bacterium similar to X. fastidiosa was isolated on ‘periwinkle wilt’ (PW medium. Selected isolates induced symptoms similar to those caused by X. fastidiosa when inoculated on Nicotiana tabacum, seedlings of almond and grapevine under greenhouse conditions. DAS-ELISA and PCR confirmed the identity of the isolated bacteria. On the basis of disease symptoms, graft transmission, isolation on specific X. fastidiosa culture medium, pathogenicity tests and positive reactions in DAS-ELISA and PCR, X. fastidiosa is associated with almond leaf scorch and Pierce’s disease in grapevine in Iran. This is the first report on the presence of X. fastidiosa in the Middle East and western Asia.

  20. Oxidative stress homeostasis in grapevine (Vitis vinifera L.

    Directory of Open Access Journals (Sweden)

    Luisa C Carvalho

    2015-03-01

    Full Text Available Plants can maintain growth and reproductive success by sensing changes in the environment and reacting through mechanisms at molecular, cellular, physiological and developmental levels. Each stress condition prompts a unique response although some overlap between the reactions to abiotic stress (drought, heat, cold, salt or high light and to biotic stress (pathogens does occur. A common feature in the response to all stresses is the onset of oxidative stress, through the production of reactive oxygen species (ROS. As hydrogen peroxide and superoxide are involved in stress signaling, a tight control in ROS homeostasis requires a delicate balance of systems involved in their generation and degradation. If the plant lacks the capacity to generate scavenging potential, this can ultimately lead to death. In grapevine, antioxidant homeostasis can be considered at whole plant levels and during the development cycle. The most striking example lies in berries and their derivatives, such as wine, with nutraceutical properties associated with their antioxidant capacity. Antioxidant homeostasis is tightly regulated in leaves, assuring a positive balance between photosynthesis and respiration, explaining the tolerance of many grapevine varieties to extreme environments.In this review we will focus on antioxidant metabolites, antioxidant enzymes, transcriptional regulation and cross-talk with hormones prompted by abiotic stress conditions. We will also discuss three situations that require specific homeostasis balance: biotic stress, the oxidative burst in berries at veraison and in vitro systems. The genetic plasticity of the antioxidant homeostasis response put in evidence by the different levels of tolerance to stress presented by grapevine varieties will be addressed. The gathered information is relevant to foster varietal adaptation to impending climate changes, to assist breeders in choosing the more adapted varieties and to suitable viticulture

  1. Imaging of Norway spruce early somatic embryos with the ESEM, Cryo-SEM and laser scanning microscope

    Czech Academy of Sciences Publication Activity Database

    Neděla, Vilém; Hřib, Jiří; Havel, L.; Hudec, Jiří; Runštuk, Jiří

    2016-01-01

    Roč. 84, May 2016 (2016), s. 67-71 ISSN 0968-4328 R&D Projects: GA ČR(CZ) GA14-22777S Institutional support: RVO:68081731 Keywords : ESEM * Cryo-SEM * bright field/dark field microscopy * extracellular matrix * Picea abies * somatic embryogenesis Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 1.980, year: 2016

  2. Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor.

    Science.gov (United States)

    Florez, Sergio L; Erwin, Rachel L; Maximova, Siela N; Guiltinan, Mark J; Curtis, Wayne R

    2015-05-16

    Theobroma cacao, the chocolate tree, is an important economic crop in East Africa, South East Asia, and South and Central America. Propagation of elite varieties has been achieved through somatic embryogenesis (SE) but low efficiencies and genotype dependence still presents a significant limitation for its propagation at commercial scales. Manipulation of transcription factors has been used to enhance the formation of SEs in several other plant species. This work describes the use of the transcription factor Baby Boom (BBM) to promote the transition of somatic cacao cells from the vegetative to embryonic state. An ortholog of the Arabidopsis thaliana BBM gene (AtBBM) was characterized in T. cacao (TcBBM). TcBBM expression was observed throughout embryo development and was expressed at higher levels during SE as compared to zygotic embryogenesis (ZE). TcBBM overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. While transient ectopic expression of TcBBM provided only moderate enhancements in embryogenic potential, constitutive overexpression dramatically increased SE proliferation but also appeared to inhibit subsequent development. Our work provides validation that TcBBM is an ortholog to AtBBM and has a specific role in both somatic and zygotic embryogenesis. Furthermore, our studies revealed that TcBBM transcript levels could serve as a biomarker for embryogenesis in cacao tissue. Results from transient expression of TcBBM provide confirmation that transcription factors can be used to enhance SE without compromising plant development and avoiding GMO plant production. This strategy could compliment a hormone-based method of reprogramming somatic cells and lead to more precise manipulation of SE at the regulatory level of transcription factors. The technology would benefit the propagation of elite varieties with low regeneration potential as well as the production of transgenic plants, which

  3. Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.

    Science.gov (United States)

    Ribeiro, Eduardo de Souza; Gerger, Renato Pereira da Costa; Ohlweiler, Lain Uriel; Ortigari, Ivens; Mezzalira, Joana Cláudia; Forell, Fabiana; Bertolini, Luciana Relly; Rodrigues, José Luiz; Ambrósio, Carlos Eduardo; Miglino, Maria Angélica; Mezzalira, Alceu; Bertolini, Marcelo

    2009-09-01

    Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.

  4. ROOTSTOCK-SCION INTERACTION: 1. EFFECT ON THE YIELD COMPONENTS OF CABERNET SAUVIGNON GRAPEVINE

    Directory of Open Access Journals (Sweden)

    ALBERTO MIELE

    Full Text Available ABSTRACT The interaction between rootstock, scion and environment can induce different responses to the grapevine physiology. Thus, the aim of this study was to determine the rootstock effect on the yield components of Cabernet Sauvignon (CS grapevine grown in the Serra Gaúcha viticultural region. The experimental design was completely randomized blocks, with 15 treatments, three replicates and ten vines per plot. The results show that all variables evaluated were significantly affected by the year and the rootstock. The CS/Solferino was among other combinations influenced by the year and had higher significant yield/ vine. Indeed, it was higher than that CS/Rupestris du Lot, CS/101-14 Mgt., CS/3309 C, CS/5BB K, CS/161- 49 C, CS/1103 P. and CS/Isabel. The number of clusters/bud, per burst bud and per vine and the weight of clusters were affected by the rootstock as well. Pruning weight/vine, yield/pruning weight, leaf area/vine, leaf area index and leaf area/fresh fruit weight are variables related to the physiology of grapevine which were also affected by the rootstock. In general, rootstocks had adapted well to the environment where the experiment was carried out, giving vigor and high yield to Cabernet Sauvignon grapevine, which means that they may be used by grape growers in this region. However, the choice of the right rootstock depends on various aspects, such as those related to the soil characteristics, climate conditions, grape varieties, and even clones, and production purposes.

  5. In vivo somatic mutation systems in the mouse

    International Nuclear Information System (INIS)

    Russell, L.B.

    1979-01-01

    In an effort to meet the need for a fast and cheap in vivo prescreen for inherited mammalian point mutations, a somatic forward-mutation method, originally developed in an x-ray experiment, has more recently been tested in work with chemical mutagens. The method makes use of coat-color mutations because the gene product is usually locally expressed, mosaics can be detected with minimal effort, and opportunities for making comparison with induction of germinal point mutations are greatest.--Following treatment of embryos that are heterozygous at specific coat-color loci, various induced genetic changes can result in expression of the recessive (RS) in clones derived from mutant melanocyte precursor cells. However, other events, such as decrease in the number of precursor cells, or disturbed differentiation, can also result in spots, which with careful classification can usually be distinguished from RS's on the basis of their location and color. When this is done, the relative RS frequencies for a series of compounds at least roughly parallel the relative spermatogonial mutation rates. The fact that easily measurable (though low) RS rates are obtained with compounds that have yielded negative results in spermatogonial tests is not surprising in view of the fact that RS's can be caused by several mechanisms besides point mutation.--In spite of the parallelism observed in one laboratory, the usefulness of the in vivo somatic mutation method as a prescreen could come to be doubted because of major discrepancies between results of similar experiments at different laboratories. However, It appears probable that at least some of these discrepancies are due to failure to discriminate between spots that probably resulted from melanocyte insufficiency and spots that resulted from expression of the recessive.--Reverse somatic mutation systems can potentially avoid some of the pitfalls of forward mutation systems. Such system are still in developmental stages

  6. In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat.

    Science.gov (United States)

    Ohkoshi, Katsuhiro; Takahashi, Seiya; Koyama, Shin-Ichiro; Akagi, Satoshi; Adachi, Noritaka; Furusawa, Tadashi; Fujimoto, Jun-Ichiro; Takeda, Kumiko; Kubo, Masanori; Izaike, Yoshiaki; Tokunaga, Tomoyuki

    2003-01-01

    The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.

  7. Viability of bovine demi embryo after splitting of fresh and frozen thawed embryo derived from in vitro embryo production

    Directory of Open Access Journals (Sweden)

    M Imron

    2007-06-01

    Full Text Available In vivo embryo production was limited by number of donor, wide variability respond due to superovulation program and also immunoactifity of superovulation hormone (FSH. Splitting technology could be an alternative to increase the number of transferrable embryos into recipien cows. Splitting is done with cutting embryo becoming two equal pieces (called demi embrio base on ICM orientation. The objective of this research was to determine the viability of demi embryo obtained from embryo splitting of fresh and frozen thawed embryo. The results showed that demi embryos which performed blastocoel reexpansion 3 hours after embryo splitting using fresh and frozen thawed embryos were 76.9 and 76.2% respectively. Base on existention of inner cell mass (ICM, the number of demi embryos developed with ICM from fresh and frozen thawed embryos were not significantly different (90.6 and 85.7% respectively. The cell number of demi embryo from fresh embryos splitting was not different compared with those from frozen thawed embryos (36.1 and 35.9 respectively. These finding indicated that embryo splitting can be applied to frozen thawed embryos with certain condition as well as fresh embryos.

  8. cDNA cloning, genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis).

    Science.gov (United States)

    Zhang, Li-Feng; Li, Wan-Feng; Han, Su-Ying; Yang, Wen-Hua; Qi, Li-Wang

    2013-10-15

    A full-length cDNA and genomic sequences of a translationally controlled tumor protein (TCTP) gene were isolated from Japanese larch (Larix leptolepis) and designated LaTCTP. The length of the cDNA was 1, 043 bp and contained a 504 bp open reading frame that encodes a predicted protein of 167 amino acids, characterized by two signature sequences of the TCTP protein family. Analysis of the LaTCTP gene structure indicated four introns and five exons, and it is the largest of all currently known TCTP genes in plants. The 5'-flanking promoter region of LaTCTP was cloned using an improved TAIL-PCR technique. In this region we identified many important potential cis-acting elements, such as a Box-W1 (fungal elicitor responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), a CGTCA-motif (cis-acting regulatory element involved in MeJA-responsiveness), a GT1-motif (light responsive element), a Skn-1-motif (cis-acting regulatory element required for endosperm expression) and a TGA-element (auxin-responsive element), suggesting that expression of LaTCTP is highly regulated. Expression analysis demonstrated ubiquitous localization of LaTCTP mRNA in the roots, stems and needles, high mRNA levels in the embryonal-suspensor mass (ESM), browning embryogenic cultures and mature somatic embryos, and low levels of mRNA at day five during somatic embryogenesis. We suggest that LaTCTP might participate in the regulation of somatic embryo development. These results provide a theoretical basis for understanding the molecular regulatory mechanism of LaTCTP and lay the foundation for artificial regulation of somatic embryogenesis. © 2013.

  9. De novo formation of nucleoli in developing mouse embryos originating from enucleolated zygotes.

    Science.gov (United States)

    Kyogoku, Hirohisa; Fulka, Josef; Wakayama, Teruhiko; Miyano, Takashi

    2014-06-01

    The large, compact oocyte nucleoli, sometimes referred to as nucleolus precursor bodies (NPBs), are essential for embryonic development in mammals; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygote or embryo, leading to developmental failure. It has been convincingly documented that zygotes inherit the oocyte nucleolar material and form NPBs again in pronuclei. It is commonly accepted that during early embryonic development, the original compact zygote NPBs gradually transform into reticulated nucleoli of somatic cells. Here, we show that zygote NPBs are not required for embryonic and full-term development in the mouse. When NPBs were removed from late-stage zygotes by micromanipulation, the enucleolated zygotes developed to the blastocyst stage and, after transfer to recipients, live pups were obtained. We also describe de novo formation of nucleoli in developing embryos. After removal of NPBs from zygotes, they formed new nucleoli after several divisions. These results indicate that the zygote NPBs are not used in embryonic development and that the nucleoli in developing embryos originate from de novo synthesized materials. © 2014. Published by The Company of Biologists Ltd.

  10. Method validation for determination of metals in Vitis labrusca L. grapevine leaf extracts by inductively coupled plasma mass spectrometry (ICP-MS

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    LIANE V.V. BOKOWSKI

    Full Text Available ABSTRACT Vitis labrusca L. is the main species used for wine and juice production in Brazil. The grapevine leaves can be used both as functional foods and as cheapest sources for the extraction of phenolic compounds. Besides the antioxidant activity, grapevine leaves exhibited significant anti-inflammatory activity. Therefore, the aim of this study was to develop and validate an analytical methodology to determine the metals selenium (96Se, chromium (53Cr, nickel (62Ni, cadmium (111Cd and lead (206Pb in 30 samples of grapevine leaf extracts (Vitis labrusca, Bordo cultivar using inductively coupled plasma mass spectrometry (ICP-MS. To obtain the grapevine leaf extracts the samples were milled, weighed and digested in microwave oven with nitric acid. The method showed linearity, precision, accuracy and limits of quantification and detection acceptable for INMETRO protocol validation of analytical methods. Therefore, the method using ICP-MS was developed and validated to determine metals concentrations in grapevine leaves of Vitis labrusca L. and the proposed method could be applied in routine analytical laboratory.

  11. 5-Azacytidine combined with 2,4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels.

    Science.gov (United States)

    Fraga, Hugo P F; Vieira, Leila N; Caprestano, Clarissa A; Steinmacher, Douglas A; Micke, Gustavo A; Spudeit, Daniel A; Pescador, Rosete; Guerra, Miguel P

    2012-12-01

    DNA methylation is an epigenetic regulatory mechanism of gene expression which can be associated with developmental phases and in vitro morphogenetic competence in plants. The present work evaluated the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2,4-D) on Acca sellowiana somatic embryogenesis (SE) and global DNA methylation levels by high-performance liquid chromatography mass spectrometry (HPLC/MS/MS). 2,4-D-free treatments revealed no somatic embryo formation in both accessions tested. Treatments supplemented with 2,4-D pulse plus AzaC in the culture medium resulted in increased embryo formation. In AzaC-free treatment, HPLC/MS/MS analysis showed a gradual increase in methylation levels in cultures of both accessions tested during SE induction. Treatment with AzaC and 2,4-D-free resulted in a marked decrease in methylation for both accessions, ranging from 37.6 to 20.8 %. In treatment with 2,4-D and AzaC combined, the 85 accession showed increasing global methylation levels. Otherwise, the 101X458 accession, in the same treatment, showed a decrease between 10 and 20 days, followed by an increase after 30 days (39.5, 36.2 and 41.6 %). These results indicate that 2,4-D pulse combined with AzaC improves SE induction. However, the conversion phase showed that although positively influencing SE induction, AzaC had a dysregulatory effect on the stage of autotrophic plant formation, resulting in significantly lower conversion rates. The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and global DNA methylation dynamics are related to morphogenetic response. 5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.

  12. Micropropagation of Citrus spp. by organogenesis and somatic embryogenesis.

    Science.gov (United States)

    Chiancone, Benedetta; Germanà, Maria Antonietta

    2013-01-01

    Citrus spp., the largest fruit crops produced worldwide, are usually asexually propagated by cuttings or grafting onto seedling rootstocks. Most of Citrus genotypes are characterized by polyembryony due to the occurrence of adventive nucellar embryos, which lead to the production of true-to-type plants by seed germination. Tissue culture and micropropagation, in particular, are valuable alternatives to traditional propagation to obtain a high number of uniform and healthy plants in a short time and in a small space. Moreover, in vitro propagation provides a rapid system to multiply the progeny obtained by breeding programs, allows the use of monoembryonic and seedless genotypes as rootstocks, and it is very useful also for breeding and germplasm preservation.In this chapter, two protocols regarding organogenesis of a rootstock and somatic embryogenesis of a cultivar have been described.

  13. The effect of reclaimed wastewater on the quality and growth of grapevines.

    Science.gov (United States)

    Mendoza-Espinosa, L G; Cabello-Pasini, A; Macias-Carranza, V; Daessle-Heuser, W; Orozco-Borbón, M V; Quintanilla-Montoya, A L

    2008-01-01

    The effect of the use of treated wastewater on the growth of cabernet sauvignon and merlot grapes from the Guadalupe Valley, Mexico was evaluated. Secondary advanced effluent was used to irrigate the grapevines at a rate of 66 L/vine/week. Wastewater quality results confirmed that all parameters complied with Mexican legislation for crop irrigation as well as reuse in activities in which the public would be in direct or indirect contact with the reclaimed water. Results showed that the number of leaves per shoot and the overall biomass increased in plants irrigated with wastewater and grape production per plant was 20% higher. The concentration of carbohydrates, organic acids and pH were similar in grapes from vines irrigated with wastewater to those irrigated with groundwater. Throughout the experiment, no fecal coliform bacteria were detected in the cultivated grapes. The wastewater caused an increase in the biomass of the grapevines and there was no presence of microbial indicators in the final product so a higher wine production could be achieved without an increase in health risk related problems. If 200 L/s of reclaimed wastewater would be returned to be used for grapevine irrigation in Valle de Guadalupe (the same amount that is currently being sent as drinking water to Ensenada), assuming an irrigation application of 6,000-7.500 m3/ha/year, approximately 837-1046 hectares (ha) of grapevines could be irrigated. Part of ongoing research includes an economical analysis of the best options for Ensenada and the Valle de Guadalupe in order to establish the optimum volume of water to be returned, the cost of its transportation, as well as the cost of irrigation. (c) IWA Publishing 2008.

  14. Evaluation of pollen dispersal and cross pollination using transgenic grapevine plants.

    Science.gov (United States)

    Harst, Margit; Cobanov, Beatrix-Axinja; Hausmann, Ludger; Eibach, Rudolf; Töpfer, Reinhard

    2009-01-01

    Public debate about the possible risk of genetically modified plants often concerns putative effects of pollen dispersal and out-crossing into conventional fields in the neighborhood of transgenic plants. Though Vitis vinifera (grapevine) is generally considered to be self-pollinating, it cannot be excluded that vertical gene transfer might occur. For monitoring pollen flow and out-crossing events, transgenic plants of Vitis vinifera cv. 'Dornfelder' harboring the gus-int gene were planted in the center of a field experiment in Southwest Germany in 1999. The rate of pollen dispersal was determined by pollen traps placed at radial distances of 5-150 m from the pollen-donor plants, at 1.00 and 1.80 m above ground. Transgenic pollen was evaluated by GUS staining, and could clearly be distinguished from pollen originating from non-transgenic grapevine plants. Transgenic pollen was observed up to 150 m from the pollen donors. The rate of out-crossing was determined by sampling seeds of selected grapevines at a distance of 10 m to the pollen source, and of a sector at 20 m distance, respectively, followed by GUS analysis of seedlings. The average cross-pollination rate during the experiment (2002-2004) was 2.7% at a distance of 20 m. The results of this first pilot study present a good base for further assessment under the conditions of normal viticulture practice.

  15. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    Science.gov (United States)

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

  16. THE BIOTECHNOLOGY OF EMBRYOGENIC CELL LINES OBTAINING AND PLANTLETS OF CONIFEROUS SPECIES IN SIBERIA IN CULTURE IN VITRO

    Directory of Open Access Journals (Sweden)

    Tretiakova I.

    2012-08-01

    Full Text Available Experiments of culturing the immature isolated embryos and megagamethophytes of Siberian coniferous species were carried out on different modified media: ½ LV medium for Pinus sibirica and Pinus pumila, MSG and AI media (patent № 2431651 for Larix sibirica and Larix gmelinii, DCR medium for Picea ajanensis. For induction of embryogenic callus every species needs the optimized medium supplemented with L-glutamine, casein hydrolysate, ascorbic acid and hormones with different concentrations and their different proportions. The active proliferation of embryonal masses is observed on the same medium with reduced concentration of cytokinins. The proliferation of embryonal masses was significantly improved when they were subcultured after dispersing in liquid medium. The somatic embryos from embryonal masses are matured on basal medium with ABA (60-120 mM and PEG. In spite of species specificity the embryogenesis of morphogenic structures had the same scheme: elongation and asymmetric division of somatic cells, formation of initial cells and embryonal tubes, development of globular, torpedo and bipolar somatic embryos, embryos maturation and germination. However, not all donor-plants of coniferous species can form the embryogenic cell lines and somatic embryos. The active development of embryonal masses and formation of somatic embryos are observed from zygotic embryo of hybrid seeds of P. sibirica and L. sibirica. The obtained embryogenic lines were characterized by different proliferative activity. During 10 months cultivation the value of embryonal masses in different lines was 140-570 g. The number of somatic embryos varies from 210 to 410 per 100 mg of callus fresh weight. Decreasing proliferation activity did not observed during 24-45 months cultivation. However, development of somatic embryos in long cultivated lines decreased. Maturation of somatic embryos and development of plantlets were established in L. sibirica and P. pumila 50

  17. A complex protein derivative acts as biogenic elicitor of grapevine resistance against powdery mildew under field conditions

    Directory of Open Access Journals (Sweden)

    Andrea eNesler

    2015-09-01

    Full Text Available Powdery mildew caused by Erysiphe necator is one of the most important grapevine diseases in several viticulture areas, and high fungicide input is required to control it. However, numerous synthetic chemical pesticides are under scrutiny due to concerns about their impact on human health and the environment. Biopesticides, such as biogenic elicitors, are a promising alternative to chemical fungicides. Although several studies have reported on effective elicitors against grapevine diseases, their efficacy under field conditions has not been investigated extensively or has occurred at rather limited levels. Our goal was to examine the efficacy of a protein-based composition, namely nutrient broth (NB, against powdery mildew under field conditions and to characterize its mechanism of action. Weekly treatments with NB was highly effective in controlling powdery mildew on grapevine across seasons with different disease pressures. The level of disease control achieved with NB was comparable to standard fungicide treatments both on leaves and bunches across three different years. NB has no direct toxic effect on the germination of E. necator conidia, and it activates plant resistance with both systemic and translaminar effect in experiments with artificial inoculation under controlled conditions. NB induced the expression of defense-related genes in grapevine, demonstrating stimulation of plant defense mechanisms, prior to and in the early stages of pathogen infection. NB is a natural derivative from meat and yeast, substances that tend not to raise concerns about toxicological and ecotoxicological properties. NB represents a valid control tool for integrated plant protection programs against powdery mildew, to reduce the use of synthetic pesticides on grapevine.

  18. A complex protein derivative acts as biogenic elicitor of grapevine resistance against powdery mildew under field conditions.

    Science.gov (United States)

    Nesler, Andrea; Perazzolli, Michele; Puopolo, Gerardo; Giovannini, Oscar; Elad, Yigal; Pertot, Ilaria

    2015-01-01

    Powdery mildew caused by Erysiphe necator is one of the most important grapevine diseases in several viticulture areas, and high fungicide input is required to control it. However, numerous synthetic chemical pesticides are under scrutiny due to concerns about their impact on human health and the environment. Biopesticides, such as biogenic elicitors, are a promising alternative to chemical fungicides. Although several studies have reported on effective elicitors against grapevine diseases, their efficacy under field conditions has not been investigated extensively or has occurred at rather limited levels. Our goal was to examine the efficacy of a protein-based composition, namely nutrient broth (NB), against powdery mildew under field conditions and to characterize its mechanism of action. Weekly treatments with NB was highly effective in controlling powdery mildew on grapevine across seasons with different disease pressures. The level of disease control achieved with NB was comparable to standard fungicide treatments both on leaves and bunches across three different years. NB has no direct toxic effect on the germination of E. necator conidia, and it activates plant resistance with both systemic and translaminar effect in experiments with artificial inoculation under controlled conditions. NB induced the expression of defense-related genes in grapevine, demonstrating stimulation of plant defense mechanisms, prior to and in the early stages of pathogen infection. NB is a natural derivative from meat and yeast, substances that tend not to raise concerns about toxicological and ecotoxicological properties. NB represents a valid control tool for integrated plant protection programs against powdery mildew, to reduce the use of synthetic pesticides on grapevine.

  19. Selection of grapevine leaf varieties for culinary process based on phytochemical composition and antioxidant properties.

    Science.gov (United States)

    Lima, Adriano; Bento, Albino; Baraldi, Ilton; Malheiro, Ricardo

    2016-12-01

    Grapevine leaves are an abundant sub-product of vineyards which is devalued in many regions. The objective of this work is to study the antioxidant activity and phytochemical composition of ten grapevine leaf varieties (four red varieties: Tinta Amarela, Tinta Roriz, Touriga Franca, and Touriga Nacional; and six white varieties: Côdega do Larinho, Fernão Pires, Gouveio, Malvasia Fina, Rabigato, and Viosinho) to select varieties to be used as food ingredients. White grapevine leaves revealed higher antioxidant potential. Malvasia Fina reported better antioxidant properties contrasting with Touriga Franca. Phenolic content varied between 112 and 150mgGAEg(-1) of extract (gallic acid equivalents), hydroxycinnamic acid derivatives and flavonols varied between 76 and 108mgCAEg(-1) of extract (caffeic acid equivalents) and 39 and 54mgQEg(-1) of extract (quercetin equivalents). Malvasia Fina is a good candidate for culinary treatment due to its antioxidant properties and composition in bioactive compounds. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Embryo density and medium volume effects on early murine embryo development.

    Science.gov (United States)

    Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

    1992-10-01

    One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

  1. Somatic symptom disorder

    Science.gov (United States)

    ... related disorders; Somatization disorder; Somatiform disorders; Briquet syndrome; Illness anxiety disorder References American Psychiatric Association. Somatic symptom disorder. Diagnostic and Statistical Manual of Mental Disorders . ...

  2. Molecular characterization and evolution studies of a SERK like gene transcriptionally induced during somatic embryogenesis in Phoenix Dactylifera L v Deglet Nour

    Directory of Open Access Journals (Sweden)

    Rekik Imen

    2015-01-01

    Full Text Available A somatic embryogenesis receptor kinase like (SERKL cDNA, designated PhSERKL, was isolated from date palm (Phoenix Dactylifera L using RACE PCR. PhSERKL protein shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. Phylogenetic analyses using PHYLIP and Notung 2.7 programs suggest that the SERK proteins of some plant species resulted from relatively ancient duplication events. We predict an ancestor protein of monocots and dicots SERK using FASTML program. Somatic embryogenic cultures of date palm were established following transfer of callus cultures to medium containing 2, 4-dichlorophenoxyacetic acid. The role of PhSERKL gene during establishment of somatic embryogenesis in culture was investigated using quantitative real-time PCR. PhSERKL gene was highly expressed during embryogenic competence acquisition and globular embryo formation in culture. Overall, levels of expression of PhSERKL gene were lower in nonembryogenic tissues and organs than in embryogenic callus.

  3. A molecular approach to study the arbuscular mycorrhizal fungi community in a typical Piedmont grapevine cultivar

    Science.gov (United States)

    Magurno, F.; Bughi Peruglia, G.; Lumini, E.; Bianciotto, V.; Balestrini, R.

    2009-04-01

    Viticulture and wine production represent one of the most relevant agro-food sectors for the Piedmont Region (Italy) in terms of value, with more than 400 millions € a year (12 % of total agricultural production of the Region and the 10 % of the national grape and wine production). The soil where grapevines (Vitis spp.) grow is one of the first parameters influencing the complex grapevine-wine chain. Arbuscular mycorrhizal fungi (AMFs), a main component of soil microbiota in most agrosystems, are considered crucial biomarkers of soil quality because of their biofertilisers role. As mutualistic symbionts, they colonize the roots of the majority of plants. Benefits in symbiosis are well showed as an improvement in shoot/root growth, mineral transport, water-stress tolerance and resistance to certain diseases. Grapevines roots are often heavily colonized by AMFs under field conditions and in some cases AMFs appear to be necessary for their normal growth and survival. Even so, little information are until now available about composition of AMFs communities living in the vineyards soil and in associations with grapevine roots, mainly related to morphological characterization. Vineyard of Nebbiolo, one of the most important Piedmont cultivar, was selected in order to study the AMFs community using a molecular approach. Soil samples and roots from an experimental vineyard located in Lessona (Biella, Piedmont, Italy) were analyzed using AM fungal-specific primers to partially amplify the small subunit (SSU) of the ribosomal DNA genes. Much more than 650 clones were sequenced. Phylogenetic analyses identified 32 OTUs from soil, clustered into Glomus groups Aa, Ab, Ad and B, Diversisporaceae and Gigasporaceae families. Thirteen OTUs from roots were determined, clustered into Glomus groups Ab, Ad and B, and Gigasporaceae family. In particular, Glomus group Ad was the best represented in both compartments, suggesting a correlation between intra and extra radical communities

  4. Phytotoxins Produced by Fungi Associated with Grapevine Trunk Diseases

    Science.gov (United States)

    Andolfi, Anna; Mugnai, Laura; Luque, Jordi; Surico, Giuseppe; Cimmino, Alessio; Evidente, Antonio

    2011-01-01

    Up to 60 species of fungi in the Botryosphaeriaceae family, genera Cadophora, Cryptovalsa, Cylindrocarpon, Diatrype, Diatrypella, Eutypa, Eutypella, Fomitiporella, Fomitiporia, Inocutis, Phaeoacremonium and Phaeomoniella have been isolated from decline-affected grapevines all around the World. The main grapevine trunk diseases of mature vines are Eutypa dieback, the esca complex and cankers caused by the Botryospheriaceae, while in young vines the main diseases are Petri and black foot diseases. To understand the mechanism of these decline-associated diseases and the symptoms associated with them, the toxins produced by the pathogens involved in these diseases were isolated and characterised chemically and biologically. So far the toxins of only a small number of these decline fungi have been studied. This paper presents an overview of the toxins produced by the most serious of these vine wood pathogens: Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and some taxa in the Botryosphaeriaceae family, and examines how these toxins produce decline symptoms. The chemical structure of these metabolites and in some cases their vivotoxin nature are also discussed. PMID:22295177

  5. Phytotoxins Produced by Fungi Associated with Grapevine Trunk Diseases

    Directory of Open Access Journals (Sweden)

    Antonio Evidente

    2011-12-01

    Full Text Available Up to 60 species of fungi in the Botryosphaeriaceae family, genera Cadophora, Cryptovalsa, Cylindrocarpon, Diatrype, Diatrypella, Eutypa, Eutypella, Fomitiporella, Fomitiporia, Inocutis, Phaeoacremonium and Phaeomoniella have been isolated from decline-affected grapevines all around the World. The main grapevine trunk diseases of mature vines are Eutypa dieback, the esca complex and cankers caused by the Botryospheriaceae, while in young vines the main diseases are Petri and black foot diseases. To understand the mechanism of these decline-associated diseases and the symptoms associated with them, the toxins produced by the pathogens involved in these diseases were isolated and characterised chemically and biologically. So far the toxins of only a small number of these decline fungi have been studied. This paper presents an overview of the toxins produced by the most serious of these vine wood pathogens: Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and some taxa in the Botryosphaeriaceae family, and examines how these toxins produce decline symptoms. The chemical structure of these metabolites and in some cases their vivotoxin nature are also discussed.

  6. The grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis.

    Science.gov (United States)

    Martins, Viviana; Carneiro, Filipa; Conde, Carlos; Sottomayor, Mariana; Gerós, Hernâni

    2017-12-01

    The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca 2+ , Na + and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca 2+ homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca 2+ , demonstrating a role in Ca 2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na + , Li + , Mn 2+ and Cu 2+ in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca 2+ , and Na + in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.

  7. Physical mapping in highly heterozygous genomes: a physical contig map of the Pinot Noir grapevine cultivar

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    Jurman Irena

    2010-03-01

    Full Text Available Abstract Background Most of the grapevine (Vitis vinifera L. cultivars grown today are those selected centuries ago, even though grapevine is one of the most important fruit crops in the world. Grapevine has therefore not benefited from the advances in modern plant breeding nor more recently from those in molecular genetics and genomics: genes controlling important agronomic traits are practically unknown. A physical map is essential to positionally clone such genes and instrumental in a genome sequencing project. Results We report on the first whole genome physical map of grapevine built using high information content fingerprinting of 49,104 BAC clones from the cultivar Pinot Noir. Pinot Noir, as most grape varieties, is highly heterozygous at the sequence level. This resulted in the two allelic haplotypes sometimes assembling into separate contigs that had to be accommodated in the map framework or in local expansions of contig maps. We performed computer simulations to assess the effects of increasing levels of sequence heterozygosity on BAC fingerprint assembly and showed that the experimental assembly results are in full agreement with the theoretical expectations, given the heterozygosity levels reported for grape. The map is anchored to a dense linkage map consisting of 994 markers. 436 contigs are anchored to the genetic map, covering 342 of the 475 Mb that make up the grape haploid genome. Conclusions We have developed a resource that makes it possible to access the grapevine genome, opening the way to a new era both in grape genetics and breeding and in wine making. The effects of heterozygosity on the assembly have been analyzed and characterized by using several complementary approaches which could be easily transferred to the study of other genomes which present the same features.

  8. Management of somatic symptoms

    DEFF Research Database (Denmark)

    Schröder, Andreas; Dimsdale, Joel

    2014-01-01

    on the recognition and effective management of patients with excessive and disabling somatic symptoms. The clinical presentation of somatic symptoms is categorized into three groups of patients: those with multiple somatic symptoms, those with health anxiety, and those with conversion disorder. The chapter provides...

  9. Induction of Somatic Embryogenesis and Regeneration of Secondary Embryogenesis of Oil Palm%油棕体细胞胚的诱导和次生胚的增殖研究

    Institute of Scientific and Technical Information of China (English)

    邹积鑫; 潘登浪; 林位夫

    2016-01-01

    油棕体细胞胚的诱导是油棕体胚发生技术体系中最关键的培养过程,通过建立油棕次生胚增殖培养方法可有效地实现油棕组培苗快速的增殖繁育。本研究通过系统的对油棕体细胞胚发生及次生胚再生途径中若干影响因子的比较研究,建立油棕体细胞胚和次生胚再生体系。结果表明:油棕体细胞胚再生体系在不同基因型的油棕品种内诱导率存在较大差别,其中植物外源激素二氯苯氧乙酸(2,4-D)对油棕胚诱导效果较好;油棕次生胚增殖在不同基因型的油棕品种内差异不显著,木本植物培养基(WPM)在油棕次生胚增殖中培养效果最好。%Induction of oil palm somatic embryogenesis is the most critical cultural process in oil palm tissue culture. Rapid proliferation of oil palm can be effectively realized through the establishment of oil palm secondary embryogenesis regeneration in tissue culture. Several factors that affecting induction of oil palm somatic embryos and regeneration of secondary embryogenesis were compared systematically to establish a secondary oil palm somatic embryo regeneration system. The results showed that there is great difference in induction rate of somatic embryogenesis between different oil palm genotypes, and the woody plant medium (WPM) is the best basic medium in proliferation of oil palm secondary embryos.

  10. Identification of a novel vitivirus from grapevines in New Zealand.

    Science.gov (United States)

    Blouin, Arnaud G; Keenan, Sandi; Napier, Kathryn R; Barrero, Roberto A; MacDiarmid, Robin M

    2018-01-01

    We report a sequence of a novel vitivirus from Vitis vinifera obtained using two high-throughput sequencing (HTS) strategies on RNA. The initial discovery from small-RNA sequencing was confirmed by HTS of the total RNA and Sanger sequencing. The new virus has a genome structure similar to the one reported for other vitiviruses, with five open reading frames (ORFs) coding for the conserved domains described for members of that genus. Phylogenetic analysis of the complete genome sequence confirmed its affiliation to the genus Vitivirus, with the closest described viruses being grapevine virus E (GVE) and Agave tequilana leaf virus (ATLV). However, the virus we report is distinct and shares only 51% amino acid sequence identity with GVE in the replicase polyprotein and 66.8% amino acid sequence identity with ATLV in the coat protein. This is well below the threshold determined by the ICTV for species demarcation, and we propose that this virus represents a new species. It is provisionally named "grapevine virus G".

  11. Biochemical method for fast affinity diagnosis in grape-vine transplantation

    International Nuclear Information System (INIS)

    Lilov, D.

    1977-01-01

    Long term experiments have proved the affinity of cv. Mavroud in transplantations on various root stocks. Best affinity was observed in the combination cv. Mavroud X Riparia tomanteau, followed, in a descending order, by the combinations Mavroud X Mavroud (autotransplantation), Mavroud X Berlandieri X Riparia Kobber SBB and Mavroud X Riparia 33 EM. In view to establish indices for predicting the transplantation affinity a great number of physiological-biochemical and morphological-anatomical studies were carried out. The results obtained showed that a most clearly expressed positive, statistically significant correlation exists between the amount of 15 N transported from the root stock to the scions, shoots and leaves. As a result, a biochemical method for fast affinity diagnosis in grape-vine transplantation has been developed. The reliability of the method has been checked up also with other cultivars. Up to the present no such method was known in grape-vine science and practice. (author)

  12. Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.

    Science.gov (United States)

    Locati, Mauro D; Pagano, Johanna F B; Ensink, Wim A; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Zhu, Kongju; Spaink, Herman P; Girard, Geneviève; Rauwerda, Han; Jonker, Martijs J; Dekker, Rob J; Breit, Timo M

    2017-04-01

    5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci. © 2017 Locati et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  13. Further evidence for a broader concept of somatization disorder using the somatic symptom index.

    Science.gov (United States)

    Hiller, W; Rief, W; Fichter, M M

    1995-01-01

    Somatization syndromes were defined in a sample of 102 psychosomatic inpatients according to the restrictive criteria of DSM-III-R somatization disorder and the broader diagnostic concept of the Somatic Symptom Index (SSI). Both groups showed a qualitatively similar pattern of psychopathological comorbidity and had elevated scores on measures of depression, hypochondriasis, and anxiety. A good discrimination between mild and severe forms of somatization was found by using the SSI criterion. SSI use accounted for a substantial amount of comorbidity variance, with rates of 15%-20% for depression, 16% for hypochondriasis, and 13% for anxiety. The results provide further evidence for the validity of the SSI concept, which reflects the clinical relevance of somatization in addition to the narrow definition of somatization disorder.

  14. Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos

    DEFF Research Database (Denmark)

    Alexopoulos, Natalie I.; Maddox-Hyttel, Poul; Tveden-Nyborg, Pernille Yde

    2008-01-01

    , immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were...

  15. Is somatic comorbidity associated with more somatic symptoms, mental distress, or unhealthy lifestyle in elderly cancer survivors?

    Science.gov (United States)

    Grov, Ellen Karine; Fosså, Sophie D; Dahl, Alv A

    2009-06-01

    The associations of lifestyle factors, somatic symptoms, mental distress, and somatic comorbidity in elderly cancer survivors have not been well studied. This study examines these associations among elderly cancer survivors (age >or=65 years) in a population-based sample. A cross-sectional comparative study of Norwegian elderly cancer survivors. Combining information from The Norwegian Cancer Registry, and by self-reporting, 972 elderly cancer survivors were identified, of whom 632 (65%) had somatic comorbidity and 340 did not. Elderly cancer survivors with somatic comorbidity had significantly higher BMI, more performed minimal physical activity, had more somatic symptoms, used more medication, and had more frequently seen a medical doctor than survivors without somatic comorbidity. In multivariable analyses, unhealthy lifestyle and higher somatic symptoms scores were significantly associated with cancer cases with somatic comorbidity. In univariate analyses those with somatic comorbidity were significantly older, had lower levels of education, higher proportions of BMI >or= 30, less physical activity, poorer self-rated health, higher somatic symptoms score, more mental distress, had more frequently seen a medical doctor last year, and more frequently used daily medication. Our outcome measures of lifestyle, somatic symptoms and mental distress were all significantly associated with somatic comorbidity in elderly cancer survivors, however only lifestyle and somatic symptoms were significant in multivariable analyses. In elderly cancer survivors not only cancer, but also somatic comorbidity, deserve attention. Such comorbidity is associated with unhealthy lifestyles, more somatic symptoms and mental distress which should be evaluated and eventually treated.

  16. Vegetative, productive and qualitative performance of grapevine "Cabernet Sauvignon" according to the use of winter cover crops

    OpenAIRE

    Bettoni, Jean Carlos; Feldberg, Nelson Pires; Nava, Gilberto; Veiga, Milton da; Wildner, Leandro do Prado

    2016-01-01

    ABSTRACT To study the effect of winter cover crops on the vegetative, productive and qualitative behavior of "Cabernet Sauvignon" grapevines, an experiment was conducted in two wine harvests by sowing different species of winter cover crops and additional treatments with manual weeding and mechanical mowing in an experimental vineyard located at the Experimental Station of Epagri in Videira, state of Santa Catarina, Brazil. Plant attributes of the grapevine, such as number of rods and weight ...

  17. Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

    Science.gov (United States)

    Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

    2009-09-01

    Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

  18. Gating in grapevine: Relationship between application of the fungicide fludioxonil and circadian rhythm on photosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Petit, Anne-Noelle [Laboratoire de Stress, Defenses et Reproduction des Plantes, URVVC-SE EA 2069, Universite de Reims Champagne-Ardenne, UFR Sciences Exactes et Naturelles, Batiment 18, Moulin de la Housse, BP 1039, F-51687 REIMS Cedex 2 (France)], E-mail: petit081@etudiant.univ-reims.fr; Fontaine, Florence [Laboratoire de Stress, Defenses et Reproduction des Plantes, URVVC-SE EA 2069, Universite de Reims Champagne-Ardenne, UFR Sciences Exactes et Naturelles, Batiment 18, Moulin de la Housse, BP 1039, F-51687 REIMS Cedex 2 (France)], E-mail: florence.fontaine@univ-reims.fr; Clement, Christophe [Laboratoire de Stress, Defenses et Reproduction des Plantes, URVVC-SE EA 2069, Universite de Reims Champagne-Ardenne, UFR Sciences Exactes et Naturelles, Batiment 18, Moulin de la Housse, BP 1039, F-51687 REIMS Cedex 2 (France)], E-mail: christophe.clement@univ-reims.fr; Vaillant-Gaveau, Nathalie [Laboratoire de Stress, Defenses et Reproduction des Plantes, URVVC-SE EA 2069, Universite de Reims Champagne-Ardenne, UFR Sciences Exactes et Naturelles, Batiment 18, Moulin de la Housse, BP 1039, F-51687 REIMS Cedex 2 (France)], E-mail: nathalie.vaillant-gaveau@univ-reims.fr

    2009-01-15

    The aim of this study was to determine the impact of the fludioxonil (fdx) fungicide on the diurnal fluctuation in grapevine photosynthesis. Therefore, fdx treatment was performed at the end of flowering, at 8 am, 12 am or 7 pm. The study was performed in experimental field and several photosynthesis parameters were followed one day after treatment. Morning fdx treatment induced (i) a significant and simultaneous drop of both photosynthesis (Pn) and stomatal conductance between 8 am and 4 pm and (ii) an increase of intercellular CO{sub 2} concentration when compared to control plants. On the contrary, evening fdx treatment did not affect Pn whereas midday treatment caused Pn increase after 4 pm. These data suggest that (i) morning fdx treatment results in a non-stomatal limitation of Pn, (ii) midday treatment is more suitable to treat grapevine with fdx and (iii) a phenomenon of gating was noticed. - The period of fdx spraying was an important parameter in stress response: the midday fdx treatment is more suitable to treat grapevine with fdx.

  19. Gating in grapevine: Relationship between application of the fungicide fludioxonil and circadian rhythm on photosynthesis

    International Nuclear Information System (INIS)

    Petit, Anne-Noelle; Fontaine, Florence; Clement, Christophe; Vaillant-Gaveau, Nathalie

    2009-01-01

    The aim of this study was to determine the impact of the fludioxonil (fdx) fungicide on the diurnal fluctuation in grapevine photosynthesis. Therefore, fdx treatment was performed at the end of flowering, at 8 am, 12 am or 7 pm. The study was performed in experimental field and several photosynthesis parameters were followed one day after treatment. Morning fdx treatment induced (i) a significant and simultaneous drop of both photosynthesis (Pn) and stomatal conductance between 8 am and 4 pm and (ii) an increase of intercellular CO 2 concentration when compared to control plants. On the contrary, evening fdx treatment did not affect Pn whereas midday treatment caused Pn increase after 4 pm. These data suggest that (i) morning fdx treatment results in a non-stomatal limitation of Pn, (ii) midday treatment is more suitable to treat grapevine with fdx and (iii) a phenomenon of gating was noticed. - The period of fdx spraying was an important parameter in stress response: the midday fdx treatment is more suitable to treat grapevine with fdx

  20. Poly(lactic-co-glycolic) acid nanoparticles uptake by Vitis vinifera and grapevine-pathogenic fungi

    International Nuclear Information System (INIS)

    Valletta, Alessio; Chronopoulou, Laura; Palocci, Cleofe; Baldan, Barbara; Donati, Livia; Pasqua, Gabriella

    2014-01-01

    Poly(lactic-co-glycolic) acid (PLGA)-based NPs are currently considered among the most promising drug carriers, nevertheless their use in plants has never been investigated. In this work, for the first time, we demonstrated the ability of PLGA NPs to cross the plant cell wall and membrane of Vitis vinifera cell cultures and grapevine-pathogenic fungi. By means of fluorescence microscopy, we established that PLGA NPs can enter in grapevine leaf tissues through stomata openings and that they can be absorbed by the roots and transported to the shoot through vascular tissues. TEM analysis on cultured cells showed that NPs ≤ 50 nm could enter cells, while bigger ones remained attached to the cell wall. Viability tests demonstrated that PLGA NPs were not cytotoxic for V. vinifera-cultured cells. The cellular uptake of PLGA NPs by some important grapevine-pathogenic fungi has also been observed, thus suggesting that PLGA NPs could be used to deliver antifungal compounds within fungal cells. Overall the results reported suggest that such NPs may play a key role in future developments of agrobiotechnologies, as it is currently happening in biomedicine

  1. Poly(lactic-co-glycolic) acid nanoparticles uptake by Vitis vinifera and grapevine-pathogenic fungi

    Energy Technology Data Exchange (ETDEWEB)

    Valletta, Alessio [“Sapienza” University of Rome, Department of Environmental Biology (Italy); Chronopoulou, Laura; Palocci, Cleofe, E-mail: cleofe.palocci@uniroma1.it [“Sapienza” University of Rome, Department of Chemistry (Italy); Baldan, Barbara [University of Padua, Department of Biology (Italy); Donati, Livia; Pasqua, Gabriella [“Sapienza” University of Rome, Department of Environmental Biology (Italy)

    2014-12-15

    Poly(lactic-co-glycolic) acid (PLGA)-based NPs are currently considered among the most promising drug carriers, nevertheless their use in plants has never been investigated. In this work, for the first time, we demonstrated the ability of PLGA NPs to cross the plant cell wall and membrane of Vitis vinifera cell cultures and grapevine-pathogenic fungi. By means of fluorescence microscopy, we established that PLGA NPs can enter in grapevine leaf tissues through stomata openings and that they can be absorbed by the roots and transported to the shoot through vascular tissues. TEM analysis on cultured cells showed that NPs ≤ 50 nm could enter cells, while bigger ones remained attached to the cell wall. Viability tests demonstrated that PLGA NPs were not cytotoxic for V. vinifera-cultured cells. The cellular uptake of PLGA NPs by some important grapevine-pathogenic fungi has also been observed, thus suggesting that PLGA NPs could be used to deliver antifungal compounds within fungal cells. Overall the results reported suggest that such NPs may play a key role in future developments of agrobiotechnologies, as it is currently happening in biomedicine.

  2. The Role of Peroxisome Proliferator-Activated Receptors in the Development and Physiology of Gametes and Preimplantation Embryos

    Directory of Open Access Journals (Sweden)

    Jaou-Chen Huang

    2008-01-01

    Full Text Available In several species, a family of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs composed of three isotypes, is expressed in somatic cells and germ cells of the ovary as well as the testis. Invalidation of these receptors in mice or stimulation of these receptors in vivo or in vitro showed that each receptor has physiological roles in the gamete maturation or the embryo development. In addition, synthetic PPARγ ligands are recently used to induce ovulation in women with polycystic ovary disease. These results reveal the positive actions of PPAR in reproduction. On the other hand, xenobiotics molecules (in herbicides, plasticizers, or components of personal care products, capable of activating PPAR, may disrupt normal PPAR functions in the ovary or the testis and have consequences on the quality of the gametes and the embryos. Despite the recent data obtained on the biological actions of PPARs in reproduction, relatively little is known about PPARs in gametes and embryos. This review summarizes the current knowledge on the expression and the function of PPARs as well as their partners, retinoid X receptors (RXRs, in germ cells and preimplantation embryos. The effects of natural and synthetic PPAR ligands will also be discussed from the perspectives of reproductive toxicology and assisted reproductive technology.

  3. Bioavailability of potentially toxic elements in soil-grapevine (leaf, skin, pulp and seed) system and environmental and health risk assessment.

    Science.gov (United States)

    Milićević, Tijana; Urošević, Mira Aničić; Relić, Dubravka; Vuković, Gordana; Škrivanj, Sandra; Popović, Aleksandar

    2018-06-01

    Monitoring of potentially toxic elements in agricultural soil represents the first measure of caution regarding food safety, while research into element bioavailability should be a step forward in understanding the element transportation chain. This study was conducted in the grapevine growing area ("Oplenac Wine Route") for investigating element bioavailability in the soil-grapevine system accompanied by an assessment of the ecological implications and human health risk. Single extraction procedures (CH 3 COOH, Na 2 EDTA, CaCl 2 , NH 4 NO 3 and deionised H 2 O) and digestion were performed to estimate the bioavailability of 22 elements (Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Sb, Sr, V and Zn) from the topsoil (0-30 cm) and subsoil (30-60 cm) to the grapevine parts (leaf, skin, pulp and seed) and wine. The extractants were effective comparing to the pseudo-total concentrations in following order Na 2 EDTA ˃ CH 3 COOH ˃ NH 4 NO 3  ˃ CaCl 2 , H 2 O 2 h and 16 h. The most suitable extractants for assessing the bioavailability of the elements from the soil to the grapevine parts were CaCl 2 , NH 4 NO 3 and Na 2 EDTA, but deionised H 2 O could be suitable, as well. The results showed that Ba was the most bioavailable element in the soil-grapevine system. Contamination factor implied a moderate contamination (1  1), the influence of atmospheric deposition on the aerial grapevine parts (leaves and grape skin) was observed. Nevertheless, low adverse health risk effects (HI wine consumers. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Development of degenerate and species-specific primers for the differential and simultaneous RT-PCR detection of grapevine-infecting nepoviruses of subgroups A, B and C.

    Science.gov (United States)

    Digiaro, Michele; Elbeaino, Toufic; Martelli, Giovanni Paolo

    2007-04-01

    Based on the nucleotide sequence homology of RNA-1 and RNA-2 of nepoviruses isolated from grapevines, three sets of degenerate primers, one for each of the three subgroups of the genus (A, B and C), were designed and proved effective for RT-PCR detection of subgroups in infected grapevines and herbaceous hosts. Primers designed specifically for detecting subgroup A species amplified a fragment of 255 bp from samples infected by Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV) and Grapevine deformation virus (GDefV), but not from samples infected by other nepovirus species. Similarly, primers for detection of subgroup B nepoviruses amplified a 390 bp product from samples infected by Grapevine chrome mosaic virus (GCMV), Tomato black ring virus (TBRV), Grapevine Anatolian ringspot virus (GARSV) and Artichoke Italian latent virus (AILV). The third set of primers amplified a 640 bp fragment, only from samples infected by subgroup C nepoviruses, i.e Tomato ringspot virus (ToRSV) Grapevine Bulgarian latent virus (GBLV), and Grapevine Tunisian ringspot virus (GTRSV). These primers were able to detect simultaneously all viral species belonging to the same subgroup and to discriminate species of different subgroups. Multiplex-PCR detection of subgroup A and B nepoviruses was obtained using a specific primer (sense for subgroup A and antisense for subgroup B) for each of the species of the same subgroup in combination with the degenerate subgroup-specific primers. In this way it was possible to detect four different viral species in single samples containing mixtures of viruses of the same subgroup. In particular, for viruses of subgroup A (TRSV, GFLV, ArMV and GDefV) amplicons of 190, 259, 301 and 371 bp were obtained, whereas amplicons of 190, 278, 425 and 485 bp, respectively, were obtained from samples infected with viruses of subgroup B (GCMV, AILV, GARSV and TBRV).

  5. Hope for restoration of dead valuable bulls through cloning using donor somatic cells isolated from cryopreserved semen.

    Directory of Open Access Journals (Sweden)

    Naresh L Selokar

    Full Text Available Somatic cells were isolated from cryopreserved semen of 4 buffalo bulls, 3 of which had died over 10 years earlier, and were established in culture. The cells expressed cytokeratin-18, keratin and vimentin indicating that they were of epithelial origin. The cells were used as nuclear donors for hand-made cloning for producing buffalo embryos. The blastocyst rate and quality, as indicated by apoptotic index, were comparable among embryos produced using cells obtained from fresh or frozen-thawed semen or those obtained from conventional cell sources such as skin. Examination of the epigenetic status revealed that the global level of H3K27me3 but not that of H3K9/14ac and H4K5ac differed significantly (P<0.05 among cloned embryos from different bulls. The relative mRNA abundance of HDAC1, DNMT1, P53 and CASPASE 3 but not that of DNMT3a differed in cells and in cloned embryos. Following transfer of 24 cloned embryos produced from fresh semen-derived cells to 12 recipients, one calf weighing 55 kg, which is now 6 months of age and is normal, was born through normal parturition. Following transfer of 20 embryos produced from frozen-thawed semen-derived cells to 10 recipients, 2 became pregnant, one of which aborted in the first trimester; the calf born was severely underweight (17 kg, and died 12 h after birth. The ability of cells derived from fresh and frozen-thawed semen to produce live offspring confirms the ability of these cells to be reprogrammed. Our findings pave the way for restoration of highly precious progeny-tested bulls, which has immense economic importance, and can also be used for restoration of endangered species.

  6. Hope for restoration of dead valuable bulls through cloning using donor somatic cells isolated from cryopreserved semen.

    Science.gov (United States)

    Selokar, Naresh L; Saini, Monika; Palta, Prabhat; Chauhan, Manmohan S; Manik, Radheysham; Singla, Suresh K

    2014-01-01

    Somatic cells were isolated from cryopreserved semen of 4 buffalo bulls, 3 of which had died over 10 years earlier, and were established in culture. The cells expressed cytokeratin-18, keratin and vimentin indicating that they were of epithelial origin. The cells were used as nuclear donors for hand-made cloning for producing buffalo embryos. The blastocyst rate and quality, as indicated by apoptotic index, were comparable among embryos produced using cells obtained from fresh or frozen-thawed semen or those obtained from conventional cell sources such as skin. Examination of the epigenetic status revealed that the global level of H3K27me3 but not that of H3K9/14ac and H4K5ac differed significantly (Pcloned embryos from different bulls. The relative mRNA abundance of HDAC1, DNMT1, P53 and CASPASE 3 but not that of DNMT3a differed in cells and in cloned embryos. Following transfer of 24 cloned embryos produced from fresh semen-derived cells to 12 recipients, one calf weighing 55 kg, which is now 6 months of age and is normal, was born through normal parturition. Following transfer of 20 embryos produced from frozen-thawed semen-derived cells to 10 recipients, 2 became pregnant, one of which aborted in the first trimester; the calf born was severely underweight (17 kg), and died 12 h after birth. The ability of cells derived from fresh and frozen-thawed semen to produce live offspring confirms the ability of these cells to be reprogrammed. Our findings pave the way for restoration of highly precious progeny-tested bulls, which has immense economic importance, and can also be used for restoration of endangered species.

  7. Hope for Restoration of Dead Valuable Bulls through Cloning Using Donor Somatic Cells Isolated from Cryopreserved Semen

    Science.gov (United States)

    Selokar, Naresh L.; Saini, Monika; Palta, Prabhat; Chauhan, Manmohan S.; Manik, Radheysham; Singla, Suresh K.

    2014-01-01

    Somatic cells were isolated from cryopreserved semen of 4 buffalo bulls, 3 of which had died over 10 years earlier, and were established in culture. The cells expressed cytokeratin-18, keratin and vimentin indicating that they were of epithelial origin. The cells were used as nuclear donors for hand-made cloning for producing buffalo embryos. The blastocyst rate and quality, as indicated by apoptotic index, were comparable among embryos produced using cells obtained from fresh or frozen-thawed semen or those obtained from conventional cell sources such as skin. Examination of the epigenetic status revealed that the global level of H3K27me3 but not that of H3K9/14ac and H4K5ac differed significantly (Pcloned embryos from different bulls. The relative mRNA abundance of HDAC1, DNMT1, P53 and CASPASE 3 but not that of DNMT3a differed in cells and in cloned embryos. Following transfer of 24 cloned embryos produced from fresh semen-derived cells to 12 recipients, one calf weighing 55 kg, which is now 6 months of age and is normal, was born through normal parturition. Following transfer of 20 embryos produced from frozen-thawed semen-derived cells to 10 recipients, 2 became pregnant, one of which aborted in the first trimester; the calf born was severely underweight (17 kg), and died 12 h after birth. The ability of cells derived from fresh and frozen-thawed semen to produce live offspring confirms the ability of these cells to be reprogrammed. Our findings pave the way for restoration of highly precious progeny-tested bulls, which has immense economic importance, and can also be used for restoration of endangered species. PMID:24614586

  8. ADVANCES IN PROPAGATION OF GRAPEVINE IN THE WORLD

    OpenAIRE

    GROHS, DANIEL SANTOS; ALMANÇA, MARCUS ANDRÉ KURTZ; FAJARDO, THOR VINICIUS MARTINS; HALLEEN, FRANCOIS; MIELE, ALBERTO

    2017-01-01

    ABSTRACT Grapevine production by classical grafting methods and in commercial scale emerged over 130 years. This system remained handmade until the mid-1950s, when the first international certification programs aimed at obtaining mother plants with high viral sanity emerged. The necessity to increase the scale of production on industrial model and plant material production based on minimum morphological standards appeared at the end of the 1960s. Along the 1970s, research unlocked knowledge ...

  9. Shoot growth of Merlot and Cabernet Sauvignon grapevine varieties

    Directory of Open Access Journals (Sweden)

    Marcelo Borghezan

    2012-02-01

    Full Text Available The objective of this work was to evaluate shoot growth of the grapevine varieties Merlot and Cabernet Sauvignon, during 2006/2007, and Cabernet Sauvignon, during 2008/2009, in São Joaquim, SC, Brazil. The experiment was carried out in a commercial vineyard trained on a vertical trellis system. The shoots of the central part of the plants were selected, and the lengths from the base to the apex of 20 shoots per cultivar were evaluated. In 2006/2007, monitoring began at pruning, on 9/15/2006, and ended on 2/6/2007, totalizing 144 days of evaluation. During the 2008/2009 cycle, phenology and shoot growth for 'Cabernet Sauvignon' were assessed from grape development (1/13/2009 (pea-sized grapes until shoot vegetative growth had ceased. Budburst occurred in the second half of September, and shoot-growth cessation occurred during ripening. Higher growth rates (about 4 cm per day were observed in pre- and post-flowering, followed by reduction due to the competition for photosynthates for the formation of flowers and bunches. Temperature and photoperiod induce grapevine shoots to cease growth in the highland regions of Santa Catarina State, Brazil.

  10. Biotechnology of temperate fruit trees and grapevines.

    Science.gov (United States)

    Laimer, Margit; Mendonça, Duarte; Maghuly, Fatemeh; Marzban, Gorji; Leopold, Stephan; Khan, Mahmood; Balla, Ildiko; Katinger, Hermann

    2005-01-01

    Challenges concerning fruit trees and grapevines as long lived woody perennial crops require adapted biotechnological approaches, if solutions are to be found within a reasonable time frame. These challenges are represented by the need for correct identification of genetic resources, with the foreseen use either in conservation or in breeding programmes. Molecular markers provide most accurate information and will be the major solution for questions about plant breeders rights. Providing healthy planting material and rapid detection of newly introduced pathogens by reliable methods involving serological and molecular biological tools will be a future challenge of increases importance, given the fact that plant material travels freely in the entire European Union. But also new breeding goals and transgenic solutions are part of the biotechnological benefits, e.g. resistance against biotic and abiotic stress factors, modified growth habits, modified nutritional properties and altered processing and storage qualities. The successful characterization of transgenic grapevines and stone fruit trees carrying genes of viral origin in different vectors constructed under ecological consideration, will be presented. Beyond technical feasibility, efficiency of resistance, environmental safety and Intellectual Property Rights, also public acceptance needs consideration and has been addressed in a specific project. The molecular determination of internal quality parameters of food can also be addressed by the use of biotechnological tools. Patient independent detection tools for apple allergens have been developed and should allow to compare fruits from different production systems, sites, and genotypes for their content of health threatening compounds.

  11. Endophytic bacterial community of grapevine leaves influenced by sampling date and phytoplasma infection process.

    Science.gov (United States)

    Bulgari, Daniela; Casati, Paola; Quaglino, Fabio; Bianco, Piero A

    2014-07-21

    Endophytic bacteria benefit host plant directly or indirectly, e.g. by biocontrol of the pathogens. Up to now, their interactions with the host and with other microorganisms are poorly understood. Consequently, a crucial step for improving the knowledge of those relationships is to determine if pathogens or plant growing season influence endophytic bacterial diversity and dynamic. Four healthy, four phytoplasma diseased and four recovered (symptomatic plants that spontaneously regain a healthy condition) grapevine plants were sampled monthly from June to October 2010 in a vineyard in north-western Italy. Metagenomic DNA was extracted from sterilized leaves and the endophytic bacterial community dynamic and diversity were analyzed by taxon specific real-time PCR, Length-Heterogeneity PCR and genus-specific PCR. These analyses revealed that both sampling date and phytoplasma infection influenced the endophytic bacterial composition. Interestingly, in June, when the plants are symptomless and the pathogen is undetectable (i) the endophytic bacterial community associated with diseased grapevines was different from those in the other sampling dates, when the phytoplasmas are detectable inside samples; (ii) the microbial community associated with recovered plants differs from that living inside healthy and diseased plants. Interestingly, LH-PCR database identified bacteria previously reported as biocontrol agents in the examined grapevines. Of these, Burkholderia, Methylobacterium and Pantoea dynamic was influenced by the phytoplasma infection process and seasonality. Results indicated that endophytic bacterial community composition in grapevine is correlated to both phytoplasma infection and sampling date. For the first time, data underlined that, in diseased plants, the pathogen infection process can decrease the impact of seasonality on community dynamic. Moreover, based on experimental evidences, it was reasonable to hypothesize that after recovery the restructured

  12. Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Piyatrakul Piyanuch

    2012-12-01

    Full Text Available Abstract Background Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. Results The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. Conclusions This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult.

  13. In vitro manipulation techniques of porcine embryos: a meta-analysis related to transfers, pregnancies and piglets.

    Science.gov (United States)

    Liu, Ying; Li, Juan; Løvendahl, Peter; Schmidt, Mette; Larsen, Knud; Callesen, Henrik

    2015-03-01

    During the last 17 years, considerable advancements have been achieved in the production of pigs, transgenic and non-transgenic, by methods of somatic cell nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection, microinjection and sperm-mediated gene transfer by artificial insemination. Therefore, a review of the overall efficiency for the developmental competence of embryos produced by these in vitro methods would be useful in order to obtain a more thorough overview of this growing area with respect to its development and present status. In this review a meta-analysis was used to analyse data collected from all published articles with a focus on zygotes and embryos for transfer, pregnancy, full-term development and piglets born. It was generally concluded that an increasing level of in vitro manipulation of porcine embryos decreased the overall efficiency for production of piglets. The techniques of nuclear transfer have been developed markedly through the increasing number of studies performed, and the results have become more stable. Prolonged in vitro culture period did not lead to any negative effect on nuclear transfer embryos after their transfer and it resulted in a similar or even higher litter size. More complete information is needed in future scientific articles about these in vitro manipulation techniques to establish a more solid basis for the evaluation of their status and to reveal and further investigate any eventual problems.

  14. Genetic structure of the fungal grapevine pathogen Eutypa lata from four continents

    Science.gov (United States)

    The generalist ascomycete fungus Eutypa lata causes Eutypa dieback of grapevine (Vitis vinifera) worldwide. To decipher the cosmopolitan distribution of this fungus, the population genetic structure of 17 geographic samples was investigated from four continental regions (Australia, California, Europ...

  15. Novel embryo selection techniques to increase embryo implantation in IVF attempts.

    Science.gov (United States)

    Sigalos, George Α; Triantafyllidou, Olga; Vlahos, Nikos F

    2016-11-01

    The final success of an IVF attempt depends on several steps and decisions taken during the ovarian stimulation, the oocyte retrieval, the embryo culture and the embryo transfer. The final selection of the embryos most likely to implant is the final step in this process and the responsibility of the lab. Apart from strict morphologic criteria that historically have been used in embryo selection, additional information on genetic, metabolomic and morphokinetic characteristics of the embryo is recently combined to morphology to select the embryo most likely to produce a pregnancy. In this manuscript, we review the most recent information on the current methods used for embryo selection presenting the predictive capability of each one. A literature search was performed on Pubmed, Medline and Cochrane Database of Systematic Reviews for published studies using appropriate key words and phrases with no limits placed on time. It seems that the combination of morphologic criteria in conjunction to embryo kinetics as documented by time-lapse technology provides the most reliable information on embryo quality. Blastocyst biopsy with subsequent comprehensive chromosome analysis allows the selection of the euploid embryos with the higher implantation potential. Embryo time-lapse imaging and blastocyst biopsy combined to comprehensive chromosome analysis are the most promising technologies to increase pregnancy rates and reduce the possibility of multiple pregnancies. However, further studies will demonstrate the capability of routinely using these technologies to significantly improve IVF outcomes.

  16. Root-associated bacteria promote grapevine growth: from the laboratory to the field

    KAUST Repository

    Rolli, Eleonora; Marasco, Ramona; Saderi, Stefano; Corretto, Erika; Mapelli, Francesca; Cherif, Ameur; Borin, Sara; Valenti, Leonardo; Sorlini, Claudia; Daffonchio, Daniele

    2016-01-01

    of different geographical origins derived from different crop plants can colonize grapevine to gain a beneficial outcome for the plant leading to promote growth at the field scale. Methods: To link the ecological functions of bacteria to the promotion of plant

  17. Association of RGA-SSCP markers with resistance to downy mildew and anthracnose in grapevines.

    Science.gov (United States)

    Tantasawat, P A; Poolsawat, O; Prajongjai, T; Chaowiset, W; Tharapreuksapong, A

    2012-07-02

    Downy mildew (Plasmopara viticola) and anthracnose (Sphaceloma ampelinum) are two major diseases that severely affect most grapevine (Vitis vinifera) cultivars grown commercially in Thailand. Progress of conventional breeding programs of grapevine for improved resistance to these diseases can be speeded up by selection of molecular markers associated with resistance traits. We evaluated the association between 13 resistance gene analog (RGA)-single-strand conformation polymorphism (SSCP) markers with resistance to downy mildew and anthracnose in 71 segregating progenies of seven cross combinations between susceptible cultivars and resistant lines. F(1) hybrids from each cross were assessed for resistance to downy mildew and anthracnose (isolates Nk4-1 and Rc2-1) under laboratory conditions. Association of resistance traits with RGA-SSCP markers was evaluated using simple linear regression analysis. Three RGA-SSCP markers were found to be significantly correlated with anthracnose resistance, whereas significant correlation with downy mildew resistance was observed for only one RGA-SSCP marker. These results demonstrate the usefulness of RGA-SSCP markers. Four candidate markers with significant associations to resistance to these two major diseases of grapevine were identified. However, these putative associations between markers and resistance need to be verified with larger segregating populations before they can be used for marker-assisted selection.

  18. Deep amplicon sequencing reveals mixed phytoplasma infection within single grapevine plants

    DEFF Research Database (Denmark)

    Nicolaisen, Mogens; Contaldo, Nicoletta; Makarova, Olga

    2011-01-01

    The diversity of phytoplasmas within single plants has not yet been fully investigated. In this project, deep amplicon sequencing was used to generate 50,926 phytoplasma sequences from 11 phytoplasma-infected grapevine samples from a PCR amplicon in the 5' end of the 16S region. After clustering ...

  19. A DNA based method to detect the grapevine root-rotting fungus Roesleria subterranea in soil and root samples

    Directory of Open Access Journals (Sweden)

    S. Neuhauser

    2009-05-01

    Full Text Available Roesleria subterranea causes root rot in grapevine and fruit trees. The fungus has long been underestimated as a weak parasite, but during the last years it has been reported to cause severe damages in German vineyards. Direct, observation-based detection of the parasite is time consuming and destructive, as large parts of the rootstocks have to be uprooted and screened for the tiny, stipitate, hypogeous ascomata of R. subterranea. To facilitate rapid detection in vineyards, protocols to extract DNA from soil samples and grapevine roots, and R.-subterranea-specific PCR primers were designed. Twelve DNA-extraction protocols for soil samples were tested in small-scale experiments, and selected parameters were optimised. A protocol based on ball-mill homogenization, DNA extraction with SDS, skim milk, chloroform, and isopropanol, and subsequent purifi cation of the raw extracts with PVPP-spin-columns was most effective. This DNA extraction protocol was found to be suitable for a wide range of soil-types including clay, loam and humic-rich soils. For DNA extraction from grapevine roots a CTAB-based protocol was more reliable for various grapevine rootstock varieties. Roesleria-subterranea-specific primers for the ITS1-5.8S-ITS2 rDNA region were developed and tested for their specifi city to DNA extracts from eleven R. subterranea strains isolated from grapevine and fruit trees. No cross reactions were detected with DNA extracts from 44 different species of fungi isolated from vineyard soils. The sensitivity of the species-specifi c primers in combination with the DNA extraction method for soil was high: as little as 100 fg μl-1 R.-subterranea-DNA was suffi cient for a detection in soil samples and plant material. Given that specifi c primers are available, the presented method will also allow quick and large-scale testing for other root pathogens.

  20. Identification and utilization of a new Erysiphe necator isolate NAFU1 to quickly evaluate powdery mildew resistance in wild Chinese grapevine species using detached leaves.

    Science.gov (United States)

    Gao, Yu-Rong; Han, Yong-Tao; Zhao, Feng-Li; Li, Ya-Juan; Cheng, Yuan; Ding, Qin; Wang, Yue-Jin; Wen, Ying-Qiang

    2016-01-01

    The most economically important disease of cultivated grapevines worldwide is powdery mildew caused by the biotrophic fungal pathogen Erysiphe necator. To integrate effective genetic resistance into cultivated grapevines, numerous disease resistance screens of diverse Vitis germplasm, including wild species, have been conducted to identify powdery mildew resistance, but the results have been inconsistent. Here, a new powdery mildew isolate that is infectious on grapevines, designated Erysiphe necator NAFU1 (En. NAFU1), was identified and characterized by phylogeny inferred from the internal transcribed spacer (ITS) of pathogen ribosomal DNA sequences. Three classical methods were compared for the maintenance of En. NAFU1, and the most convenient method was maintenance on detached leaves and propagation by contact with infected leaves. Furthermore, controlled inoculations of En. NAFU1 were performed using detached leaves from 57 wild Chinese grapevine accessions to quickly evaluate powdery mildew resistance based on trypan blue staining of leaf sections. The results were compared with previous natural epidemics in the field. Among the screened accessions inoculated with En. NAFU1, 22.8% were resistant, 33.3% were moderately resistant, and 43.9% were susceptible. None of the accessions assessed herein were immune from infection. These results support previous findings documenting the presence of race-specific resistance to E. necator in wild Chinese grapevine. The resistance of wild Chinese grapevine to En. NAFU1 could be due to programmed cell death. The present results suggest that En. NAFU1 isolate could be used for future large-scale screens of resistance to powdery mildew in diverse Vitis germplasms and investigations of the interaction between grapevines and pathogens. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  1. Hypochondriasis and somatization.

    Science.gov (United States)

    Kellner, R

    1987-11-20

    Between 60% and 80% of healthy individuals experience somatic symptoms in any one week. About 10% to 20% of a random sample of people worry intermittently about illness. A substantial proportion of patients present physicians with somatic complaints for which no organic cause can be found. Patients who are hypochondriacal do not understand the benign nature of functional somatic symptoms and interpret these as evidence of disease. Hypochondriacal concerns range from common short-lived worries to persistent and distressing fears or convictions of having a disease. Hypochondriasis can be secondary to other psychiatric disorders (eg, melancholia or panic disorder), and hypochondriacal attitudes remit when the primary disorder is successfully treated. Patients with primary hypochondriasis are also anxious or depressed, but the fear of disease, or the false belief of having a disease, persists and is the most important feature of their psychopathology. There are substantial differences among hypochondriacal patients in their personalities and psychopathologies. Psychotherapy as well as psychotropic drugs are effective in the treatment of functional somatic symptoms. There are no adequate controlled studies on the value of psychotherapy in hypochondriasis; the recommended guidelines are based on uncontrolled studies of hypochondriasis and on controlled studies of the psychotherapy in similar disorders. The prognosis of functional somatic symptoms as well as that of hypochondriasis is good in a substantial proportion of patients.

  2. How Streptomyces anulatus Primes Grapevine Defenses to Cope with Gray Mold: A Study of the Early Responses of Cell Suspensions

    Directory of Open Access Journals (Sweden)

    Parul Vatsa-Portugal

    2017-06-01

    Full Text Available Gray mold, caused by Botrytis cinerea, is one of the most destructive diseases of grapevine and is controlled with an intense application of fungicides. As alternatives to chemicals, beneficial microbes may promote plant health by stimulating the plant’s immune system. An actinomycete, Streptomyces anulatus S37, has been screened from the rhizosphere microbiome of healthy Vitis vinifera on the basis of its ability to promote grapevine growth and to induce resistance against various phytopathogens, including B. cinerea. However, molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain unknown. This study focuses on local defense events induced in grapevine cells during interactions with S. anulatus S37 before and after pathogen challenge. We demonstrated that S. anulatus S37 induced early responses including oxidative burst, extracellular alkalinization, activation of protein kinases, induction of defense gene expression and phytoalexin accumulation, but not the programmed cell death. Interestingly, upon challenge with the B. cinerea, the S. anulatus S37 primed grapevine cells for enhanced defense reactions with a decline in cell death. In the presence of the EGTA, a calcium channel inhibitor, the induced oxidative burst, and the protein kinase activity were inhibited, but not the extracellular alkalinization, suggesting that Ca2+ may also contribute upstream to the induced defenses. Moreover, desensitization assays using extracellular pH showed that once increased by S. anulatus S37, cells became refractory to further stimulation by B. cinerea, suggesting that grapevine cells perceive distinctly beneficial and pathogenic microbes.

  3. Comparison Effects of Sucrose and Date Palm Syrup on Somatic Embryogenesis of Date Palm (Phoenix dactylifera L )

    OpenAIRE

    Abdullatif Alkhateeb

    2008-01-01

    The effect of different concentration of date palm syrup (2, 4, 6, 8 and 10%) and sucrose at concentration of 30 and 60 g/l in addition to the control (without carbon source) on the micro propagation of date palm "cv. Suckary" were investigated. The results indicated that date syrup was taken up from the media as shown by the increase in total dry weight of culture. Addition of sucrose at 60 g/l produced the highest number of somatic embryos and longest shoot equal to that produced by date sy...

  4. Cell lineage patterns in the shoot meristem of the sunflower embryo in the dry seed

    International Nuclear Information System (INIS)

    Jegla, D.E.; Sussex, I.M.

    1989-01-01

    We mapped the fate of cells in the shoot meristem of the dry-seed embryo of sunflower, Helianthus annuus L. cv. Peredovic, using irradiation-induced somatic sectors. We analyzed 249 chlorophyll-deficient or glabrous (hairless) sectors generated in 236 plants. Most sectors observed in the inflorescence extended into vegetative nodes. Thus cell lineages that ultimately gave rise to reproductive structures also contributed to vegetative structures. No single sector extended the entire length of the shoot. Thus the shoot is not derived from one or a few apical initials. Rather, the position, vertical extent, and width of the sectors at different levels of the shoot suggest that the shoot is derived from three to four circumferential populations of cells in each of three cell layers of the embryo meristem. Sectors had no common boundaries even in plants with two or three independent sectors, but varied in extent and overlapped along the length of the shoot. Thus individual cells in a single circumferential population behaved independently to contribute lineages of different vertical extents to the growing shoot. The predicted number of circumferential populations of cells as well as the apparent cell number in each population was consistent with the actual number of cells in the embryo meristem observed in histological sections

  5. VULNERABILITY TO CAVITATION IN GRAPEVINES HAS BEEN OVERESTIMATED BY THE CENTRIFUGE TECHNIQUE

    Science.gov (United States)

    Grapevines are considered among the most vulnerable woody plant species to water stress-induced cavitation with embolism forming at slight tensions. However, we found that native embolism in stems of field grown Vitis vinifera cv. Chardonnay never exceeded 30% despite xylem water potentials ('x) rea...

  6. Long-term effect on in vitro cloning efficiency after treatment of somatic cells with Xenopus egg extract in the pig.

    Science.gov (United States)

    Liu, Ying; Ostrup, Olga; Li, Rong; Li, Juan; Vajta, Gábor; Kragh, Peter M; Schmidt, Mette; Purup, Stig; Hyttel, Poul; Klærke, Dan; Callesen, Henrik

    2014-08-01

    In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7-22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.

  7. Electronic nose as an innovative tool for the diagnosis of grapevine crown gall

    Energy Technology Data Exchange (ETDEWEB)

    Blasioli, S., E-mail: sonia.blasioli@unibo.it [Dipartimento di Scienze e Tecnologie Agroambientali, Universita di Bologna, V.le Fanin, 44, 40127 Bologna (Italy); Biondi, E., E-mail: erbiondi@tin.it [Dipartimento di Scienze e Tecnologie Agroambientali, Universita di Bologna, V.le Fanin, 44, 40127 Bologna (Italy); Braschi, I., E-mail: ilaria.braschi@unibo.it [Dipartimento di Scienze e Tecnologie Agroambientali, Universita di Bologna, V.le Fanin, 44, 40127 Bologna (Italy); Mazzucchi, U., E-mail: umberto.mazzucchi@unibo.it [Dipartimento di Scienze e Tecnologie Agroambientali, Universita di Bologna, V.le Fanin, 44, 40127 Bologna (Italy); Bazzi, C., E-mail: carlo.bazzi@unibo.it [Dipartimento di Scienze e Tecnologie Agroambientali, Universita di Bologna, V.le Fanin, 44, 40127 Bologna (Italy); Gessa, C.E., E-mail: carloemanuele.gessa@unibo.it [Dipartimento di Scienze e Tecnologie Agroambientali, Universita di Bologna, V.le Fanin, 44, 40127 Bologna (Italy)

    2010-07-05

    For the first time, a portable electronic nose was used to discriminate between healthy and galled grapevines, experimentally inoculated with two tumourigenic strains of Agrobacterium vitis. The volatile profile of target cutting samples was analysed by headspace solid phase microextraction coupled with gas chromatography-mass spectrometry. Spectra from tumoured samples revealed the presence of styrene which is compatible with decarboxylation of cinnamic acid involved in secondary metabolism of plants. Principal Component Analysis confirmed the difference in volatile profiles of infected vines and their healthy controls. Linear Discriminant Analysis allowed the correct discrimination between healthy and galled grapevines (83.3%, cross-validation). Although a larger number of samples should be analysed to create a more robust model, our results give novel interesting clues to go further with research on the diagnostic potential of this innovative system associated with multi-dimensional chemometric techniques.

  8. The nucleotide sequence of satellite RNA in grapevine fanleaf virus, strain F13.

    Science.gov (United States)

    Fuchs, M; Pinck, M; Serghini, M A; Ravelonandro, M; Walter, B; Pinck, L

    1989-04-01

    The nucleotide sequence of cDNA copies of grapevine fanleaf virus (strain F13) satellite RNA has been determined. The primary structure obtained was 1114 nucleotides in length, excluding the poly(A) tail, and contained only one long open reading frame encoding a 341 residue, highly hydrophilic polypeptide of Mr37275. The coding sequence was bordered by a leader of 14 nucleotides and a 3'-terminal non-coding region of 74 nucleotides. No homology has been found with small satellite RNAs associated with other nepoviruses. Two limited homologies of eight nucleotides have been detected between the satellite RNA in grapevine fanleaf virus and those in tomato black ring virus, and a consensus sequence U.G/UGAAAAU/AU/AU/A at the 5' end of nepovirus RNAs is reported. A less extended consensus exists in this region in comovirus and picornavirus RNA.

  9. [Somatization disorders of the urogenital tract].

    Science.gov (United States)

    Günthert, E A

    2002-11-01

    Diffuse symptoms in the urogenital region can frequently be explained by somatization disorders. Since they cannot be proven either by laboratory tests or with common technical diagnostic methods, somatization disorders should always be taken into consideration. Somatization disorders are to be considered functional disorders. Since somatization disorders due to muscular tension prevail in the urogenital region, the functional disturbance can be explained by the muscular tension. Subsequently, muscular tension causes the pathophysiological development of symptoms. As a rule they appear as myofascial pain or disorder. Muscular tension can have a psychic origin. The absence of urological findings is typical. Males and females between the ages of 16 and 75 can be affected by somatization disorders in the urogenital region. Somatization disorders due to muscular tension belong to the large group of symptoms due to tension. Diagnostic and therapeutic procedures as well as the pathophysiology of somatization disorders due to muscular tension are illustrated by two detailed case-reports.

  10. Lodgepole pine: the first evidence of seed-based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees.

    Science.gov (United States)

    Park, So-Young; Klimaszewska, Krystyna; Park, Ji-Young; Mansfield, Shawn D

    2010-11-01

    Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus.

  11. Climate vs grapevine pests and diseases worldwide: the first results of a global survey

    Directory of Open Access Journals (Sweden)

    Benjamin Bois

    2017-05-01

    Methods and results: Based on the answer of respondent about the main reported diseases/pests in their region, a severity index was calculated. Each region was geolocalised and data were compared to the WorldClim gridded climate database to document the range of climate conditions (growing season temperature and rainfall associated to the main diseases/pests. The potential climatic-induced changes of grapevine disease and pest geography by 2050 are assessed using agro-climate projections from the ARPEGE CNRM model, using the RCP 4.5 scenario. The preliminary results allow to determine the distribution of diseases as function of agroclimatic indicators. Conclusion: While the distribution of diseases differs according to the region of the world, the current analysis suggests that mildews remain the major phytosanitary threat in most of the regions. Powdery mildew, trunk diseases and viruses were reported in extremely diverse climatic conditions, including intermediate and wet regions.  Significance and impact of the study: This paper present an original methodology to address the relationship between grapevine disease and pest occurrences and climate. Such documentation is scarce in the current literature. Further analysis is currently being performed, including additional survey answers, climate indices and supplementary data collected (spatial extension, frequency of treatments… to better depict the challenges of grapevine phytosanitary management in a changing climate.

  12. Sesquiterpene volatile organic compounds (VOCs are markers of elicitation by sulfated laminarine in grapevine

    Directory of Open Access Journals (Sweden)

    Malik eChalal

    2015-05-01

    Full Text Available Inducing resistance in plants by application of elicitors of defense reactions is an attractive plant protection strategy, especially for grapevine (Vitis vinifera which is susceptible to severe fungal diseases. Though induced resistance (IR can be successful in controlled conditions, under outdoor conditions IR is in most cases not effective enough for practical disease control. Progress in the application of IR requires a better understanding of grapevine defense mechanisms and the ability to monitor defense markers in order to identify factors (physiological, environmental… that can impact IR in the vineyard.Volatile organic compounds (VOCs are well-known plant defenses compounds that have only received little or no attention in the case of grape-pathogen interactions to date. This prompted us to investigate whether an elicitor, the sulfated laminarin (PS3, actually induces the production of VOCs in grapevine. Online analysis (PTR-QMS of VOC emissions in dynamic cuvettes and passive sampling in gas tight bags with solid phase micro extraction (SPME-GC-MS under greenhouse conditions showed that PS3 elicited emission of VOCs. Some of them (as (E,E-α-farnesene might be good candidates as biomarkers of elicitor-IR whereas methyl salicylate appears to be rather a biomarker of downy mildew infection. A negative correlation between VOC emission and disease severity suggests a positive role of VOCs in grape defense against diseases.

  13. Differential Effect of Medium on the Ratio of ICM/TE of Bovine Embryos in a Co-culture System

    Directory of Open Access Journals (Sweden)

    Mohsen Forouzanfar

    2010-01-01

    Full Text Available Background: This study was undertaken to investigate the efficiency of two differentembryo somatic cell co-culture conditions, tissue culture medium 199 (TCM199–vero cellsand Menezo B2 (B2-vero cells, for the in vitro developmental quantity and quality of bovineembryos.Materials and Methods: Bovine oocytes were allowed to mature and subsequently undergofertilization in vitro. Their presumptive zygotes were cultured in either TCM199 or B2 culturemedia in conjunction with vero cells for up to nine days. The culture media were refreshedevery two days and the proportion of embryos that cleaved and further developed to themorula and blastocyst (early, expand and hatched stages were recorded. Hatched blastocystsunderwent differential staining in order to determine the numbers of inner cell mass (ICMand tropho ectoderm (TE and total cell number (TCN.Results: Of the two groups, no significant difference was seen between the proportions ofthe presumptive zygotes cleaved, those which developed to 8-16 cells, morula and reacheddays 7or 8 blastocyst stage or hatched. However, the values for TCN and TE of the TCM199-vero embryos were significantly greater than those of B2-vero embryos. The values for ICM/TCN and ICM/TE were significantly greater in the B2-vero group versus the TCM199-verogroup.Conclusion: Both TCM199 and B2 culture media in conjunction with vero cells were ofthe same efficiency when used for in vitro development of bovine presumptive zygotes.However, TCM199 was superior in providing embryos with more embryo cell numbers,whereas B2 medium was superior in providing embryos with greater ICM/TE and ICM/TCN ratios.

  14. Radicinin from Cochliobolus sp. inhibits Xylella fastidiosa, the causal agent of Pierce's Disease of grapevine.

    Science.gov (United States)

    Aldrich, Thomas J; Rolshausen, Philippe E; Roper, M Caroline; Reader, Jordan M; Steinhaus, Matthew J; Rapicavoli, Jeannette; Vosburg, David A; Maloney, Katherine N

    2015-08-01

    The fastidious phytopathogenic bacterium, Xylella fastidiosa, poses a substantial threat to many economically important crops, causing devastating diseases including Pierce's Disease of grapevine. Grapevines (Vitis vinifera L.) planted in an area under Pierce's Disease pressure often display differences in disease severity and symptom expression, with apparently healthy vines growing alongside the dying ones, despite the fact that all the vines are genetic clones of one another. Under the hypothesis that endophytic microbes might be responsible for this non-genetic resistance to X. fastidiosa, endophytic fungi were isolated from vineyard cvs. 'Chardonnay' and 'Cabernet Sauvignon' grown under high Pierce's Disease pressure. A Cochliobolus sp. isolated from a Cabernet Sauvignon grapevine inhibited the growth of X. fastidiosa in vitro. Bioassay-guided isolation of an organic extract of Cochliobolus sp. yielded the natural product radicinin as the major active compound. Radicinin also inhibited proteases isolated from the culture supernatant of X. fastidiosa. In order to assess structure-activity relationships, three semi-synthetic derivatives of radicinin were prepared and tested for activity against X. fastidiosa in vitro. Assay results of these derivatives are consistent with enzyme inactivation by conjugate addition to carbon-10 of radicinin, as proposed previously. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Characterization of the Xylella fastidiosa PD1311 gene mutant and its suppression of Pierce's disease on grapevines.

    Science.gov (United States)

    Hao, Lingyun; Johnson, Kameka; Cursino, Luciana; Mowery, Patricia; Burr, Thomas J

    2017-06-01

    Xylella fastidiosa causes Pierce's disease (PD) on grapevines, leading to significant economic losses in grape and wine production. To further our understanding of X. fastidiosa virulence on grapevines, we examined the PD1311 gene, which encodes a putative acyl-coenzyme A (acyl-CoA) synthetase, and is highly conserved across Xylella species. It was determined that PD1311 is required for virulence, as the deletion mutant, ΔPD1311, was unable to cause disease on grapevines. The ΔPD1311 strain was impaired in behaviours known to be associated with PD development, including motility, aggregation and biofilm formation. ΔPD1311 also expressed enhanced sensitivity to H 2 O 2 and polymyxin B, and showed reduced survival in grapevine sap, when compared with wild-type X. fastidiosa Temecula 1 (TM1). Following inoculation, ΔPD1311 could not be detected in grape shoots, which may be related to its altered growth and sensitivity phenotypes. Inoculation with ΔPD1311 2 weeks prior to TM1 prevented the development of PD in a significant fraction of vines and eliminated detectable levels of TM1. In contrast, vines inoculated simultaneously with TM1 and ΔPD1311 developed disease at the same level as TM1 alone. In these vines, TM1 populations were distributed similarly to populations in TM1-only inoculated plants. These findings suggest that, through an indirect mechanism, pretreatment of vines with ΔPD1311 suppresses pathogen population and disease. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  16. A Fundamental Step in IPM on Grapevine: Evaluating the Side Effects of Pesticides on Predatory Mites

    Directory of Open Access Journals (Sweden)

    Alberto Pozzebon

    2015-10-01

    Full Text Available Knowledge on side effects of pesticides on non-target beneficial arthropods is a key point in Integrated Pest Management (IPM. Here we present the results of four experiments conducted in vineyards where the effects of chlorpyrifos, thiamethoxam, indoxacarb, flufenoxuron, and tebufenozide were evaluated on the generalist predatory mites Typhlodromus pyri Scheuten and Amblyseius andersoni (Chant, key biocontrol agents of herbivorous mites on grapevines. Results show that indoxacarb and tebufenozide had a low impact on the predatory mites considered here, while a significant impact was observed for chlorpyrifos, flufenoxuron, and thiamethoxam. The information obtained here should be considered in the design of IPM strategies on grapevine.

  17. Structural Changes Induced in Grapevine (Vitis vinifera L. DNA by Femtosecond IR Laser Pulses: A Surface-Enhanced Raman Spectroscopic Study

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    Nicoleta E. Dina

    2016-05-01

    Full Text Available In this work, surface-enhanced Raman spectra of ten genomic DNAs extracted from leaf tissues of different grapevine (Vitis vinifera L. varieties, respectively, are analyzed in the wavenumber range 300–1800 cm−1. Furthermore, structural changes induced in grapevine genomic nucleic acids upon femtosecond (170 fs infrared (IR laser pulse irradiation (λ = 1100 nm are discussed in detail for seven genomic DNAs, respectively. Surface-enhanced Raman spectroscopy (SERS signatures, vibrational band assignments and structural characterization of genomic DNAs are reported for each case. As a general observation, the wavenumber range between 1500 and 1660 cm−1 of the spectra seems to be modified upon laser treatment. This finding could reflect changes in the base-stacking interactions in DNA. Spectral shifts are mainly attributed to purines (dA, dG and deoxyribose. Pyrimidine residues seem to be less affected by IR femtosecond laser pulse irradiation. Furthermore, changes in the conformational properties of nucleic acid segments are observed after laser treatment. We have found that DNA isolated from Feteasca Neagra grapevine leaf tissues is the most structurally-responsive system to the femtosecond IR laser irradiation process. In addition, using unbiased computational resources by means of principal component analysis (PCA, eight different grapevine varieties were discriminated.

  18. Arsenic and trace elements in soil, water, grapevine and onion in Jáchal, Argentina.

    Science.gov (United States)

    Funes Pinter, Iván; Salomon, M Victoria; Gil, Raúl; Mastrantonio, Leandro; Bottini, Rubén; Piccoli, Patricia

    2018-02-15

    Contamination by trace elements (TE) is an increasing concern worldwide. In some areas, crop production could be limited by the presence of metals and metalloids, so it is important to determine their concentrations and mobility. The region of Jáchal, province of San Juan, Argentina, has good growing conditions for onion and grapevine production, but their quality and yield are affected by high TE concentration in soils and water. Soils, water, grapevine and onion were sampled and TE content determined. In soils elevated As, B, Cr, Hg, and Tl concentrations were detected (506±46, 149±3, 2714±217, 16±7, and 12±3μgg -1 , respectively, for maximum values measured), and physicochemical properties of the soil promotes these elements mobility. Water samples had high As, B, Cr, and Fe concentrations (1438±400, 10,871±471, 11,516±2363, and 3071±257μgL -1 , respectively, for maximum values measured) while in onion bulbs and grapevine berries, As, Cr, Cu, and Fe (92±7 and 171±20, 1412±18 and 2965±32, 17±3 and 126±88, and 418±204 and 377±213μgg -1 , respectively, for maximum values measured) exceeded the limits for food consumption established by Argentinian law. Correlation analyses indicated that: i) there is a common source of TE in this area, ii) each elements concentration in plants is associated with different soil variables and different soils depths, and iii) the lack of correlation between soil and water indicates that concentration in water is not constant over the time and/or there exists a differential accumulation of elements in soils depending on their own properties. Data obtained demonstrate very high concentration of TE in soil, grapevines, and onion plants in Jáchal region, and different remediation techniques are necessary to stabilize and minimize the bioavailability of these elements. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Fatty Acid Methyl Ester (FAME) analyses for characterization and detection of grapevine pathogens

    Science.gov (United States)

    Grapevines can become infected by a variety of devastating pathogens, including the bacterium Xylella fastidiosa and canker fungi. Multiple strains of Xylella fastidiosa exist, each causing different diseases on various hosts. Although sequence-based genotyping can assist in distinguishing these str...

  20. A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines

    DEFF Research Database (Denmark)

    Polizzotto, Rachele; Andersen, Birgitte; Martini, Marta

    2012-01-01

    A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions.Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a...

  1. Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

    Science.gov (United States)

    Wellner, Karen L.

    2014-01-01

    In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

  2. [Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

    Science.gov (United States)

    Zhao, H; Teng, X M; Li, Y F

    2017-11-25

    Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all Pembryo group were significantly higher than those in classⅡ frozen embryo group (both Pembryos of the better quality embryo are higher.

  3. Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation.

    Science.gov (United States)

    Wessels, Cara; Penrose, Lindsay; Ahmad, Khaliq; Prien, Samuel

    2017-11-01

    Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term

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    Brochard Vincent

    2009-02-01

    Full Text Available Abstract Background Genome reprogramming in early mouse embryos is associated with nuclear reorganization and particular features such as the peculiar distribution of centromeric and pericentric heterochromatin during the first developmental stage. This zygote-specific heterochromatin organization could be observed both in maternal and paternal pronuclei after natural fertilization as well as in embryonic stem (ES cell nuclei after nuclear transfer suggesting that this particular type of nuclear organization was essential for embryonic reprogramming and subsequent development. Results Here, we show that remodeling into a zygotic-like organization also occurs after somatic cell nuclear transfer (SCNT, supporting the hypothesis that reorganization of constitutive heterochromatin occurs regardless of the source and differentiation state of the starting material. However, abnormal nuclear remodeling was frequently observed after SCNT, in association with low developmental efficiency. When transient treatment with the histone deacetylase inhibitor trichostatin A (TSA was tested, we observed improved nuclear remodeling in 1-cell SCNT embryos that correlated with improved rates of embryonic development at subsequent stages. Conclusion Together, the results suggest that proper organization of constitutive heterochromatin in early embryos is involved in the initial developmental steps and might have long term consequences, especially in cloning procedures.

  5. Monitoring Milk Somatic Cell Counts

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    Gheorghe Şteţca

    2014-11-01

    Full Text Available The presence of somatic cells in milk is a widely disputed issue in milk production sector. The somatic cell counts in raw milk are a marker for the specific cow diseases such as mastitis or swollen udder. The high level of somatic cells causes physical and chemical changes to milk composition and nutritional value, and as well to milk products. Also, the mastitic milk is not proper for human consumption due to its contribution to spreading of certain diseases and food poisoning. According to these effects, EU Regulations established the maximum threshold of admitted somatic cells in raw milk to 400000 cells / mL starting with 2014. The purpose of this study was carried out in order to examine the raw milk samples provided from small farms, industrial type farms and milk processing units. There are several ways to count somatic cells in milk but the reference accepted method is the microscopic method described by the SR EN ISO 13366-1/2008. Generally samples registered values in accordance with the admissible limit. By periodical monitoring of the somatic cell count, certain technological process issues are being avoided and consumer’s health ensured.

  6. Mediators between bereavement and somatic symptoms

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    Konkolÿ Thege Barna

    2012-06-01

    Full Text Available Abstract Background In our research we examined the frequency of somatic symptoms among bereaved (N = 185 and non-bereaved men and women in a national representative sample (N = 4041 and investigated the possible mediating factors between bereavement status and somatic symptoms. Methods Somatic symptoms were measured by the Patient Health Questionnaire (PHQ-15, anxiety with a four-point anxiety rating scale, and depression with a nine-item shortened version of the Beck Depression Inventory. Results Among the bereaved, somatic symptoms proved to be significantly more frequent in both genders when compared to the non-bereaved, as did anxiety and depression. On the multivariate level, the results show that both anxiety and depression proved to be a mediator between somatic symptoms and bereavement. The effect sizes indicated that for both genders, anxiety was a stronger predictor of somatic symptoms than depression. Conclusions The results of our research indicate that somatic symptoms accompanying bereavement are not direct consequences of this state but they can be traced back to the associated anxiety and depression. These results draw attention to the need to recognize anxiety and depression looming in the background of somatic complaints in bereavement and to the importance of the dissemination of related information.

  7. Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.

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    Jin Akagi

    Full Text Available Zebrafish (Danio rerio has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP. The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

  8. Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish.

    Science.gov (United States)

    Lehner, Adam; Kaszas, Zita; Murber, Akos; Rigo, Janos; Urbancsek, Janos; Fancsovits, Peter

    2017-08-01

    Culturing embryos in groups is a common practice in mammalian embryology. Since the introduction of different microwell dishes, it is possible to identify oocytes or embryos individually. As embryo density (embryo-to-volume ratio) may affect the development and viability of the embryos, the purpose of this study was to assess the effect of different embryo densities on embryo quality. Data of 1337 embryos from 228 in vitro fertilization treatment cycles were retrospectively analyzed. Embryos were cultured in a 25 μl microdrop in a microwell group culture dish containing 9 microwells. Three density groups were defined: Group 1 with 2-4 (6.3-12.5 μl/embryo), Group 2 with 5-6 (4.2-5.0 μl/embryo), and Group 3 with 7-9 (2.8-3.6 μl/embryo) embryos. Proportion of good quality embryos was higher in Group 2 on both days (D2: 18.9 vs. 31.5 vs. 24.7%; p Culturing 5-6 embryos together in a culture volume of 25 μl may benefit embryo quality. As low egg number, position, and distance of the embryos may influence embryo quality, results should be interpreted with caution.

  9. Phylogeny of geminivirus coat protein sequences and digital PCR aid in identifying Spissistilus festinus (Say) as a vector of Grapevine red blotch-associated virus

    Science.gov (United States)

    Grapevine red blotch-associated virus (GRBaV) is a newly identified virus of grapevines, and a putative member of a new genus within the family Geminiviridae. This virus is associated with red blotch disease that was first reported in California in 2008. It affects the profitability of vineyards by ...

  10. Synthetic seeds of a wild passionfruit species with ornamental potential

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    Maurecilne Lemes Silva

    2015-12-01

    Full Text Available Passiflora cincinnata is a wild species of passion fruit with a wide geographical distribution. It has vigorous growth, climbing habit and very showy and fragrant flowers. The aim of the present investigation was to obtain synthetic seeds from encapsulated zygotic and somatic embryos of P. cincinnata, cultivated under different conditions. Precotyledonary and cotyledonary stage embryos were obtained from zygotic embryos cultivated on MS medium supplemented with 18.1 μM of 2,4-Acid-dichlorophenoxyacetic (2,4-D and 4.5 μM of Benzyladenine (BA. Zygotic embryos and somatic embryos stages were encapsulated using sodium alginate (2.5% w v-1 and CaCl2.2H2O (1 mM as complexing agent. The zygotic and somatic embryos were encapsulated in a matrix containing (I sodium alginate, (II sodium alginate + artificial endosperm and (III sodium alginate + artificial endosperm supplemented with activated charcoal (0.15% w/v. Zygotic embryos encapsulated in the matrix (I, matrix (II and matrix (III and cultivated in flasks, germinated at rates of 79%, 76% and 86% respectively. The cotyledonary somatic embryos encapsulated in the 3 different matrices showed better germination rates when cultivated on cellulose plugs, with more than 50% of embryos converted into plants. Precotyledonary somatic embryos did not germinated regardless the matrix and cultivation. When cultivating the alginate beads ex vitro, both substrate Plantmax and Florialite showed low number of germinated embryos, and the best result (12.67% were obtained using Florialite and embryos encapsulated in the matrix (I.

  11. Osteoporosis and Somatization of Anxiety

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    Maria Papanikou

    2013-12-01

    Full Text Available Chronic stress can now be physiologically traced as a significant player in the creation of osteoporotic bones. The present pilot study involved 100 women (N = 42 have been diagnosed with osteopenia, N = 21 have been diagnosed with osteoporosis, N = 37 had a non-osteoporotic condition who participated in the Hellenic Society of Osteoporosis Association Support. Correlations between somatic symptoms of anxiety and osteoporosis, and among medications and somatization in women were explored. Assessments were based on a self-report demographic questionnaire and on the Short Anxiety Screening Test (SAST administered for detection of anxiety disorder and somatization. Statistical analysis detected non-significant differences regarding the correlation between anxiety symptomatology or somatization due to osteoporosis and osteopenia diagnosis. The same pattern is observed among women’s age group, the occupational and marital status. Hypothesis that the osteoporosis and osteopenia group would manifest significant relationships with the age group and medicines was confirmed, as well as between somatization and medicines that women with osteoporosis and osteopenia undertake. The results suggest that women are not prone to manifest anxiety or somatization in relation to the osteoporosis condition. However, the majority of women with osteoporosis and osteopenia consume more than two medicines other than those for osteoporosis. This quantity and combination they undertake appear to contribute and deteriorate their anxiety/somatization symptomatology. Further research based on a larger sample would give more definite results.

  12. Characterization of Campylocarpon pseudofasciculare associated with black foot of grapevine in southern Brazil

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    Ricardo Feliciano dos SANTOS

    2015-01-01

    Full Text Available The incidence and severity of black foot has been recently increasing in nurseries and vineyards of southern Brazil. The goal of the present study was to characterize Campylocarpon isolates associated with black foot of grapevines (Vitis spp. using multi-gene DNA analysis (internal transcribed spacers [ITS rDNA], β-tubulin and histone H3 and morphological characteristics, and to test the pathogenicity of the isolates in grapevine (Vitis labrusca cv. Bordô. The three DNA regions analyzed indicated that all the isolates belonged to Campylocarpon pseudofasciculare. Their morphology was similar to descriptions published for the species. Isolates exhibited umber- to chestnut-coloured colonies and macroconidia (38.0 × 7.0 μm predominantly with three septa. All the isolates inoculated in V. labrusca cv. Bordô caused typical symptoms of black foot. This is the first report of Campylocarpon pseudofasciculare in southern Brazil.

  13. In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

    Science.gov (United States)

    Yakovlev, Igor A; Fossdal, Carl G

    2017-01-01

    Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21-22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21-22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic

  14. In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce

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    Igor A. Yakovlev

    2017-09-01

    Full Text Available Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs and other small non-coding RNAs (sRNAs play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C. We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21–22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21–22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be

  15. Protocol for in vitro somatic embryogenesis and regeneration of rice (Oryza sativa L.).

    Science.gov (United States)

    Verma, Dipti; Joshi, Rohit; Shukla, Alok; Kumar, Pramod

    2011-12-01

    Development of highly efficient and reproducible plant regeneration system has tremendous potential to provide improved technology to assist in genetic transformation of indica rice cultivars for their further exploitation in selection. For the development of a highly reproducible regeneration system through somatic embryogenesis, mature embryos of highly popular rice cultivars i.e., Govind (for rainfed areas), Pusa Basmati-1 (aromatic basmati) and Jaya (for irrigated areas) were used. Optimum callus formation (%) to MS medium supplemented with 2, 4-D was obtained at 12.0 microM in Govind, 14.0 microM in Jaya and 15.0 microM in Pusa Basmati-1. All the cultivars showed good proliferation on MS medium without hormone. In Govind, highest embryogenic response was observed in MS medium supplemented with 2, 4-D (0.4 microM) + kinetin (0.4 microM), while in Pusa Basmati-1 with 2, 4-D (0.4 microM) + kinetin (2.0 microM) and in Jaya on hormone-free MS medium. Excellent embryo regeneration in Govind was observed on MS medium supplemented with low concentrations (1.1 microM) of BAP or hormone-free MS medium, while in Pusa Basmati-1 and Jaya embryogenesis was observed on MS medium supplemented with higher concentration of BAP (2.2 microM). Similarly, maximum plantlets with proliferated roots were observed in Govind on hormone-free MS medium, while in Pusa Basmati-1 and Jaya on MS medium supplemented with high concentration of NAA (4.0 microM). Developed plantlets were further successfully acclimatized and grown under pot culture up to maturity. Further the yield potential of in vitro developed plants was accessed at par to the direct seeded one under pot culture. Present, protocol standardizes somatic embryogenesis and efficient regeneration of agronomically important, high yielding and diverse indica rice cultivars which can be utilized as an efficient tool for molecular studies and genetic transformation in future.

  16. The impact of UV-B irradiation applied at different phases of somatic embryo development in Norway spruce on polyamine metabolism

    Czech Academy of Sciences Publication Activity Database

    Cvikrová, Milena; Vondráková, Zuzana; Eliášová, Kateřina; Pešek, Bedřich; Trávníčková, Alena; Vágner, Martin

    2016-01-01

    Roč. 30, č. 1 (2016), s. 113-124 ISSN 0931-1890 R&D Projects: GA MŠk(CZ) LD13051; GA MŠk(CZ) LD13050 Institutional support: RVO:61389030 Keywords : Picea abies * Putrescine * Somatic embryogenesis Subject RIV: EF - Botanics Impact factor: 1.842, year: 2016

  17. Resistance to Plasmopara viticola in a grapevine segregating population is associated with stilbenoid accumulation and with specific host transcriptional responses

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    Delledonne Massimo

    2011-08-01

    Full Text Available Abstract Background Downy mildew, caused by the oomycete Plasmopara viticola, is a serious disease in Vitis vinifera, the most commonly cultivated grapevine species. Several wild Vitis species have instead been found to be resistant to this pathogen and have been used as a source to introgress resistance into a V. vinifera background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity is closely related to the specific compound. The aim of this study was to assess the resistance response to P. viticola of the Merzling × Teroldego cross by profiling the stilbenoid content of the leaves of an entire population and the transcriptome of resistant and susceptible individuals following infection. Results A three-year analysis of the population's response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of trans-resveratrol, trans-piceid, trans-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine. Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response. A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis. A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-P. viticola incompatible interaction. Functional annotation of these transcripts

  18. Influence of Maternal Aging on Mitochondrial Heterogeneity, Inheritance, and Function in Oocytes and Preimplantation Embryos

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    Dori C. Woods

    2018-05-01

    Full Text Available Contrasting the equal contribution of nuclear genetic material from maternal and paternal sources to offspring, passage of mitochondria, and thus mitochondrial DNA (mtDNA, is uniparental through the egg. Since mitochondria in eggs are ancestral to all somatic mitochondria of the next generation and to all cells of future generations, oocytes must prepare for the high energetic demands of maturation, fertilization and embryogenesis while simultaneously ensuring that their mitochondrial genomes are inherited in an undamaged state. Although significant effort has been made to understand how the mtDNA bottleneck and purifying selection act coordinately to prevent silent and unchecked spreading of invisible mtDNA mutations through the female germ line across successive generations, it is unknown if and how somatic cells of the immediate next generation are spared from inheritance of detrimental mtDNA molecules. Here, we review unique aspects of mitochondrial activity and segregation in eggs and early embryos, and how these events play into embryonic developmental competency in the face of advancing maternal age.

  19. Handmade Cloned Buffalo (Bubalus bubalis) Embryos Produced from Somatic Cells Isolated from Milk and Ear Skin Differ in Their Developmental Competence, Epigenetic Status, and Gene Expression.

    Science.gov (United States)

    Jyotsana, Basanti; Sahare, Amol A; Raja, Anuj K; Singh, Karn P; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat

    2015-10-01

    We compared the cloning efficiency of buffalo embryos produced by handmade cloning (HMC) using ear skin- and milk-derived donor cells. The blastocyst rate was lower (p  milk-derived blastocysts and that of NANOG was (p  milk-derived > skin-derived blastocysts. The expression level of all these genes, except NANOG, was lower (p < 0.05) in milk- than in skin-derived or IVF blastocysts. In conclusion, milk-derived cells can be used for producing HMC embryos of quality similar to that of skin-derived embryos, although with a lower blastocyst rate.

  20. Survey of potential sharpshooter and spittlebug vectors of Xylella fastidiosa to grapevines at the São Francisco River Valley, Brazil

    Directory of Open Access Journals (Sweden)

    Rudiney Ringenberg

    2014-06-01

    Full Text Available Survey of potential sharpshooter and spittlebug vectors of Xylella fastidiosa to grapevines at the São Francisco River Valley, Brazil. Pierce's disease of grapevines, caused by Xylella fastidiosa, is a serious problem in some regions of North America, not yet reported in Brazil. In this study, a survey of potential sharpshooter (Hemiptera, Cicadellidae, Cicadellinae and spittlebug (Hemiptera, Cercopidae vectors of X. fastidiosa was conducted in vineyards at the São Francisco River Valley, a major grape growing region in Brazil. Four vineyards of Vitis vinifera L. were sampled fortnightly from June/2005 to June/2007, using yellow sticky cards, each placed at two different heights (45 cm aboveground and 45 cm above the crop canopy in 10 sampling localities. A total of 4,095 specimens of sharpshooters were collected, nearly all from 3 Proconiini species, Homalodisca spottii Takiya, Cavichioli & McKamey, 2006 (96.8% of the specimens, Tapajosa fulvopunctata (Signoret, 1854 (3.1%, and Tretogonia cribrata Melichar, 1926 (1 specimen. Hortensia similis (Walker, 1851 (2 specimens was the only Cicadellini species. Only 1 cercopid specimen, belonging to Aeneolamia colon (Germar, 1821, was trapped. Even though they are not considered potential Xylella vectors, 2 Gyponini leafhoppers were collected: Curtara samera DeLong & Freytag, 1972 (11 specimens and Curtara inflata DeLong & Freytag, 1976 (1 specimen. Homalodisca spottii was observed feeding and mating on green branches of grapevines, in addition to egg masses. Because of its prevalence on the crop canopy, occurrence throughout the year (with peaks from February to August, and ability to colonize grapevines, H. spottii could be an important vector if a X. fastidiosa strain pathogenic to grapevines becomes introduced at the São Francisco River Valley.

  1. The somatic symptom scale-8 (SSS-8): a brief measure of somatic symptom burden.

    Science.gov (United States)

    Gierk, Benjamin; Kohlmann, Sebastian; Kroenke, Kurt; Spangenberg, Lena; Zenger, Markus; Brähler, Elmar; Löwe, Bernd

    2014-03-01

    Somatic symptoms are the core features of many medical diseases, and they are used to evaluate the severity and course of illness. The 8-item Somatic Symptom Scale (SSS-8) was recently developed as a brief, patient-reported outcome measure of somatic symptom burden, but its reliability, validity, and usefulness have not yet been tested. To investigate the reliability, validity, and severity categories as well as the reference scores of the SSS-8. A national, representative general-population survey was performed between June 15, 2012, and July 15, 2012, in Germany, including 2510 individuals older than 13 years. The SSS-8 mean (SD), item-total correlations, Cronbach α, factor structure, associations with measures of construct validity (Patient Health Questionnaire-2 depression scale, Generalized Anxiety Disorder-2 scale, visual analog scale for general health status, 12-month health care use), severity categories, and percentile rank reference scores. The SSS-8 had excellent item characteristics and good reliability (Cronbach α = 0.81). The factor structure reflects gastrointestinal, pain, fatigue, and cardiopulmonary aspects of the general somatic symptom burden. Somatic symptom burden as measured by the SSS-8 was significantly associated with depression (r = 0.57 [95% CI, 0.54 to 0.60]), anxiety (r = 0.55 [95% CI, 0.52 to 0.58]), general health status (r = -0.24 [95% CI, -0.28 to -0.20]), and health care use (incidence rate ratio, 1.12 [95% CI, 1.10 to 1.14]). The SSS-8 severity categories were calculated in accordance with percentile ranks: no to minimal (0-3 points), low (4-7 points), medium (8-11 points), high (12-15 points), and very high (16-32 points) somatic symptom burden. For every SSS-8 severity category increase, there was a 53% (95% CI, 44% to 63%) increase in health care visits. The SSS-8 is a reliable and valid self-report measure of somatic symptom burden. Cutoff scores identify individuals with low, medium, high, and very high somatic

  2. Temperature dependent RNA metabolism in Xylella fastidiosa during cold stress and grapevine infection

    Science.gov (United States)

    Re-occurrence of Pierce’s disease of grapes, caused by Xylella fastidiosa, is known to be influenced by environmental factors, particularly cold temperatures during overwintering. Grapevines in colder regions are often cured of X. fastidiosa infection over the winter season, depending on cultivar, t...

  3. The distribution and symptomatology of grapevine trunk disease pathogens are influenced by climate

    NARCIS (Netherlands)

    van Niekerk, J.M.; Bester, W.; Halleen, F.; Crous, P.W.; Fourie, P.H.

    2011-01-01

    Grapevine trunk diseases, caused by a range of phytopathogenic fungi, represent a serious impediment to wine and table grape production wherever these crops are cultivated. Previous studies have shown that the distribution of these pathogens is influenced by climate and that they are associated with

  4. Ecophysiological modeling of grapevine water stress in Burgundy terroirs by a machine-learning approach

    Directory of Open Access Journals (Sweden)

    Luca eBrillante

    2016-06-01

    Full Text Available In a climate change scenario, successful modeling of the relationships between plant-soil-meteorology is crucial for a sustainable agricultural production, especially for perennial crops. Grapevines (Vitis vinifera L. cv Chardonnay located in eight experimental plots (Burgundy, France along a hillslope were monitored weekly for three years for leaf water potentials, both at predawn (Ψpd and at midday (Ψstem. The water stress experienced by grapevine was modeled as a function of meteorological data (minimum and maximum temperature, rainfall and soil characteristics (soil texture, gravel content, slope by a gradient boosting machine. Model performance was assessed by comparison with carbon isotope discrimination (δ13C of grape sugars at harvest and by the use of a test-set. The developed models reached outstanding prediction performance (RMSE < 0.08 MPa for Ψstem and < 0.06 MPa for Ψpd, comparable to measurement accuracy. Model predictions at a daily time step improved correlation with δ13C data, respect to the observed trend at a weekly time scale. The role of each predictor in these models was described in order to understand how temperature, rainfall, soil texture, gravel content and slope affect the grapevine water status in the studied context. This work proposes a straight-forward strategy to simulate plant water stress in field condition, at a local scale; to investigate ecological relationships in the vineyard and adapt cultural practices to future conditions.

  5. Ecophysiological Modeling of Grapevine Water Stress in Burgundy Terroirs by a Machine-Learning Approach.

    Science.gov (United States)

    Brillante, Luca; Mathieu, Olivier; Lévêque, Jean; Bois, Benjamin

    2016-01-01

    In a climate change scenario, successful modeling of the relationships between plant-soil-meteorology is crucial for a sustainable agricultural production, especially for perennial crops. Grapevines (Vitis vinifera L. cv Chardonnay) located in eight experimental plots (Burgundy, France) along a hillslope were monitored weekly for 3 years for leaf water potentials, both at predawn (Ψpd) and at midday (Ψstem). The water stress experienced by grapevine was modeled as a function of meteorological data (minimum and maximum temperature, rainfall) and soil characteristics (soil texture, gravel content, slope) by a gradient boosting machine. Model performance was assessed by comparison with carbon isotope discrimination (δ(13)C) of grape sugars at harvest and by the use of a test-set. The developed models reached outstanding prediction performance (RMSE < 0.08 MPa for Ψstem and < 0.06 MPa for Ψpd), comparable to measurement accuracy. Model predictions at a daily time step improved correlation with δ(13)C data, respect to the observed trend at a weekly time scale. The role of each predictor in these models was described in order to understand how temperature, rainfall, soil texture, gravel content and slope affect the grapevine water status in the studied context. This work proposes a straight-forward strategy to simulate plant water stress in field condition, at a local scale; to investigate ecological relationships in the vineyard and adapt cultural practices to future conditions.

  6. Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars

    Czech Academy of Sciences Publication Activity Database

    Drábek, A.; Smolíková, M.; Kalendar, R.; Pinto, F. A. L.; Pavloušek, P.; Klepárník, Karel; Frébort, I.

    2016-01-01

    Roč. 37, 23-24 (2016), s. 3059-3067 ISSN 0173-0835 Institutional support: RVO:68081715 Keywords : grapevine DNA analysis * multiplex PCR * STRs * Vitis vinifera L Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.744, year: 2016

  7. Endogenous target mimics down-regulate miR160 mediation of ARF10, -16 and -17 cleavage during somatic embryogenesis in Dimocarpus longan Lour.

    Directory of Open Access Journals (Sweden)

    yuling elin

    2015-11-01

    Full Text Available MicroRNA160 plays a critical role in plant development by negatively regulating the auxin response factors ARF10, -16 and -17. However, the ways in which miR160 expression is regulated at the transcriptional level, and how miR160 interacts with its targets during plant embryo development, remain unknown. Here, we studied the regulatory relationships among endogenous target mimics (eTMs, and miR160 and its targets, and their involvement in hormone signaling and somatic embryogenesis (SE in Dimocarpus longan. We identified miR160 family members and isolated the miR160 precursor, primary transcript, and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, abscisic acid, salicylic acid and heat stress. The pri-miR160 was down-regulated in response to salicylic acid but up-regulated by gibberellic acid, ethylene, and methyl jasmonate treatment, suggesting that pri-miR160 was associated with hormone transduction. Dlo-miR160a, -a* and -d* reached expression peaks in torpedo-shaped embryos, globular embryos and cotyledonary embryos, respectively, but were barely detectable in embryogenic callus. This suggests that they have expression-related and functional diversity, especially during the middle and later developmental stages of SE. Four potential eTMs for miR160 were identified. Two of them, glucan endo-1,3-beta- glucosidase-like protein 2-like and calpain-type cysteine protease DEK1, were confirmed to control the corresponding dlo-miR160a* expression level. This suggests that they may function to abolish the binding between dlo-miR160a* and its targets. These two eTMs also participated in auxin and ABA signal transduction. DlARF10, -16, and -17 targeting by dlo-miR160a was confirmed; their expression levels were higher in friable-embryogenic callus and incomplete compact pro-embryogenic cultures and responded to 2,4-D, suggesting they may play a major role in the early stages of longan SE dependent on 2,4-D. The e

  8. Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

    Science.gov (United States)

    Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A; Larsen, Martin Jakob

    2016-01-01

    Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

  9. Comparative response of six grapevine rootstocks to inoculation with arbuscular mycorrhizal fungi based on root traits

    Science.gov (United States)

    Pogiatzis, Antreas; Bowen, Pat; Hart, Miranda; Holland, Taylor; Klironomos, John

    2017-04-01

    Arbuscular mycorrhizal (AM) symbiosis has been proven to be essential in grapevines, sustaining plant growth especially under abiotic and biotic stressors. The mycorrhizal growth response of young grapevines varies among rootstock cultivars and the underlying mechanisms involved in this variation are unknown. We predicted that this variation in mycorrhizal response may be explained by differences in root traits among rootstocks. We analyzed the entire root system of six greenhouse-grown rootstocks (Salt Creek, 3309 Couderc, Riparia Gloire, 101-14 Millardet et de Grasset, Swarzmann, Teleki 5C), with and without AM fungal inoculation (Rhizophagus irregularis) and characterized their morphological and architectural responses. Twenty weeks after the inoculation, aboveground growth was enhanced by AM colonization. The rootstock varieties were distinctly different in their response to AM fungi, with Salt Creek receiving the highest growth benefit, while Schwarzmann and 5C Teleki receiving the lowest. Plant responsiveness to AM fungi was negatively correlated with branching intensity (fine roots per root length). Furthermore, there was evidence that mycorrhizas can influence the expression of root traits, inducing a higher branching intensity and a lower root to shoot ratio. The results of this study will help to elucidate how interactions between grapevine rootstocks and AM fungi may benefit the establishment of new vineyards.

  10. Nitrogen fertilization of Cabernet Sauvignon grapevines: yield, total nitrogen content in the leaves and must composition

    Directory of Open Access Journals (Sweden)

    Felipe Lorensini

    2015-08-01

    Full Text Available Grapevines grown on sandy soils are subjected to the application of supplemental nitrogen (N; however, there is little information available regarding the impact of these applications on yield, plant nutritional state and must composition. The aim of this study was to evaluate the yield, nutritional state and must composition of grapevines subjected to N fertilization. Cabernet Sauvignon grapevines were subjected to annual applications of 0, 10, 15, 20, 40, 80 and 120 kg N ha-1 in 2008, 2009 and 2010. During the 2008/09, 2009/10 and 2010/11 harvest seasons, leaves were collected during full flowering and when the berries changed color, and the total N content was analyzed. The grape yield and the enological characteristics of the must were evaluated. The response to applied N was low, and the highest Cabernet Sauvignon grape yield was obtained in response to an application of 20 kg N ha-1 year-1. The application of N increased the nutrient content in the leaf collected at full flowering, but it had little effect on the total nutrient content in the must, and it did not affect the enological characteristics of the must, such as soluble solids, pH, total acidity, malic acid and tartaric acid.

  11. [Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

    Science.gov (United States)

    Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

    2014-06-01

    To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

  12. Proteomic analysis of grapevine resistance induced by Trichoderma harzianum T39 reveals specific defence pathways activated against downy mildew

    Science.gov (United States)

    Perazzolli, Michele

    2012-01-01

    Downy mildew is caused by the oomycete Plasmopara viticola and is one of the most serious diseases of grapevine. The beneficial microorganism Trichoderma harzianum T39 (T39) has previously been shown to induce plant-mediated resistance and to reduce the severity of downy mildew in susceptible grapevines. In order to better understand the cellular processes associated with T39-induced resistance, the proteomic and histochemical changes activated by T39 in grapevine were investigated before and 1 day after P. viticola inoculation. A comprehensive proteomic analysis of T39-induced resistance in grapevine was performed using an eight-plex iTRAQ protocol, resulting in the identification and quantification of a total of 800 proteins. Most of the proteins directly affected by T39 were found to be involved in signal transduction, indicating activation of a complete microbial recognition machinery. Moreover, T39-induced resistance was associated with rapid accumulation of reactive oxygen species and callose at infection sites, as well as changes in abundance of proteins involved in response to stress and redox balance, indicating an active defence response to downy mildew. On the other hand, proteins affected by P. viticola in control plants mainly decreased in abundance, possibly reflecting the establishment of a compatible interaction. Finally, the high-throughput iTRAQ protocol allowed de novo peptide sequencing, which will be used to improve annotation of the Vitis vinifera cv. Pinot Noir proteome. PMID:23105132

  13. Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

    Science.gov (United States)

    Noli, Laila; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

    2017-03-01

    Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non

  14. Water stress exacerbates the severity of Botryosphaeria dieback in grapevines infected by Neofusicoccum parvum

    Science.gov (United States)

    Botryosphaeria dieback (causal fungus Neofusicoccum parvum) is a detrimental grapevine trunk disease, causing internal wood degradation, killing shoots, and reducing yields. We examined the interactive effects of drought and N. parvum infection, common vineyard stresses, on wood-lesion development. ...

  15. Changes in protein composition and protein phosphorylation during ...

    African Journals Online (AJOL)

    Changes in protein profiles and protein phosphorylation were studied in various stages of germinating somatic and zygotic embryos. Many proteins, which were expressed in cotyledonary stage somatic embryos, were also present in the zygotic embryos obtained from mature dry seed. The intensity of 22 kDa protein was ...

  16. Responses of grapevine rootstocks to drought through altered root system architecture and root transcriptomic regulations.

    Science.gov (United States)

    Yıldırım, Kubilay; Yağcı, Adem; Sucu, Seda; Tunç, Sümeyye

    2018-06-01

    Roots are the major interface between the plant and various stress factors in the soil environment. Alteration of root system architecture (RSA) (root length, spread, number and length of lateral roots) in response to environmental changes is known to be an important strategy for plant adaptation and productivity. In light of ongoing climate changes and global warming predictions, the breeding of drought-tolerant grapevine cultivars is becoming a crucial factor for developing a sustainable viticulture. Root-trait modeling of grapevine rootstock for drought stress scenarios, together with high-throughput phenotyping and genotyping techniques, may provide a valuable background for breeding studies in viticulture. Here, tree grafted grapevine rootstocks (110R, 5BB and 41B) having differential RSA regulations and drought tolerance were investigated to define their drought dependent root characteristics. Root area, root length, ramification and number of root tips reduced less in 110R grafted grapevines compared to 5BB and 41B grafted ones during drought treatment. Root relative water content as well as total carbohydrate and nitrogen content were found to be much higher in the roots of 110R than it was in the roots of other rootstocks under drought. Microarray-based root transcriptome profiling was also conducted on the roots of these rootstocks to identify their gene regulation network behind drought-dependent RSA alterations. Transcriptome analysis revealed totally 2795, 1196 and 1612 differentially expressed transcripts at the severe drought for the roots of 110R, 5BB and 41B, respectively. According to this transcriptomic data, effective root elongation and enlargement performance of 110R were suggested to depend on three transcriptomic regulations. First one is the drought-dependent induction in sugar and protein transporters genes (SWEET and NRT1/PTR) in the roots of 110R to facilitate carbohydrate and nitrogen accumulation. In the roots of the same rootstock

  17. Biostimulation of grapevine : mode of action and possible agronomic uses

    OpenAIRE

    Krzyzaniak, Yuko; Trouvelot, Sophie; Heloir, Marie-Claire; Fourquez, P.; Magnin-Robert, Jean-Bernard; Randoux, B.; Siah, A.; Halama, Patrice; Moreau, Emmanuelle; Adrian, Marielle

    2016-01-01

    Although there is a growing interest for the use of biostimulants in agriculture, only few methods allowing a precise description of their effects on plants have been reported. In the IRIS+ FUI project, two major and highly different worldwide crops, wheat (annual, monocotyledon) and grapevine (perennial, broadleaf), were chosen to deepen our knowledge of such compounds and explore their potential additional interest. The first objective is to develop in greenhouse conditions, a panel of tool...

  18. Grapevine rootstock effects on scion sap phenolic levels, resistance to Xylella fastidiosa infection, and progression of Pierce’s disease

    Directory of Open Access Journals (Sweden)

    Christopher Michael Wallis

    2013-12-01

    Full Text Available The xylem-limited bacterium Xylella fastidiosa (Xf causes Pierce’s disease (PD, an important disease of grapevine, Vitis vinifera L.. Grapevine rootstocks were developed to provide increased resistance to root disease, but rootstock effects on cane and vine diseases remain unclear. Grapevines that consisted of Cabernet Sauvignon or Chardonnay grafted to 13 different rootstocks were inoculated with Xf and evaluated for PD severity and Xf titer after six months. A subset of six rootstock/scion combinations had xylem sap phenolic levels assessed in non-infected and Xf-infected grapevines. Vigor also was analyzed by measuring root lengths and masses. Cabernet Sauvignon grafted to 101-14MG, 1103P, 420A, or Schwarzmann had reduced PD severity compared to Cabernet Sauvignon grafted to 110R, 5BB, or SO4. Chardonnay grafted to Salt Creek or Freedom had reduced PD severity compared to Chardonnay grafted to RS3 or Schwarzmann. Chardonnay grafted to RS3 had greater Xf titer than Chardonnay grafted to 101-14MG, Freedom, or Salt Creek. No other differences in Xf titer among rootstocks were observed. Of the six scion/rootstock combinations which had xylem sap phenolics analyzed, Chardonnay/ RS3 had the highest levels of most phenolics whereas Cabernet Sauvignon/101-14MG had the lowest phenolic levels. However, Chardonnay/101-14MG, which had mild PD symptoms, had greater sap levels of caftaric acid than other scion/rootstock combinations. Sap levels of caftaric acid, methyl salicylate, a procyanidin trimer, and quinic acid were greater in Xf-infected versus non-infected grapevines. Chardonnay on 101-14MG or Salt Creek had greater root mass than Chardonnay on RS3. Cabernet Sauvignon on 101-14MG had greater root mass than Cabernet Sauvignon on 110R. These results identified rootstocks with the capacity for reducing PD symptom progression. Rootstocks also were shown to affect Xf titer, xylem sap phenolic levels, and plant vigor.

  19. Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

    Directory of Open Access Journals (Sweden)

    Anne Bruun Krøigård

    Full Text Available Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

  20. Protocols for Callus and Somatic Embryo Initiation for Hibiscus sabdariffa L. (Malvaceae): Influence of Explant Type, Sugar, and Plant Growth Regulators

    Science.gov (United States)

    A significant work on callus induction and somatic embryogenesis was realized for Hibiscus sabdariffa. Two genotypes (Hibiscus sabdariffa and Hibiscus sabdariffa var. altissima) two sugars (sucrose and glucose) and three concentrations (1 %, 2%, 3%) of each sugar, 3 explant types (root, hypocotyl, c...

  1. Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

    International Nuclear Information System (INIS)

    Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen

    2016-01-01

    The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

  2. Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen, E-mail: sodmergn@pku.edu.cn

    2016-05-27

    The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

  3. Shoot growth of Merlot and Cabernet Sauvignon grapevine varieties

    OpenAIRE

    Marcelo Borghezan; Olavo Gavioli; Hamilton Justino Vieira; Aparecido Lima da Silva

    2012-01-01

    The objective of this work was to evaluate shoot growth of the grapevine varieties Merlot and Cabernet Sauvignon, during 2006/2007, and Cabernet Sauvignon, during 2008/2009, in São Joaquim, SC, Brazil. The experiment was carried out in a commercial vineyard trained on a vertical trellis system. The shoots of the central part of the plants were selected, and the lengths from the base to the apex of 20 shoots per cultivar were evaluated. In 2006/2007, monitoring began at pruning, on 9/15/2006, ...

  4. New insights into thermal growing conditions of Portuguese grapevine varieties under changing climates

    Science.gov (United States)

    Santos, João A.; Costa, Ricardo; Fraga, Helder

    2018-03-01

    New decision support tools for Portuguese viticulture are urging under a climate change context. In the present study, heat and chilling accumulation conditions of a collection of 44 grapevine cultivars currently grown in Portugal are assessed at very high spatial resolution ( 1 km) and for 1981-2015. Two bioclimatic indices that incorporate non-linear plant-temperature relationships are selected for this purpose: growing degree hours—GDH (February-October) and chilling portions—CP (October-February). The current thermal growing conditions of each variety are examined and three clusters of grapevine cultivars are identified based on their GDH medians, thus assembling varieties with close heat accumulation requirements and providing more physiologically consistent information when compared to previous studies, as non-linear plant-temperature relationships are herein taken into account. These new clusters are also a complement to previous bioclimatic zoning. Ensemble mean projections under two anthropogenic-driven scenarios (RCP4.5 and RCP8.5, 2041-2070), from four EURO-CORDEX simulations, reveal a widespread increase of GDH and decrease of CP, but with spatial heterogeneities. The spatial variability of these indices throughout Portugal is projected to decrease (strongest increases of GDH in the coolest regions of the northeast) and to increase (strongest decreases of CP in the warmest regions of the south and west), respectively. The typical heat accumulation conditions of each cluster are projected to gradually shift north-eastwards and to higher-elevation areas, whereas insufficient chilling may represent a new challenge in warmer future climates. An unprecedented level of detail for a large collection of grapevine varieties in Portugal is provided, thus promoting a better planning of climate change adaptation measures.

  5. Evaluation of biocontrol agents for grapevine pruning wound protection against trunk pathogen infection.

    Directory of Open Access Journals (Sweden)

    Charl KOTZE

    2011-12-01

    Full Text Available Trunk diseases of grapevine are caused by numerous pathogens, including Eutypa lata, Phaeomoniella chlamydospora, and species of Botryosphaeriaceae (incl. Botryosphaeria and aggregate genera, Phomopsis and Phaeoacremonium. Since infections occur mainly through pruning wounds, that have been shown by previous research to stay susceptible for up to 16 weeks after pruning, long-term pruning wound protection is required for prevention of infection. This study evaluated several biocontrol agents against a range of trunk disease pathogens in dual plate laboratory trials to determine macroscopic and microscopic interactions. The biocontrol agents had a substantial effect on all the pathogens, with a wide range of macroscopic and microscopic interactions observed. The best performing biocontrol agents were tested in two field trials. Fresh pruning wounds were treated with benomyl, Trichoderma products (Biotricho®, Vinevax® and ECO 77® and isolates (USPP-T1 and -T2, identified as T. atroviride and Bacillus subtilis. Seven days after treatment the pruning wounds were inoculated by spraying with spore suspensions of Neofusicoccum australe, N. parvum, Diplodia seriata, Lasiodiplodia theobromae, Eutypa lata, Phaeomoniella chlamydospora or Phomopsis viticola. Eight months after inoculation, the treatments were evaluated by isolation onto potato dextrose agar. The efficacy of the biocontrol agents was in most cases similar or superior to that observed for benomyl. Isolate USPP-T1, in particular, was very effective, reducing incidence of Ph. viticola, E. lata, Pa. chlamydospora, N. australe, N. parvum, D. seriata and L. theobromae by 69, 76, 77, 78, 80, 85 and 92%, respectively. This is the first report of biological protection of grapevine pruning wounds against this group of grapevine trunk disease pathogens.

  6. Embryos, individuals, and persons: an argument against embryo creation and research.

    Science.gov (United States)

    Tollefsen, C

    2001-01-01

    One strategy for arguing that it should be legally permissible to create human embryos, or to use spare human embryos, for scientific research purposes involves the claim that such embryos cannot be persons because they are not human individuals while twinning may yet take place. Being a human individual is considered to be by most people a necessary condition for being a human person. I argue first that such an argument against the personhood of embryos must be rationally conclusive if their destruction in public places such as laboratories is to be countenanced. I base this argument on a popular understanding of the role that the notion of privacy plays in abortion laws. I then argue that such arguments against personhood are not rationally conclusive. The claim that the early embryos is not a human individual is not nearly as obvious as some assert.

  7. Single-embryo transfer versus multiple-embryo transfer.

    Science.gov (United States)

    Gerris, Jan

    2009-01-01

    Despite the progress made in assisted reproductive technology, live birth rates remain disappointingly low. Multiple-embryo transfer has been an accepted practice with which to increase the success rate. This has led to a higher incidence of multiple-order births compared with natural conception, which not only increase the risk of mortality and morbidity to both mother and children but are also associated with social and economic consequences. Elective single-embryo transfer (eSET) was developed in an effort to increase singleton pregnancies in assisted reproduction. Studies comparing eSET with multiple-embryo transfer highlight the benefit of this approach and suggest that, with careful patient selection and the transfer of good-quality embryos, the risk of a multiple-order pregnancy can be reduced without significantly decreasing live birth rates. Although the use of eSET has gradually increased in clinical practice, its acceptance has been limited by factors such as availability of funding and awareness of the procedure. An open discussion of eSET is warranted in an effort to enable a broader understanding by physicians and patients of the merits of this approach. Ultimately, eSET may provide a more cost-effective, potentially safer approach to patients undergoing assisted reproduction technology.

  8. Mouse Embryo Compaction.

    Science.gov (United States)

    White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

    2016-01-01

    Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. © 2016 Elsevier Inc. All rights reserved.

  9. Extending a structural model of somatization to South Koreans: Cultural values, somatization tendency, and the presentation of depressive symptoms.

    Science.gov (United States)

    Zhou, Xiaolu; Min, Seongho; Sun, Jiahong; Kim, Se Joo; Ahn, Joung-Sook; Peng, Yunshi; Noh, Samuel; Ryder, Andrew G

    2015-05-01

    Somatization refers to the tendency to emphasize somatic symptoms when experiencing a psychiatric disturbance. This tendency has been widely reported in patients from East Asian cultural contexts suffering from depression. Recent research in two Chinese samples have demonstrated that the local cultural script for depression, involving two aspects-the experience and expression of distress (EED) and conceptualization and communication of distress (CCD)-can be evoked to help explain somatization. Given the beliefs and practices broadly shared across Chinese and South Korean cultural contexts, the current study seeks to replicate this explanatory model in South Koreans. Our sample included 209 psychiatric outpatients from Seoul and Wonju, South Korea. Self-report questionnaires were used to assess somatization tendency, adherence to traditional values, and psychological and somatic symptoms of depression. Results from SEM showed that the EED and CCD factors of somatization tendency were differently associated with cultural values and somatic symptoms, replicating our previous findings in Chinese outpatients. The reliance on a brief self-report measure of somatization tendency, not originally designed to assess separate EED and CCD factors, highlights the need for measurement tools for the assessment of cultural scripts in cross-cultural depression research. The replication of the Chinese structural model of somatization in South Korea lends empirical support to the view that somatization can be understood as the consequence of specific cultural scripts. These scripts involve the experience and expression of distress as well as culturally meaningful ways in which this distress is conceptualized and communicated to others. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Stable transformation via particle bombardment in two different soybean regeneration systems.

    Science.gov (United States)

    Sato, S; Newell, C; Kolacz, K; Tredo, L; Finer, J; Hinchee, M

    1993-05-01

    The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

  11. Dissipation of glyphosate from grapevine soils in Sonora, Mexico

    Directory of Open Access Journals (Sweden)

    Norma J. Salazar López

    2016-10-01

    Full Text Available Grapevine is one of the important crops in Sonora, due to revenue generation from its export to foreign countries. Among the most widely used herbicides for this crop is glyphosate, which is considered moderately toxic and persistent. The present research evaluates the dissipation of glyphosate in grapevine planted soil at three depths (5, 30 and 60 cm. Sampling was carried out before glyphosate application, and 5, 10, 18, 27, and 65 days after. Glyphosate was extracted from soil samples using ammonium hydroxide. The derivate extracts were partitioned with dichloromethane and analyzed using gas chromatography with pulsed flame photometric detector (PFPD. The results showed that average glyphosate residues are significantly greater at 5 cm (0.09 mg kg-1 than the other depths (30 and 60 cm, having a difference of 0.078 mg kg-1 between them (P < 0.03. Glyphosate concentration time profiles were similar; it reached maximum soil concentration in a range of 10 to 18 days after application. The half-life of glyphosate in soil has an average of 39 days at all depths. Our data suggests that the release in soil of glyphosate applied to weeds delays its transference to soil by 14 days, and extends residue half life to 55 days after application. These results could be the basis for further research, including more environmental parameters that could affect the dissipation or degradation process in soil.

  12. Impact of an arbuscular mycorrhizal fungus versus a mixed microbial inoculum on the transcriptome reprogramming of grapevine roots.

    Science.gov (United States)

    Balestrini, Raffaella; Salvioli, Alessandra; Dal Molin, Alessandra; Novero, Mara; Gabelli, Giovanni; Paparelli, Eleonora; Marroni, Fabio; Bonfante, Paola

    2017-07-01

    Grapevine, cultivated for both fruit and beverage production, represents one of the most economically important fruit crops worldwide. With the aim of better understanding how grape roots respond to beneficial microbes, a transcriptome sequencing experiment has been performed to evaluate the impact of a single arbuscular mycorrhizal (AM) fungal species (Funneliformis mosseae) versus a mixed inoculum containing a bacterial and fungal consortium, including different AM species, on Richter 110 rootstock. Results showed that the impact of a single AM fungus and of a complex microbial inoculum on the grapevine transcriptome differed. After 3 months, roots exclusively were colonized after the F. mosseae treatment and several AM marker genes were found to be upregulated. The mixed inoculum led only to traces of colonization by AM fungi, but elicited an important transcriptional regulation. Additionally, the expression of genes belonging to categories such as nutrient transport, transcription factors, and cell wall-related genes was significantly altered in both treatments, but the exact genes affected differed in the two conditions. These findings advance our understanding about the impact of soil beneficial microbes on the root system of a woody plant, also offering the basis for novel approaches in grapevine cultivation.

  13. Assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

    International Nuclear Information System (INIS)

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Miller, L.; Samuels, S.J.; Chang, R.J.; Overstreet, J.W.

    1988-01-01

    We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos

  14. Quality information from the grapevine.

    Science.gov (United States)

    Laszlo, Pierre

    2010-07-01

    A letter by Lucien Herr, a highly regarded leading French intellectual at the time of World War I, provides capsule portraits of chemists such as Gabriel Bertrand, Paul Lebeau, Charles Moureu, and Georges Urbain. It makes us better aware of who they were and of how their contemporaries saw their work, which had much to do with their personalities, whether congenial or abrasive. This article is concerned with the kind of information carried by the so-called grapevine. It can be invaluable to the historian, for the light it sheds on the character of a scientist. The document drawn upon, from World War I (1915), depicts graphically the personalities of some of the French chemists engaged in the rush to design and produce chemical weapons. It is a frank and even brutal appraisal of their strengths and weaknesses. This is the kind of evaluation that scientists routinely engage in, but devoid of the hyperbole, pro or con, which usually flavours it.

  15. Nitrogen nutrition of the grape-vine (Vitis vinifera spp)

    International Nuclear Information System (INIS)

    Conradie, W.J.

    1985-12-01

    A thorough knowledge concerning the nitrogen relationship in the grape-vine is essential in order to appreciate how different patterns of uptake, assimilation, storage and utilisation of nitrogen might be advantageous in particular environmental situations. The 15 N-isotope technique has been used to determine the uptake and distribution of nitrogen absorbed during early spring, early summer and autumn. Apart from the total N fraction, protein N and soluble N were determined as well. The utilisation of labelled N applied in the field, was determined for vineyards on heavier and lighter soils

  16. Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

    Science.gov (United States)

    Kim, Eun Young; Park, Min Jee; Park, Hyo Young; Noh, Eun Ji; Noh, Eun Hyung; Park, Kyoung Sik; Lee, Jun Beom; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

    2012-08-01

    In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

  17. Pembentukan Embrio Endospermik Sekunder Mangga (Mangifera indica L. Gedong Gincu Klon 289

    Directory of Open Access Journals (Sweden)

    Irni Furnawanthi Hindaningrum

    2014-09-01

    Full Text Available ABSTRACTThe improvement of Mangifera indica L. by conventional breeding approaches has been confounded by the long generation cycle, low fruit set, single seed per fruit and high degree of cross pollination. Biotechnology complements conventional breeding and expedite the mango improvement programs. Endosperm culture is a direct method to produce triploid plants. This study aimed  to obtain embryo from endosperm culture. The system of secondary somatic embriogenesis in mango described here represents a source of embryogenic material may be used for mass propagation and genetic manipulation of this crop. The method consisted of induction, proliferation, maturation, germination, and histological analysis of the obtaimed embryos. A protocol for plantlet regeneration was developed for Gedong Gincu mango clone 289 through secondary somatic embryogenesis. Primary somatic embryos (proembryo and cotyledonary embryos were cultured in induction medium to induce the secondary somatic embryos. The best proliferation rate was 0.22 in medium with 1 g L-1 Poly Vinyl Pyrrolidone (PVP for multiplication of secondary somatic embryos. Maturation of inoculum derived from the proliferation medium supplemented with 2 g L-1 of activated charcoal on medium containing 0.4 mg L-1 BAP provides the average 2.39 embryo formation of cotyledonari phase. The highest germination frequency (20% was obtained in media with GA3 1.5 mg L-1.Keywords: endosperm, Gedong Gincu, Mangifera indica L, secondary endospermic embrio

  18. Effects of Drought Stress and Rewatering on some Morphological and Physiological Properties of Three Grapevine Cultivars

    Directory of Open Access Journals (Sweden)

    Mehdi Aran

    2017-12-01

    Full Text Available Introduction: Most plants have developed morphological and physiological mechanisms which allow them to cope with drought stress. Almost all the studies conducted on grapevines (Vitisvinifera L. responses to drought conditions have focused on physiological responses such as stomatal reactions, photosynthesis and osmotic adjustment, and biochemical responses like carbohydrates and proline. According to these studies, physiological and biochemical responses of grapevines to water stress are quite variable. This variability could be related to cultivar, time of the year, previous water stress level, intensity of stress, and environmental conditions. Osmotic adjustment in terms of compatible solutes accumulation has been considered as an important physiological adaptation for plant to resist drought, which facilitates the extraction of water from dry soils and maintenance of cell turgor, gas exchange and growth in very dry environments. Acting as compatible solutes as well as antioxidants, a significant rise in proline amount was observed in grapevine leaves under water stress conditions, suggesting that this amino acid has a protective role against the formation of excessive reactive oxygen species (ROS. Plants, in order to overcome oxidative stress, have developed enzymatic and non-enzymatic antioxidant defense mechanisms against scavenge ROS. Materials and Methods: This research was conducted to assess the effect of different levels of irrigation on some characteristics of three cultivars of grapevine (Yaghooti, Bidanesefid and Askari, as a factorial based on a randomized complete block design in two years with four replications. The experiment started in June 21, 2014 and 2015. Water treatments were applied in four levels including: control plant (100% FC, moderate stress (60% FC, severe stress (30% FC and rewatering treatment after severe stress treatment. Increase height, leaf number, stem diameter, leaf fresh and dry weight, stem dry weight

  19. Somatic symptom profiles in the general population

    DEFF Research Database (Denmark)

    Eliasen, Marie; Jørgensen, Torben; Schröder, Andreas

    2017-01-01

    PURPOSE: The aim of this study was to identify and describe somatic symptom profiles in the general adult population in order to enable further epidemiological research within multiple somatic symptoms. METHODS: Information on 19 self-reported common somatic symptoms was achieved from a population...

  20. MMPI screening scales for somatization disorder.

    Science.gov (United States)

    Wetzel, R D; Brim, J; Guze, S B; Cloninger, C R; Martin, R L; Clayton, P J

    1999-08-01

    44 items on the MMPI were identified which appear to correspond to some of the symptoms in nine of the 10 groups on the Perley-Guze checklist for somatization disorder (hysteria). This list was organized into two scales, one reflecting the total number of symptoms endorsed and the other the number of organ systems with at least one endorsed symptom. Full MMPIs were then obtained from 29 women with primary affective disorder and 37 women with somatization disorder as part of a follow-up study of a consecutive series of 500 psychiatric clinic patients seen at Washington University. Women with the diagnosis of somatization disorder scored significantly higher on the somatization disorder scales created from the 44 items than did women with only major depression. These new scales appeared to be slightly more effective in identifying somatization disorder than the use of the standard MMPI scales for hypochondriasis and hysteria. Further development is needed.

  1. Migration and growth of protoplanetary embryos. I. Convergence of embryos in protoplanetary disks

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiaojia; Lin, Douglas N. C. [Department of Astronomy and Astrophysics, University of California, Santa Cruz, CA 95064 (United States); Liu, Beibei [Kavli Institute for Astronomy and Astrophysics and Department of Astronomy, School of Physics, Peking University, Beijing 100871 (China); Li, Hui, E-mail: xzhang47@ucsc.edu [Los Alamos National Laboratory, Los Alamos, NM 87545 (United States)

    2014-12-10

    According to the core accretion scenario, planets form in protostellar disks through the condensation of dust, coagulation of planetesimals, and emergence of protoplanetary embryos. At a few AU in a minimum mass nebula, embryos' growth is quenched by dynamical isolation due to the depletion of planetesimals in their feeding zone. However, embryos with masses (M{sub p} ) in the range of a few Earth masses (M {sub ⊕}) migrate toward a transition radius between the inner viscously heated and outer irradiated regions of their natal disk. Their limiting isolation mass increases with the planetesimals surface density. When M{sub p} > 10 M {sub ⊕}, embryos efficiently accrete gas and evolve into cores of gas giants. We use a numerical simulation to show that despite stream line interference, convergent embryos essentially retain the strength of non-interacting embryos' Lindblad and corotation torques by their natal disks. In disks with modest surface density (or equivalently accretion rates), embryos capture each other in their mutual mean motion resonances and form a convoy of super-Earths. In more massive disks, they could overcome these resonant barriers to undergo repeated close encounters, including cohesive collisions that enable the formation of massive cores.

  2. Identification of Agrobacterium vitis as a causal agent of grapevine crown gall in Serbia

    Directory of Open Access Journals (Sweden)

    Kuzmanović N.

    2012-01-01

    Full Text Available In 2010, a serious outbreak of crown gall disease was observed on grapevines (Vitis vinifera L. cv. Cabernet Sauvignon in several commercial vineyards located in the Vojvodina province, Serbia. Bacteria were isolated from the young tumor tissue on nonselective YMA medium and five representative strains were selected for further identification. Tumorigenic (Ti plasmid was detected in all strains by PCR using primers designed to amplify the virC pathogenicity gene, producing a 414-bp PCR product. The strains were identified as Agrobacterium vitis using differential physiological and biochemical tests, and a multiplex PCR assay targeting 23S rRNA gene sequences. In the pathogenicity assay, all strains induced characteristic symptoms on inoculated tomato and grapevine plants. They were less virulent on tomato plants in comparison to the reference strains of A. tumefaciens and A. vitis. [Ministarstva nauke Republike Srbije, br. III46008: Development of integrated management of harmful organisms in plant production in order to overcome resistance and to improve food quality and safety

  3. Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.).

    Science.gov (United States)

    Cavalcante Alves, J M; Sihachakr, D; Allot, M; Tizroutine, S; Mussio, I; Servaes, A; Ducreux, G

    1994-05-01

    The potential of somatic embryogenesis was evaluated for 10 cultivars of sweet potato through extensive embryogenic response and isozyme analysis. Embryogenic callus was induced by incubating lateral buds on Murashige and Skoog medium containing 10 μM 2,4-dichlorophenoxyacetic acid for 6-8 weeks. The frequency of embryogenic response was low, and varied with genotypes, ranging from 0 to 17%. Embryo to plantlet formation could be enhanced by the use of the combination of 2,4-dichlorophenoxyacetic acid with kinetin, both used at 0.01 μM. Embryogenic callus with its potential of plantlet formation has constantly been maintained for over two years. However, after several subcultures, 0.5 to 12% of embryogenic callus reverted irreversibly into friable fast-growing non-embryogenic callus whose ability to regenerate shoots was then definitively lost. The isozymes of esterase, peroxidase, glutamate oxaloacetate transaminase and acid phosphatase investigated in this study were found appropriate to distinguish compact embryogenic from friable non-embryogenic callus in sweet potato. In fact, the callus reversion was associated with a loss of bands or a decline in isozyme activity. On the contrary, very small changes in isozyme activity or no specific changes at all were observed during the differentiation of embryogenic callus into globular embryos.

  4. Screening of Brazilian soybean genotypes with high potential for somatic embryogenesis and plant regeneration Seleção de genótipos brasileiros de soja com alto potencial para embriogênese somática e regeneração de plantas

    Directory of Open Access Journals (Sweden)

    Annette Droste

    2010-07-01

    Full Text Available The aim of this work was to identify Brazilian soybean (Glycine max genotypes with potential to respond to in vitro culture stimuli for primary somatic embryo induction, secondary embryo proliferation and plant regeneration. Differences among eight tested cultivars were observed at each stage. Two cultivars, IAS-5 and BRSMG 68 Vencedora, were selected for the evaluation of the capacity for embryo differentiation and plant regeneration. These cultivars had high embryo induction frequencies, repetitive embryogenic proliferation, and low precocious embryo germination in the initial experiment. The effect of abscisic acid (ABA and charcoal addition on plant regeneration was investigated. The addition of ABA to proliferation medium and of ABA and activated charcoal to maturation medium increased embryo differentiation rates, which resulted in a higher number of regenerated plants. The BRSMG 68 Vencedora cultivar was found to have a high potential for embryo induction, embryo proliferation and plant regeneration. The potential of this cultivar for somatic embryogenesis was similar to that observed for cultivar IAS-5, which is currently used for soybean transformation in Brazil. BRSMG 68 Vencedora may be a good alternative genotype for soybean genetic engineering via somatic embryogenesis protocols.O objetivo deste trabalho foi identificar cultivares brasileiras de soja (Glycine max com capacidade de resposta aos estímulos da cultura in vitro para a indução de embriões somáticos primários, proliferação de embriões secundários e regeneração de plantas. Foram observadas diferenças para cada estádio entre as oito cultivares testadas. Foram selecionadas duas cultivares, 'IAS-5' e BRSMG 68 Vencedora, para avaliação do potencial dos embriões quanto à diferenciação e conversão em plantas. Essas cultivares tiveram altas taxas de indução, proliferação embriogênica repetitiva e baixa germinação precoce dos embriões, no experimento

  5. Who abandons embryos after IVF?

    LENUS (Irish Health Repository)

    Walsh, A P H

    2010-04-01

    This investigation describes features of in vitro fertilisation (IVF) patients who never returned to claim their embryos following cryopreservation. Frozen embryo data were reviewed to establish communication patterns between patient and clinic; embryos were considered abandoned when 1) an IVF patient with frozen embryo\\/s stored at our facility failed to make contact with our clinic for > 2 yrs and 2) the patient could not be located after a multi-modal outreach effort was undertaken. For these patients, telephone numbers had been disconnected and no forwarding address was available. Patient, spouse and emergency family contact\\/s all escaped detection efforts despite an exhaustive public database search including death records and Internet directory portals. From 3244 IVF cycles completed from 2000 to 2008, > or = 1 embryo was frozen in 1159 cases (35.7%). Those without correspondence for > 2 yrs accounted for 292 (25.2%) patients with frozen embryos; 281 were contacted by methods including registered (signature involving abandoned embryos did not differ substantially from other patients. The goal of having a baby was achieved by 10\\/11 patients either by spontaneous conception, adoption or IVF. One patient moved away with conception status unconfirmed. The overall rate of embryo abandonment was 11\\/1159 (< 1%) in this IVF population. Pre-IVF counselling minimises, but does not totally eliminate, the problem of abandoned embryos. As the number of abandoned embryos from IVF accumulates, their fate urgently requires clarification. We propose that clinicians develop a policy consistent with relevant Irish Constitutional provisions to address this medical dilemma.

  6. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

    Science.gov (United States)

    Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

    2013-10-01

    Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The

  7. Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the grapevine Vitis vinifera

    International Nuclear Information System (INIS)

    Atkinson, Sarah C.; Dogovski, Con; Newman, Janet; Dobson, Renwick C. J.; Perugini, Matthew A.

    2011-01-01

    Dihydrodipicolinate synthase from the common grapevine V. vinifera has been cloned, expressed, purified and crystallized in the presence of the substrate pyruvate by in-drop hexahistidine-tag cleavage. A diffraction data set has been collected to a resolution of 2.2 Å. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS from the grapevine Vitis vinifera (Vv-DHDPS). Following in-drop cleavage of the hexahistidine tag, cocrystals of Vv-DHDPS with the substrate pyruvate were grown in 0.1 M Bis-Tris propane pH 8.2, 0.2 M sodium bromide, 20%(w/v) PEG 3350. X-ray diffraction data in space group P1 at a resolution of 2.2 Å are presented. Preliminary diffraction data analysis indicated the presence of eight molecules per asymmetric unit (V M = 2.55 Å 3 Da −1 , 52% solvent content). The pending crystal structure of Vv-DHDPS will provide insight into the molecular evolution in quaternary structure of DHDPS enzymes

  8. Characterization of the largest relic Eurasian wild grapevine reservoir in Southern Iberian Peninsula

    Energy Technology Data Exchange (ETDEWEB)

    Arroyo-García, R.; Cantos, M.; Lara, M.; López, M.A.; Gallardo, A.; Ocete, C.A.; Pérez, A.; Bánáti, B.; García, J.L.; Ocete, R.

    2016-11-01

    Wild grapevine is becoming a threatened species in the Iberian Peninsula due to human impacts. The aim of this work was to carry out a holistic study for six years of the largest wild grapevine population found up to date in SW Iberian Peninsula. This population has 115 vines. Ampelographic and soil characteristics have been studied. Evaluation of its environment has also been studied by describing the main parasitic species and natural enemies of pests. The ability of this plant material for its micropropagation and storage in slow-growth conditions has been tested. Microvinification resulted in a wine with good acidity and medium color intensity, two interesting characteristics under a warm climatology. Finally, the identification of private alleles in this wild population, absent in other locations from the Northern and Southern Iberian territories, is a very valuable feature and confirms the importance of establishing conservation programs. The population here studied is genetically unique and potentially useful for commercial rootstocks and cultivars breeding that would improve viticulture and enology. (Author)

  9. Grapevine tissues and phenology differentially affect soluble carbohydrates determination by capillary electrophoresis.

    Science.gov (United States)

    Moreno, Daniela; Berli, Federico; Bottini, Rubén; Piccoli, Patricia N; Silva, María F

    2017-09-01

    Soluble carbohydrates distribution depends on plant physiology and, among other important factors, determines fruit yield and quality. In plant biology, the analysis of sugars is useful for many purposes, including metabolic studies. Capillary electrophoresis (CE) proved to be a powerful green separation technique with minimal sample preparation, even in complex plant tissues, that can provide high-resolution efficiency. Matrix effect refers to alterations in the analytical response caused by components of a sample other than the analyte of interest. Thus, the assessment and reduction of the matrix factor is fundamental for metabolic studies in different matrices. The present study evaluated the source and levels of matrix effects in the determination of most abundant sugars in grapevine tissues (mature and young leaves, berries and roots) at two phenological growth stages. Sucrose was the sugar that showed the least matrix effects, while fructose was the most affected analyte. Based on plant tissues, young leaves presented the smaller matrix effects, irrespectively of the phenology. These changes may be attributed to considerable differences at chemical composition of grapevine tissues with plant development. Therefore, matrix effect should be an important concern for plant metabolomics. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  10. Viable calves produced by somatic cell nuclear transfer using meiotic-blocked oocytes.

    Science.gov (United States)

    De Bem, Tiago H C; Chiaratti, Marcos R; Rochetti, Raquel; Bressan, Fabiana F; Sangalli, Juliano R; Miranda, Moysés S; Pires, Pedro R L; Schwartz, Kátia R L; Sampaio, Rafael V; Fantinato-Neto, Paulo; Pimentel, José R V; Perecin, Felipe; Smith, Lawrence C; Meirelles, Flávio V; Adona, Paulo R; Leal, Cláudia L V

    2011-10-01

    Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

  11. Personality characteristics in patients with somatized disorder

    Directory of Open Access Journals (Sweden)

    Ekaterina Anatolyevna Tolkach

    2010-01-01

    Full Text Available Objective: to study personality characteristics, behavioral style, and modes of relations with their people in patients with somatized disorder. Subjects and methods. Eighty-six patients diagnosed as having somatized disorder were examined using Leary's interpersonal diagnosis system. Results. The author revealed the following personality characteristics and behavioral styles: a depressed need for authoritarianism, dominance, autonomy, aggressiveness, a display of qualities, such as superfriendliness, benevolence, submissiveness, dependency, and suspiciousness. These characteristics give an insight into the development of somatization in patients with somatized disorder.

  12. Embriogênese somática e regeneração de plantas a partir de embrião maduro de aveia Somatic embryogenesis and plant regeneration derived from mature embryos of oat

    Directory of Open Access Journals (Sweden)

    Caren Regina Cavichioli Lamb

    2002-02-01

    Full Text Available Calo embriogênico tem sido o tecido-alvo mais utilizado para transformação genética de cereais. O objetivo deste trabalho foi investigar o estabelecimento de calos embriogênicos e a regeneração de plantas in vitro a partir de embriões maduros de genótipos de aveia (Avena sativa L.. Embriões maduros foram retirados das sementes e colocados em meio MS (Murashige & Skoog, contendo 30,0 g L-1 de sacarose e 2,0 mg L-1 de ácido 2,4-diclorofenoxiacético (2,4-D. Após o período de indução de calos, agregados embriogênicos foram isolados e subcultivados a cada 21 dias para meio fresco. Os calos embriogênicos foram então transferidos para meio de indução de parte aérea, e, na seqüência, as partes aéreas foram transferidas para meio de indução de raízes. Houve diferenças entre genótipos quanto à capacidade de embriogênese somática e regeneração de plantas in vitro a partir de embrião maduro. Este explante permitiu a indução de calos embriogênicos, que se multiplicaram, e que regeneraram in vitro um grande número de plantas de genótipos como UFRGS 7 e UFRGS 19, o que o faz passível de ser utilizado na transformação genética da aveia.Embryogenic callus has been the most used target tissue for cereal genetic transformation. Therefore, the objective of this study was to investigate the establishment of embryogenic calli and the in vitro plant regeneration from mature embryos of oat genotypes (Avena sativa L.. Mature embryos were taken out of the seeds and placed on a culture medium MS (Murashige & Skoog, containing 30,0 mg L-1 of sucrose and 2,0 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D. From the induction period, embryogenic aggregates were isolated and subcultivated each 21 days into a fresh medium. After this period, embryogenic calli were transferred to a medium for shoot regeneration. Subsequently, the shoot was transferred to a medium for root induction. There was variability among genotypes for somatic

  13. Proteomic analysis of shoot tissue during photoperiod induced growth cessation in V. riparia Michx. grapevines

    Directory of Open Access Journals (Sweden)

    Victor Kim J

    2010-08-01

    Full Text Available Abstract Background Growth cessation, cold acclimation and dormancy induction in grapevines and other woody perennial plants native to temperate continental climates is frequently triggered by short photoperiods. The early induction of these processes by photoperiod promotes winter survival of grapevines in cold temperate zones. Examining the molecular processes, in particular the proteomic changes in the shoot, will provide greater insight into the signaling cascade that initiates growth cessation and dormancy induction. To begin understanding transduction of the photoperiod signal, Vitis riparia Michx. grapevines that had grown for 35 days in long photoperiod (long day, LD, 15 h were subjected to either a continued LD or a short photoperiod (short day, SD, 13 h treatment. Shoot tips (4-node shoot terminals were collected from each treatment at 7 and 28 days of LD and SD for proteomic analysis via two-dimensional (2D gel electrophoresis. Results Protein profiles were characterized in V. riparia shoot tips during active growth or SD induced growth cessation to examine physiological alterations in response to differential photoperiod treatments. A total of 1054 protein spots were present on the 2D gels. Among the 1054 proteins, 216 showed differential abundance between LD and SD (≥ two-fold ratio, p-value ≤ 0.05. After 7 days, 39 protein spots were more abundant in LD and 30 were more abundant in SD. After 28 days, 93 protein spots were more abundant in LD and 54 were more abundant in SD. MS/MS spectrometry was performed to determine the functions of the differentially abundant proteins. Conclusions The proteomics analysis uncovered a portion of the signal transduction involved in V. riparia grapevine growth cessation and dormancy induction. Different enzymes of the Calvin-Benson cycle and glutamate synthetase isoforms were more abundant either in LD or SD treatments. In LD tissues the significantly differentially more abundant proteins

  14. Proteomic analysis of shoot tissue during photoperiod induced growth cessation in V. riparia Michx. grapevines

    Science.gov (United States)

    2010-01-01

    Background Growth cessation, cold acclimation and dormancy induction in grapevines and other woody perennial plants native to temperate continental climates is frequently triggered by short photoperiods. The early induction of these processes by photoperiod promotes winter survival of grapevines in cold temperate zones. Examining the molecular processes, in particular the proteomic changes in the shoot, will provide greater insight into the signaling cascade that initiates growth cessation and dormancy induction. To begin understanding transduction of the photoperiod signal, Vitis riparia Michx. grapevines that had grown for 35 days in long photoperiod (long day, LD, 15 h) were subjected to either a continued LD or a short photoperiod (short day, SD, 13 h) treatment. Shoot tips (4-node shoot terminals) were collected from each treatment at 7 and 28 days of LD and SD for proteomic analysis via two-dimensional (2D) gel electrophoresis. Results Protein profiles were characterized in V. riparia shoot tips during active growth or SD induced growth cessation to examine physiological alterations in response to differential photoperiod treatments. A total of 1054 protein spots were present on the 2D gels. Among the 1054 proteins, 216 showed differential abundance between LD and SD (≥ two-fold ratio, p-value ≤ 0.05). After 7 days, 39 protein spots were more abundant in LD and 30 were more abundant in SD. After 28 days, 93 protein spots were more abundant in LD and 54 were more abundant in SD. MS/MS spectrometry was performed to determine the functions of the differentially abundant proteins. Conclusions The proteomics analysis uncovered a portion of the signal transduction involved in V. riparia grapevine growth cessation and dormancy induction. Different enzymes of the Calvin-Benson cycle and glutamate synthetase isoforms were more abundant either in LD or SD treatments. In LD tissues the significantly differentially more abundant proteins included flavonoid

  15. Comparing Kaolin and Pinolene to Improve Sustainable Grapevine Production during Drought

    OpenAIRE

    Brillante, Luca; Belfiore, Nicola; Gaiotti, Federica; Lovat, Lorenzo; Sansone, Luigi; Poni, Stefano; Tomasi, Diego

    2016-01-01

    Viticulture is widely practiced in dry regions, where the grapevine is greatly exposed to water stress. Optimizing plant water use efficiency (WUE) without affecting crop yield, grape and wine quality is crucial to limiting use of water for irrigation and to significantly improving viticulture sustainability. This study examines the use in vineyards of particle film technology (engineered kaolin) and compares it to a film-forming antitranspirant (pinolene), traditionally used to limit leaf wa...

  16. Somatic tinnitus prevalence and treatment with tinnitus retraining therapy.

    Science.gov (United States)

    Ostermann, K; Lurquin, P; Horoi, M; Cotton, P; Hervé, V; Thill, M P

    2016-01-01

    Somatic tinnitus originates from increased activity of the dorsal cochlear nucleus, a cross-point between the somatic and auditory systems. Its activity can be modified by auditory stimulation or somatic system manipulation. Thus, sound enrichment and white noise stimulation might decrease tinnitus and associated somatic symptoms. The present uncontrolled study sought to determine somatic tinnitus prevalence among tinnitus sufferers, and to investigate whether sound therapy with counselling (tinnitus retraining therapy; TRT) may decrease tinnitus-associated somatic symptoms. To determine somatic tinnitus prevalence, 70 patients following the TRT protocol completed the Jastreboff Structured Interview (JSI) with additional questions regarding the presence and type of somatic symptoms. Among 21 somatic tinnitus patients, we further investigated the effects of TRT on tinnitus-associated facial dysesthesia. Before and after three months of TRT, tinnitus severity was evaluated using the Tinnitus Handicap Inventory (THI), and facial dysesthesia was assessed with an extended JSI-based questionnaire. Among the evaluated tinnitus patients, 56% presented somatic tinnitus-including 51% with facial dysesthesia, 36% who could modulate tinnitus by head and neck movements, and 13% with both conditions. Self-evaluation indicated that TRT significantly improved tinnitus and facial dysesthesia in 76% of patients. Three months of TRT led to a 50% decrease in mean THI and JSI scores regarding facial dysesthesia. Somatic tinnitus is a frequent and underestimated condition. We suggest an extension of the JSI, including specific questions regarding somatic tinnitus. TRT significantly improved tinnitus and accompanying facial dysesthesia, and could be a useful somatic tinnitus treatment.

  17. Annotation of differentially expressed genes in the somatic embryogenesis of musa and their location in the banana genome.

    Science.gov (United States)

    Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-Graciamedrano, Rosa Maria

    2013-01-01

    Analysis of cDNA-AFLP was used to study the genes expressed in zygotic and somatic embryogenesis of Musa acuminata Colla ssp. malaccensis, and a comparison was made between their differential transcribed fragments (TDFs) and the sequenced genome of the double haploid- (DH-) Pahang of the malaccensis subspecies that is available in the network. A total of 253 transcript-derived fragments (TDFs) were detected with apparent size of 100-4000 bp using 5 pairs of AFLP primers, of which 21 were differentially expressed during the different stages of banana embryogenesis; 15 of the sequences have matched DH-Pahang chromosomes, with 7 of them being homologous to gene sequences encoding either known or putative protein domains of higher plants. Four TDF sequences were located in all Musa chromosomes, while the rest were located in one or two chromosomes. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed. Thus the availability of the genome of Musa and the information of TDFs sequences presented here opens new possibilities for an in-depth study of the molecular and biochemical research of zygotic and somatic embryogenesis of Musa.

  18. The Role of Somatic Symptoms in Sexual Medicine: Somatization as Important Contextual Factor in Male Sexual Dysfunction.

    Science.gov (United States)

    Fanni, Egidia; Castellini, Giovanni; Corona, Giovanni; Boddi, Valentina; Ricca, Valdo; Rastrelli, Giulia; Fisher, Alessandra Daphne; Cipriani, Sarah; Maggi, Mario

    2016-09-01

    An important feature of somatic symptom disorder is the subjective perception of the physical symptoms and its maladaptive interpretation. Considering that psychological distress is often expressed through somatic symptoms, it is possible that they underlie at least a part of the symptoms in subjects complaining of sexual dysfunction. Nevertheless, studies on the impact of somatoform disorders in sexual dysfunction are scanty. To define the psychological, relational, and organic correlates of somatic symptoms in a large sample of patients complaining of sexual problems. A consecutive series of 2833 men (mean age 50.2 ± 13.5 years) was retrospectively studied. Somatic symptoms were assessed using the "somatized anxiety symptoms" subscale of the Middlesex Hospital Questionnaire (MHQ-S). Several clinical, biochemical, psychological, and relational parameters were studied. Patients were interviewed with the previously validated Structured Interview on Erectile Dysfunction (SIEDY), and ANDROTEST (a structured interview for the screening of hypogonadism in patients with sexual dysfunction). Among the 2833 patients studied, subjects scoring higher on somatic symptoms were older, more obese, reporting unhealthy lifestyle (current smoking, alcohol consumption), and a lower education (all P sexuality more often, including erectile problems (spontaneous or sexual-related), low sexual desire, decreased frequency of intercourse, and perceived reduction of ejaculate volume (all P sexual dysfunction. High levels of somatic symptoms in subjects with sexual dysfunction can be related to the sexual symptom itself. The consequences of this pattern have great clinical relevance in a sexual medicine setting, considering their severe impact on sexuality. Copyright © 2016 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.

  19. Frozen-Thawed Embryo Transfer Cycles Have a Lower Incidence of Ectopic Pregnancy Compared With Fresh Embryo Transfer Cycles.

    Science.gov (United States)

    Zhang, Xinyu; Ma, Caihong; Wu, Zhangxin; Tao, Liyuan; Li, Rong; Liu, Ping; Qiao, Jie

    2017-01-01

    To evaluate the risk of ectopic pregnancy of embryo transfer. A retrospective cohort study on the incidence of ectopic pregnancy in fresh and frozen-thawed embryo transfer cycles from January 1 st , 2010, to January 1 st , 2015. Infertile women undergoing frozen-thawed transfer cycles or fresh transfer cycles. In-vitro fertilization, fresh embryo transfer, frozen-thawed embryo transfer, ectopic pregnancy. Ectopic pregnancy rate and clinical pregnancy rate. A total of 69 756 in vitro fertilization-embryo transfer cycles from 2010 to 2015 were analyzed, including 45 960 (65.9%) fresh and 23 796 (34.1%) frozen-thawed embryo transfer cycles. The clinical pregnancy rate per embryo transfer was slightly lower in fresh embryo transfer cycles compared with frozen-thawed embryo transfer cycles (40.8% vs 43.1%, P cycles, blastocyst transfer shows a significantly lower incidence of ectopic pregnancy (0.8% vs 1.8%, P = .002) in comparison with day 3 cleavage embryo transfer. The risk of ectopic pregnancy is lower in frozen-thawed embryo transfer cycles than fresh embryo transfer cycles, and blastocyst transfer could further decrease the ectopic pregnancy rate in frozen-thawed embryo transfer cycles.

  20. Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR.

    Science.gov (United States)

    Bulgari, Daniela; Casati, Paola; Brusetti, Lorenzo; Quaglino, Fabio; Brasca, Milena; Daffonchio, Daniele; Bianco, Piero Attilio

    2009-08-01

    Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.