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.

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

Science.gov (United States)

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.

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

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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.

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

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

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

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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.

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

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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

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 ...

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.

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

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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.

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...

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

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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.

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....

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.

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

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...

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...

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

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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.

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.

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 ...

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.

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.

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.

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) ...

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

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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.

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)

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.

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...

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

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.

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 ...

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

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

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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

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)

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

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.

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.

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...

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.

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

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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

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

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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.

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

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...

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.

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.

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

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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.

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.

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.

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.

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

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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.

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 ...

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

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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.

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....

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

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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.

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.

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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

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

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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.

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

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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.

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

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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.

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.

Direct somatic embryogenesis in Swietenia macrophylla King

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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

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

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

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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.

Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

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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.

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

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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.

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

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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.

Hemoglobin promotes somatic embryogenesis in peanut cultures.

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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%).

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

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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.

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.

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

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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.

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

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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.

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.

In vitro somatic embryogenesis and plant regeneration of cassava.

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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.

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.

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.

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....

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

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

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.

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

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.

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.

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

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.

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.

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.

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.

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.

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

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.

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.

Anatomy of somatic embryogenesis in Carica papaya L.

Directory of Open Access Journals (Sweden)

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.

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

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.

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.

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

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.

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.

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.

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)

Replication of somatic micronuclei in bovine enucleated oocytes

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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

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.

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...

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)

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.

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

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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.

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

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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

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.

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.

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.

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.

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...

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...

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

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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.

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.

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.

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.

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)

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.

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.

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.

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

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

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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

[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.

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

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 ...

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

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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.

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

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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

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...

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.

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)

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.

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

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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

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

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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.

Somatic Embryogenesis in Yam (Dioscorea rotundata

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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.

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.

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...

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.

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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

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.

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.

PECTIMORF and BIOBRAS-16 utilization in the potato somatic embryogenesis

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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

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

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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.

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.

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.

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.

The role of chromatin modifications in somatic embryogenesis in plants

Directory of Open Access Journals (Sweden)

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.

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.

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...

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

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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.

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

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.

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.

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.

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)

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.

Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

Science.gov (United States)

Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

2012-07-01

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

[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.

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

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.

Protein phosphorylation during coconut zygotic embryo development

International Nuclear Information System (INIS)

Islas-Flores, I.; Oropeza, C.; Hernandez-Sotomayor, S.M.T.

1998-01-01

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species

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.

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 ...

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...

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.

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....

What Drives Embryo Development? Chromosomal Normality or Mitochondria?

Directory of Open Access Journals (Sweden)

A. Bayram

2017-01-01

Full Text Available Objective. To report the arrest of euploid embryos with high mtDNA content. Design. A report of 2 cases. Setting. Private fertility clinic. Patients. 2 patients, 45 and 40 years old undergoing IVF treatment. Interventions. Mature oocytes were collected and vitrified from two ovarian stimulations. Postthaw, survived mature oocytes underwent fertilization by intracytoplasmic sperm injection (ICSI. Preimplantation genetic screening (PGS and mitochondrial DNA (mtDNA copy number were done using next generation sequencing (NGS. The only normal embryo among the all-biopsied embryos had the highest “Mitoscore” value and was the only arrested embryo in both cases. Therefore, the embryo transfer was cancelled. Main Outcome Measures. Postthaw survival and fertilization rate, embryo euploidy, mtDNA copy number, and embryo development. Results. In both patients, after PGS only 1 embryo was euploid. Both embryos had the highest mtDNA copy number from all tested embryos and both embryos were arrested on further development. Conclusions. These cases clearly demonstrate the lack of correlation between mtDNA value (Mitoscore and chromosomal status of embryo.

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...

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

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.

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.

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

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.).

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.

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.

Enhancement of NMRI Mouse Embryo Development In vitro

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Abedini, F.

2013-12-01

Full Text Available Most of the systematic studies used in the development of human embryo culture media have been done first on mouse embryos. The general use of NMRI outbred mice is a model for toxicology, teratology and pharmacology. NMRI mouse embryo exhibit the two-cell block in vitro. The objective of this study was to evaluate and compare the effects of four kinds of culture media on the development of zygotes (NMRI after embryo vitrification. One-cell mouse embryos were obtained from NMRI mice after superovulation and mating with adult male NMRI mice. And then randomly divided into 4 groups for culture in four different cultures media including: M16 (A, DMEM/Ham, F-12 (B, DMEM/Ham's F-12 co-culture with Vero cells(C and DMEM/Ham's F-12 co-culture with MEF cells (D. Afterward all of the embryos were vitrified in EFS40 solution and collected. Results of our study revealed, more blastocysts significantly were developed with co-culture with MEF cells in DMEM/Ham's F-12 medium. More research needed to understand the effect of other components of culture medium, and co-culture on NMRI embryo development.

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

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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.

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.

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...

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'.

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....

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.

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.

Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae).

Science.gov (United States)

González-Gutiérrez, Alejandra G; Gutiérrez-Mora, Antonia; Rodríguez-Garay, Benjamín

2014-01-01

Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer's-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded.

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.

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 ...

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

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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.

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.

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

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

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

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.

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

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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.

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

[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.

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

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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.

The Roles of Glutathione Peroxidases during Embryo Development.

Science.gov (United States)

Ufer, Christoph; Wang, Chi Chiu

2011-01-01

Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency - in contrast to all other GPx family members - leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on

Effect of Somatic Cell Types and Culture Medium on in vitro Maturation, Fertilization and Early Development Capability of Buffalo Oocytes

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H. Jamil*, H. A. Samad, N. Rehman, Z. I. Qureshi and L. A. Lodhi

2011-04-01

Full Text Available This study was designed to evaluate the efficacy of different somatic cell types and media in supporting in vitro maturation (IVM, in vitro fertilization (IVF and early embryonic development competence of buffalo follicular oocytes. Cumulus oocyte complexes were collected for maturation from follicles (>6mm of buffalo ovaries collected at the local abattoir. Oocytes were co-cultured in tissue culture medium (TCM-199 with either granulosa cells, cumulus cells, or buffalo oviductal epithelial cells (BOEC @ 3x106 cells/ml or in TCM-199 without helper cells (control at 39°C and 5%CO2 in humidified air. Fresh semen was prepared in modified Ca++ free Tyrode medium. Fertilization was carried out in four types of media: i Tyrode lactate albumin pyruvate (TALP, ii TALP+BOEC, iii modified Ca++ free Tyrode and iv modified Ca++ free Tyrode+BOEC. Fertilized oocytes were cultured for early embryonic development in TCM-199 with and without BOEC. Higher maturation rates were observed in the granulosa (84.24% and cumulus cells (83.44% than BOEC co culture system (73.37%. Highest fertilization rate was obtained in modified Ca++ free Tyrode with BOEC co culture (70.42%, followed by modified Ca++ free Tyrode alone (63.77%, TALP with BOEC (36.92% and TALP alone (10.94%. Development of early embryos (8-cell stage improved in TCM-199 with BOEC co culture than TCM-199 alone. From the results of this study, it can be concluded that addition of somatic cells (granulosa cells, cumulus cells results in higher maturation rates of buffalo follicular oocytes than BOEC co culture system, while fertilization rate improved in modified Ca++ free Tyrode with and without BOEC. Addition of BOEC to TCM-199 improved the developmental capacity of early embryo.

Sex determination of duck embryos: observations on syrinx development

Science.gov (United States)

Wilson, Robert E.; Sonsthagen, Sarah A.; Franson, J. Christian

2013-01-01

Ducks exhibit sexual dimorphism in vocal anatomy. Asymmetrical ossification of the syrinx (bulla syringealis) is discernable at about 10 days of age in male Pekin duck (Anas platyrhynchos domestica) embryos, but information is lacking on the early development of the bulla in wild ducks. To evaluate the reliability of this characteristic for sexing developing embryos, we examined the syrinx of dead embryos and compared results with molecular sexing techniques in high arctic nesting Common Eiders (Somateria mollissima). Embryos 8 days or older were accurately (100%) sexed based on the presence/absence of a bulla, 2 days earlier than Pekin duck. The use of the tracheal bulla can be a valuable technique when sex identification of embryos or young ducklings is required.

Effect of localized hypoxia on Drosophila embryo development.

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Zhinan Wang

Full Text Available Environmental stress, such as oxygen deprivation, affects various cellular activities and developmental processes. In this study, we directly investigated Drosophila embryo development in vivo while cultured on a microfluidic device, which imposed an oxygen gradient on the developing embryos. The designed microfluidic device enabled both temporal and spatial control of the local oxygen gradient applied to the live embryos. Time-lapse live cell imaging was used to monitor the morphology and cellular migration patterns as embryos were placed in various geometries relative to the oxygen gradient. Results show that pole cell movement and tail retraction during Drosophila embryogenesis are highly sensitive to oxygen concentrations. Through modeling, we also estimated the oxygen permeability across the Drosophila embryonic layers for the first time using parameters measured on our oxygen control device.

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.

Role of melatonin in embryo fetal development

OpenAIRE

Voiculescu, SE; Zygouropoulos, N; Zahiu, CD; Zagrean, AM

2014-01-01

Melatonin is an indoleamine produced by the pineal gland and secreted in a circadian manner. In the past few decades, research over this topic has been enhanced. Melatonin has many important roles in the human physiology: regulator of the circadian rhythms, sleep inducer, antioxidant, anticarcinogenic. This paper reviews the involvement of melatonin in embryo fetal development. The pineal gland develops completely postpartum, so both the embryo and the fetus are dependent on the maternal mela...

Rape embryogenesis. III. Embryo development in time

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Teresa Tykarska

2014-01-01

Full Text Available It was found that the growth curve of the rape embryo axis is of triple sigmoid type. Embryo growth occurs in 3 phases corresponding to 3 different periods of development. Phase I includes growth of the apical cell up to it's division into two layers of octants. Phase II comprises the increase of the spherical proembryo to the change of its symmetry from radial to bilateral. Phase III includes, growth of the embryo from the heart stage up to the end of embryogenesis. In each phase the relative growth rate increases drastically and then diminishes. The differences in growth intensity during the same phase are several-fold. The growth intensity maximum of the embryo axis occurs in phase II. The phasic growth intensity maxima occur: in phase I during apical cell elongation, :before its division, and in phases II and III in the periods of cell division ;growth in globular and torpedo-shaped -shaped embryos.

Effects of UV-C irradiation on development of goldfish embryos

International Nuclear Information System (INIS)

Wu Jian; Dai Guifu; Zhang Fengqiu; Lu Lei

2005-01-01

Goldfish embryos at five different developmental stages, from fertilized eggs to heat beating stage, were irradiated by UV rays, and hatching rate, darkly pigmented eye rate and abnormal embryo rate of the irradiated embryos were investigated. Being subjected to very low amount (≤3 min.) of the UV irradiation, the embryos earlier than gastrula stage showed hormesis. However, the embryos at gastrula or heart beating stage were very sensitive to UV irradiation, showing just damage effect, which was very strong even at very low amount of the UV irradiation. The results also showed that development of the gastrula embryos irradiated by the UV rays stopped before darkly pigmented eye state, whereas embryos irradiated at heart beating stage by the UV rays could develop to the darkly pigmented eye stage, though they could not hatch out. (authors)

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

Assessing embryo development using swept source optical coherence tomography

Science.gov (United States)

Caujolle, S.; Cernat, R.; Silvestri, G.; Marques, M. J.; Bradu, A.; Feuchter, T.; Robinson, G.; Griffin, D.; Podoleanu, A.

2018-03-01

A detailed assessment of embryo development would assist biologists with selecting the most suitable embryos for transfer leading to higher pregnancy rates. Currently, only low resolution microscopy is employed to perform this assessment. Although this method delivers some information on the embryo surface morphology, no specific details are shown related to its inner structure. Using a Master-Slave Swept-Source Optical Coherence Tomography (SS-OCT), images of bovine embryos from day 7 after fertilization were collected from different depths. The dynamic changes inside the embryos were examined, in detail and in real-time from several depths. To prove our ability to characterize the morphology, a single embryo was imaged over 26 hours. The embryo was deprived of its life support environment, leading to its death. Over this period, clear morphological changes were observed.

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.

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.

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.

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.

Proteomics of desiccation tolerance during development and germination of maize embryos

DEFF Research Database (Denmark)

Huang, Hui; Møller, Ian Max; Song, Song-Quan

2012-01-01

Maize seeds were used to identify the key embryo proteins involved in desiccation tolerance during development and germination. Immature maize embryos (28N) during development and mature embryos imbibed for 72 h (72HN) are desiccation sensitive. Mature maize embryos (52N) during development...... pattern. We infer that these eleven proteins are involved in seed desiccation tolerance. We conclude that desiccation-tolerant embryos make more economical use of their resources to accumulate protective molecules and antioxidant systems to deal with maturation drying and desiccation treatment........ are desiccation tolerant. Thiobarbituric acid reactive substance and hydrogen peroxide contents decreased and increased with acquisition and loss of desiccation tolerance, respectively. A total of 111 protein spots changed significantly (1.5 fold increase/decrease) in desiccation-tolerant and -sensitive embryos...

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.

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

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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.

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)

Fruit, seed and embryo development of different cassava (Manihot ...

African Journals Online (AJOL)

Fruit, seed and embryo developments of different cassava (Manihot esculenta Crantz) genotypes, as well as embryo rescue, were investigated. The fruits of three genotypes after uncontrolled open pollination presented the same progressive development with similar sizes at different stages. There are large differences in ...

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.

Effect of embryo density on in vitro development and gene expression in bovine in vitro-fertilized embryos cultured in a microwell system.

Science.gov (United States)

Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Aikawa, Yoshio; Ohtake, Masaki; Matsuda, Hideo; Kobayashi, Shuji; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

2013-01-01

To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 μl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

The efficacy of the well of the well (WOW) culture system on development of bovine embryos in a small group and the effect of number of adjacent embryos on their development.

Science.gov (United States)

Kang, Sung-Sik; Ofuji, Sosuke; Imai, Kei; Huang, Weiping; Koyama, Keisuke; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

2015-06-01

The aim of the present study was to clarify the efficacy of the well of the well (WOW) culture system for a small number of embryos and the effect of number of adjacent embryos in a WOW dish on blastocyst development. In conventional droplet culture, embryos in the small-number group (5-6 embryos/droplet) showed low blastocyst development compared with a control group (25-26 embryos/droplet). However, small and large numbers of embryos (5-6 and 25 embryos, respectively) in a WOW dish showed no significant differences in cleavage, blastocyst rates, and mean cell number in blastocysts compared with the control group (25-30 embryos/droplet). In addition, the number of adjacent embryos in a WOW dish did not affect the development to blastocysts and cell number in blastocysts. In conclusion, a WOW dish can provide high and stable blastocyst development in small group culture wherever embryos are placed in microwells of the WOW dish.

Preimplantation development of embryos in women of advanced maternal age

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O. V. Chaplia

2014-04-01

Full Text Available In order to reveal the influence of genetic component on the early embryo development, the retrospective study of morphokinetic characteristics of 717 embryos subjected to preimplantation genetic testing was conducted. Blastomere biopsy for FISH-based preimplantation genetic screening of 7 chromosomes was performed on the third day of culture, while embryo developmental potential and morphological features at the cleavage and blastulation stage were studied regarding maternal age particularly in the group of younger women and patients older than 36. Results of genetic testing revealed that euploid embryos rate gradually decreased with maternal age comprising 39.9% in young women group and 25.3% of specimen belonging to elder patients. At the cleavage stage, morphological characteristics of aneuploid and euploid embryos didn’t differ significantly regardless of the age of patients that could be accounted for the transcriptional silence of embryo genome till the third day of its development. However, in case of prolonged culture chromosomally balanced embryos rarely faced developmental arrest (in 7.9% and formed blastocysts half more frequently compared to aberrant embryos (respectively 75.6 versus 49.8%. Nevertheless, no substantial difference was found between blastocyst formation rate among embryos with similar genetic component regardless of the maternal age. Taking into consideration high rate of chromosomally unbalanced embryos specific to patients of advanced maternal age, the relative proportion of aneuplouid blastocysts was significantly higher in this group of embryos. Thus, without genetic screening there is a possibility of inaccurate selection of embryos for women of advanced reproductive age for transfer procedure even in case of prolonged culture. Consequently, increase of aneuploid embryos frequency associated with permanent preimplantation natural selection effectiveness along with the postimplantation natural selection failure

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.

Influence of the radiation (Co60) in pre-implants rabbit embryos: effect on atypic mitotic index and embryo pole development

International Nuclear Information System (INIS)

Approbato, Mario S.; Oliveira Moura, Katia K.V. de; Souza Florencio, Rodopiano de; Garcia, Ricardo; Faria, Renato S.; Benedetti, Leonardo N.; Goulart, Flamarion B.

1995-01-01

We studied the effect of ionizing irradiation on 12 New Zealand rabbits (65 embryos), at three different times: at match time (zero hour), two days after and four days after, with two different irradiation doses: five c Gy and ten c Gy. Six rabbits (36 blastocysts) were used as controls. the matching instant was the zero hour. Exactly six days after (± 60 minutes) the embryos of each rabbit was picked up by flushing the uterus with culture media. the embryos were fixed in methanol for 48 hours, and colored with acid Mayer hematoxylin. The following embryo parameters were studied: embryo pole development; percentage of abnormal mitotic figures. irradiation time was associated with lower scores of embryo pole development, but not with irradiation dose. There were no gross abnormalities of embryo pole. The abnormal mitotic cells was affected both by the time and dose of irradiation. (author)

Protein Expression Landscape of Mouse Embryos during Pre-implantation Development

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Yawei Gao

2017-12-01

Full Text Available Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst. We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.

Effect of ambient light exposure of media and embryos on development and quality of porcine parthenogenetically activated embryos.

Science.gov (United States)

Li, Rong; Liu, Ying; Pedersen, Hanne Skovsgaard; Callesen, Henrik

2015-06-01

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time. In conclusion, exposure to ambient light can be harmful to embryo development, both when medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed for >1 h. In practice, it is therefore recommended to protect both culture medium and porcine embryos against ambient light during in vitro handling in the laboratory.

In vitro culture and somatic cell nuclear transfer affect imprinting of SNRPN gene in pre- and post-implantation stages of development in cattle

Directory of Open Access Journals (Sweden)

Goff Alan K

2009-02-01

Full Text Available Abstract Background Embryo in vitro manipulations during early development are thought to increase mortality by altering the epigenetic regulation of some imprinted genes. Using a bovine interspecies model with a single nucleotide polymorphism, we assessed the imprinting status of the small nuclear ribonucleoprotein polypeptide N (SNRPN gene in bovine embryos produced by artificial insemination (AI, in vitro culture (IVF and somatic cell nuclear transfer (SCNT and correlated allelic expression with the DNA methylation patterns of a differentially methylated region (DMR located on the SNRPN promoter. Results In the AI group, SNRPN maternal expression is silenced at day 17 and 40 of development and a third of the alleles analyzed are methylated in the DMR. In the IVF group, maternal transcripts were identified at day 17 but methylation levels were similar to the AI group. However, day-40 fetuses in the IVF group showed significantly less methylation when compared to the AI group and SNRPN expression was mostly paternal in all fetal tissues studied, except in placenta. Finally, the SCNT group presented severe loss of DMR methylation in both day-17 embryos and 40 fetuses and biallelic expression was observed in all stages and tissues analyzed. Conclusion Together these results suggest that artificial reproductive techniques, such as prolonged in vitro culture and SCNT, lead to abnormal reprogramming of imprinting of SNRPN gene by altering methylation levels at this locus.

Role of melatonin in embryo fetal development.

Science.gov (United States)

Voiculescu, S E; Zygouropoulos, N; Zahiu, C D; Zagrean, A M

2014-01-01

Melatonin is an indoleamine produced by the pineal gland and secreted in a circadian manner. In the past few decades, research over this topic has been enhanced. Melatonin has many important roles in the human physiology: regulator of the circadian rhythms, sleep inducer, antioxidant, anticarcinogenic. This paper reviews the involvement of melatonin in embryo fetal development. The pineal gland develops completely postpartum, so both the embryo and the fetus are dependent on the maternal melatonin provided transplacentally. Melatonin appears to be involved in the normal outcome of pregnancy beginning with the oocyte quality and finishing with the parturition. Its pregnancy night-time concentrations increase after 24 weeks of gestation, with significantly high levels after 32 weeks. Melatonin receptors are widespread in the embryo and fetus since early stages. There is solid evidence that melatonin is neuroprotective and has a positive effect on the outcome of the compromised pregnancies. In addition, chronodisruption leads to a reproductive dysfunction. Thus, the influence of melatonin on the developing human fetus may not be limited to the entertaining of circadian rhythmicity, but further studies are needed.

Expression of Aquaporins in Human Embryos and Potential Role of AQP3 and AQP7 in Preimplantation Mouse Embryo Development

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Yun Xiong

2013-05-01

Full Text Available Background/Aims: Water channels, also named aquaporins (AQPs, play crucial roles in cellular water homeostasis. Methods: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. Results: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. Conclusion: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

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

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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.

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

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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.

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

DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

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Christine Couldrey

Full Text Available Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT. Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5 during development of cattle generated either by artificial insemination (AI or in vitro fertilization (IVF and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic

Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

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Binder, N K; Evans, J; Gardner, D K; Salamonsen, L A; Hannan, N J

2014-10-10

Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and

Somatic embryogenesis in cell cultures of Glycine species.

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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.

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.

Beneficial effect of two culture systems with small groups of embryos on the development and quality of in vitro-produced bovine embryos.

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Cebrian-Serrano, A; Salvador, I; Silvestre, M A

2014-02-01

Currently, in vitro-produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one-third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU-IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF-ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF-ITS (EGF-ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF-ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF-ITS improved the embryo quality when smaller groups of embryos were cultured. © 2013 Blackwell Verlag GmbH.

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.

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

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...

The development of ovary in quail's embryo | Rong | African Journal ...

African Journals Online (AJOL)

The experiment was conducted to study the development of ovary in quails' embryos which were incubated for 4 to 17 days and incubated out for 1 day. The quails' embryos or gonads were cut out and HE staining was carried out. The results showed that when embryo was hatched for 4 days, lots of primordial germ cells ...

The effect of flurbiprofen on the development of anencephaly in early stage chicken embryos.

Science.gov (United States)

Özeren, Ersin; Er, Uygur; Güvenç, Yahya; Demirci, Adnan; Arıkök, Ata Türker; Şenveli, Engin; Ergün, Rüçhan Behzat

2015-04-01

The study investigated the effect of flurbiprofen on the development of anencephaly in early stage chicken embryos. We looked at four groups with a total of 36 embryos. There was a control group, a normal saline group, a normal-dose group and a high-dose group with ten, ten, eight and eight eggs with embryo respectively. Two embryos in the control group, studied with light microscopy at 48 h, were consistent with 28-29 hours' incubation in the Hamburger-Hamilton System. They had open neural tubes. The other embryos in this group were considered normal. One embryo in the normal saline group was on the occlusion stage at 48 h. One embryo showed an open neural tube. They were compatible with 28-29 hours' incubation in the Hamburger-Hamilton system. The remaining eight embryos showed normal development. In the normal dose group, one embryo showed underdevelopment of the embryonic disc and the embryo was dead. In four embryos, the neural tubes were open. One cranial malformation was found that was complicated with anencephaly in one embryo. In two embryos the neural tubes were closed, as they showed normal development, and they reached their expected stages according to the Hamburger-Hamilton classification. There was no malformation or growth retardation. Four experimental embryos were anencephalic in the high dose group, and three embryos had open neural tubes. One embryo exhibited both anencephaly and a neural tube closure defect. None of the embryos in this group showed normal development. Even the usual therapeutic doses of flurbiprofen increased the risk of neural tube defect. Flurbiprofen was found to significantly increase the risk of anencephaly. The provision of improved technical materials and studies with larger sample sizes will reveal the stage of morphological disruption during the development of embryos.

Embryo sac development in some representatives of the tribe Cynodonteae (Poaceae

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A. Strydom

1994-10-01

Full Text Available Chloris virgata Sw., Cynodon dactylon (L. Pers., Harpochloa falx (L. f. Kuntze, and Tragus berteronianus Schult. have a Polygonum type of embryo sac development. Unreduced embryo sacs were found in Eustachys paspaloides (Vahl Lanza & Mattei,  Harpochloa falx, and  Rendlia altera (Rendle Chiov. Both facultative and obligate apomixis were observed. The Hieracium type of embryo sac development was observed in the aposporic specimens.

Embryo sac development in some representatives of the tribe Cynodonteae (Poaceae)

OpenAIRE

A. Strydom; J. J. Spies

1994-01-01

Chloris virgata Sw., Cynodon dactylon (L.) Pers., Harpochloa falx (L. f.) Kuntze, and Tragus berteronianus Schult. have a Polygonum type of embryo sac development. Unreduced embryo sacs were found in Eustachys paspaloides (Vahl) Lanza & Mattei,  Harpochloa falx, and  Rendlia altera (Rendle) Chiov. Both facultative and obligate apomixis were observed. The Hieracium type of embryo sac development was observed in the aposporic specimens.

Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

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Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

2015-11-01

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

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

Long-distance transportation of primate embryos developing in culture: a preliminary study.

Science.gov (United States)

Nichols, Stephanie; Harvey, Alexandra; Gierbolini, Lynette; Gonzalez-Martinez, Janis; Brenner, Carol; Bavister, Barry

2010-03-01

Non-human primate embryos are invaluable for conducting research relevant to human infertility and stem cells, but their availability is restricted. In this preliminary study, rhesus monkey embryos were produced by IVF at the Caribbean Primate Research Centre and shipped in tubes of gassed culture medium within a battery-powered transport incubator by overnight courier to Wayne State University in Michigan. Upon arrival, the embryos were incubated in fresh culture medium to evaluate further development. In 11 shipments comprising 98 cleavage-stage embryos developing from oocytes that were mature (MII) upon collection, 51 (52%) reached advanced preimplantation stages (morula to hatched blastocyst) during prolonged culture following transportation. However, most embryos produced from oocytes that were immature (MI) at collection arrested and only 5/51 (10%) reached advanced stages of development. This study demonstrates that non-cryopreserved primate embryos can be routinely transported between distant sites without loss of developmental ability. In this way, the processes of production and study of non-cryopreserved primate embryos need not be restricted to the same or nearby laboratories. This will expand the use of these embryos for research and facilitate generation of translationally relevant information. Published by Elsevier Ltd.

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

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Xinyu Liu

Full Text Available Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF failure due to poor embryo quality.

Embryo splitting

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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.

Fibroblast growth factor signaling is required for early somatic gonad development in zebrafish.

Science.gov (United States)

Leerberg, Dena M; Sano, Kaori; Draper, Bruce W

2017-09-01

The vertebrate ovary and testis develop from a sexually indifferent gonad. During early development of the organism, primordial germ cells (the gamete lineage) and somatic gonad cells coalesce and begin to undergo growth and morphogenesis to form this bipotential gonad. Although this aspect of development is requisite for a fertile adult, little is known about the genetic regulation of early gonadogenesis in any vertebrate. Here, we provide evidence that fibroblast growth factor (Fgf) signaling is required for the early growth phase of a vertebrate bipotential gonad. Based on mutational analysis in zebrafish, we show that the Fgf ligand 24 (Fgf24) is required for proliferation, differentiation, and morphogenesis of the early somatic gonad, and as a result, most fgf24 mutants are sterile as adults. Additionally, we describe the ultrastructural elements of the early zebrafish gonad and show that distinct somatic cell populations can be identified soon after the gonad forms. Specifically, we show that fgf24 is expressed in an epithelial population of early somatic gonad cells that surrounds an inner population of mesenchymal somatic gonad cells that are in direct contact with the germ cells, and that fgf24 is required for stratification of the somatic tissue. Furthermore, based on gene expression analysis, we find that differentiation of the inner mesenchymal somatic gonad cells into functional cell types in the larval and early juvenile-stage gonad is dependent on Fgf24 signaling. Finally, we argue that the role of Fgf24 in zebrafish is functionally analogous to the role of tetrapod FGF9 in early gonad development.

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

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

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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.

Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

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Binghua Xue

Full Text Available Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

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

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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.

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos

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B Cisterna

2009-08-01

Full Text Available In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in premRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos.

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Cisterna, B; Flach, F; Vecchio, L; Barabino, S M L; Battistelli, S; Martin, T E; Malatesta, M; Biggiogera, M

2008-01-01

In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

The effect of unilateral ovariectomy on early embryonic survival and embryo development in rabbits

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R. Peiró

2014-06-01

Full Text Available Unilateral ovariectomy can be used to study uterine capacity in rabbits because an overcrowding of the functional uterine horn is produced. Due to the uterus duplex, the rabbit is the ideal model for such studies. However, this technique may affect embryo survival. The aim of this work is to study the effect of unilateral ovariectomy on early embryo survival and development in rabbit. A total of 101 unilateral ovariectomised females and 52 intact females were compared after slaughter at 30 h post-mating. Early embryo survival was estimated as the ratio between number of embryo recovered and ovulation rate. No differences were found between intact and unilaterally ovariectomised females in this trait. Unilateral ovariectomy did not change embryo development, measured as the number of embryo cells. Variability of embryo development was not affected either. At 30 h post-mating, the majority of embryos (86.2% were 4-cell stage. Embryo quality was evaluated according to morphological criteria. No difference in embryo quality between intact and unilaterally ovariectomised females was found. Therefore, unilateral ovariectomy performed before puberty in rabbit does not modify early embryo survival and development.

Selection for rapid embryo development correlates with embryo exposure to maternal androgens among passerine birds.

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Schwabl, Hubert; Palacios, Maria G; Martin, Thomas E

2007-08-01

Greater offspring predation favors evolution of faster development among species. We hypothesized that greater offspring predation exerts selection on mothers to increase levels of anabolic androgens in egg yolks to achieve faster development. Here, we tested whether (1) concentrations of yolk androgens in passerine species were associated with offspring predation and (2) embryo and nestling development rates were associated with yolk androgen concentrations. We examined three androgens that increase in potency along the synthesis pathway: androstenedione (A(4)) to testosterone (T) to 5 alpha -dihydrotestosterone (5 alpha -DHT). Concentrations of none of these steroids were related to clutch size; only A(4) was allometrically related to egg volume. Species that experience greater predation showed higher yolk concentrations of T and 5 alpha -DHT. Higher concentrations of T and particularly 5 alpha -DHT were strongly correlated with faster development during the embryo period and less so during the nestling period. Development rates were most strongly correlated with 5 alpha -DHT, suggesting that potency increases along the androgen synthesis pathway and that effects are mediated by the androgen receptor pathway. These results are consistent with the hypothesis that selection for faster development by time-dependent offspring mortality may be achieved epigenetically by varying embryo exposure to maternal anabolic steroids.

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

The Early Stages of Heart Development: Insights from Chicken Embryos

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Johannes G. Wittig

2016-04-01

Full Text Available The heart is the first functioning organ in the developing embryo and a detailed understanding of the molecular and cellular mechanisms involved in its formation provides insights into congenital malformations affecting its function and therefore the survival of the organism. Because many developmental mechanisms are highly conserved, it is possible to extrapolate from observations made in invertebrate and vertebrate model organisms to humans. This review will highlight the contributions made through studying heart development in avian embryos, particularly the chicken. The major advantage of chick embryos is their accessibility for surgical manipulation and functional interference approaches, both gain- and loss-of-function. In addition to experiments performed in ovo, the dissection of tissues for ex vivo culture, genomic, or biochemical approaches is straightforward. Furthermore, embryos can be cultured for time-lapse imaging, which enables tracking of fluorescently labeled cells and detailed analysis of tissue morphogenesis. Owing to these features, investigations in chick embryos have led to important discoveries, often complementing genetic studies in mice and zebrafish. As well as including some historical aspects, we cover here some of the crucial advances made in understanding early heart development using the chicken model.

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

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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.

[Effect of krypton-containing gas mixture on Japanese quail embryo development].

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Kussmaul', A R; Gur'eva, T S; Dadasheva, O A; Pavlov, N B; Pavlov, B N

2008-01-01

Investigated were effects of gas mixture with up to 3.0 kgs/cm2 of krypton on the embryonic development of domesticated Japanese quail (Coturnix coturnix japonica dom.). Results demonstrated absence of a serious krypton effect on Japanese quail embryos. Development of embryos proceeded in due course; morphometrically the experimental embryos were essentially similar to controls. It should be noted that despite exposure to acute hypoxic hypoxia during the initial 12 hours of development in the krypton-containing gas mixture, viability of quail embryos was high enough which can be ascribed to the krypton protective action. Besides, an additional experiment showed that krypton partial pressure of 5-5.5 kgs/cm2 produces the narcotic effect on adult Japanese quails.

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.

Cardio-respiratory development in bird embryos: new insights from a venerable animal model

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Warren W. Burggren

Full Text Available ABSTRACT The avian embryo is a time-honored animal model for understanding vertebrate development. A key area of extensive study using bird embryos centers on developmental phenotypic plasticity of the cardio-respiratory system and how its normal development can be affected by abiotic factors such as temperature and oxygen availability. Through the investigation of the plasticity of development, we gain a better understanding of both the regulation of the developmental process and the embryo's capacity for self-repair. Additionally, experiments with abiotic and biotic stressors during development have helped delineate not just critical windows for avian cardio-respiratory development, but the general characteristics (e.g., timing and dose-dependence of critical windows in all developing vertebrates. Avian embryos are useful in exploring fetal programming, in which early developmental experiences have implications (usually negative later in life. The ability to experimentally manipulate the avian embryo without the interference of maternal behavior or physiology makes it particularly useful in future studies of fetal programming. The bird embryo is also a key participant in studies of transgenerational epigenetics, whether by egg provisioning or effects on the germline that are transmitted to the F1 generation (or beyond. Finally, the avian embryo is heavily exploited in toxicology, in which both toxicological testing of potential consumer products as well as the consequences of exposure to anthropogenic pollutants are routinely carried out in the avian embryo. The avian embryo thus proves useful on numerous experimental fronts as an animal model that is concurrently both of adequate complexity and sufficient simplicity for probing vertebrate cardio-respiratory development.

Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.

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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.

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

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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.

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

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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.

Fertilization and Embryo Development of Fresh and Cryopreserved Sibling Oocytes

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Robert F. Casper

2010-01-01

Full Text Available Background: Oocyte cryopreservation is potentially the best way to preserve female fertility forunmarried women or young girls at risk of losing ovarian function. The aim of this study was tocompare fertilization and embryo development in frozen-thawed oocytes to their fresh siblings inwomen undergoing in vitro fertilization (IVF and embryo transfer (ET.Materials and Methods: Eleven infertile women undergoing infertility treatment, between theages of 24 to 37 years (mean ± SD = 31.6 ± 3.5, were included in this study. Mature oocytesfrom each patient were randomized into cryopreserved and fresh groups prior to intracytoplasmicsperm injection (ICSI. One hundred and thirty nine oocytes were retrieved, of which 105 were atmetaphase II (MII. Forty- five fresh MII oocytes were kept in culture whereas their sibling 60 MIIoocytes were cryopreserved using a slow cooling protocol. The frozen oocytes remained in LN2for 2 hours before thawing. ICSI was performed 1-2 hours after thawing for frozen oocytes and 4-5hours after retrieval for fresh oocytes. Fertilization and embryo development were compared.Results: Following thawing, 31 oocytes (51.6 % survived and 22 fertilized (79% while 32 freshoocytes fertilized upon ICSI (71%. The mean ± SE scores for embryos developing from frozenthawedoocytes were significantly lower at 48 and 72 hours post-ICSI than for embryos resultingfrom fresh oocytes (p<0.05.Conclusion: Our data demonstrated that oocyte freezing resulted in acceptable survival ratesfollowing cryopreservation, and similar fertilization rates following ICSI as compared to the freshsibling oocytes. However the number of blastomeres and the embryo quality on day three wassuperior in embryos from fresh oocytes when compared to the frozen oocytes.

THE BIOTECHNOLOGY OF EMBRYOGENIC CELL LINES OBTAINING AND PLANTLETS OF CONIFEROUS SPECIES IN SIBERIA IN CULTURE IN VITRO

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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

The Effects of Progesterone on Oocyte Maturation and Embryo Development

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Saeed Zavareh

2013-01-01

Full Text Available Oocyte maturation and embryo development are controlled by intra-ovarian factors suchas steroid hormones. Progesterone (P4 exists in the follicular fluid that contributes tonormal mammalian ovarian function and has several critical functions during embryodevelopment and implantation, including endometrial receptivity, embryonic survivalduring gestation and transformation of the endometrial stromal cells to decidual cells.It is well known that the physiological effects of P4 during the pre-implantation stages ofsome mammal’s embryos are mediated by P4 receptors and their gene expression is determined.The effects of P4 on oocytes and embryo development have been assessed bysome investigations, with contradictory results. P4, a dominant steroid in follicular fluidat approximately 18 hours after the luteinizing hormone (LH surge may have a criticalrole in maturation of oocytes at the germinal stage. However, it has been shown that differentconcentrations of P4 could not improve in vitro maturation rates of germinal vesicles(GV in cumulus oocyte complexes (COCs and cumulus denuded oocytes (CDOs.Culture media supplemented with P4 significantly improved mouse embryo development.In addition, an in vivo experimental design has shown high blastocyst survival andimplantation rates in P4-treated mice.In this review we explain some of the findings that pertain to the effects of P4 onoocyte maturation and embryo development both in vitro and in vivo.

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

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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

A cutin fluorescence pattern in developing embryos of some angiosperms

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Ewa Szczuka

2014-01-01

Full Text Available A cuticle visualized by auramine O fluorescence appears on the developing embryos of 9 species belonging to Cruciferae, Caryophyllaceae, Plantaginaceae, Linaceae and Papilionaceae. In the investigated species the formation and extent of fluorescing and non-fluorescing embryonic areas follow a similar pattern. At first the cutin fluorescing layer is formed on the apical part of the proembryo without delimited protoderm. This layer extends and at the late globular stage envelops the embryo proper, except for a cell adjoining the suspensor. Fluorescing cutin persists during the heart stage but disappears from the torpedo embryo. During these stages there is no cutine fluorescence on suspensorial cells. Continuous cutin fluorescence appears again on the surface of the whole embryo by the late torpedo stage. Then fluorescence disappears from the radicular part of U-shaped embryos, but persists on the shoot apex, cotyledons and at least on the upper part of hypocotyl. It is assumed that polarization and nutrition of the embryo may be influenced by cuticular changes.

Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds

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Miguel A. Ibeas

2017-12-01

Full Text Available Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

Chronology of early embryonic development and embryo uterine migration in alpacas.

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Picha, Y; Tibary, A; Memon, M; Kasimanickam, R; Sumar, J

2013-03-01

The objectives were to: (1) describe the chronology of early embryonic development from ovulation to entry into the uterus; and (2) to determine the timing of embryo migration to the left uterine horn when ovulation occurred from the right ovary. The experiment was conducted in Peru. Females (n = 132) were randomly assigned to 15 experimental groups. All females were mated to an intact male, given 50 μg GnRH im (Cystorelin) and ovulation time determined by transrectal ultrasonography, conducted every 6 hours, starting 24 hours postmating. Animals were slaughtered at a specific intervals postovulation and reproductive tracts were recovered and subjected to oviductal and uterine flushing for females slaughtered between 1 and 6 days postovulation (dpo; Day 0 = ovulation) and uterine flushing for females slaughtered from 7 to 15 dpo for recovery of oocytes/embryos. Season of mating did not influence the interval from mating to ovulation (winter: 29 ± 6 hours vs. summer: 30 ± 6 hours; P = 0.49). Ovulation rates for females mated during winter and summer were 92% versus 100%, respectively (P = 0.05). Fertilization rates for winter and summer mated females were 72% and 82% (P = 0.29). Unfertilized ova were not retained in the uterine tube. All embryos collected were in the uterine tube ipsilateral to the side of ovulation between 1 and 5 dpo. Embryos reached the uterus on 6 dpo. Embryos began to elongate on 9 dpo; at this time, 83% of embryos derived from right-ovary ovulations were collected from the left uterine horn. Embryos occupied the entire uterine cavity by 10 dpo. In conclusion, we characterized early embryo development and location of embryo during its early developmental stages in alpaca. This was apparently the first report regarding chronology of embryo development and migration to the left horn in alpaca which merits further investigation regarding its role in maternal recognition of pregnancy. Copyright © 2013 Elsevier Inc. All rights reserved.

Toxicity of beauvericin on porcine oocyte maturation and preimplantation embryo development

NARCIS (Netherlands)

Schoevers, Eric J; Santos, Regiane R; Fink-Gremmels, Johanna; Roelen, Bernard A J

2016-01-01

Beauvericin (BEA) is one of many toxins produced by Fusarium species that contaminate feed materials. The aim of this study was to assess its effects on porcine oocyte maturation and preimplantation embryo development. Cumulus-oocyte-complexes and developing embryos were exposed to BEA and cultured

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

Pro-apoptotic Effect of Pifithrin-α on Preimplantation Porcine Fertilized Embryo Development

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Brendan Mulligan

2012-12-01

Full Text Available The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-α (PFT-α, on preimplantation porcine in vitro fertilized (IVF embryo development in culture. Treatment of PFT-α was administered at both early (0 to 48 hpi, and later stages (48 to 168 hpi of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3, was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-α, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-α treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-α administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-α treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-α may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-α as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

The effect of insecticide Deltamethrin on development of chick embryos

International Nuclear Information System (INIS)

Al-Naal, R.; Bassal, M. Osman, M.

1997-04-01

This study was conducted to evaluate the cyto and the embryo toxicity of Deltamethrin and its commercial formulation DECIS 50 EC in chick embryo during its critical embryonic development period before and in the organogenesis. The embryos were incubated in well closed plastic caps containing the complete egg composition at 38 o. the Deltamethrin and DECIS were found to cause histological and morphological malformations, specially in the brain, also they reduced the majority of the synthetic activities of the DNA, RNA, and proteins in the embryonic and the vascular areas. The flow cytometric analysis showed alterations in frequency of cells in both embryonic and vascular areas in the treated embryo during the cell cycle phases. Our study also showed that the DECIS had greater cyto and embryo toxicity than the Seltamethrin for analysis (author). 149 refs., 36 figs., 16 tabs

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.

In vitro development rate of preimplantation rabbit embryos cultured with different levels of melatonin.

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Mehaisen, Gamal Mohamed Kamel; Saeed, Ayman Moustafa

2015-02-01

This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2-4 cells, 8-16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10(-3) M, 10(-6) M or 10(-9) M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10(-3) M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10(-3) M versus 10(-9) M, 10(-6) M and control, respectively, P< 0.05). At the morula stage, melatonin at 10-3 M significantly increased the in vitro development of embryos (92% for 10(-3) M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16-30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10(-6) M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10-3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

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.

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.

Optimized ex-ovo culturing of chick embryos to advanced stages of development.

Science.gov (United States)

Cloney, Kellie; Franz-Odendaal, Tamara Anne

2015-01-24

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

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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.

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.

Effect of pollen irradiation on hybridization of wheat and Eltrigia intermedia and their embryo development

International Nuclear Information System (INIS)

Li Guiying; Wang Linqing; Shi Jinguo

2005-01-01

Spikes of Eltrigia intermedia were radiated with 5-100 Gy γ-ray during anthesis, and then their pollens were collected to pollinate to the common wheat 'J-11' and 'Chinese Spring'. The effects of pollen irradiation on the seed setting, embryo development, embryo culture and plantlet rate were studied. The results showed that low dose (5-9 Gy) of irradiation enhanced the seed setting for Chinese spring x E. intermedia, but no such effect for J-11xE. intermedia. Irradiation with all doses damaged embryo development, percentage of seeds with embryos; rate of immature hybrid embryos developing into plantlets decreases with the increased doses. Percentage of seeds with abnormal embryos increased significantly with the doses. 12.9%-14.5% of embryos could develop into plants in 30 Gy treatment, which seldom occur to wheat. Embryos in 50 Gy-100 Gy treatment were affected so serious that even none of them could develop into plants in vitro culture. It may be an effective approach to obtaining more mutants and enhancing induced mutation breeding by using combination of pollen irradiation, immature embryo rescue and anther culture of the resultant progenies. (authors)

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.

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.

Phospholipid transfer activities in toad oocytes and developing embryos

International Nuclear Information System (INIS)

Rusinol, A.; Salomon, R.A.; Bloj, B.

1987-01-01

The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing 14 C-labeled phospholipids and 3 H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth

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).

Cucumber (Cucumis sativus L.) embryo development in situ after pollination with irradiated pollen

International Nuclear Information System (INIS)

Faris, N.M.; Niemirowicz-Szczytt, K.

1999-01-01

Embryological studies were undertaken to compare the normal development of cucumber endosperm and embryo with that observed after pollination with gamma-irradiated pollen (0.1 and 0.3 kGy). Delayed penetration of the pollen tube occurred at both irradiation doses. Endosperm and embryo development was also delayed, but was initiated within 6 days after pollination in 100% of embryo sacs at 0.1 kGy and in 70-80% at 0.3 kGy. Various abnormalities in endosperm and embryo cell structure confirmed progressive degeneration, which occurred earlier with the higher dose of irradiation. Degeneration increased dramatically; only 30-40% of the embryos reached the globular stage 15 days after pollination. (author)

Effects of N, N-dimethylglycine on the development of in vitro produced bovine embryos.

Science.gov (United States)

Takahashi, Toshikiyo; Itoh, Ryu; Nagai, Takashi

2009-06-01

This study investigated the effects of N, N-Dimethylglycine (DMG) on the development of in vitro produced (IVP) bovine embryos. IVP embryos were obtained by in vitro fertilization of in vitro matured oocytes for 6 h. In Experiment 1, IVP embryos were cultured in mSOFaa supplemented with bovine serum albumin but without glucose (SOF1) for 4 days, transferred to mSOFaa (with 5% fetal bovine serum and 1.5 mM glucose; SOF2) supplemented with 0 (control), 0.1,1 or 10 microM DMG and cultured for an additional 7 days (11 days in total) to assess their development in vitro. When cultured in the medium with 0.1 microM DMG, a significantly higher number of IVP embryos developed to the blastocyst and hatched blastocyst stages (40.3 and 40.8%, respectively) compared with the other groups (18.7-31.0% and 15.0-28.7%, respectively; PDMG for 4 days, transferred to SOF2 with or without 0.1 microM DMG and further cultured as in Experiment 1; DMG was added to either SOF1 or SOF2 and to both of them to assess its exposure effects on embryo development. When cultured continuously with DMG for 11 days, significantly higher rates of IVP embryos developed into blastocyst and hatched blastocyst stages (39.0 and 47.7%, respectively) compared with the other groups (31.0-32.2% and 29.5-31.0%, respectively; PDMG to IVC medium after 7 days of IVC. When DMG was added to IVC medium, the ratio of embryos developed to advanced developmental stages (No. of embryos developed to the blastocyst and expanded blastocyst stages/No. of embryos developed to the morula stage) was 28.7% (86/3) and 7 times higher than that of those cultured without DMG, 4.0% (52/13). These results suggest that addition of 0.1 microM DMG to mSOFaa during IVC of IVP bovine embryos has a promoting effect on their development.

Soybean roots retain the seed urease isozyme synthesized during embryo development

International Nuclear Information System (INIS)

Torisky, R.S.; Polacco, J.C.

1990-01-01

Roots of young soybean (Glycine max [L.] Merr.) plants (up to 25 days old) contain two distinct urease isozymes, which are separable by hydroxyapatite chromatography. These two urease species (URE1 and URE2) differ in: (a) electrophoretic mobility in native gels, (b) pH dependence, and (c) recognition by a monoclonal antibody specific for the seed (embryo-specific) urease. By these parameters root URE1 urease is similar to the abundant embryo-specific urease isozyme, while root URE2 resembles the ubiquitous urease which has previously been found in all soybean tissues examined (leaf, embryo, seed coat, and cultured cells). The embryo-specific and ubiquitous urease isozymes are products of the Eu1 and Eu4 structural genes, respectively. Roots of the eu1-sun/eu1-sun genotype, which lacks the embryo-specific urease (i.e. seed urease-null), contain no URE1 urease activity. Roots of eu4/eu4, which lacks ubiquitous urease, lack the URE2 (leaflike) urease activity. From these genetic and biochemical criteria, then, we conclude that URE1 and URE2 are the embryo-specific and ubiquitous ureases, respectively. Adventitious roots generated from cuttings of any urease genotype lack URE1 activity. In seedling roots the seedlike (URE1) activity declines during development. Roots of 3-week-old plants contain 5% of the total URE1 activity of the radicle of 4-day-old seedlings, which, in turn, has approximately the same urease activity level as the dormant embryonic axis. The embryo-specific urease incorporates label from [ 35 S]methionine during embryo development but not during germination, indicating that there is no de novo synthesis of the embryo-specific (URE1) urease in the germinating root

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

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

The influence of zygote pronuclear morphology on in vitro human embryo development

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Lidija Križančić-Bombek

2007-09-01

Full Text Available Background: The selection of embryos with largest implantation potential is an important part in assisted reproduction. Besides the embryo or blastocyst morphology, selection criteria such as position and orientation of pronuclei (PN in relation to polar body positioning and the number, size and distribution of nucleolar precursor bodies (NPB have been proposed. In our study, a correlation between PN and NBP morphology with the development of early embryos (day 2 of cultivation and blastocysts (day 5 was investigated.Methods: 653 zygotes from 113 IVF (in vitro fertilization and ICSI (intracytoplasmic sperm injection patients, younger than 40 years, were assessed 18–20 hours post-insemination. Optimal zygotes (Z1 had thouching centrally located PN with equall numbers of alligned NPB. Other zygote types differred from Z1 in having scattered NPB in both PN (Z2 or alligned NPB in one PN (Z3 or in PN beeing distant from one another (Z4. For each zygote type a percentage of normal early embryos and blastocysts was calculated.Results: Among 653 assessed zygotes 21.8 % were Z1; 29.1 % Z2, 34.6 % Z3 and 14.5 % Z4. The percentage of normal early embryos decreased from Z1 to Z4 zygote type (70.4 % vs. 55.3 % vs. 59.7 % vs.45.3 %; p < 0.05 as well as the percentage of developed blastocysts (63.4 % vs. 55.3 % vs. 58.8 % vs. 43.2 %. However, the percentages of optimal blastocysts in the four groups did not differ (11.3 % vs. 11.1 % vs. 8.4 % vs. 6.3 %.Conclusions: Best grade zygotes result in batter early embryo and blastocyst development suggesting that zygote morphology can be used in combination with embryo and/or blastocyst evaluation as a method for embryo selection prior to embryo transfer.

Characterization of the onset of embryonic control and early development in the bovine embryo

International Nuclear Information System (INIS)

Barnes, F.L.

1988-01-01

Bovine embryos were used to determine if morphological and molecular features of early development are similar to in vivo recovered bovine embryos and to determine at what level early bovine development is regulated. Radiolabeling of IVP embryos and in vivo recovered embryos with 35 S-methionine for SDS-polyacrylamide gel electrophoresis reveals that these embryos are equivalent. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between late 8-cells and morulae. This transition is α-amanitin sensitive therefore due to de novo embryonic transcription. Embryonic transcription is partially responsible for terminating the post-transcriptionally regulated period of early bovine development. Argentophillic nucleolar organizing regions (Ag-NORs) indicate onset of nucleolar activation. Ag-NORs were absent in 2- and 4-cell IVP embryos and rarely occurred in 8-cell IVP embryos cultured in vitro. IVP 1- and 2-cell embryos cultured to blastocysts in sheep oviducts demonstrated Ag-NORs. Thus the lack of nucleolar activation of IVP embryos cultured in vitro is culture induced between the 2- and 8-cell stage

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.)

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

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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.

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

Science.gov (United States)

van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

2017-11-25

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

Effect of ambient light exposure of media and embryos on development and quality of porcine parthenogenetically activated embryos

DEFF Research Database (Denmark)

Li, Rong; Liu, Ying; Callesen, Henrik

2015-01-01

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos...... was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased...... the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time...

Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model.

Science.gov (United States)

Bobek, Vladimir; Plachy, Jiri; Pinterova, Daniela; Kolostova, Katarina; Boubelik, Michael; Jiang, Ping; Yang, Meng; Hoffman, Robert M

2004-01-01

The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.

Bio-electrosprayed multicellular zebrafish embryos are viable and develop normally

International Nuclear Information System (INIS)

Clarke, Jonathan D W; Jayasinghe, Suwan N

2008-01-01

Bio-electrosprays are rapidly emerging as a viable protocol for directly engineering living cells. This communication reports the bio-electrospraying of multicellular organisms, namely zebrafish embryos. The results demonstrate that the bio-electrospray protocol fails to induce any embryological perturbations. In addition to analysing overall embryo morphology, we use transgenic embryos that express green fluorescent protein in specific brain neurons to determine that neuronal numbers and organization are completely normal. These results demonstrate that the bio-electrospraying protocol does not interfere with the complex gene regulation and cell movements required for the development of a multicellular organism. (communication)

Changes in RNA Splicing in Developing Soybean (Glycine max Embryos

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Delasa Aghamirzaie

2013-11-01

Full Text Available Developing soybean seeds accumulate oils, proteins, and carbohydrates that are used as oxidizable substrates providing metabolic precursors and energy during seed germination. The accumulation of these storage compounds in developing seeds is highly regulated at multiple levels, including at transcriptional and post-transcriptional regulation. RNA sequencing was used to provide comprehensive information about transcriptional and post-transcriptional events that take place in developing soybean embryos. Bioinformatics analyses lead to the identification of different classes of alternatively spliced isoforms and corresponding changes in their levels on a global scale during soybean embryo development. Alternative splicing was associated with transcripts involved in various metabolic and developmental processes, including central carbon and nitrogen metabolism, induction of maturation and dormancy, and splicing itself. Detailed examination of selected RNA isoforms revealed alterations in individual domains that could result in changes in subcellular localization of the resulting proteins, protein-protein and enzyme-substrate interactions, and regulation of protein activities. Different isoforms may play an important role in regulating developmental and metabolic processes occurring at different stages in developing oilseed embryos.

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.

Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

Energy Technology Data Exchange (ETDEWEB)

Osychenko, A A; Zalesskii, A D; Krivokharchenko, A S; Zhakhbazyan, A K; Nadtochenko, V A [N N Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow (Russian Federation); Ryabova, A V [A M Prokhorov General Physics Institute, Russian Academy of Sciences, Moscow (Russian Federation)

2015-05-31

Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated. (extreme light fields and their applications)

Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

Science.gov (United States)

Beck, Aaron P; Watt, Roland M; Bonner, Jennifer

2014-02-28

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Development of aromatic giant-embryo rice by molecular marker-assisted selection

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ZHU Yingdong

2013-12-01

Full Text Available Aromatic rice is loved for its distinctive aroma when cooking and eating.In this research,aromatic normal-embryo rice and non-aromatic giant-embryo rice,"Shangshida No.5",both bred by our laboratory,were selected as the parents for the hybridization.We used conventional breeding techniques as well as fragrance gene marker-assisted selection to derive new aromatic giant-embryo rice "Shangshida No.8".By comparing the agronomic and yield characters of "Shangshida No.5" and "Shangshida No.8",the average of filled grains per panicle of "Shangshida No.5" exceeds "Shangshida No.8" very significantly,while the average of effective panicles of "Shangshida No.8" is slightly more than "Shangshida No.5".Also,in the weight of thousand grains "Shangshida No.8" is slightly heavier than "Shangshida No.5".Thus,their grain weights per plant are close,29.10 g and 28.92 g respectively.By comparing the traits of rice embryo,there is no significant difference in embryo weight and volume.Also,there is no significant difference in weight ratio and volume ratio of embryo.This research has laid a solid foundation for further market development and application of aromatic giant-embryo rice.

Protein nutrition and metabolism during early development of the chick embryo

International Nuclear Information System (INIS)

Klein, N.W.

1976-01-01

Cultures of intact early chick embryos have been used as a model system in which to study the nutrition and metabolism of proteins during early embryonic development. Previous studies have shown that these embryos require nutrient proteins for growth and development. The protein requirement was found to be specific in that at least two proteins were essential; one a transferrin (either conalbumin or yolk transferrin) and the other either ovalbumin or lipovitellin. Variations in the quantity or type of protein provided in the medium altered the growth of embryo regions through regionally specific changes in protein breakdown. This was confirmed through protein synthetic studies with isolated polyribosomes. More recently such variations in protein nutrition have been shown also to affect the actual patterns of proteins synthesized by regions of the embryo. These observed responses to protein nutrition have been difficult to reconcile with our observation that proteins as such did not reach the embryo proper but were first degraded to amine acids within the yolk-sac membrane. Studies on the synthesis of serum proteins by the yolk-sac membrane have provided a possible explanation in that the relative synthesis of individual serum proteins was dramatically influenced by the protein composition of the culture medium. We are currently attempting to demonstrate that serum proteins are indeed the mediators of the response of embryos to protein nutrition. (author)

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.

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.

Endogenous electric fields in embryos during development, regeneration and wound healing

International Nuclear Information System (INIS)

Nuccitelli, R.

2003-01-01

All embryos that have been investigated drive ionic currents through themselves and these currents will generate internal electric fields. Here, those examples in which such fields have been measured directly are discussed. The first such measurements were made in chick embryos and about 20 mV mm -1 was measured near the posterier intestinal portal in 2-4-day-old embryos. This electric field is important for the development of tail structures because reducing its magnitude results in abnormal tail development. The second embryonic electric field measured directly was in the axolotl, where a rostral-caudal field of about the same magnitude was detected. Modification of this field during neurulation but not gastrulation caused developmental abnormalities. Most recently, the development of left-right asymmetry in frog and chick embryos was found to require a voltage difference between blastomeres at a very early developmental stage. This field was measured in the chick embryo to be 10-20 mV mm -1 across the primitive streak. Mammalian skin wounds generate 150 mV mm -1 fields lateral to the wound and corneal epidermal wounds exhibit lateral fields of 40 mV mm -1 . The presence of these endogenous fields would suggest that exposures to external electric fields should be limited to magnitudes of less than 0.1 V m -1 . (author)

Endogenous electric fields in embryos during development, regeneration and wound healing

Energy Technology Data Exchange (ETDEWEB)

Nuccitelli, R

2003-07-01

All embryos that have been investigated drive ionic currents through themselves and these currents will generate internal electric fields. Here, those examples in which such fields have been measured directly are discussed. The first such measurements were made in chick embryos and about 20 mV mm^-1 was measured near the posterier intestinal portal in 2-4-day-old embryos. This electric field is important for the development of tail structures because reducing its magnitude results in abnormal tail development. The second embryonic electric field measured directly was in the axolotl, where a rostral-caudal field of about the same magnitude was detected. Modification of this field during neurulation but not gastrulation caused developmental abnormalities. Most recently, the development of left-right asymmetry in frog and chick embryos was found to require a voltage difference between blastomeres at a very early developmental stage. This field was measured in the chick embryo to be 10-20 mV mm^-1 across the primitive streak. Mammalian skin wounds generate 150 mV mm^-1 fields lateral to the wound and corneal epidermal wounds exhibit lateral fields of 40 mV mm^-1. The presence of these endogenous fields would suggest that exposures to external electric fields should be limited to magnitudes of less than 0.1 V m^-1. (author)

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

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

Science.gov (United States)

de Vega-Bartol, José J; Simões, Marta; Lorenz, W Walter; Rodrigues, Andreia S; Alba, Rob; Dean, Jeffrey F D; Miguel, Célia M

2013-08-30

It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in

In vitro development of embryos from experimentally Kerack-addicted Mice

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Elham Mohammadzadeh

2017-08-01

Full Text Available Background: Prenatal drug exposure, as a common public health concern, is associated with an increased risk of adverse effects on early embryo development. Objective: To investigate the in vitro development of - embryo from experimentally Kerack-addicted mice. Materials and Methods: Twenty-five female mice were studied in five groups: control, vehicle, and three experimental groups of Kerack-dependent mice (I, II, and III which received different doses of Kerack for 14 days. After the establishment of addiction model (7 days, experimental groups I, II, and III were given Kerack intraperitoneally at the doses of 5, 35, and 70 mg/kg, twice a day for a period of 7 days, respectively. The vehicle group received normal saline and lemon juice whilst the control group just received water and food. Morulae were obtained through oviduct flashing. The survived embryos were cultured in T6+ 5mg/ml bovine serum albumin. The developmental rates up to hatched stage daily and embryo quality (differential staining and Tunnel staining were also assessed Results: The developmental potential of embryos obtained from the addicted mother was significantly decreased in comparison with control group. There was a significant reduction in the rate of blastocyst formation in the high dose Kerack dependent group. However, in addicted mice there was reduction in the total cell number (40.92% vs. 65.08% in control and, inner cell mass percentage (17.17% vs. 26.15% in control while apoptotic cells numbers were increased (7.17 vs. 1.46 in control (p<0.05. Conclusion: The Kerack addiction during pregnancy retards preimplantation development and induces apoptosis.

EFFECT OF GAMMA IRRADIATION ON THE GROWTH AND DEVELOPMENT OF SAGO PALM (Metroxylon sagu Rottb. CALLI

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Imron Riyadi

2017-01-01

Full Text Available The application of gamma irradiation on plant materials may increase the genetic variation of the offspring with useful traits. The experiment was conducted to determine the effect of irradiation dosage of gamma ray on growth and development of sago palm (Metroxylon sagu calli. Friable calli of sago palm derived from suspension culture were used as a material source. The primary calli were initiated from apical meristematic tissues of sago palm suckers of Alitir variety from Merauke, Papua. The treatments used were dosage of gamma ray irradiation at 0, 5, 10, 15, 20 and 25 Gy. The treated calli were then subcultured on modified Murashige and Skoog (MMS solid medium containing 3% sucrose and 0.1% activated charcoal and added with 1 mg l-1 2,4-D and 0.1 mg l-1 kinetin. The results showed that at all irradiation dosages, calli biomass increased significantly. The highest proliferation of calli biomass of 5.33 folds from the initial culture after 4 weeks was achieved at gamma irradiation of 25 Gy, whereas the lowest proliferation of calli biomass of 3.4 folds was achieved at control. The best development of embryogenic calli was obtained at 10 Gy that produced 100% somatic embryos, whereas the lowest somatic embryo formation at 0% was obtained at 0 and 25 Gy after one subculture. High response of somatic embryo induction to gamma irradiation at 10 Gy may increase production of somatic embryos. These results can be used in in vitro breeding of sago palm via mutagenesis to create new elite varieties.

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

. brizantha cv. Marandu do not have epiblast and are classified as Panicum-type. Under in vitro culture conditions, embryogenic calli and somatic embryos of U. brizantha cv. Marandu develop from meristematic cells of the scutellum.

Parental Overprotection Predicts the Development of Functional Somatic Symptoms in Young Adolescents

OpenAIRE

Janssens, Karin A. M.; Oldehinkel, Albertine J.; Rosmalen, Judith G. M.

2009-01-01

Objective To examine whether parental overprotection contributes to die development of functional somatic symptoms (FSS) in young adolescents. In addition, we aimed to study whether this potential effect of parental overprotection is mediated by parenting distress and/or moderated by the adolescent's sex. Study design FSS were measured in 2230 adolescents (ages 10 to 12 years from the Tracking Adolescents' Individual Lives Survey) by the Somatic Complaints subscale of the Youth Self Report at...

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...

Nickel affects gill and muscle development in oriental fire-bellied toad (Bombina orientalis) embryos

Energy Technology Data Exchange (ETDEWEB)

Park, Chan Jin; Song, Sang Ha; Kim, Dae Han; Gye, Myung Chan, E-mail: mcgye@hanyang.ac.kr

2017-01-15

Highlights: • Nickel inhibited the development of external gill in B. orientalis embryos. • The 168 h LC{sub 50} and EC{sub 50} values of nickel were 33.8 and 5.4 μM, respectively, in embryos. • Nickel induced abnormal tail development of embryos. • NF stage 26–31 was the most sensitive window for embryos to nickel exposure. • Nickel affected the calcium-dependent myogenic gene expression in embryos. - Abstract: The developmental toxicity of nickel was examined in the embryos of Bombina orientalis, a common amphibian in Korea. Based on a standard frog embryo teratogenesis assay, the LC{sub 50} and EC{sub 50} for malformation of nickel after 168 h of treatment were 33.8 μM and 5.4 μM, respectively. At a lethal concentration (100 μM), nickel treatment decreased the space between gill filaments and caused epithelial swelling and abnormal fusion of gill filaments. These findings suggest that nickel affects the functional development of gills, leading to embryonic death. At sublethal concentrations (1–10 μM), nickel produced multiple embryonic abnormalities, including bent tail and tail dysplasia. At 10 μM, nickel significantly decreased tail length and tail muscle fiber density in tadpoles, indicating inhibition of myogenic differentiation. Before hatching, the pre-muscular response to muscular response stages (stages 26–31) were the most sensitive period to nickel with respect to tail muscle development. During these stages, MyoD mRNA was upregulated, whereas myogenic regulatory factor 4 mRNA was downregulated by 0.1 μM nickel. Calcium-dependent kinase activities in muscular response stage embryos were significantly decreased by nickel, whereas these activities were restored by exogenous calcium. In tadpoles, 10 μM nickel significantly decreased the expression of the myosin heavy chain and the 12/101 muscle marker protein in the tail. Expression was restored by exogenous calcium. Our results indicate that nickel affects muscle development by

Regional localization of suspensor mRNAs during early embryo development.

Science.gov (United States)

Weterings, K; Apuya, N R; Bi, Y; Fischer, R L; Harada, J J; Goldberg, R B

2001-11-01

We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/beta-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote.

Regional Localization of Suspensor mRNAs during Early Embryo Development

Science.gov (United States)

Weterings, Koen; Apuya, Nestor R.; Bi, Yuping; Fischer, Robert L.; Harada, John J.; Goldberg, Robert B.

2001-01-01

We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/β-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote. PMID:11701878

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.

Impact of GnRH analogues on oocyte/embryo quality and embryo development in in vitro fertilization/intracytoplasmic sperm injection cycles: a case control study

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Rigó János

2009-09-01

Full Text Available Abstract Background Despite the clinical outcomes of ovarian stimulation with either GnRH-agonist or GnRH-antagonist analogues for in vitro fertilization (IVF being well analysed, the effect of analogues on oocyte/embryo quality and embryo development is still not known in detail. The aim of this case-control study was to compare the efficacy of a multiple-dose GnRH antagonist protocol with that of the GnRH agonist long protocol with a view to oocyte and embryo quality, embryo development and IVF treatment outcome. Methods Between October 2001 and December 2008, 100 patients were stimulated with human menopausal gonadotrophin (HMG and GnRH antagonist in their first treatment cycle for IVF or intracytoplasmic sperm injection (ICSI. One hundred combined GnRH agonist + HMG (long protocol cycles were matched to the GnRH antagonist + HMG cycles by age, BMI, baseline FSH levels and by cause of infertility. We determined the number and quality of retrieved oocytes, the rate of early-cleavage embryos, the morphology and development of embryos, as well as clinical pregnancy rates. Statistical analysis was performed using Wilcoxon's matched pairs rank sum test and McNemar's chi-square test. P Results The rate of cytoplasmic abnormalities in retrieved oocytes was significantly higher with the use of GnRH antagonist than in GnRH agonist cycles (62.1% vs. 49.9%; P Conclusion Antagonist seemed to influence favourably some parameters of early embryo development dynamics, while other morphological parameters seemed not to be altered according to GnRH analogue used for ovarian stimulation in IVF cycles.

Viability of bovine demi embryo after splitting of fresh and frozen thawed embryo derived from in vitro embryo production

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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.

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.

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.

The Well-of-the-Well system: an efficient approach to improve embryo development.

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Vajta, Gábor; Korösi, Tamás; Du, Yutao; Nakata, Kumiko; Ieda, Shoko; Kuwayama, Masashige; Nagy, Zsolt Peter

2008-07-01

Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.

Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

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Dawei Yu

Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality.

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Ricaurte Lopera-Vásquez

Full Text Available To evaluate the effect of conditioned media (CM and Extracellular Vesicles (EVs derived from bovine oviduct epithelial cell (BOEC lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E, together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5 EVs/mL, 1.5x10(5 EVs/mL or 7.5x10(4 EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2 and blastocyst development (Day 7-9 was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the

EMBRYO DEVELOPMENT OF YELLOWFIN TUNA (Thunnus albacares AT DIFFERENT INCUBATION TEMPERATURE

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Jhon Harianto Hutapea

2007-12-01

Full Text Available The experiment was conducted in order to figure out the effect of incubation temperature on embryonic development of yellowfin tuna, Thunnus albacares eggs. Five different incubation temperatures were applied as treatments, i.e.: 24°C, 26°C, 28°C, 30°C, and 32°C with 3 replicate each. Ten micro plates with lid (IWAKI, Japan were used; each has 6 well and 10 mL volumes. Five micro plates were used for experiment and five for balance on shaker. Three well of each micro plate were filled with 8 mL ultra violet sterilized sea water and 50 fertilized eggs. Temperature was set using Multi Thermo Incubator which has 5 level racks. Temperatures were set from the lowest to the highest on bottom to upper rack order. To maintain eggs dispersed in the medium, shaker on each rack was operated at 150 RPM. The embryo was monitored every 30-60 minutes depends on embryonic stage development using Microscope which was connected to Digital Camera DXM 1200F. Image analyses by Image Analyzer Program. The results showed, incubation temperature was significantly affect (P<0.05 embryonic development and hatching time of yellowfin tuna (Thunnus albacares eggs. Optimum incubation temperature for embryo development and hatching was 28°C. Decreased on incubation temperature slows down embryo development at all stages, and vice versa, increased on incubation temperature accelerates embryo development.

/sup 31/P nuclear-magnetic-resonance studies an the developing embryos of Xenopus laevis

Energy Technology Data Exchange (ETDEWEB)

Gadian, D G [Oxford Univ. (UK). Dept. of Biochemistry; Colman, A [Oxford Univ. (UK). Dept. of Zoology

1976-01-01

The concentrations of nucleoside triphosphate, inorganic phosphate and yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus laevis by /sup 31/P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5 - 4.5 nmol/embryo at least until the 'spontaneous movement' stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfertilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid-phosphate and protein-phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein-phosphate resonance disappears at a greater rate than the lipid-phosphate peak. The total phosphorus content of the embryo (ca. 200 nmol/embryo) is shown biochemically to remain constant during development; however, the total amount of phosphorus observed by NMR decreases by about 40% during development. From the resonance positions of their ..cap alpha.., ..beta.. and ..gamma.. phosphate groups is is deduced that the nucleoside triphosphate molecules are liganded in vivo to a divalent cation which is not manganese, but could be either magnesium or calcium. From the position of the inorganic phosphate resonance it is deduced that the internal pH of embryos where this resonance is evident is 6.8 +- 0.2.

Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring

DEFF Research Database (Denmark)

Ingerslev, Hans Jakob; Hindkjær, Johnny Juhl; Kirkegaard, Kirstine

2012-01-01

recently demonstrated to occur from first cleavage cycle in mice using time-lapse microscopy, with the largest impact on the pre-compaction stages. However, embryonic development in mice differs in many aspects from human embryonic development. The objective of this retrospective, descriptive study...... was to evaluate the influence of oxygen tension on human pre-implantation development using time-lapse monitoring. Materials and methods: Human embryos were cultured to the blastocyst stage in a time-lapse incubator (EmbryoScope™) in 20% O2 (group 1), 20% O2 for 24 hours followed by culture in 5% O2 (group 2......) or in 5% O2 (group 3). Eligible were patients with age 8 oocytes retrieved. Group 1 consisted of 120 IVF/ICSI embryos from 26 patients recruited to a study conducted to evaluate the safety of the time-lapse incubator by randomising 1:1 embryos from a patient to culture...

Rethinking In Vitro Embryo Culture: New Developments in Culture Platforms and Potential to Improve Assisted Reproductive Technologies1

Science.gov (United States)

Smith, Gary D.; Takayama, Shuichi; Swain, Jason E.

2011-01-01

ABSTRACT The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future. PMID:21998170

Differential Effect of Medium on the Ratio of ICM/TE of Bovine Embryos in a Co-culture System

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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.

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.

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

Development of the epaxial muscles in the human embryo

NARCIS (Netherlands)

Mekonen, Hayelom K.; Hikspoors, Jill P. J. M.; Mommen, Greet; Eleonore KÖhler, S.; Lamers, Wouter H.

2016-01-01

Although the intrinsic muscles of the back are defined by their embryological origin and innervation pattern, no detailed study on their development is available. Human embryos (5-10 weeks development) were studied, using Amira3D® reconstruction and Cinema4D® remodeling software for visualization.

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.

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

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.

HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

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Levesque-Tremblay, Gabriel; Müller, Kerstin; Mansfield, Shawn D; Haughn, George W

2015-03-01

Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy. © 2015 American Society of Plant Biologists. All Rights Reserved.

Exploring polyamines: Functions in embryo/fetal development

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Tarique Hussain

2017-03-01

Full Text Available Polyamines such as putrescine, spermidine, spermine and agmatine are aliphatic polycationic compounds present in all living cells, and are derived from amino acids, intestinal bacteria, exfoliated enterocytes and supported from diet. Polyamines as the key compounds play essential role in cell proliferation, growth and differentiation. They also exert significant effects on embryonic development, implantation, embryonic diapause, placentation, angiogensis and fetal development. This review paper summarizes the functions of polyamines and embryo/fetus development and its regulatory mechanism which should help to provide some evidences for clinic.

Sildenafil citrate (Viagra) impairs fertilization and early embryo development in mice.

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Glenn, David R J; McClure, Neil; Cosby, S Louise; Stevenson, Michael; Lewis, Sheena E M

2009-03-01

To determine the effects of sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor known to affect sperm function, on fertilization and early embryo cleavage. This acute mammal study included male and female mice assigned randomly, the females sacrificed after mating and their oocytes/embryos evaluated at four time periods after treatment. Academic research environment. Male and female CBAB(6) mice. Female mice were injected intraperitoneally with 5 IU gonadotropin (hCG) to stimulate follicular growth and induce ovulation. They were each caged with a male that had been gavaged with sildenafil citrate (0.06 mg/0.05 mL) and allowed to mate. After 12, 36, 60, and 84 h, females were killed, their oviducts were dissected out, and retrieved embryos were assessed for blastomere number and quality. Fertilization rates and numbers of embryos were evaluated after treatment. Fertilization rates (day 1) were markedly reduced (-33%) in matings where the male had taken sildenafil citrate. Over days 2-4, the numbers of embryos developing in the treated group were significantly fewer than in the control group. There was also a trend for impaired cleavage rates within those embryos, although this did not reach significance. The impairments to fertility caused by sildenafil citrate have important implications for infertility centers and for couples who are using this drug precoitally while attempting to conceive.

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.

Transcriptomic profiling of bovine IVF embryos revealed candidate genes and pathways involved in early embryonic development

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Yandell Brian S

2010-01-01

Full Text Available Abstract Background Early embryonic loss is a large contributor to infertility in cattle. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. The objective of this study was to identify genes differentially expressed between blastocysts and degenerative embryos at early stages of development. Results Using microarrays, genome-wide RNA expression was profiled and compared for in vitro fertilization (IVF - derived blastocysts and embryos undergoing degenerative development up to the same time point. Surprisingly similar transcriptomic profiles were found in degenerative embryos and blastocysts. Nonetheless, we identified 67 transcripts that significantly differed between these two groups of embryos at a 15% false discovery rate, including 33 transcripts showing at least a two-fold difference. Several signaling and metabolic pathways were found to be associated with the developmental status of embryos, among which were previously known important steroid biosynthesis and cell communication pathways in early embryonic development. Conclusions This study presents the first direct and comprehensive comparison of transcriptomes between IVF blastocysts and degenerative embryos, providing important information for potential genes and pathways associated with early embryonic development.

Vitamin D receptor signaling is required for heart development in zebrafish embryo

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Kwon, Hye-Joo, E-mail: hjkwon@pnu.edu.sa [Biology Department, Texas A& M University, College Station, TX77843-3258 (United States); Biology Department, Princess Nourah University, Riyadh 11671 (Saudi Arabia)

2016-02-12

Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects. Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development. - Highlights: • VDR signaling is involved in embryonic heart development. • Knockdown of vdrb, but not vdra, causes decreased heart rate in zebrafish embryo. • Loss of vdr results in cardiac laterality defects. • Loss of vdra/b alters atrioventricular boundary formation. • Loss of vdra/b causes abnormal cardiac looping.