Genetic transformation via Agrobacterium tumefaciens in embryogenic cell suspensions of the plantain hybrid cultivar FHIA 21 (AAAB

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Boris Chong

2002-01-01

Full Text Available The genetic improvement of plantain and banana is of great importance for its level of consumption on a world-wide scale. Genetic transformation constitutes one alternative for genetic improvement and has complemented traditional techniques. In this work some parameters of genetic transformation of plantain were studied by means of transient expression of â-glucoronidase in the hybrid cultivar FHIA-21(AAAB by Agrobacterium tumefaciens. A study with the β-Agrobacterium tumefaciens strains AT-2260 and EHA-105, both with the plasmid pCAMBIA-3301 was done. A comparison between the time of infection and time of co-culture was studied. The strain of better behavior was EHA-105. In the study of the time of infection and co-culture, the best combination resulted to be the one with two hours of infection and six days of co-culture. Key words: At-2260, EHA-105, β-glucoronidase, Musa

Agrobacterium tumefaciens-mediated transformation of biofuel plant ...

African Journals Online (AJOL)

Establishment of an efficient transformation system is a prerequisite for genetic improvement of Jatropha curcas, a promising biodiesel feedstock plant, by transgenic approach. In this study an efficient Agrobacterium-mediated transformation protocol using cotyledon explants from J. curcas seeds was developed.

Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

KAUST Repository

Kumar, Nitish

2010-07-01

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M NAA, and 0.25 mg l(-1) activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was

Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

KAUST Repository

Kumar, Nitish; Vijay Anand, K.G.; Pamidimarri, D.V.N. Sudheer; Sarkar, Tanmoy; Reddy, Muppala P.; Radhakrishnan, T.; Kaul, Tanushri; Reddy, M.K.; Sopori, Sudhir K.

2010-01-01

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M NAA, and 0.25 mg l(-1) activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was

Transformation of medicinal plants using Agrobacterium tumefaciens

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Katarzyna Bandurska

2016-12-01

Full Text Available For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall. This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.

Transformation of medicinal plants using Agrobacterium tumefaciens.

Science.gov (United States)

Bandurska, Katarzyna; Berdowska, Agnieszka; Król, Małgorzata

2016-12-20

For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall). This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.

Agrobacterium tumefaciens-mediated transformation for investigating pathogenicity genes of the phytopathogenic fungus Colletotrichum sansevieriae.

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Nakamura, Masayuki; Kuwahara, Hideto; Onoyama, Keisuke; Iwai, Hisashi

2012-08-01

Agrobacterium tumefaciens-mediated transformation (AtMT) has become a common technique for DNA transformation of yeast and filamentous fungi. In this study, we first established a protocol of AtMT for the phytopathogenic fungus Colletotrichum sansevieriae. Binary T-DNA vector containing the hygromycin B phosphotransferase gene controlled by the Aspergillus nidulans gpdA promoter and the trpC terminator was constructed with pCAMBIA0380 and used with three different strains LBA4404, GV3101, and GV2260 of A. tumefaciens. Transformants were most effectively obtained when GV2260 and C. sansevieriae Sa-1-2 were co-cultivated; there were about 320 transformants per 10(6) spores. When 1,048 transformants were inoculated on Sansevieria trifasciata, three transformants were found to have completely lost their pathogenicity and two transformants displayed reduced pathogenicity. All of the five transformants had a single copy of T-DNA in their genomes. The three pathogenicity-deficient transformants were subjected to thermal asymmetric interlaced polymerase chain reaction and the reaction allowed us to amplify the sequences flanking the left and/or right borders. The flanking sequences of the two transformants, M154 and M875, showed no homology to any sequences in databases, but the sequences of M678 contained motifs of alpha-1,3-glucan synthase, suggesting that the gene might contribute to the pathogenicity of C. sansevieriae. This study describes a useful method for investigating pathogenicity genes in C. sansevieriae.

Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens.

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Michelmore, R; Marsh, E; Seely, S; Landry, B

1987-12-01

Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R2 seedlings on media containing G418.

Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis.

Science.gov (United States)

Lu, Yuanyuan; Xiao, Shuqin; Wang, Fen; Sun, Jiaying; Zhao, Likun; Yan, Libin; Xue, Chunsheng

2017-02-01

An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis. Copyright © 2016 Elsevier B.V. All rights reserved.

Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens.

Science.gov (United States)

Manfroi, Ernandes; Yamazaki-Lau, Elene; Grando, Magali F; Roesler, Eduardo A

2015-12-01

Low transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciens overgrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future.

Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

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Wang Quan

2012-06-01

Full Text Available Abstract Background As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus produces many terpenoid indole alkaloids (TIAs, such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. Results To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS gene and a selectable marker neomycin phosphotransferase II gene (NTPII. The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC

Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation

DEFF Research Database (Denmark)

Frandsen, Rasmus John Normand; Frandsen, Mette; Giese, Nanna Henriette

2012-01-01

-step cloning strategies for construction of vectors for Agrobacterium tumefaciens-mediated transformation (ATMT). Targeted genome modifications require integration by a homologous double crossover event, which is achieved by placing target sequences on either side of a selection marker gene in the vector....... Protocols are given for two single-step vector construction techniques. The In-Fusion cloning technique is independent of compatible restriction enzyme sites in the vector and the fragment to be cloned. The method can be directly applied to any vector of choice and it is possible to carry out four fragment...... cloning without the need for subcloning. The cloning efficiency is not always as high as desired, but it still presents an efficient alternative to restriction enzyme and ligase-based cloning systems. The USER technology offers a higher four fragment cloning efficiency than In-Fusion, but depends...

Transgenic carnation plants obtained by Agrobacterium tumefaciens mediated transformation of petal explants

NARCIS (Netherlands)

Altvorst, van A.C.; Koehorst, H.; Jong, de J.; Dons, M.M.

1996-01-01

Transgenic carnation plants were obtained after infection of petal explants with the supervirulent Agrobacterium tumefaciens strain AGLO. Southern blot techniques confirmed the transgenic nature of four transformed plants. The expression of the gus gene was verified in these plants by histochemical

OPTIMIZATION OF FACTORS AFFECTING THE Agrobacterium tumefaciens- MEDIATED TRANSFORMATION OF Eucalyptus saligna

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Yohana de Oliveira-Cauduro

2018-02-01

Full Text Available ABSTRACT This study aimed to evaluate the effect of factors that may affect the genetic transformation of cotiledonary explants of Eucalyptus saligna mediated by EHA105 strain of Agrobacterium tumefaciens. The vector pBI121 carrying gus gene under control of 35S CaMV promoter was used. The effect of the following factors was evaluated: explant pre-culture, use of different antibiotics and presence of acetosyringone (AS in co-culture media. An antioxidant solution was also used during excision, containing ascorbic acid (250mg.L-1, citric acid (25mg.L-1 and PVP-40 (1g.L-1. Pre-culture of the explants before the co-culture with bacteria was done over a 4-day period in MS culture medium supplemented with 4.4µM BAP and 2.7ìM NAA. After theco-culture period, three concentrations of kanamycin (12.5;25 and 50mg.L-1 combined with 300mg.L-1 Augmentin® in the culture medium were tested The influence of the antibiotic was also evaluated by keeping the explants in a medium containing 50mg.L-1 Km and 300mg.L-1 Augmentin® or 500mg.L-1 cefotaxime. It was concluded that Augmentin® stimulates organogenesis, that a Km concentration of 12.5mg.L-1 allows selection of explants transformed with gus gene and, finally, the addition of AS (50ìM to the liquid and solid co-culture media has a positive effect on gus gene expression. Moreover, the use of an antioxidant solution during cotyledon excision is dispensable and the pre-culture of the explants has no effect on bud regeneration or gus gene expression. A transformation efficiency of 1.5% was reached.

Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

Directory of Open Access Journals (Sweden)

Ernandes Manfroi

2015-01-01

Full Text Available AbstractLow transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037. Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciensovergrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future.

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species.

Science.gov (United States)

Dai, Ziyu; Deng, Shuang; Culley, David E; Bruno, Kenneth S; Magnuson, Jon K

2017-08-01

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces

Genetic transformation of switchgrass.

Science.gov (United States)

Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu

2009-01-01

Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species

Energy Technology Data Exchange (ETDEWEB)

Dai, Ziyu; Deng, Shuang; Culley, David E.; Bruno, Kenneth S.; Magnuson, Jon K.

2017-06-19

Background: Because of interest in the production of renewable bio-hydrocarbon fuels, various living organisms have been explored for their potential use in producing fuels and chemicals. The oil-producing (oleaginous) yeast Lipomyces starkeyi is the subject of active research regarding the production of lipids using a wide variety of carbon and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements using the tools of synthetic biology and metabolic engineering. However, using these tools for strain improvement requires the establishment of effective and reliable transformation methods with suitable selectable markers (antibiotic resistance or auxotrophic marker genes) and the necessary genetic elements (promoters and terminators) for expression of introduced genes. Chemical-based methods have been published, but suffer from low efficiency or the requirement for targeting to rRNA loci. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. Results: In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species and that the introduced DNA can be reliably integrated into the chromosomes of these species. The gene deletion of Ku70 and Pex10 was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial -glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1 promoter was also stably expressed in seven different Lipomyces species. Conclusion: The results from this study clearly demonstrate that Agrobacterium-mediated transformation is a reliable genetic tool for gene deletion and integration and expression of heterologous genes in L. starkeyi and other Lipomyces species.

A Novel Phenolic Compound, Chloroxynil, Improves Agrobacterium-Mediated Transient Transformation in Lotus japonicus.

Science.gov (United States)

Kimura, Mitsuhiro; Cutler, Sean; Isobe, Sachiko

2015-01-01

Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 μM chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops.

Genetic transformation of carnation (Dianthus caryophylus L.).

Science.gov (United States)

Nontaswatsri, Chalermsri; Fukai, Seiichi

2010-01-01

This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

Agrobacterium tumefaciens mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

NARCIS (Netherlands)

Zheng, S.J.; Khrustaleva, L.I.; Henken, G.; Sofiari, E.; Jacobsen, E.; Kik, C.; Krens, F.A.

2001-01-01

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development

Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.

Science.gov (United States)

Song, Geun Cheol; Lee, Soohyun; Hong, Jaehwa; Choi, Hye Kyung; Hong, Gun Hyong; Bae, Dong-Won; Mysore, Kirankumar S; Park, Yong-Soon; Ryu, Choong-Min

2015-07-01

Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Genetic transformation of extremophilic fungi Acidea extrema and Acidothrix acidophila

Czech Academy of Sciences Publication Activity Database

Hršelová, Hana; Hujslová, Martina; Gryndler, Milan

2015-01-01

Roč. 60, č. 4 (2015), s. 365-371 ISSN 0015-5632 R&D Projects: GA ČR(CZ) GAP504/11/0484 Institutional support: RVO:61388971 Keywords : TUMEFACIENS-MEDIATED TRANSFORMATION * AGROBACTERIUM-TUMEFACIENS * FILAMENTOUS FUNGI Subject RIV: EE - Microbiology, Virology Impact factor: 1.335, year: 2015

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

Science.gov (United States)

Han, Guomin; Shao, Qian; Li, Cuiping; Zhao, Kai; Jiang, Li; Fan, Jun; Jiang, Haiyang; Tao, Fang

2018-05-01

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 10 6 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

Sonication assisted Agrobacterium -mediated transformation of ...

African Journals Online (AJOL)

In this study, a protocol was developed to obtain stable lines of the Spring Dendrobium cultivar 'Sanya' via sonication assisted Agrobacterium-mediated transformation (SAAT) of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strain LBA4404 was used with the binary vector AG205 containing the chalcone ...

Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

OpenAIRE

Nonaka, Satoko; Ezura, Hiroshi

2014-01-01

Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-pro...

Barley Transformation Using Agrobacterium-Mediated Techniques

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Harwood, Wendy A.; Bartlett, Joanne G.; Alves, Silvia C.; Perry, Matthew; Smedley, Mark A.; Leyland, Nicola; Snape, John W.

Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.

BINARY VECTOR CONSTRUCTION OF KAPPA(κ-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION

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Muh Alias L. Rajamuddin

2016-11-01

Full Text Available Increasing of kappa (κ-carrageenan content in Kappaphycus alvarezii seaweed is potentially be achieved by applying transgenesis technology. This study was performed to obtain a construction of  κ-Carrageenase gene and Agrobacterium tumefaciens to carry those construction genes.  The κ-Carrageenase (κ-Car gene was involved in κ-carrageenan biosynthesis. The κ-Car gene sequence was ligated between the 35S CaMV promoter and tNos terminator sequences to generate pMSH/κ-Car expression vector. Transformation of pMSH/κ-Car plasmid to Escherichia coli was performed by heat-shock method, and to Agrobacterium tumefaciens by tri-parental mating method. The results showed that several colonies of E. coli and A. tumefaciens grew in the selective culture mediums containing antibiotic. PCR analysis using primers 35S-Forward and tNos-Reverse with DNA template from those bacterial colonies resulted DNA fragment of about 2,000 bp, the same as the total length of 35S CaMV promoter, κ-Car gene and tNos terminator sequences. Therefore, the construction of pMSH/κ-Car gene was succeeded and a colony of A. tumefaciens transformant carrying pMSH/κ-Car plasmid was successfully produced.                                                                                   Keywords:  Agrobacterium tumefaciens, kappa(κ-Carrageenase gene, transgenesis, vector

Transgene expression in tick cells using agrobacterium tumefaciens

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Ticks transmit infectious diseases to humans and other animals. Genetic manipulation of these arthropods would allow the development of alternative disease control strategies. Interestingly, Agrobacterium tumefaciens (At) mediated T-DNA transfer has been recently shown to promote the genetic modific...

Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

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Ibrahim, Evra Raunie

2014-01-01

Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

Genetic Transformation of Metroxylon sagu (Rottb. Cultures via Agrobacterium-Mediated and Particle Bombardment

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Evra Raunie Ibrahim

2014-01-01

Full Text Available Sago palm (Metroxylon sagu is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L. Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance.

Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration

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Ningxia Du; Paula M. Pijut

2009-01-01

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion...

Identification of pathogenicity-related genes in the vascular wilt fungus verticillium dahliae by agrobacterium tumefaciens-mediated t-DNA insertional mutagenesis.

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Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that underpin pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transform...

Improvement of Agrobacterium-mediated transformation and rooting of black cherry

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Ying Wang; Paula M. Pijut

2014-01-01

An improved protocol for Agrobacterium-mediated transformation of an elite, mature black cherry genotype was developed. To increase transformation efficiency, vacuum infiltration, sonication, and a combination of the two treatments were applied during the cocultivation of leaf explants with Agrobacterium tumefaciens strain EHA105...

Robust regeneration protocol for the Agrobacterium tumefaciens mediated transformation of Solanum tuberosum

International Nuclear Information System (INIS)

Abbasi, A.; Bilal, M.; Hussain, J.; Shah, M. M.; Hassan, A.

2016-01-01

Plant genetic transformation requires robust regeneration system. Plant growth regulators (PGRs) such as cytokinins (CKs) play a pivotal role in organogenesis; however, CKs are the most expensive PGRs. In the current study, an efficient yet economical protocol for regeneration of potato plant was developed. Stem inter-nodal and leaf explants were cultured on different regeneration media supplemented with varying concentration of different CKs such as kinetin and zeatin. Murashige- Skoog media added with zeatin (1, 1.5 mg/L) was designated as RZ1, RZ1.5, respectively or kinetin (1.5, 2 mg/L) was designated as RK1.5 and RK2, respectively, however, concentrations of other hormones such as NAA (1-Naphthaleneacetic acid) and GA3 (Gibberellic acid A3) were kept same. RZ1 and RZ1.5 gave significantly better Results as compared to RK-type media in all aspects studied such as callus initiation, days to first shoot emergence, number of shoots per explants. RZ1 medium was then selected as regeneration media for Agrobacterium-mediated transformation of potato plants with cyanobacterial phosphoenol pyruvate carboxylase gene, which provided multiple putative transformants on selection media. The transformants were further confirmed through PCR. The current protocol is found to be cost effective and efficient for the regeneration of Solanum tuberosum and can be successfully implied for the Agrobacterium-mediated transformation. (author)

Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

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Michielse, C.B.; Ram, A.F.J.; Hooykaas, P.J.J.; Hondel, C.A.M.J.J. van den

2004-01-01

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins

AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION OF SORGHUM USING TISSUE CULTURE-BASED AND POLLEN-MEDIATED APPROACHES

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Elkonin L.A.

2012-08-01

Full Text Available Genetic transformation is a powerful tool for genetic improvement of arable crops. Genetic engineering approaches are especially important for modification of starch and protein contents, vitamin and micronutrient concentration, improvement of nutritive value of protein fractions, and increase tolerance to environmental stresses. Application of transgenic technologies for genetic improvement of sorghum, a highly productive heat tolerant and drought resistant crop, is extremely important since climate aridization in many regions all over the globe hampers sustainable production of traditional cereals, such as wheat, maize and barley. However, sorghum, in spite of great number of investigations, is one of the most recalcitrant crop species to genetic modification. The most frequently reported problems are a low frequency of transformation and silencing of transgenes. Using the A. tumefaciens strain AGL0/p35SGIB with the bar and gus-intron genes under the nos and CaMV35S promoters, respectively, we studied different methods of Agrobacterium-mediated genetic transformation of the grain sorghum: in vitro culture-based techniques, by inoculation of immature embryos or embryo-derived calli, and pollen-mediated approach, by inoculation of flowering panicles. Four lines of grain sorghum – Milo-10, [9E] Milo-10 (CMS-line, KVV-114, and KVV-45 – were used. In both approaches, for activation of vir-genes agrobacterial cell suspension was grown in the AB or modified AB media with acetosyringone at room temperature. In vitro culture approach was effective for obtaining transgenic plants in the lines Milo-10 and KVV-45, which were able to produce embryogenic callus from immature embryos after their co-cultivation with agrobacterial cell suspension. Callus cultures tolerant to glufosinate ammonium (GA and capable to plant regeneration were obtained. The frequency of immature embryos producing PCR-positive transgenic plants varied in different experiments

Genetic transformation of Eucalyptus camaldulensis by agrobalistic method

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Evânia Galvão Mendonça

2013-06-01

Full Text Available Eucalyptus stands in the setting of worldwide forestry due to its adaptability, rapid growth, production of high-quality and low cost of wood pulp fibers. The eucalyptus convetional breeding is impaired mainlly by the long life cycle making the genetic transformation systems an important tool for this purpose. However, this system requires in vitro eficient protocols for plant induction, regeneration and seletion, that allow to obtain transgenic plants from the transformed cell groups. The aim of this work was to evaluate the callus formation and to optimize the leaves and callus genetic transformation protocol by using the Agrobacterium tumefaciens system. Concerning callus formation, two different culture media were evaluated: MS medium supplemented with auxin, cytokinin (M1 and the MS medium with reduced nitrogen concentration and supplemented with auxin, cytokinin coconut water (M2. To establish the leave genetic transformation, those were exposed to agrobiolistics technique (gene gun, to tissue injury, and A. tumesfasciens EHA 105 contening the vetor pCambia 3301 (35S::GUS::NOS, for gene transference and to establish the callus transformation thoses were exposed only to A. tumefasciens. For both experiments, the influence of different infection periods was evaluated. The M2 medium provided the best values for callus sizea and fresh and dry weight. The leaves genetic transformation using the agrobiolistics technique was effective, the gus gene transient expression could be observed. No significant differences were obtained in the infection periods (4, 6 and 8 minutes. The callus genetic transformation with A. tumefaciens also promotend the gus gene transient expression on the callus co-cultiveted for 15 e 30 minutes. The transformed callus was transfered to a regeneration and selection medium and transformed plants were obtained.

Genetic transformation in two potato cultivars with T-DNA from disarmed Agrobacterium

NARCIS (Netherlands)

Ooms, G.; Burrell, M.M.; Karp, A.; Bevan, M.; Hille, J.

1987-01-01

Derivatives of potato (Solanum tuberosum cv.'s 'Maris Bard' and 'Desiree') transformed with disarmed T-DNA from genetically engineered Agrobacterium tumefaciens strains were isolated. The transformed plants were recovered from shoot-forming tumours induced by infection of wounds with mixed-cultures

Effective elimination of chimeric tissue in transgenics for the stable genetic transformation of lesquerella fendleri

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In order to improve the potential of Lesquerella fendleri as a valuable industrial oilseed crop, a stable genetic transformation system was developed. Genetic transformation was performed by inoculating leaf segments with an Agrobacterium tumefaciens strain AGL1 carrying binary vector pCAMBIA 1301.1...

Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene.

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Li, D D; Shi, W; Deng, X X

2003-12-01

Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.

Establishment of an Indirect Genetic Transformation Method for Arabidopsis thaliana ecotype Bangladesh

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Bulbul AHMED

2011-11-01

Full Text Available Arabidopsis thaliana is a small flowering plant belonging to the Brassicaceae family, which is adopted as a model plant for genetic research. Agrobacterium tumifaciensmediated transformation method for A. thaliana ecotype Bangladesh was established. Leaf discs of A. thaliana were incubated with A. tumefaciens strain LBA4404 containing chimeric nos. nptII. nos and intron-GUS genes. Following inoculation and co-cultivation, leaf discs were cultured on selection medium containing 50 mg/l kanamycin + 50 mg/l cefotaxime + 1.5 mg/l NAA and kanamycin resistant shoots were induced from the leaf discs after two weeks. Shoot regeneration was achieved after transferring the tissues onto fresh medium of the same combination. Finally, the shoots were rooted on MS medium containing 50 mg/l kanamycin. Incorporation and expression of the transgenes were confirmed by PCR analysis. Using this protocol, transgenic A. thaliana plants can be obtained and indicates that genomic transformation in higher plants is possible through insertion of desired gene. Although Agrobacterium mediated genetic transformation is established for A. thaliana, this study was the conducted to transform A. thaliana ecotype Bangladesh.

Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

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2011-01-01

Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated

Agrobacterium-mediated genetic transformation of Coffea arabica (L. is greatly enhanced by using established embryogenic callus cultures

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Lashermes Philippe

2011-05-01

Full Text Available Abstract Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%. At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our

Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes.

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Rudder, Steven; Doohan, Fiona; Creevey, Christopher J; Wendt, Toni; Mullins, Ewen

2014-04-07

Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT).

AGROBACTERIUM-MEDIATED TRANSFORMATION OF COMPOSITAE PLANTS. I. CONSTRUCTION OF TRANSGENIC PLANTS AND «HAIRY» ROOTS WITH NEW PROPERTIES

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N. A.Matvieieva

2013-02-01

Full Text Available The review explores some of the recent advances and the author's own researchs concerning biotechnological approaches for Agrobacterium tumefaciens- and A. rhizogenes-mediated transformation of Compositae family plants. This paper reviews the results of genetic transformation of Compositae plants, including edible (Cichorium intybus, Lactuca sativa, oil (Helianthus annuus, decorative (Gerbera hybrida, medical (Bidens pilosa, Artemisia annua, Artemisia vulgaris, Calendula officinalis, Withania somnifera etc. plant species. Some Compositae genetic engineering areas are considered including creation of plants, resistant to pests, diseases and herbicides, to the effect of abiotic stress factors as well as plants with altered phenotype. The article also presents the data on the development of biotechnology for Compositae plants Cynara cardunculus, Arnica montana, Cichorium intybus, Artemisia annua "hairy" roots construction.

A comparison study of Agrobacterium-mediated transformation methods for root-specific promoter analysis in soybean.

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Li, Caifeng; Zhang, Haiyan; Wang, Xiurong; Liao, Hong

2014-11-01

Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean. An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.

Agrobacterium-mediated transformation as a tool for functional genomics in fungi

NARCIS (Netherlands)

Michielse, C.B.; Hooykaas, P.J.J.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.

2005-01-01

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and

Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).

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Song, Guo-Qing; Sink, K C

2004-12-01

Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

Agrobacterium-mediated genetic transformation of Pogostemon cablin (Blanco) Benth. Using leaf explants: bactericidal effect of leaf extracts and counteracting strategies.

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Paul, Anamika; Bakshi, Souvika; Sahoo, Debee Prasad; Kalita, Mohan Chandra; Sahoo, Lingaraj

2012-04-01

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 μM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait.

Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

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Ravanfar, Seyed Ali; Aziz, Maheran Abdul; Saud, Halimi Mohd; Abdullah, Janna Ong

2015-11-01

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

Transformation of Mortierella alpina (fatty acid supplier myceliums via AMT system (Agrobacterium Mediated Transformation

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Aida Javanmard

2016-09-01

Full Text Available Introduction: Mortierella alpina is one of the most important fungi in food industry because of having ability of synthesizing unsaturated fatty acids, particularly Arashidonic Acid. This is a precursor of Eicosanoidregulate-lipoprotein metabolism which is involved in blood rheology, platelet activation and leukocyte-function, and the functional characteristics of the cell membrane. Materials and methods: In this study genetic transformation of M. alpina CBS754.68 fungus was evaluated via Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacteriums containing pBI121 vector were used for transformation of three days of old mycelia. Three days old hyphae were exposed to the bacteria with three level of time (one, two and three hours in the present of acetosyringone. Mitotic stability of the third generation of transgenic (T2 was confirmed by GUS assay and amplification of CaMV 35S promoter by polymerase chain reaction. Results: The highest percentage of transformation and mitotic stability were obtained by using A. tumefaciens and A. rhizogenese, respectively. Discussion and conclusion: The results showed that to obtain more efficient and more stable transformation, the fundamental factor is the use of suitable species of Agrobacterium. It is the first report for transformation of autothroph strain of M. alpine via Agrobacterium.

Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum.

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Zhang, Tao; Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

2015-08-01

The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by Pseudogymnoascus destructans (Pd). Recent studies indicated that apart from Pd, there is an abundance of other Pseudogymnoascus species in the hibernacula soil. We developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system for Pd and a related fungus Pseudogymnoascus pannorum (Pp) to advance experimental studies. URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular genetic analyses. A Uracil-Specific Excision Reagent (USER) Friendly pRF-HU2 vector containing Pd or Pp ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by electroporation and the resulting strains were co-cultivated with conidia of Pd or Pp for various durations and temperatures to optimize the ATMT system. Overall, 680 Pd (0.006%) and 1800 Pp (0.018%) transformants were obtained from plating of 10(7) conidia; their recoveries were strongly correlated with the length of the incubation period (96h for Pd; 72h for Pp) and with temperature (15-18°C for Pd; 25°C for Pp). The homologous recombination in transformants was 3.1% for Pd and 16.7% for Pp. The availability of a standardized ATMT system would allow future molecular genetic analyses of Pd and related cold-adapted fungi. Copyright © 2015. Published by Elsevier Inc.

T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus Transferencia de T-DNA de Agrobacterium tumefaciens al hongo ectomicorrícico Pisolithus microcarpus

Directory of Open Access Journals (Sweden)

A.G. Pardo

2005-06-01

Full Text Available The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.El hongo ectomicorrícico modelo Pisolithus microcarpus aislamiento 441 fue transformado utilizando Agrobacterium tumefaciens LBA 1100 y AGL-1. El marcador de selección fue el gen Shble de Streptoallotecius hidustanus, el cual confiere resistencia a fleomicina, bajo el control del promotor y terminador del gen gpd de Schizophyllum commune. La transformación resultó en clones resistentes a fleomicina comprobándose por PCR la presencia del transgen. La transferencia génica mediada por Agrobacterium podría permitir el desarrollo de la tecnología de interferencia por ARN en P. microcarpus.

Complementation of a threonine dehydratase-deficient Nicotiana plumbaginifolia mutant after Agrobacterium tumefaciens-mediated transfer of the Saccharomyces cerevisiae ILV1 gene.

OpenAIRE

Colau, D; Negrutiu, I; Van Montagu, M; Hernalsteens, J P

1987-01-01

The Saccharomyces cerevisiae ILV1 gene, encoding threonine dehydratase (EC 4.2.1.16) was fused to the transferred DNA nopaline synthase promoter and the 3' noncoding region of the octopine synthase gene. It was introduced, by Agrobacterium tumefaciens-mediated gene transfer, into an isoleucine-requiring Nicotiana plumbaginifolia auxotroph deficient in threonine dehydratase. Functional complementation by the ILV1 gene product was demonstrated by the selection of several transformed lines on a ...

An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.

Science.gov (United States)

Zhang, Jin jing; Shi, Liang; Chen, Hui; Sun, Yun qi; Zhao, Ming wen; Ren, Ang; Chen, Ming jie; Wang, Hong; Feng, Zhi yong

2014-01-01

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species. Copyright © 2014 Elsevier GmbH. All rights reserved.

Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene

NARCIS (Netherlands)

Heidekamp, F.; Dirkse, W.G.; Hille, J.; Ormondt, H. van

1983-01-01

The nucleotide sequence of the tmr gene, encoded by the octopine Ti plasmid from Agrobacterium tumefaciens (pTiAch5), was determined. The T-DNA, which encompasses this gene, is involved in tumor formation and maintenance, and probably mediates the cytokinin-independent growth of transformed plant

Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOGR1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens

Science.gov (United States)

Izawati, Abang Masli Dayang; Masani, Mat Yunus Abdul; Ismanizan, Ismail; Parveez, Ghulam Kadir Ahmad

2015-01-01

DOGR1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOGR1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l-1 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOGR1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOGR1 gene and 2-DOG for regenerating transgenic oil palm. PMID:26442041

Optimization of factors influencing microinjection method for Agrobacterium tumefaciens-mediated transformation of tomato.

Science.gov (United States)

Vinoth, S; Gurusaravanan, P; Jayabalan, N

2013-02-01

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L(-1) thidiazuron, 1.5 mg L(-1) indole-3-butyric acid, 30 mg L(-1) kanamycin, and 0-1.5 mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

Establishment of an efficient plant regeneration culture protocol and achievement of successful genetic transformation in Jatropha curcas L.

Science.gov (United States)

Liu, Ying; Liu, Guoxuan; Yang, Yali; Niu, Sufang; Yang, Fuguang; Yang, Shaoxia; Tang, Jianian; Chen, Jianping

2017-12-01

An efficient and reproducible protocol is described for shoot-bud regeneration and Agrobacterium tumefaciens-mediated genetic transformation of J. curcas. Treating the explants with high concentrations (5-120 mg/L) of TDZ for short durations (5-80 min) before inoculation culture increased significantly the regeneration frequency and improved the quality of the regenerated buds. The highest shoot-buds induction rate (87.35%) was achieved when petiole explants were treated with 20 mg/L TDZ solution for 20 min and inoculated on hormone-free MS medium for 30 days. Regenerated shoots of 0.5 cm or a little longer were isolated and grafted to seedling stocks of the same species, and then the grafted plantlets were planted on half-strength MS medium containing 0.1 mg/L IBA and 2 mg/L sodium nitroprusside (SNP). This grafting strategy was found to be very effective, to obtain that healthy grafted plantlets ready for acclimatization within 20 days. By the above mentioned protocol and with general Agrobacterium - mediated genetic transformation methods only 65 days were needed to obtain intact transgenic plants.

VirD2 of Agrobacterium tumefaciens : functional domains and biotechnological applications

NARCIS (Netherlands)

Kregten, Maartje van

2011-01-01

Agrobacterium tumefaciens is a pathogenic bacterium, which can genetically transform plants and other organisms. It does so by translocating a part of its DNA, the T-strand, in complex with the relaxase protein VirD2. We have shown that VirD2 is the determinant of translocation of the VirD2-T-strand

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

Science.gov (United States)

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

Directory of Open Access Journals (Sweden)

Hiroaki Mano

Full Text Available The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium. We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP. Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholinoethanesulfonic acid (MES buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max and pea (Pisum sativum. The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability*

Science.gov (United States)

Song, Zhang-yue; Tian, Jing-luan; Fu, Wei-zhe; Li, Lin; Lu, Ling-hong; Zhou, Lian; Shan, Zhi-hui; Tang, Gui-xiang; Shou, Hui-xia

2013-01-01

The Agrobacterium-mediated transformation system is the most commonly used method in soybean transformation. Screening of soybean genotypes favorable for Agrobacterium-infection and tissue regeneration is the most important step to establish an efficient genetic transformation system. In this study, twenty soybean genotypes that originated from different soybean production regions in China were screened for transient infection, regeneration capacity, and stable transgenic efficiency. Three genotypes, Yuechun 04-5, Yuechun 03-3, and Tianlong 1, showed comparable stable transgenic efficiencies with that of the previously reported American genotypes Williams 82 and Jack in our experimental system. For the Tianlong 1, the average stable transformation efficiency is 4.59%, higher than that of control genotypes (Jack and Williams 82), which is enough for further genomic research and genetic engineering. While polymerase chain reaction (PCR), LibertyLink strips, and β-glucuronidase (GUS) staining assays were used to detect the insertion and expression of the transgene, leaves painted with 135 mg/L Basta could efficiently identify the transformants. PMID:23549846

Relationship between expression level of hygromycin B-resistant gene and Agrobacterium tumefaciens-mediated transformation efficiency in Beauveria bassiana JEF-007.

Science.gov (United States)

Nai, Y S; Lee, M R; Kim, S; Lee, S J; Kim, J C; Yang, Y T; Kim, J S

2017-09-01

Agrobacterium tumefaciens-mediated transformation (AtMT) is an effective method for generation of entomopathogenic Beauveria bassiana transformants. However, some strains grow on the selective medium containing hygromycin B (HygB), which reduces the selection efficiency of the putative transformants. In this work, a relationship between HygB resistance gene promoter and AtMT efficiency was investigated to improve the transformant selection. Ten B. bassiana isolates were grown on 800 μg ml -1 HygB medium, but only JEF-006, -007 and -013 showed susceptibility to the antibiotics. Particularly, JEF-007 showed the most dose-dependent susceptibility. Two different Ti-Plasmids, pCeg (gpdA promoter based) and pCambia-egfp (CaMV 35S promoter based), were constructed to evaluate the promoters on the expression of HygB resistance gene (hph) at 100, 150 and 200 μg ml -1 HygB medium. Eight days after the transformation, wild type, AtMT/pCeg and AtMT/pCambia-egfp colonies were observed on 100 μg ml -1 HygB, but significantly larger numbers of colonies were counted on AtMT/pCeg plates. At higher HygB concentration (150 μg ml -1 ), only AtMT/pCeg colonies were further observed, but very few colonies were observed on the wild type and AtMT/pCambia-egfp plates. Putative transformants were subjected to PCR, RT-PCR and qRT-PCR to investigate the T-DNA insertion rate and gene expression level. Consequently, >80% of colonies showed successful AtMT transformation, and the hph expression level in AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies. In the HygB-susceptible B. bassianaJEF-007, gpdA promoter works better than CaMV 35S promoter in the expression of HygB resistance gene at 150 μg ml -1 HygB, consequently improving the selection efficiency of putative transformants. These results provide useful information for determining AtMT effectiveness in B. bassiana isolates, particularly antibiotic susceptibility and the role of promoters. © 2017 The

ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens

Czech Academy of Sciences Publication Activity Database

Bleho, J.; Obert, B.; Takáč, T.; Petrovská, Beáta; Heym, C.; Menzel, D.; Šamaj, J.

2012-01-01

Roč. 249, č. 1 (2012), s. 53-63 ISSN 0033-183X Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511 Keywords : Agrobacterium rhizogenes * Agrobacterium tumefaciens * Endoplasmic reticulum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.855, year: 2012

The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae.

Science.gov (United States)

Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Pham, Thu Ha; Phan, Tuan-Nghia; Tran, Van-Tuan

2016-12-01

Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

Science.gov (United States)

Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

1998-01-01

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

Effect of Pre-culture Irradiation and Explant Types on Efficiency of Brassica napus Genetic Transformation

International Nuclear Information System (INIS)

Amer, I.M.; Moustafa, H.A.M.; Azzam, C.R.

2008-01-01

The irradiated seeds of canola cv. Drakkar ( Brassica napus l. ) were germinated under aspect conditions, cotyledonary petioles and hypocotyl of 6 days old seedlings were used for Agrobacterium-mediated transformation. Agrobacterium tumefaciens has construct with the selectable marker gene (NPT II) and the desirable gene (HPPD). Direct and indirect shoot organogenesis were obtained from the both explants. Cotyledonary petioles was higher responded than hypocotyl with respective 26% and 14% of the explants producing NPT II-positive shoots after the selection on 50mg/l kanamycin. Calli might develop on and not in the agar medium were un transformation. This explains the higher number of escapes detected in hypocotyl explants than in experiments with cotyledons. The frequency of transformation plants as a function of indirect organogenesis was more than direct shoot regeneration from explants. The pre- irradiation with 75 Gy of gamma rays enhanced the genetic transformation frequencies by about 10 % as compared to that of the un-irradiated material. The obtained shoots were rooted and regenerated mature plants

An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii.

Science.gov (United States)

Pratheesh, P T; Vineetha, M; Kurup, G Muraleedhara

2014-06-01

Algal-based recombinant protein production has gained immense interest in recent years. The development of algal expression system was earlier hindered due to the lack of efficient and cost-effective transformation techniques capable of heterologous gene integration and expression. The recent development of Agrobacterium-mediated genetic transformation method is expected to be the ideal solution for these problems. We have developed an efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Pre-treatment of Agrobacterium in TAP induction medium (pH 5.2) containing 100 μM acetosyringone and 1 mM glycine betaine and infection of Chlamydomonas with the induced Agrobacterium greatly improved transformation frequency. This protocol was found to double the number of transgenic events on selection media compared to that of previous reports. PCR was used successfully to amplify fragments of the hpt and GUS genes from transformed cells, while Southern blot confirmed the integration of GUS gene into the genome of C. reinhardtii. RT-PCR, Northern blot and GUS histochemical analyses confirm GUS gene expression in the transgenic cell lines of Chlamydomonas. This protocol provides a quick, efficient, economical and high-frequency transformation method for microalgae.

Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity

Science.gov (United States)

Sarnatska, Veresa; Gladun, Hanna; Padalko, Svetlana

To investigate simulated microgravity (clinorotation) effect on plant cell transformation with Agrobacterium tumefaciens and crown gall formation, the culture of primary explants of potato and Jerusalem artichoke tubers was used. It is found that the efficiency of tumor formation and development in clinorotated explants are considerably reduced. When using the explants isolated from potato tubers clinorotated for 3, 5 and 19 days, drastic reduction of formation and development of crown gall tumors was observed. Conversely, the tumor number and their development increased when potato tubers were clinorotated for one day. As was estimated by us previously, cells of Jerusalem artichoke explants are the most sensitive to agrobacteria on 4-5 h of in vitro culturing and this time corresponds to the certain period of G1-stage of the cell cycle. We have also estimated that this period is characterized by the increase of binding of acridine orange by nuclear chromatin and increase in activity of RNA-polymerase I and II. Inoculation of explants with agrobacteria in this period was the most optimal for transformation and crown gall induction. We estimated that at four - hour clinorotation of explants the intensity of acridine orange binding to nuclei was considerably lower than on 4h in the control. At one-day clinorotation of potato tubers, a considerable increase in template accessibility of chromatin and in activity of RNA-polymerase I and II occurred. These results may serve as an evidence for the ability of plant dormant tissues to respond to microgravity. Another demonstration of dormant tissue response to changed gravity we obtained when investigating pathogenesis-related proteins (PR-proteins). PR-proteins were subjected to nondenaturing PAGE.and we have not found any effect of microgravity on PR-proteins of potato explants with normal or tumorous growth. We may suggest that such response derives from the common effects of two stress factors - wounding and changed

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.).

Science.gov (United States)

Han, J-S; Kim, C K; Park, S H; Hirschi, K D; Mok, I- G

2005-03-01

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

Agrobacterium-mediated transformation of watermelon ( Citrullus ...

African Journals Online (AJOL)

Transformation of watermelon (Citrullus lanatus cv. Zaojia) using Agrobacterium tumefaciens strain EHA105 containing the plasmid pRD400 carrying Pti4 gene was studied in this work. Proximal cotyledons as explants were pre-cultivated for two day in the dark and it was found that the best condition for transformation of ...

Transformation of different barley (Hordeum vulgare L.) cultivars by Agrobacterium tumefaciens infection of in vitro cultured ovules

DEFF Research Database (Denmark)

Holme, Inger Bæksted; Brinch-Pedersen, Henrik; Lange, Mette

2008-01-01

, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary...

An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae.

Science.gov (United States)

Wang, Sheng; Xing, Haiying; Hua, Chenlei; Guo, Hui-Shan; Zhang, Jie

2016-06-01

The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae.

SacB-SacR gene cassette as the negative selection marker to suppress Agrobacterium overgrowth in Agrobacterium-mediated plant transformation

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Yiming Liu

2016-10-01

Full Text Available Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transfor-mation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium sup¬plemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of Agrobacterium tumefaciens in the plant tissue culture process. We generated a mutant Agrobacterium tumefaciens strain GV2260 (recA-SacB/R that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcrip¬tion factor.

Differential roles of glucosinolates and camalexin at different stages of Agrobacterium-mediated transformation.

Science.gov (United States)

Shih, Po-Yuan; Chou, Shu-Jen; Müller, Caroline; Halkier, Barbara Ann; Deeken, Rosalia; Lai, Erh-Min

2018-03-02

Agrobacterium tumefaciens is the causal agent of crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. Agrobacterium tumefaciens is capable of transferring its T-DNA into different plant parts at different developmental stages for transient and stable transformation. However, the plant genes and mechanisms involved in these transformation processes are not well understood. We used Arabidopsis thaliana Col-0 seedlings to reveal the gene expression profiles at early time points during Agrobacterium infection. Common and differentially expressed genes were found in shoots and roots. A gene ontology analysis showed that the glucosinolate (GS) biosynthesis pathway was an enriched common response. Strikingly, several genes involved in indole glucosinolate (iGS) modification and the camalexin biosynthesis pathway were up-regulated, whereas genes in aliphatic glucosinolate (aGS) biosynthesis were generally down-regulated, on Agrobacterium infection. Thus, we evaluated the impacts of GSs and camalexin during different stages of Agrobacterium-mediated transformation combining Arabidopsis mutant studies, metabolite profiling and exogenous applications of various GS hydrolysis products or camalexin. The results suggest that the iGS hydrolysis pathway plays an inhibitory role on transformation efficiency in Arabidopsis seedlings at the early infection stage. Later in the Agrobacterium infection process, the accumulation of camalexin is a key factor inhibiting tumour development on Arabidopsis inflorescence stalks. In conclusion, this study reveals the differential roles of GSs and camalexin at different stages of Agrobacterium-mediated transformation and provides new insights into crown gall disease control and improvement of plant transformation. © 2018 THE AUTHORS. MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD.

Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.

Science.gov (United States)

Sparks, Caroline A; Doherty, Angela; Jones, Huw D

2014-01-01

The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.).

Study on rice transformation mediated by low energy ion beam implantation

International Nuclear Information System (INIS)

Li Hong; Wu Lifang; Yu Zengliang

2001-01-01

Delivery of foreign DNA into rice via ion beam was first reported in 1994. In recent years we have aimed to set up efficient transformation system mediated by low energy ion beam. The factors that influence the transformation including type of ion, parameters of ion energy, dose and dose rate, plant genotype, composition of media, concentration of hormones and antibiotics were carefully investigated. Treated with 25ke V Ar + , the transformation efficiencies of the mature embryos of rice variety 02428, Hua pei94-jian-09 and Minghui63 reached 11%, 11.4% and 7.1% measured by produced antibiotic resistant callus and l.52%, 1.87% and l.13% measured by regenerated plants respectively. PCR detection and Southern blot analysis showed that GUS report gene had inserted in rice genome. Low energy ion beam mediated gene transfer will be extended to other cereal recalcitrant to Agrobacterium tumefaciens as soon as methodological parameters were optimized. (authors)

Effect of gamma irradiation on Agrobacterium-mediated genetic transformation of Japanese lawngrass (Zoysia japonica Steud.)

International Nuclear Information System (INIS)

Zhang Lei; Anhui Agricultural Univ., Hefei; Hu Fanrong; Zhang Linlin; Wang Xueyan; Wu Dianxing; Ma Chuanxi

2004-01-01

The effects of gamma irradiation on Agrobacterium-mediated genetic transformation were investigated in the current paper, using embryonic calli derived from the mature seeds of Japanese lawngrass (Zoysia japonica Steud.). The result indicated that the GUS transient expression rates were enhanced with the increasing doses when treated by doses lower than 4 Gy, however it would be decreased when treated by doses higher than 4 Gy. Based on the survival rate and GUS transient expression rate, 2 Gy is the optimal dose for Agrobacterium-mediated genetic transformation. Further observation found that 36 hours reculture after gamma irradiation is the most appropriate for agrobacterium infection. (authors)

Methods for genetic transformation of filamentous fungi.

Science.gov (United States)

Li, Dandan; Tang, Yu; Lin, Jun; Cai, Weiwen

2017-10-03

Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of genetic transformation techniques is a precondition that enables scientists to target and modify genes efficiently and may reveal the function of target genes. The method to deliver foreign nucleic acid into cells is the sticking point for fungal genome modification. Up to date, there are some general methods of genetic transformation for fungi, including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation. This article reviews basic protocols and principles of these transformation methods, as well as their advantages and disadvantages.

Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White)

Science.gov (United States)

Conditions were optimized for transient transformation of Lilium longiflorum cv. Nellie White using Agrobacterium tumefaciens. Bulb scale and basal meristem explants were inoculated with A. tumefaciens strain AGL1 containing the binary vector pCAMBIA 2301 which has the uidA gene that codes for ß-gl...

Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes

OpenAIRE

Aarrouf, Jawad; Mallard, Stephanie; Caromel, Bernard; Lizzi, Y.; Lefebvre, Véronique

2012-01-01

Pepper is known to be a recalcitrant species to genetic transformation via Agrobacterium tumefaciens. A. rhizogenes-mediated transformation offers an alternative and rapid possibility to study gene functions in roots. In our study, we developed a new and efficient system for A. rhizogenes transformation of the cultivated species Capsicum annuum. Hypocotyls and foliar organs (true leaves and cotyledons) of Yolo Wonder (YW) and Criollo de Morelos 334 (CM334) pepper cultivars were inoculated wit...

Production of herbicide-resistant coffee plants (Coffea canephora P.) via Agrobacterium tumefaciens-mediated transformation

OpenAIRE

Ribas, Alessandra Ferreira; Kobayashi, Adilson Kenji; Pereira, Luiz Filipe Protasio; Vieira, Luiz Gonzaga Esteves

2006-01-01

Transgenic plants of Coffea canephora P. resistant to the herbicide ammonium glufosinate were regenerated from leaf explants after co-culture with Agrobacterium tumefaciens strain EHA105 harboring pCambia3301, a plasmid that contains the bar and the uidA genes both under control of 35S promoter. Direct somatic embryogenesis was induced on basal medium contained ¼ strength macro salts and half strength micro salts of MS medium, organic constituents of B5 medium and 30 g.L-1 sucrose supp...

Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.

Science.gov (United States)

Tang, Wei; Newton, Ronald J; Weidner, Douglas A

2007-01-01

An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies.

Conversion of BAC Clones into Binary BAC (BIBAC) Vectors and Their Delivery into Basidiomycete Fungal Cells Using Agrobacterium tumefaciens

KAUST Repository

Ali, Shawkat

2014-09-19

The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.

Efficient transformation and expression of gfp gene in Valsa mali var. mali.

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Chen, Liang; Sun, Gengwu; Wu, Shujing; Liu, Huixiang; Wang, Hongkai

2015-01-01

Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.

Establishment of an efficient transformation system for Pleurotus ostreatus.

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Lei, Min; Wu, Xiangli; Zhang, Jinxia; Wang, Hexiang; Huang, Chenyang

2017-11-21

Pleurotus ostreatus is widely cultivated worldwide, but the lack of an efficient transformation system regarding its use restricts its genetic research. The present study developed an improved and efficient Agrobacterium tumefaciens-mediated transformation method in P. ostreatus. Four parameters were optimized to obtain the most efficient transformation method. The strain LBA4404 was the most suitable for the transformation of P. ostreatus. A bacteria-to-protoplast ratio of 100:1, an acetosyringone (AS) concentration of 0.1 mM, and 18 h of co-culture showed the best transformation efficiency. The hygromycin B phosphotransferase gene (HPH) was used as the selective marker, and EGFP was used as the reporter gene in this study. Southern blot analysis combined with EGFP fluorescence assay showed positive results, and mitotic stability assay showed that more than 75% transformants were stable after five generations. These results showed that our transformation method is effective and stable and may facilitate future genetic studies in P. ostreatus.

Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.

Science.gov (United States)

Tang, Wei; Lin, Jinxing; Newton, Ronald J

2007-05-01

Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.

Regeneration and Agrobacterium -mediated transformation studies ...

African Journals Online (AJOL)

Leaf explants of carnation (Dianthus caryophyllus L. cv. Turbo) were used for the transformation of gene performed by the EHA 105 strain of Agrobacterium tumefaciens harboring the binary vector, pGA482GG. This vector carries the marker genes, neomycin phosphotansferase II (npt II) that determine resistance to ...

In planta transformation method for T-DNA transfer in orchids

Science.gov (United States)

Semiarti, Endang; Purwantoro, Aziz; Mercuriani, Ixora S.; Anggriasari, Anida M.; Jang, Seonghoe; Suhandono, Sony; Machida, Yasunori; Machida, Chiyoko

2014-03-01

Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro∷PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1α2 pro∷GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.

Optimization of genetic transformation protocol mediated by biolistic ...

African Journals Online (AJOL)

Abhay Kumar

2013-02-06

Feb 6, 2013 ... We report here an efficient genotype-independent genetic transformation system in wheat. Highly regenerable embryogenic calli obtained from mature seeds were employed as the target tissue for the genetic transformation of three bread wheat varieties viz C306, HDR77 and PBW343 representing.

Agrobacterium mediated transformation of Tunisian Cucumis melo ...

African Journals Online (AJOL)

Transgenic Cucumis melo cv. Maazoun containing the neomycin phosphotransferase II (NPT II) chimeric gene conferring resistance to kanamycin were obtained from cotyledons explants inoculated with Agrobacterium tumefaciens (GV3101) that contained the binary vector plasmid pADI. Transformed shoots were obtained ...

Coat protein-mediated resistance against an Indian isolate of the ...

Indian Academy of Sciences (India)

Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were ...

(IS136) of Agrobacterium tumefaciens

Indian Academy of Sciences (India)

Agrobacterium strains harbour insertion sequences, which are known to transpose into genomes as well as into Ti plasmids. In this study we report the inactivation of a transgene due to transposition of the A. tumefaciens insertion sequence IS136. The transposition was discovered following transformation of plant tissues, ...

Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).

Science.gov (United States)

Mhatre, Minal

2013-01-01

Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern.

Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.

Science.gov (United States)

Yadav, Reena; Mehrotra, Meenakshi; Singh, Aditya K; Niranjan, Abhishek; Singh, Rani; Sanyal, Indraneel; Lehri, Alok; Pande, Veena; Amla, D V

2017-01-01

Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99 % inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014 μg/g), chlorogenic acid (0.0978 ± 0.0046 μg/g), and quercetin (0.0971 ± 0.0046 μg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8 h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.

An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars.

Science.gov (United States)

Shri, Manju; Rai, Arti; Verma, Pankaj Kumar; Misra, Prashant; Dubey, Sonali; Kumar, Smita; Verma, Sikha; Gautam, Neelam; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

2013-04-01

Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of L-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD600 nm = 0.5-0.8) promoted the highest frequency of transformation (83.04 %) in medium containing L-cysteine (400 mg l(-1)). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of L-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.

Efficient and genotype-independent Agrobacterium--mediated tomato transformation.

Science.gov (United States)

Park, Sung Hun; Morris, Jay L; Park, Jung Eun; Hirschi, Kendal D; Smith, Roberta H

2003-10-01

An efficient method to transform five cultivars of tomato (Lycopersicon esculentum), Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported. A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and on 11 different media with cocultivation. Although all cultivars and explants formed callus and regenerated on the initial 7 media, cocultivation with A. tumefaciens significantly reduced the callus induction and regeneration. From these experiments, a transformation methodology using either hypocotyls or cotyledons cultured for one day on BA 1 mgL-1, NAA 0.1 mgL-1 and 3 days cocultivation with the Agrobacterium on this same medium followed by a transfer to a medium with zeatin 2 mgL-1 and IAA 0.1 mgL-1 for 4-6 weeks resulted in a greater than 20% transformation frequency for all five cultivars tested. In this transformation method, no feeder layers of tobacco, petunia or tomato suspension cultures were used, and the subculture media was minimal. Stable integration and transmission of the transgene in T1 generation plants were confirmed by Southern blot analysis. This procedure represents a simple, efficient and general means of transforming tomato.

Functions and regulation of quorum-sensing in Agrobacterium tumefaciens

Directory of Open Access Journals (Sweden)

Denis eFaure

2014-01-01

Full Text Available In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS. The determinants of the QS-system belong to the LuxR/LuxI class. The LuxI-like protein TraI synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last twenty years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/LuxI type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids.

Optimization of Agrobacterium-Mediated Transformation in Soybean

Science.gov (United States)

Li, Shuxuan; Cong, Yahui; Liu, Yaping; Wang, Tingting; Shuai, Qin; Chen, Nana; Gai, Junyi; Li, Yan

2017-01-01

High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide

Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.

Science.gov (United States)

González-Mula, Almudena; Lang, Julien; Grandclément, Catherine; Naquin, Delphine; Ahmar, Mohammed; Soulère, Laurent; Queneau, Yves; Dessaux, Yves; Faure, Denis

2018-07-01

Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Optimisation de la transformation génétique de la pomme de terre par Agrobacterium tumefaciens. Utilisation de la résistance à l'hygromycine comme marqueur sélectif

Directory of Open Access Journals (Sweden)

Rouvière C.

2003-01-01

Full Text Available Optimization of potato genetic transformation by Agrobacterium tumefaciens using hygromycin resistance as selective marker. The objective of this work is the optimization of a genetic transformation and a regeneration protocol of transgenic plants in Solanum tuberosum cv Désirée using hygromycin resistance as selective marker. The gene Cat2 of Nicotiana plumbaginifolia and the gene SU2 of Gossypium hirsutum, both coding for catalases, have been used. Two antibiotic concentrations (5 and 10 mg . l -1 of culture medium combined with two preculture periods (5 and 20 days on non-selective medium were tested. Coculture medium was supplemented with 10 mg . l -1 of acetosyringone to test its effect on potato transformation. The addition of this phenolic compound to the coculture medium affected positively the regeneration aptitude of transgenic SU2 plants. Only the combination of 5 mg . l -1 of hygromycin and 20 days of preculture without antibiotic allowed the obtention of plants transformed with the gene SU2. Screening of the rooted shoots with PCR showed 45% of transgenic plants for both molecular constructions used.

An Efficient Agrobacterium-Mediated Transformation System for Poplar

Science.gov (United States)

Movahedi, Ali; Zhang, Jiaxin; Amirian, Rasoul; Zhuge, Qiang

2014-01-01

Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone “Nanlin895” (Populus deltoides × P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis. PMID:24933641

Studies on Agrobacterium-mediated genetic transformation of ...

African Journals Online (AJOL)

SERVER

2008-03-04

Mar 4, 2008 ... Sweet potato (Ipomoea batatas) is the sixth most impor- tant crop in the world after ... mediated transformation system does not involve sophis- .... (w/v) agarose gel. .... This work was supported by National Natural Science.

The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner)

NARCIS (Netherlands)

Zheng Sijun, S.J.; Henken, B.; Ahn, Y.K.; Krens, F.A.; Kik, C.

2004-01-01

This paper describes the development of a reliable transformation system for garlic (Allium sativum L.) and its application in producing insect resistant GM garlic lines. The transformation system is based on Agrobacterium tumefaciens as a vector, using young callus derived from different callus

Production of herbicide-resistant coffee plants (Coffea canephora P. via Agrobacterium tumefaciens-mediated transformation

Directory of Open Access Journals (Sweden)

Alessandra Ferreira Ribas

2006-01-01

Full Text Available Transgenic plants of Coffea canephora P. resistant to the herbicide ammonium glufosinate were regenerated from leaf explants after co-culture with Agrobacterium tumefaciens strain EHA105 harboring pCambia3301, a plasmid that contains the bar and the uidA genes both under control of 35S promoter. Direct somatic embryogenesis was induced on basal medium contained ¼ strength macro salts and half strength micro salts of MS medium, organic constituents of B5 medium and 30 g.L-1 sucrose supplemented with 5µM N6 - (2-isopentenyl-adenine (2-iP. Ten µM ammonium glufosinate was used for putative transgenic somatic embryos selection. Presence and integration of the bar gene were confirmed by PCR and Southern blot analysis. Selected transgenic coffee plants sprayed with up to 1600 mg.L-1 of FinaleTM, a herbicide containing glufosinate as the active ingredient, retained their pigmentation and continued to grow normally during ex vitro acclimation.Plantas transgênicas de Coffea canephora P resistentes ao herbicida glufosinato de amônio foram regeneradas a partir de explantes foliares co-cultivados com Agrobacterium tumefaciens EHA105 contendo o plasmídio pCambia3301 que contém os genes bar e uidA ambos sob controle do promotor 35S. Embriogênese somática direta foi induzida no meio contendo ¼ da concentração de macro, metade da concentração de micronutrientes do meio MS, constituintes orgânicos do meio B5 e 30 g.L-1 de sacarose suplementado com 5µM N6 - (2-isopentenil-adenina (2-iP e 10 µM de glufosinato de amônio para seleção de embriões transgênicos putativos. A presença e a integração do gene bar foram confirmados pelas análises de PCR e Southern blot. As plantas transgênicas selecionadas de café, pulverizadas com 1600 mg.L-1 do herbicida FinaleTM que contém glufosinato como ingrediente ativo, mantiveram a coloração e continuaram crescendo normalmente na aclimatação ex vitro.

Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation.

Science.gov (United States)

Kwon, Tackmin

2016-09-01

The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection.

Genetic transformation of Physcomitrella patens mediated by ...

African Journals Online (AJOL)

USER

2010-06-21

Jun 21, 2010 ... plants could be obtained after 4 generations of selective culture. PCR analysis showed that gene ... 5'-triphosphate; SDS, sodium dodecyl sulfate; YFP, your favorite protein. ..... affected the transformation rate mediated by Agro- bacterium, this was confirmed and the result showed only the gametophores ...

Genetic transformation of garlic ( Allium sativum L.) with tobacco ...

African Journals Online (AJOL)

Garlic yield and quality have decreased due to white rot disease caused by Sclerotium cepivorum Berk. A transformation protocol to introduce tobacco chitinase and glucanase genes into garlic embryogenic calli using Agrobacterium tumefaciens has been established. LBA4404 strain having pC2301CHGLU plasmid with ...

Efficient genetic transformation of Lotus corniculatus L. using a direct shoot regeneration protocol, stepwise hygromycin B selection, and a super-binary Agrobacterium tumefaciens vector

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Nikolić Radomirka

2007-01-01

Full Text Available Cotyledons from 6-day-old Lotus corniculatus cv. Bokor seedlings, transversally cut into two halves, were capa­ble of regenerating buds without intervening callus formation. The explants were co-cultivated with the Agrobacterium tumefaciens LBA4404/pTOK233 superbinary vector carrying the uidA-intron gene and the genes hpt and nptII. They were cultured for 14 days on a regeneration medium, then subjected to a stepwise hygromycin B selection procedure consisting of gradually increasing antibiotic concentrations (5-15 mg L-1 over 21 weeks. Transformed shoots were obtained within 5 months after co-cultivation. Out of 124 initially co-cultivated explants, 52 (42% plants survived hygromycin B selection. The presence of transgenes in regenerated plants was verified by β-glucuronidase histochemical assays and PCR analysis for the presence of uidA gene sequences. Hygromycin B-resistant and PCR-positive T0 plants were cultured in the greenhouse to produce flowers and seeds. The obtained data demonstrate that the reported transformation protocol could be useful for introducing agriculturally important genes into the new L. corniculatus cultivar Bokor.

Genetic transformation of Begonia tuberhybrida by Ri rol genes.

Science.gov (United States)

Kiyokawa, S; Kikuchi, Y; Kamada, H; Harada, H

1996-04-01

We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar medium supplemented with 0.1 mg/l NAA, 0.5 mg/l BA, 500 mg/l claforan and 100 mg/l kanamycin. These shoots exhibited GUS activity and Southern analysis showed a single copy insertion into the genome. When the transgenic plants were transferred to soil, they displayed the phenotype specific to the transgenic plants by A. rhizogenes such as dwarfness, delay of flowering, and wrinkled leaves and petals.

Development of efficient plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants.

Science.gov (United States)

Dan, Yinghui; Baxter, Aaron; Zhang, Song; Pantazis, Christopher J; Veilleux, Richard E

2010-08-09

Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system

Development of Efficient Plant Regeneration and Transformation System for Impatiens Using Agrobacterium tumefaciens and Multiple Bud Cultures as Explants

Directory of Open Access Journals (Sweden)

Dan Yinghui

2010-08-01

Full Text Available Abstract Background Impatiens (Impatiens walleriana is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. Results In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892 bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. Conclusion We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained

Attachment study of Agrobacterium tumefaciens to tef [ Eragrostis tef ...

African Journals Online (AJOL)

Agrobacterium tumefaciens attachment as a factor in transformation was investigated in tef (Eragrostis tef) zygotic embryos, seeds, seedlings, leaf bases and embryogenic callus; leaf discs of yam species (Dioscorea bulbifera, Dioscorea caynensis and Dioscorea alata); and leaf discs of tobacco (Nicotina tabaccum).

Stable Agrobacterium-mediated transformation of Norway spruce embryogenic tissues using somatic embryo explants

Czech Academy of Sciences Publication Activity Database

Pavingerová, Daniela; Bříza, Jindřich; Niedermeierová, Hana; Vlasák, Josef

2011-01-01

Roč. 57, č. 7 (2011), s. 277-280 ISSN 1212-4834 R&D Projects: GA MZe QH71290 Institutional research plan: CEZ:AV0Z50510513 Keywords : Agrobacterium tumefaciens * genetic engineering * GUS activity * Picea abies (L.) Karst Subject RIV: EB - Genetic s ; Molecular Biology

Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation

Science.gov (United States)

Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina

This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.

Agrobacterium-Mediated Transformation of Leaf Base Segments.

Science.gov (United States)

Gasparis, Sebastian

2017-01-01

Agrobacterium-mediated transformation has become a routine method of genetic engineering of cereals, gradually replacing the biolistic protocols. Simple integration patterns of transgenic loci, decent transformation efficiency, and technical simplicity are the main advantages offered by this method. Here we present a detailed protocol for the production of transgenic oat plants by Agrobacterium-mediated transformation of leaf base segments. The use of leaf explants as target tissues for transformation and in vitro regeneration of transgenic plants may be a good alternative for genotypes which are not susceptible to regeneration from immature or mature embryos. We also describe the biochemical and molecular analysis procedures of the transgenic plants including a GUS histochemical assay, and Southern blot, both of which are optimized for application in oat.

Agrobacterium-mediated transformation of the wild orchid Cattleya maxima Lindl

Directory of Open Access Journals (Sweden)

Augusta Yadira Cueva-Agila

2018-03-01

Full Text Available Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60 - 80 % than untransformed controls (45 - 57 %, and this contrast was maximized in hormone-free, Murashige and Skoog (MS medium (80 % of the transformed plants compared to 57 % of the untransformed ones. This finding supports the notion that SERK plays an important role in Orchid embryogenesis

Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments Transformação genética em Citrus sinensis e Citrus limonia mediada por Agrobacterium tumefaciens a partir de segmentos de epicótilo

Directory of Open Access Journals (Sweden)

Weliton Antonio Bastos de Almeida

2003-02-01

Full Text Available Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck and Rangpur lime (Citrus limonia L. Osbeck. Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 µmol L-1; Explants ends had a longitudinal terminal incision (2-3 mm; co-cultivation temperatures of 19, 23 or 27°C were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27°C, in the absence of acetosyringone.A transformação genética permite produzir cultivares com características específicas e pode, dessa forma ser associada a programas de melhoramento de citros. O objetivo deste trabalho foi estabelecer protocolos de transformação genética para as laranjas doce 'Valência' e 'Natal' (Citrus sinensis L. Osbeck, bem como para o limão 'Cravo'(Citrus limonia L. Osbeck. Segmentos de epicótilo de plântulas germinadas in vitro (três semanas no

Production of Phytophthora infestans-resistant potato (Solanum tuberosum) utilising Ensifer adhaerens OV14

DEFF Research Database (Denmark)

Wendt, Toni; Doohan, Fiona; Mullins, Ewen

2012-01-01

their gene transfer efficiencies. In this study we set out to identify alternative bacteria species that could (a) utilize vir genes for genetic transformation and (b) substitute for A. tumefaciens in existing transformation protocols, without a prerequisite for protocol modifications. To this end we...... isolated a collection (n=751) of plant-associated bacteria from the rhizosphere of commercially grown crops. Based on various screens, including plant transformation with the open-source vector pCAMBIA5105, we identified a strain of the bacterium Ensifer adhaerens with the capacity to transform both......Based on the use of Agrobacterium tumefaciens-mediated transformation commodity crop improvement through genetic engineering is the fastest adopted crop technology in the world (James 2010). However, the complexity of the Agrobacterium patent landscape remains a challenge for non-patent holders who...

Genetic transformation of peanut (Arachis hypogaea L.) using ...

Indian Academy of Sciences (India)

Madhu

tissue culture response and plant regeneration has driven researchers to develop alternate transformation systems that target axillary meristem in the cotyledonary nodes (Somers et al 2003). We report here for the first time the mode of genetic transformation using cotyledonary node (CN) as an explant in peanut mediated ...

Methods for genetic transformation in Dendrobium.

Science.gov (United States)

da Silva, Jaime A Teixeira; Dobránszki, Judit; Cardoso, Jean Carlos; Chandler, Stephen F; Zeng, Songjun

2016-03-01

The genetic transformation of Dendrobium orchids will allow for the introduction of novel colours, altered architecture and valuable traits such as abiotic and biotic stress tolerance. The orchid genus Dendrobium contains species that have both ornamental value and medicinal importance. There is thus interest in producing cultivars that have increased resistance to pests, novel horticultural characteristics such as novel flower colours, improved productivity, longer flower spikes, or longer post-harvest shelf-life. Tissue culture is used to establish clonal plants while in vitro flowering allows for the production of flowers or floral parts within a sterile environment, expanding the selection of explants that can be used for tissue culture or genetic transformation. The latter is potentially the most effective, rapid and practical way to introduce new agronomic traits into Dendrobium. Most (69.4 %) Dendrobium genetic transformation studies have used particle bombardment (biolistics) while 64 % have employed some form of Agrobacterium-mediated transformation. A singe study has explored ovary injection, but no studies exist on floral dip transformation. While most of these studies have involved the use of selector or reporter genes, there are now a handful of studies that have introduced genes for horticulturally important traits.

Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics

Directory of Open Access Journals (Sweden)

Liu Wei

2009-06-01

Full Text Available Abstract Background Transgenic approaches provide a powerful tool for gene function investigations in plants. However, some legumes are still recalcitrant to current transformation technologies, limiting the extent to which functional genomic studies can be performed on. Superroot of Lotus corniculatus is a continuous root cloning system allowing direct somatic embryogenesis and mass regeneration of plants. Recently, a technique to obtain transgenic L. corniculatus plants from Superroot-derived leaves through A. tumefaciens-mediated transformation was described. However, transformation efficiency was low and it took about six months from gene transfer to PCR identification. Results In the present study, we developed an A. rhizogenes-mediated transformation of Superroot-derived L. corniculatus for gene function investigation, combining the efficient A. rhizogenes-mediated transformation and the rapid regeneration system of Superroot. The transformation system using A. rhizogenes K599 harbouring pGFPGUSPlus was improved by validating some parameters which may influence the transformation frequency. Using stem sections with one node as explants, a 2-day pre-culture of explants, infection with K599 at OD600 = 0.6, and co-cultivation on medium (pH 5.4 at 22°C for 2 days enhanced the transformation frequency significantly. As proof of concept, Superroot-derived L. corniculatus was transformed with a gene from wheat encoding an Na+/H+ antiporter (TaNHX2 using the described system. Transgenic Superroot plants were obtained and had increased salt tolerance, as expected from the expression of TaNHX2. Conclusion A rapid and efficient tool for gene function investigation in L. corniculatus was developed, combining the simplicity and high efficiency of the Superroot regeneration system and the availability of A. rhizogenes-mediated transformation. This system was improved by validating some parameters influencing the transformation frequency, which could

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

Science.gov (United States)

Cardoza, V.; Stewart, C. N.

2003-01-01

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

An improved method for transformation of lettuce by Agrobacterium tumefaciens with a gene that confers freezing resistance

Directory of Open Access Journals (Sweden)

Pileggi Marcos

2001-01-01

Full Text Available An efficient method for constructing transgenic lettuce cultivars by Agrobacterium tumefaciens was described by Torres et al., 1993. In the present work, an improvement of the above procedure is described and applied to transform the cultivar Grand Rapids with a mutated P5CS gene. The major modifications were concerned with turning more practical the transformation and regeneration protocols. Also we tried to improve transformation steps by increasing injured area in explants and prolonging co-cultivation with Agrobacteria (in larger concentration. A more significant selective pressure was used against non-transformed plants and bacteria. In these work we were concerned to obtain T1 and T2 seeds. The P5CS gene codes for a delta¹-pyrroline-5-carboxylate synthetase, a bifunctional enzyme that catalyzes two steps of proline biosynthesis in plants (Zhang et al., 1995; Peng et al., 1996, while the mutated gene is insensitive to feedback inhibition by proline. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold due to increased intracellular levels of proline that works like an osmoprotectant. In this work could obtain and characterize transgenic lettuce lineages which are resistant to freezing temperature.

Agrobacterium-medicated transformation of mature Prunus serotina (black cherry) and regeneration of trangenic shoots

Science.gov (United States)

Xiaomei Liu; Paula Pijut

2010-01-01

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced...

Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmeria nivea (L.) Gaud].

Science.gov (United States)

An, Xia; Wang, Bo; Liu, Lijun; Jiang, Hui; Chen, Jie; Ye, Shengtuo; Chen, Leiyu; Guo, Pingan; Huang, Xing; Peng, Dingxiang

2014-05-01

In this study, leaf midribs, the elite explants, were used for the first time to develop an efficient regeneration and transformation protocol for ramie [Boehmeria nivea (L.) Gaud.] via Agrobacterium-mediated genetic transformation. Sensitivity of leaf midribs regeneration to kanamycin was evaluated, which showed that 40 mg l(-1) was the optimal concentration needed to create the necessary selection pressure. Factors affecting the ramie transformation efficiency were evaluated, including leaf age, Agrobacterium concentration, length of infection time for the Agrobacterium solution, acetosyringone concentration in the co-cultivation medium, and the co-cultivation period. The midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD600 of 0.6, 10-min immersion in the bacteria solution, an acetosyringone concentration of 50 mg l(-1) in the co-cultivation medium and a 3-day co-cultivation period produced the highest efficiencies of regeneration and transformation. In this study, the average transformation rate was 23.25%. Polymerase chain reactions using GUS and NPTII gene-specific primers, Southern blot and histochemical GUS staining analyses further confirmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of this system of Agrobacterium-mediated genetic transformation and regeneration of transgenic plants will be used not only to introduce genes of interest into the ramie genome for the purpose of trait improvement, but also as a common means of testing gene function by enhancing or inhibiting the expression of target genes.

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens.

Science.gov (United States)

Raharjo, S H; Hernandez, M O; Zhang, Y Y; Punja, Z K

1996-04-01

Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

Science.gov (United States)

Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

2015-01-01

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat

Directory of Open Access Journals (Sweden)

Doherty Angela

2005-09-01

Full Text Available Abstract Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties.

Escherichia coli can be transformed by a liposome-mediated lipofection method.

Science.gov (United States)

Kawata, Yoshikazu; Yano, Shin-ichi; Kojima, Hiroyuki

2003-05-01

Transformation of Escherichia coli is a basic technique for genetic engineering. We used a liposome-mediated lipofection method to transform electrocompetent E. coli cells which has little natural competence of foreign DNA without electroporation treatment, and got transformants with simple and quick treatment by a plasmid or a transposon and transposase complex.

Identifikasi Transgene pada Tanaman Padi (oryza sativa var. koshihikari yang Ditransformasi dengan Bantuan Agrobacterium tumefaciens, Menggunakan Metode Tanpa Teknik Kultur Jaringan.

Directory of Open Access Journals (Sweden)

I Putu Suparthana

2015-04-01

Full Text Available Metode transformasi tanaman dengan bantuan Agrobacterium tumefaciens biasa dilakukan dengan melibatkan teknik kultur jaringan, akan tetapi teknik kultur jaringan memiliki beberapa kelemahan yaitu memerlukan suatu kondisi steril, memakan banyak waktu, sering terjadi mutasi dalam proses kultur in vitro dan sejumlah tanaman bersifat rekalsitran pada tahap regenerasi. Disisi lain metode baru (in planta transformation system yang dikembangkan dalam penelitian ini mampu mengatasi kelemahan-kelemahan tersebut. Penelitian dilakukan pada tanaman padi dengan bantuan A. tumefaciens sebagai inokulum namun tidak melibatkan teknik kultur jaringan pada masa regenerasinya. Biji padi (Oryza sativa L. var. Koshihikari direndam dalam air selama 2 hari, dengan demikian embrionya yang mengandung sel apikal meristem dapat diinokulasi dengan cara menusukkan jarum yang telah dicelupkan dalam inokulum. Biji padi yang telah diinokulasi selanjutnya ditumbuhkan dilapangan selayaknya pembenihan biasa tanpa perlakuan steril. Untuk menentukan keberhasilan teknik ini, dua jenis strain mutan A. tumefaciens (M-21 dan LBA4404 digunakan dalam transformasi. Mutan M-21 mengandung Tn5 tersisip dalam gen iaaM dan mutan LBA4404 membawa binari plasmid vektor. Transgen dari mutan M-21 dapat diidentifikasi dari perubahan fenotipe pada tanaman padi transgenik sedangkan dari mutan LBA4404 dapat diidentifikasi dengan uji histokimia dan ketahanan terhadap antibitik (hygromycin B.Kata kunci: in planta transformation, A. tumefaciens, transformation method

Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

Science.gov (United States)

Masani, Mat Yunus Abdul; Noll, Gundula A.; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk

2014-01-01

Background Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. Methodology/Principal Findings We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. Conclusions/Significance We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants. PMID:24821306

Micropropagation and genetic transformation of Tylophora indica (Burm. f.) Merr.: a review.

Science.gov (United States)

Teixeira da Silva, Jaime A; Jha, Sumita

2016-11-01

This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties. Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.

In Vitro Callogenesis and Agrobacterium-Mediated Transformation of Globe Artichoke

NARCIS (Netherlands)

Menin, B.; Moglia, A.; Comino, C.; Lanteri, S.; Herpen, van T.W.J.M.; Beekwilder, M.J.

2012-01-01

Micropropagation techniques have been widely applied in globe artichoke (C. cardunculus L. var. scolymus), however, efficient protocols for the establishment of in vitro callogenesis and organogenesis, a pre-requisite for Agrobacterium-mediated genetic transformation, have not been set up so far. We

Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus

Science.gov (United States)

Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in stored bulbs. In order to study the pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B...

Maize (Zea mays L.).

Science.gov (United States)

Frame, Bronwyn; Warnberg, Katey; Main, Marcy; Wang, Kan

2015-01-01

Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector. In addition to step-by-step laboratory transformation procedures, we include extensive details in growing donor plants and caring for transgenic plants in the greenhouse.

STUDY REGARDING EFFICIENCY OF INDUCED GENETIC TRANSFORMATION IN BACILLUS LICHENIFORMIS WITH PLASMID DNA

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T. VINTILĂ

2007-05-01

Full Text Available A strain of Bacillus licheniformis was subject to genetic transformation with plasmid vectors (pLC1 and pNC61, using electroporation technique, protoplast transformation and bivalent cations (CaCl2 mediated transformation. In the case of transformation by electroporation of Bacillus licheniformis B40, the highest number of transformed colonies (3 were obtained only after a 1,79 KV electric shock, for 2,2 milliseconds. Using this transformation technique we have obtained six kanamycin resistant transformants. The frequency of Bacillus licheniformis B40 protoplasts transformation using pLC1 and pNC61 plasmid vectors is approximately 10% (TF = 10%. As a result of pLC1 plasmid integration in Bacillus licheniformis protoplasts, six kanamycin resistant transformants were obtained. The pNC61 plasmid, which confers trimethoprim resistance, does not integrate in receiver cells by protoplast transformation. The direct genetic transformation in the presence of bivalent cations (CaCl2, mediated by pLC1 and pNC61 plasmid vectors, produce a low transformation frequency. Using this technique, we have obtained three trimethoprim resistant colonies and four kanamycin resistant colonies. The chemical way of transformation is the only technique, which realizes the integration of pNC61 in B. licheniformis B40 cells.

Agro-transformation and evaluation of resistance to Phytophthora infestansin Solanum tuberosumL. variety Désirée

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Jeanette Orbegozo

2014-03-01

Full Text Available The Oomycete Phytophthora infestans (Mont. de Bary, the causal agent of the disease known as late blight, is primarily responsible for the decreased in production performance and potato crops worldwide. The integration of the complete Rgenes sequences in the potato genome using Agro-transformation appears an alternative to be considered in the fight against this pathogen. The Rpi-blb2 gene (Rgene from the wild species Solanum bulbocastanumDunal shows a broad resistance to isolates ofP. infestans,making it an important candidate for plant breeding studies. This paper reports the integration of the Rpi-blb2gene into potato var. Désirée genome by Agrobacterium tumefaciens- mediated transformation system, the molecular characterization of 29 events transformed and whole plant infection with isolate POX67 of P. infestansfrom Peru. Désirée events [Rpi-blb2] 4 and Désirée [Rpi-blb2] 30, showed a substantial resistance to P. infestansinfection confirming complete transfer of the Rpi-blb2gene from a wild species to a cultivated species by genetic transformation.

STUDY REGARDING EFFICIENCY OF INDUCED GENETIC TRANSFORMATION IN BACILLUS LICHENIFORMIS WITH PLASMID DNA

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VINTILĂ T.

2007-01-01

Full Text Available A strain of Bacillus licheniformis was subject to genetic transformation with plasmidvectors (pLC1 and pNC61, using electroporation technique, protoplasttransformation and bivalent cations (CaCl2 mediated transformation. In the case oftransformation by electroporation of Bacillus licheniformis B40, the highest numberof transformed colonies (3 were obtained only after a 1,79 KV electric shock, for 2,2milliseconds. Using this transformation technique we have obtained six kanamycinresistant transformants. The frequency of Bacillus licheniformis B40 protoplaststransformation using pLC1 and pNC61 plasmid vectors is approximately 10% (TF =10%. As a result of pLC1 plasmid integration in Bacillus licheniformis protoplasts,six kanamycin resistant transformants were obtained. The pNC61 plasmid, whichconfers trimethoprim resistance, does not integrate in receiver cells by protoplasttransformation. The direct genetic transformation in the presence of bivalent cations(CaCl2, mediated by pLC1 and pNC61 plasmid vectors, produce a lowtransformation frequency. Using this technique, we have obtained three trimethoprimresistant colonies and four kanamycin resistant colonies. The chemical way oftransformation is the only technique, which realizes the integration of pNC61 in B.licheniformis B40 cells.

Highly efficient transformation system for Malassezia furfur and Malassezia pachydermatis using Agrobacterium tumefaciens-mediated transformation

NARCIS (Netherlands)

Celis, A.M.; Vos, Aurin; Triana, S.; Medina, C.A.; Escobar Salazar, Natalia; Restrepo, S.; Wosten, Han; de Cock, Hans

2017-01-01

Malassezia spp. are part of the normal human and animal mycobiota but are also associated with a variety of dermatological diseases. The absence of a transformation system hampered studies to reveal mechanisms underlying the switch from the non-pathogenic to pathogenic life style. Here we describe,

Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules

DEFF Research Database (Denmark)

Holme, Inger; Brinch-Pedersen, Henrik; Lange, Mette

2012-01-01

Agrobacterium-mediated transformation of in vitro cultured barley ovules is an attractive alternative to well-established barley transformation methods of immature embryos. The ovule culture system can be used for transformation with and without selection and has successfully been used to transfo...

piggybac- and PhiC31-mediated genetic transformation of the Asian tiger mosquito, Aedes albopictus (Skuse.

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Geneviève M C Labbé

Full Text Available BACKGROUND: The Asian tiger mosquito, Aedes albopictus (Skuse, is a vector of several arboviruses including dengue and chikungunya. This highly invasive species originating from Southeast Asia has travelled the world in the last 30 years and is now established in Europe, North and South America, Africa, the Middle East and the Caribbean. In the absence of vaccine or antiviral drugs, efficient mosquito control strategies are crucial. Conventional control methods have so far failed to control Ae. albopictus adequately. METHODOLOGY/PRINCIPAL FINDINGS: Germline transformation of Aedes albopictus was achieved by micro-injection of embryos with a piggyBac-based transgene carrying a 3xP3-ECFP marker and an attP site, combined with piggyBac transposase mRNA and piggyBac helper plasmid. Five independent transgenic lines were established, corresponding to an estimated transformation efficiency of 2-3%. Three lines were re-injected with a second-phase plasmid carrying an attB site and a 3xP3-DsRed2 marker, combined with PhiC31 integrase mRNA. Successful site-specific integration was observed in all three lines with an estimated transformation efficiency of 2-6%. CONCLUSIONS/SIGNIFICANCE: Both piggybac- and site-specific PhiC31-mediated germline transformation of Aedes albopictus were successfully achieved. This is the first report of Ae. albopictus germline transformation and engineering, a key step towards studying and controlling this species using novel molecular techniques and genetic control strategies.

Norway spruce (Picea abies) genetic transformation with modified Cry3A gene of Bacillus thuringiensis

Czech Academy of Sciences Publication Activity Database

Bříza, Jindřich; Pavingerová, Daniela; Vlasák, Josef; Niedermeierová, Hana

2013-01-01

Roč. 60, č. 3 (2013), s. 395-400 ISSN 0001-527X R&D Projects: GA MZe QH71290; GA ČR(CZ) GAP502/11/1471 Institutional support: RVO:60077344 Keywords : Cry3A gene modification * Picea abies * Agrobacterium tumefaciens Subject RIV: EB - Genetic s ; Molecular Biology Impact factor: 1.389, year: 2013

Agrobacterium-mediated transformation of the recalcitrant Vanda Kasem's Delight orchid with higher efficiency.

Science.gov (United States)

Gnasekaran, Pavallekoodi; Antony, Jessica Jeyanthi James; Uddain, Jasim; Subramaniam, Sreeramanan

2014-01-01

The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A 600 nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 μM acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

Cellulose Synthesis in Agrobacterium tumefaciens

Energy Technology Data Exchange (ETDEWEB)

Alan R. White; Ann G. Matthysse

2004-07-31

We have cloned the celC gene and its homologue from E. coli, yhjM, in an expression vector and expressed the both genes in E. coli; we have determined that the YhjM protein is able to complement in vitro cellulose synthesis by extracts of A. tumefaciens celC mutants, we have purified the YhjM protein product and are currently examining its enzymatic activity; we have examined whole cell extracts of CelC and various other cellulose mutants and wild type bacteria for the presence of cellulose oligomers and cellulose; we have examined the ability of extracts of wild type and cellulose mutants including CelC to incorporate UDP-14C-glucose into cellulose and into water-soluble, ethanol-insoluble oligosaccharides; we have made mutants which synthesize greater amounts of cellulose than the wild type; and we have examined the role of cellulose in the formation of biofilms by A. tumefaciens. In addition we have examined the ability of a putative cellulose synthase gene from the tunicate Ciona savignyi to complement an A. tumefaciens celA mutant. The greatest difference between our knowledge of bacterial cellulose synthesis when we started this project and current knowledge is that in 1999 when we wrote the original grant very few bacteria were known to synthesize cellulose and genes involved in this synthesis were sequenced only from Acetobacter species, A. tumefaciens and Rhizobium leguminosarum. Currently many bacteria are known to synthesize cellulose and genes that may be involved have been sequenced from more than 10 species of bacteria. This additional information has raised the possibility of attempting to use genes from one bacterium to complement mutants in another bacterium. This will enable us to examine the question of which genes are responsible for the three dimensional structure of cellulose (since this differs among bacterial species) and also to examine the interactions between the various proteins required for cellulose synthesis. We have carried out one

Genetic transformation of forest trees

African Journals Online (AJOL)

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In this review, the recent progress on genetic transformation of forest trees were discussed. Its described also, different applications of genetic engineering for improving forest trees or understanding the mechanisms governing genes expression in woody plants. Key words: Genetic transformation, transgenic forest trees, ...

Highly-efficient liposome-mediated transformation system for the basidiomycetous fungus Flammulina velutipes.

Science.gov (United States)

Shi, Liang; Chen, Dongdong; Xu, Chao; Ren, Ang; Yu, Hanshou; Zhao, Mingwen

2017-07-11

Flammulina velutipes is a well-known edible mushroom cultivated all over the world. However, because of the low transformation frequency, the expensive instruments required, and the complicated, time-consuming procedures necessary, there is insufficient genetic research on F. velutipes. In this study, we report a liposome-mediated transformation (LMT) system for the genetic transformation of F. velutipes. Using the LMT system, we obtained 82 ± 4 stable F. velutipes transformants per 10 5 protoplasts, which is a clear increase in transformation frequency compared to the other methods used. We were able to detect the expression of an EGFP reporter gene in the F. velutipes transformants using fluorescence imaging assays. Furthermore, we used this method to transfer the laccase gene into F. velutipes and found that the transcriptional level and enzymatic activity increased in these transformants. Mitotic stability analysis showed that all of the selected transformants remained mitotically stable, even after five successive rounds of sub-culturing. These results demonstrate a new transgenic approach that will facilitate F. velutipes research.

Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer

NARCIS (Netherlands)

Michielse, C.B.; Salim, K.; Ragas, P.; Ram, A.F.J.; Kudla, B.; Jarry, B.; Punt, P.J.; Hondel, C.A.M.J.J. van den

2004-01-01

Two transformation systems, based on the use of CaCl2/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS+ marker from Aspergillus nidulans, and

Impact of biological amendments on Agrobacterium tumefaciens soil survival

Science.gov (United States)

Paradox, the primary walnut rootstock used in California, is susceptible to Agrobacterium tumefaciens, which causes crown gall. While A. tumefaciens is susceptible to commonly used fumigants such as methyl bromide (MeBr) and Telone-C35 (1,3-dichloropropene and chloropicrin), these fumigants also sig...

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.).

Science.gov (United States)

Yang, Jun; Bi, Hui-Ping; Fan, Wei-Juan; Zhang, Min; Wang, Hong-Xia; Zhang, Peng

2011-12-01

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l(-1) 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l(-1) hygromycin and 200 mg l(-1) cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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)

Agrobacterium-mediated transformation of modified antifreeze protein gene in strawberry

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Srisulak Dheeranupattana

2005-07-01

Full Text Available The optimum condition for shoot regeneration from leaf explants of strawberry cultivar Tiogar was investigated. It was found that the best regeneration condition was MS medium containing N6-Benzyladenine (BA and 2,4-Dichlorophenoxy acetic acid (2,4-D at concentrations of 1 mg.l-1 and 0.2 mg.l-1, respectively. Antibiotics sensitivity test found that shoot regeneration from leaf explant was inhibited more than 90% at the concentration of kanamycin (Km as low as 5 mg.l-1. The modified gene encoding antifreeze protein isoform HPLC 6 was successfully constructed using codons which were optimally expressed in the strawberry plant. The antifreeze protein genes, naturally in plasmid pSW1 and modified in plasmid BB, were transformed to strawberry leaf explants by Agrobacterium tumefaciens LBA 4404. The strawberry plants, transformed with both AFP genes, were able to root in MS media containing 50 mg.l-1 Km, while no roots grew from nontransformed plant in this condition. Polymerase chain reaction indicated that the transgenes were integrated in the genome of transformants.

Advancing Crop Transformation in the Era of Genome Editing[OPEN

Science.gov (United States)

Blechl, Ann E.; Brutnell, Thomas P.; Conrad, Liza J.; Gelvin, Stanton B.; Jackson, David P.; Kausch, Albert P.; Lemaux, Peggy G.; Medford, June I.; Orozco-Cárdenas, Martha L.; Tricoli, David M.; Van Eck, Joyce; Voytas, Daniel F.

2016-01-01

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized. PMID:27335450

Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

Science.gov (United States)

Sivanandhan, Ganeshan; Kapil Dev, Gnajothi; Theboral, Jeevaraj; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

2015-01-01

In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. PMID:25927703

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

Science.gov (United States)

Raji, Jennifer A; Frame, Bronwyn; Little, Daniel; Santoso, Tri Joko; Wang, Kan

2018-01-01

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T 0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

Agrobacterium tumefaciens is a diazotrophic bacterium

International Nuclear Information System (INIS)

Kanvinde, L.; Sastry, G.R.K.

1990-01-01

This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grown on nitrogen-free medium, reduce acetylene to ethylene, and incorporate 15 N supplied as 15 N 2 . As with most other well-characterized diazotrophic bacteria, the presence of NH 4 + in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship

Is VIP1 important for Agrobacterium-mediated transformation?

Science.gov (United States)

Shi, Yong; Lee, Lan-Ying; Gelvin, Stanton B

2014-09-01

Agrobacterium genetically transforms plants by transferring and integrating T-(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus-tagged VirE2 or Venus-tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1-Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY-2 protoplasts, regardless of whether VirE2 was co-expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium-mediated transformation or VirE2 subcellular localization. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Wheat (Triticum aestivum L.) transformation using immature embryos.

Science.gov (United States)

Ishida, Yuji; Tsunashima, Masako; Hiei, Yukoh; Komari, Toshihiko

2015-01-01

Wheat may now be transformed very efficiently by Agrobacterium tumefaciens. Under the protocol hereby described, immature embryos of healthy plants of wheat cultivar Fielder grown in a well-conditioned greenhouse were pretreated with centrifuging and cocultivated with A. tumefaciens. Transgenic wheat plants were obtained routinely from between 40 and 90 % of the immature embryos, thus infected in our tests. All regenerants were normal in morphology and fully fertile. About half of the transformed plants carried single copy of the transgene, which are inherited by the progeny in a Mendelian fashion.

[Transgenic wheat (Triticum aestivum L.) with increased resistance to the storage pest obtained by Agrobacterium tumefaciens--mediated].

Science.gov (United States)

Bi, Rui-Ming; Jia, Hai-Yan; Feng, De-Shun; Wang, Hong-Gang

2006-05-01

The transgenic wheat of improved resistance to the storage pest was production. We have introduced the cowpea trypsin inhibitor gene (CpTI) into cultured embryonic callus cells of immature embryos of wheat elite line by Agrobacterium-mediated method. Independent plantlets were obtained from the kanamycin-resistant calli after screening. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were 3 transgenic plants viz. transformed- I, II and III (T- I, T-II and T-III). The transformation frequencies were obviously affected by Agrobacterium concentration, the infection duration and transformation treatment. The segregations of CpTI in the transgenic wheat progenies were not easily to be elucidated, and some transgenic wheat lines (T- I and T-III) showed Mendelian segregations. The determinations of insect resistance to the stored grain insect of wheat viz. the grain moth (Sitotroga cerealella Olivier) indicated that the 3 transgenic wheat progeny seeds moth-resistance was improved significantly. The seed moth-eaten ratio of T- I, T-II, T-III and nontransformed control was 19.8%, 21.9%, 32.9% and 58.3% respectively. 3 transgenic wheat T1 PCR-positive plants revealed that the 3 transgenic lines had excellent agronomic traits. They supplied good germplasm resource of insect-resistance for wheat genetic improvement.

Agrobacterium tumefaciens is a diazotrophic bacterium

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Kanvinde, L.; Sastry, G.R.K. (Univ. of Leeds (England))

1990-07-01

This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grown on nitrogen-free medium, reduce acetylene to ethylene, and incorporate {sup 15}N supplied as {sup 15}N{sub 2}. As with most other well-characterized diazotrophic bacteria, the presence of NH{sub 4}{sup +} in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship.

Direct fluorescence detection of VirE2 secretion by Agrobacterium tumefaciens

Science.gov (United States)

Yaakov, Noga; Barak, Yoav; Pereman, Idan; Christie, Peter J.; Elbaum, Michael

2017-01-01

VirE2 is a ssDNA binding protein essential for virulence in Agrobacterium tumefaciens. A tetracysteine mutant (VirE2-TC) was prepared for in vitro and in vivo fluorescence imaging based on the ReAsH reagent. VirE2-TC was found to be biochemically active as it binds both ssDNA and the acidic secretion chaperone VirE1. It was also biologically functional in complementing virE2 null strains transforming Arabidopsis thaliana roots and Nicotiana tabacum leaves. In vitro experiments demonstrated a two-color fluorescent complex using VirE2-TC/ReAsH and Alexa Fluor 488 labeled ssDNA. In vivo, fluorescent VirE2-TC/ReAsH was detected in bacteria and in plant cells at time frames relevant to transformation. PMID:28403156

Improvement of tissue culture, genetic transformation, and applications of biotechnology to Brassica.

Science.gov (United States)

Ravanfar, Seyed Ali; Orbovic, Vladimir; Moradpour, Mahdi; Abdul Aziz, Maheran; Karan, Ratna; Wallace, Simon; Parajuli, Saroj

2017-04-01

Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.

High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.

Science.gov (United States)

Mehrotra, Meenakshi; Sanyal, Indraneel; Amla, D V

2011-09-01

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

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Abbas El Sahili

2015-08-01

Full Text Available Periplasmic binding proteins (PBPs in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the

A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

Science.gov (United States)

El Sahili, Abbas; Li, Si-Zhe; Lang, Julien; Virus, Cornelia; Planamente, Sara; Ahmar, Mohammed; Guimaraes, Beatriz G; Aumont-Nicaise, Magali; Vigouroux, Armelle; Soulère, Laurent; Reader, John; Queneau, Yves; Faure, Denis; Moréra, Solange

2015-08-01

Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of

Transport of nonmetabolizable opines by Agrobacterium tumefaciens

International Nuclear Information System (INIS)

Krishnan, M.; Burgner, J.W.; Chilton, W.S.; Gelvin, S.B.

1991-01-01

We have examined the uptake of [ 14 C]octopine and [ 14 C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [ 14 C]octopine and [ 14 C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid

Transient GUS gene expression in cassava (Manihot esculenta Crantz using Agrobacterium tumefaciens leaf infiltration

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Paula Díaz T.

2014-09-01

Full Text Available Objective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS. A. tumefaciens infiltration (agroinfiltration was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan and CM6438-14 (Vergara, respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.

Effective Agrobacterium–mediated transformation of pineapple with ...

African Journals Online (AJOL)

user7

Agrobacterium tumefaciens strain LBA4404 harboring a plant expression vector pUHA1 containing a ... hybridization of this self-incompatible monocot species, only a few new hybrids ... CYP1A1 is a member of the P450 gene family isolated.

Factor affecting Agrobacterium -mediated transformation of rice ...

African Journals Online (AJOL)

Potato is a very important food crop and is adversely affected by fungus. Agrobacterium-mediated transformation can play an important role in the improvement of potato. The present study was conducted to optimize the different factors affecting Agrobacterium-mediated transformation of chitinase gene. Nodes were used as ...

Plant responses to Agrobacterium tumefaciens and crown gall development

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Gohlke, Jochen; Deeken, Rosalia

2014-01-01

Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumors. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide (“omic”) approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumor formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic, and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant. PMID:24795740

Agrobacterium tumefaciens-mediated transformation of biofuel plant ...

African Journals Online (AJOL)

Yomi

2010-09-27

Sep 27, 2010 ... several plant species including Arabidopsis (Chang et al.,. 1994; Clough and Bent, ... and Technology Talents Scheme of Yunnan Province. (Grant no. .... of shoots and roots from Jatropha curcas L. explants. J. Horticult. Sci.

Jatropha (Jatropha curcas L.).

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Maravi, Devendra Kumar; Mazumdar, Purabi; Alam, Shamsher; Goud, Vaibhav V; Sahoo, Lingaraj

2015-01-01

The seed oil of Jatropha (Jatropha curcas L.) as a source of biodiesel fuel is gaining worldwide importance. Commercial-scale exploration of Jatropha has not succeeded due to low and unstable seed yield in semiarid lands unsuitable for the food production and infestation to diseases. Genetic engineering is promising to improve various agronomic traits in Jatropha and to understand the molecular functions of key Jatropha genes for molecular breeding. We describe a protocol routinely followed in our laboratory for stable and efficient Agrobacterium tumefaciens-mediated transformation of Jatropha using cotyledonary leaf as explants. The 4-day-old explants are infected with Agrobacterium tumefaciens strain EHA105 harboring pBI121 plant binary vector, which contains nptII as plant selectable marker and gus as reporter. The putative transformed plants are selected on kanamycin, and stable integration of transgene(s) is confirmed by histochemical GUS assay, polymerase chain reaction, and Southern hybridization.

Agrobacterium mediated genetic transformation of popular Indica ...

African Journals Online (AJOL)

Various parameters critical to rice transformation were optimized including callus induction medium, bacterial concentration, co-cultivation conditions, concentration of the plant growth regulator 2,4-D and the concentration of acetosyringone. The transformed lines were analyzed using PCR for marker sequence and gusA ...

Transformation of multiple soybean cultivars by infecting ...

African Journals Online (AJOL)

Transformation of multiple soybean cultivars by infecting cotyledonary-node with Agrobacterium tumefaciens. ... In our study, the combination of Nannong88-1 with EHA105 is the optimum selection for explant and bacterial inoculum in soybean transformation, which could be applied in future functional study of soybean ...

The Genetic Intractability Of Symbiodinium microadriaticum To Standard Algal Transformation Methods

KAUST Repository

Chen, Jit Ern

2017-05-23

Modern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, in order to perform CRISPR-Ca9 mediated genome editing. Plasmid vectors containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation, silica whiskers and glass bead agitation. We report that we have been unable to confer chloramphenicol resistance to our specific Symbiodinium strain. These results are intended to provide other researchers with an overview of previously attempted techniques and sequences in order to support efficient planning of future experiments in this important field.

Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora.

Science.gov (United States)

Wu, Dongliang; Navet, Natasha; Liu, Yingchao; Uchida, Janice; Tian, Miaoying

2016-09-06

As an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility. In this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion. This system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.

Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

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Buschmann, Henrik

2016-01-01

The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

Effective Agrobacterium –mediated transformation of pineapple with ...

African Journals Online (AJOL)

cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plant expression vector pUHA1 containing a human cytochrome P4501A1 (CYP1A1) gene for 3 days. Then, the infected calli were transferred to differentiation medium.

Optimization of Agrobacterium -mediated transformation parameters ...

African Journals Online (AJOL)

Agrobacterium-mediated transformation factors for sweet potato embryogenic calli were optimized using -glucuronidase (GUS) as a reporter. The binary vector pTCK303 harboring the modified GUS gene driven by the CaMV 35S promoter was used. Transformation parameters were optimized including bacterial ...

A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis

Directory of Open Access Journals (Sweden)

Polyana Kelly Martins

2015-06-01

Full Text Available The production and use of sugarcane in Brazil is very important for bioenergy production and is recognized as one of the most efficient in the world. In our laboratory, Setaria viridis is being tested as a model plant for sugarcane. S. viridis has biological attributes (rapid life cycle, small genome, diploid, short stature and simple growth requirements that make it suitable for use as a model system. We report a highly efficient protocol for Agrobacterium-mediated genetic transformation of S. viridis. The optimization of several steps in tissue culture allowed the rapid regeneration of plants and increased the rate of transformation up to 29%. This protocol could become a powerful tool for functional genomics in sugarcane.

Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybrid Aspen and Poplar Clones.

Science.gov (United States)

De Block, M

1990-07-01

Tissue culture conditions and transformation have been established for both aspen and poplar. The use of previously described culture conditions resulted in shoot tip necrosis in the shoot cultures and necrosis of stem and leaf explants. Shoot tip necrosis could be overcome by buffering the medium with 2-(N-morpholino)ethanesulfonic acid and Ca-gluconate and by growing the shoots below 25 degrees C. Necrosis of the explants was probably due to an accumulation of ammonium in the explants and could be overcome by adapting the NO(3) (-)/NH(4) (+) ratio of the media. Stem explants of established shoot cultures of the aspen hybrid Populus alba x P. tremula and of the poplar hybrid Populus trichocarpa x P. deltoides were cocultivated with Agrobacterium strains having chimeric bar and neo genes on their disarmed tDNAs. Transformed aspen shoots were obtained from 30 to 40% of the explants, while transformed poplar shoots were obtained from 10% of the explants. Extracts from the transformed trees contained high phosphinotricin acetyltransferase and neomycin phosphotransferase activities, and the trees contained one to three copies of the chimeric genes. The transformed trees were completely resistant to the commercial preparations of the herbicide phosphinotricin (glufosinate), while control trees were not.

Transformation of soybean Gy3 gene into Artemisaarenaria mediated by corona discharge

International Nuclear Information System (INIS)

Chao, Lu-meng; Na, Ri; Xue, Dan; Xu, Yongze; Liu, Teng

2013-01-01

In order to improve the protein content of desert plant, a method of genetic transformation mediated by corona discharge was established. Artemisia seeds were processed in corona electric field for 120 min at 12 kV, and then soaked in 0.1 SSC media that contained Soybean Gy3 gene DNA to incubate for 12 h at 26 °C. Finally the seeds were inoculated on the differentiation medium. Polymerase Chain Reaction (PCR) and Reverse Transcription Polymerase Chain Reaction (RT-PCR) detection showed that the Soybean Gy3 gene had been successfully introduced into genomic DNA of the regenerated plants of Artemisaarenaria. The study provided a new way for corona discharge in plant genetic modification.

Effect of pre-plant soil fumigants on Agrobacterium tumefaciens, pythiaceous species, and subsequent soil recolonization by A. tumefaciens

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Paradox (Juglans hindsii x J. regia), the dominant rootstock used in the California walnut industry, is susceptible to crown gall, caused by Agrobacterium tumefaciens. In practice, soil fumigation has been a common preplant management strategy for crown gall, but even an industry standard, methyl b...

Transformation of Botrytis cinerea by direct hyphal blasting or by wound-mediated transformation of sclerotia

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Ish - Shalom Shahar

2011-12-01

Full Text Available Abstract Background Botrytis cinerea is a haploid necrotrophic ascomycete which is responsible for 'grey mold' disease in more than 200 plant species. Broad molecular research has been conducted on this pathogen in recent years, resulting in the sequencing of two strains, which has generated a wealth of information toward developing additional tools for molecular transcriptome, proteome and secretome investigations. Nonetheless, transformation protocols have remained a significant bottleneck for this pathogen, hindering functional analysis research in many labs. Results In this study, we tested three different transformation methods for B. cinerea: electroporation, air-pressure-mediated and sclerotium-mediated transformation. We demonstrate successful transformation with three different DNA constructs using both air-pressure- and sclerotium-mediated transformation. Conclusions These transformation methods, which are fast, simple and reproducible, can expedite functional gene analysis of B. cinerea.

Genetic transformation of forest trees | Diouf | African Journal of ...

African Journals Online (AJOL)

In this review, the recent progress on genetic transformation of forest trees were discussed. Its described also, different applications of genetic engineering for improving forest trees or understanding the mechanisms governing genes expression in woody plants. Key words: Genetic transformation, transgenic forest trees, ...

In planta transformation of sorghum (Sorghum bicolor (L.) Moench)

Indian Academy of Sciences (India)

An in planta transformation protocol for sorghum (Sorghum bicolor (L.) Moench) using shoot apical meristem of germinating seedlings is reported in this study. Agrobacterium tumefaciens strain, LBA4404 with pCAMBIA1303 vector and construct pCAMBIA1303TPS1 were individually used for transformation. Since, the ...

Biolistics Transformation of Wheat

Science.gov (United States)

Sparks, Caroline A.; Jones, Huw D.

We present a complete, step-by-step guide to the production of transformed wheat plants using a particle bombardment device to deliver plasmid DNA into immature embryos and the regeneration of transgenic plants via somatic embryogenesis. Currently, this is the most commonly used method for transforming wheat and it offers some advantages. However, it will be interesting to see whether this position is challenged as facile methods are developed for delivering DNA by Agrobacterium tumefaciens or by the production of transformants via a germ-line process (see other chapters in this book).

Progress in planta transformation without tissue culture

International Nuclear Information System (INIS)

Gu Yunhong; Chinese Academy of Sciences, Hefei; Qin Guangyong; Huo Yuping; Yu Zengliang

2004-01-01

With the development of planta genetic engineering, more emphases have been laid on convenient and high efficient genetic transformation methods. And transformation without tissue culture is a prospective direction of it. In this paper, traditional transformation methods and the methods of non-tissue culture were summarized. With the exploration and application of Arabidopsis transformation mechanism, with the use of ion beam-mediated transformation invented by Chinese scientists and the development of other transformation methods, transformation methods without tissue culture and planta genetic engineering could be improved rapidly. (authors)

Agrobacterium-mediated transformation of lipomyces

Science.gov (United States)

Dai, Ziyu; Magnuson, Jon K.; Deng, Shuang; Bruno, Kenneth S.; Culley, David E.

2018-03-13

This disclosure provides Agrobacterium-mediated transformation methods for the oil-producing (oleaginous) yeast Lipomyces sp., as well as yeast produced by the method. Such methods utilize Agrobacterium sp. cells that have a T-DNA binary plasmid, wherein the T-DNA binary plasmid comprises a first nucleic acid molecule encoding a first protein and a second nucleic acid molecule encoding a selective marker that permits growth of transformed Lipomyces sp. cells in selective culture media comprising an antibiotic.

Natural genetic transformation in Acinetobacter sp. BD413 Biofilms: introducing natural genetic transformation as a tool for bioenhancement of biofilm reactors

Energy Technology Data Exchange (ETDEWEB)

Hendrickx, L

2002-07-01

This study focussed on the localization and quantification of natural genetic transformation using neutral and disadvantageous genes in monoculture biofilms to investigate gene transfer and expression of the transferred genes in the absence of a selective advantage. Data obtained by this investigation were regarded as initial steps for evaluating the applicability of adding catabolic traits into the indigenous bacterial community of biofilm reactors by in situ natural genetic transformation. Because Acinetobacter spp. strains are readily found in waste water treatment plants and because Acinetobacter sp. BD413 possesses a high effective level of competence, natural genetic transformation was investigated in monoculture Acinetobacter sp. BD413 biofilms. The genes used for transformation encoded for the green fluorescent protein (GFP) and its variants. Monitoring of transformation events were performed with the use of automated confocal laser scanning microscopy (CLSM) and semi automated digital image processing and analysis. (orig.)

Role of the VirA histidine autokinase of Agrobacterium tumefaciens in the initial steps of pathogenesis

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Yi-Han eLin

2014-05-01

Full Text Available Histidine kinases serve as critical environmental sensing modules, and despite their designation as simple two-component modules, their functional roles are remarkably diverse. In Agrobacterium tumefaciens pathogenesis, VirA serves with VirG as the initiating sensor/transcriptional activator for inter-kingdom gene transfer and transformation of higher plants. Through responses to three separate signal inputs, low pH, sugars, and phenols, A. tumefaciens commits to pathogenesis in virtually all flowering plants. However, how these three signals are integrated to regulate the response and why these signals might be diagnostic for susceptible cells across such a broad host-range remains poorly understood. Using a homology model of the VirA linker region, we provide evidence for coordinated long-range transmission of inputs perceived both outside and inside the cell through the creation of targeted VirA truncations. Further, our evidence is consistent with signal inputs weakening associations between VirA domains to position the active site histidine for phosphate transfer. This mechanism requires long-range regulation of inter-domain stability and the transmission of input signals through a common integrating domain for VirA signal transduction.

Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens

International Nuclear Information System (INIS)

Huberman, E.; Langenbach, R.

1977-01-01

We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro

Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells.

Science.gov (United States)

Matthysse, A G; Wyman, P M; Holmes, K V

1978-11-01

Kinetic, microscopic, and biochemical studies show that virulent Ti (tumor inducing)-plasmid-containing strains of Agrobacterium attach to normal tobacco and carrot tissue culture cells. Kinetic studies showed that virulent strains of A. tumefaciens attach to the plant tissue culture cells in increasing numbers during the first 1 to 2 h of incubation of the bacteria with the plant cells. Five Ti-plasmid-containing virulent Agrobacterium strains showed greater attachment to tobacco cells than did five avirulent strains. Light and scanning electron microscopic observations confirmed that virulent strains showed little attachment. Bacterial attachment was blocked by prior incubation of the plant cells with lipopolysaccharide extracted from A. tumefaciens, but not from A. radiobacter, suggesting that bacterial lipopolysaccharide is one of the components involved in the attachment process. At least one other bacterial product may be required for attachment in tissue culture because the virulent A. tumefaciens NT1, which lacks the Ti plasmid, does not itself attach to tobacco cells, but its lipopolysaccharide does inhibit the attachment of virulent strains.

Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.

Science.gov (United States)

Rolloos, Martijn; Hooykaas, Paul J J; van der Zaal, Bert J

2015-02-09

Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.

Pepper, Sweet (Capsicum annuum)

NARCIS (Netherlands)

Heidmann, I.; Boutilier, K.A.

2015-01-01

Capsicum (pepper) species are economically important crops that are recalcitrant to genetic transformation by Agrobacterium ( Agrobacterium tumefaciens ). A number of protocols for pepper transformation have been described but are not routinely applicable. The main bottleneck in pepper

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene.

Science.gov (United States)

Narendran, M; Deole, Satish G; Harkude, Satish; Shirale, Dattatray; Nanote, Asaram; Bihani, Pankaj; Parimi, Srinivas; Char, Bharat R; Zehr, Usha B

2013-08-01

Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ). Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.

Preliminary crystallographic analysis of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens

International Nuclear Information System (INIS)

Cupp-Vickery, Jill R.; Igarashi, Robert Y.; Meyer, Christopher R.

2005-01-01

Crystallization and X-ray diffraction methods for native A. tumefaciens ADP-glucose pyrophosphorylase and its selenomethionyl derivative are described. Two crystal forms are identified, both of which diffract to 2 Å

Direct genetic transformation of Hibiscus sabdariffa L.

African Journals Online (AJOL)

Administrator

After 60 days evaluation point, the assessment of the transformation by PCR revealed that H. sabdariffa line tested, carried the nptII gene. Key words: Hibiscus sabdariffa, genetic transformation. INTRODUCTION. Hibiscus sabdariffa is a crop widely cultivated in Sub. Saharan Africa, growing on sandy soils after the harvest.

Novel and potential application of cryopreservation to plant genetic transformation.

Science.gov (United States)

Wang, Biao; Zhang, Zhibo; Yin, Zhenfang; Feng, Chaohong; Wang, Qiaochun

2012-01-01

The world population now is 6.7 billion and is predicted to reach 9 billion by 2050. Such a rapid growing population has tremendously increased the challenge for food security. Obviously, it is impossible for traditional agriculture to ensure the food security, while plant biotechnology offers considerable potential to realize this goal. Over the last 15 years, great benefits have been brought to sustainable agriculture by commercial cultivation of genetically modified (GM) crops. Further development of new GM crops will with no doubt contribute to meeting the requirements for food by the increasing population. The present article provides updated comprehensive information on novel and potential application of cryopreservation to genetic transformation. The major progresses that have been achieved in this subject include (1), long-term storage of a large number of valuable plant genes, which offers a good potential for further development of novel cultivars by genetic transformation; (2), retention of regenerative capacity of embryogenic tissues and protoplasts, which ensures efficient plant regeneration system for genetic transformation; (3), improvement of transformation efficiency and plant regeneration of transformed cells; (4), long-term preservation of transgenic materials with stable expression of transgenes and productive ability of recombinant proteins, which allows transgenic materials to be stored in a safe manner before being analyzed and evaluated, and allows establishment of stable seed stocks for commercial production of homologous proteins. Data provided in this article clearly demonstrate that cryo-technique has an important role to play in the whole chain of genetic transformation. Further studies coupling cryotechnique and genetic transformation are expected to significantly improve development of new GM crops. Copyright © 2011 Elsevier Inc. All rights reserved.

One out of four: HspL but no other small heat shock protein of Agrobacterium tumefaciens acts as efficient virulence-promoting VirB8 chaperone.

Directory of Open Access Journals (Sweden)

Yun-Long Tsai

Full Text Available Alpha-crystallin-type small heat shock proteins (sHsps are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named hspL, hspC, hspAT1, and hspAT2 were identified in Agrobacterium tumefaciens, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS. HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of hspL but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the hspL deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of hspL and only modest increase of hspC, hspAT1, and hspAT2 at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays in vitro indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated in vivo, in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in A. tumefaciens. Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence

A highly efficient method for Agrobacterium mediated transformation ...

African Journals Online (AJOL)

An Agrobacterium mediated transformation method was developed for the Thai rice variety, Pathumthani 1 (PT1), and the Indian rice variety, Pokkali (PKL). Various aspects of the transformation method, including callus induction, callus age, Agrobacterium concentration and co-cultivation period were examined, in order to ...

Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes.

Science.gov (United States)

Aarrouf, J; Castro-Quezada, P; Mallard, S; Caromel, B; Lizzi, Y; Lefebvre, V

2012-02-01

Pepper is known to be a recalcitrant species to genetic transformation via Agrobacterium tumefaciens. A. rhizogenes-mediated transformation offers an alternative and rapid possibility to study gene functions in roots. In our study, we developed a new and efficient system for A. rhizogenes transformation of the cultivated species Capsicum annuum. Hypocotyls and foliar organs (true leaves and cotyledons) of Yolo Wonder (YW) and Criollo de Morelos 334 (CM334) pepper cultivars were inoculated with the two constructs pBIN-gus and pHKN29-gfp of A. rhizogenes strain A4RS. Foliar explants of both pepper genotypes infected by A4RS-pBIN-gus or A4RS-pHKN29-gfp produced transformed roots. Optimal results were obtained using the combination of the foliar explants with A4RS-pHKN29-gfp. 20.5% of YW foliar explants and 14.6% of CM334 foliar explants inoculated with A4RS-pHKN29-gfp produced at least one root expressing uniform green fluorescent protein. We confirmed by polymerase chain reaction the presence of the rolB and gfp genes in the co-transformed roots ensuring that they integrated both the T-DNA from the Ri plasmid and the reporter gene. We also demonstrated that co-transformed roots of YW and CM334 displayed the same resistance response to Phytophthora capsici than the corresponding untransformed roots. Our novel procedure to produce C. annuum hairy roots will thus support the functional analysis of potential resistance genes involved in pepper P. capsici interaction.

Construction of a gene bank and use of the chromosome walking technique for the detection of new putative agrocin genes in Agrobacterium tumefaciens strain D286

International Nuclear Information System (INIS)

Herrera, G.

1987-02-01

A gene bank of Agrobacterium tumefaciens D286 wt has been constructed by cloning D286 wt DNA partially digested with EcoRI in the cosmid vector pLAFRI. The library; composed of 1750 members with a 27.7 kb average insert size was probed with pCDTn5-3, a cosmid vector carrying a D286:: Tn5 insert from the strain D286:: Tn5 Ag-. One recombinant cosmid of the library, pCDO932, was detected. The insert DNA of pCDO932 has sequences homologous to the D286:: Tn5 insert of pCDTn5-3, therefore it carries putative wt agrocin D286 genes. The insert DNA of pCDO932 was isolated and used to probe the D286 wt gene library. Chromosome walking resulted in the detection of pCD2375. EcoRI restriction digestions and DNA homology studies of pCDO932 and pCD2375 showed that their D286 wt inserts are both composed of 4 EcoRI DNA sub-fragments totalling 21.8 and 24.8 kb respectively, with an overlapping sequence extending 3.5 kb. In order to overcome the failure to detect A. tumefaciens cells transformed with pCDO932. Vectors pSUP204-1 was constructed. Such vector has been derived from pSUP204 which were slightly altered by cloning into it a 700 bp λ DNA SalI fragmet. This resulted in insertion inactivation of the Tc r gene, allows the use of pSUP204-1 as a subcloning vector in conjugations and transformations involving pCDO932 or pCD2375 and strains D286:: Tn5 Ag- and C58 C1G. Two recombinant cosmids bearing D286 wt DNA inserts, at least one of which (pCDO932) contains DNA sequences putatively affecting agrocin D286 production, are now available for further genetic manipulations. pSUP204-1 should prove useful as a subcloning vector for transformations and conjugations involving recombinant cosmids from the D286 wt gene bank and Agrobacterium strains. Future work on the molecular biology of agrocin D286 production is discussed. The DNA probe used in this study was labelled with phosphorus 32

Laccase mediated transformation of 17β-estradiol in soil

International Nuclear Information System (INIS)

Singh, Rashmi; Cabrera, Miguel L.; Radcliffe, David E.; Zhang, Hao; Huang, Qingguo

2015-01-01

It is known that 17β-estradiol (E2) can be transformed by reactions mediated by some oxidoreductases such as laccase in water. Whether or how such reactions can happen in soil is however unknown although they may significantly impact the environmental fate of E2 that is introduced to soil by land application of animal wastes. We herein studied the reaction of E2 in a model soil mediated by laccase, and found that the reaction behaviors differ significantly from those in water partly because of the dramatic difference in laccase stability. We also examined E2 transformation in soil using 14 C-labeling in combination with soil organic matter extraction and size exclusion chromatography, which indicated that applied 14 C radioactivity was preferably bound to humic acids. The study provides useful information for understanding the environmental fate of E2 and for developing a novel soil remediation strategy via enzyme-enhanced humification reactions. - Highlights: • E2 was effectively transformed in soil through reactions mediated by laccase. • The reaction behaviors in soil differ significantly from those in water. • E2 was preferably bound to the humic acids in soil. • Laccase treatment resulted in changes in the structures of the humic acids. - E2 was effectively transformed in soil by preferably binding to the humic acids through reactions mediated by laccase

PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival

Directory of Open Access Journals (Sweden)

Amanda R. Panfil

2015-12-01

Full Text Available Human T-cell leukemia virus type-1 (HTLV-1 is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL. This disease manifests after a long clinical latency period of up to 2–3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5 on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment.

Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars.

Science.gov (United States)

Liu, Yan-Rong; Cen, Hui-Fang; Yan, Jian-Ping; Zhang, Yun-Wei; Zhang, Wan-Jun

2015-07-01

Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars. Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic "core" structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95%, and upland cultivars Blackwell and Dacotah, 50 and 76%, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8% was achieved for lowland cultivar Alamo, and 8.0% for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.

Development of antibiotic marker-free creeping bentgrass resistance against herbicides.

Science.gov (United States)

Lee, Ki-Won; Kim, Ki-Yong; Kim, Kyung-Hee; Lee, Byung-Hyun; Kim, Jin-Seog; Lee, Sang-Hoon

2011-01-01

Herbicide-resistant creeping bentgrass plants (Agrostis stolonifera L.) without antibiotic-resistant markers were produced by Agrobacterium-mediated transformation. Embryogenic callus tissues were infected with Agrobacterium tumefaciens EHA105, harboring the bar and the CP4-EPSPS genes for bialaphos and glyphosate resistance. Phosphinothricin-resistant calli and plants were selected. Soil-grown plants were obtained at 14-16 weeks after transformation. Genetic transformation of the selected, regenerated plants was validated by PCR. Southern blot analysis revealed that at least one copy of the transgene was integrated into the genome of the transgenic plants. Transgene expression was confirmed by Northern blot. CP4-EPSPS protein was detected by ELISA. Transgenic plants remained green and healthy when sprayed with Basta, containing 0.5% glufosinate ammonium or glyphosate. The optimized Agrobacterium-mediated transformation method resulted in an average of 9.4% transgenic plants. The results of the present study suggest that the optimized marker-free technique could be used as an effective and reliable method for routine transformation, which may facilitate the development of varieties of new antibiotic-free grass species.

Genetic transformation of mature citrus plants.

Science.gov (United States)

Cervera, Magdalena; Juárez, José; Navarro, Luis; Peña, Leandro

2005-01-01

Most woody fruit species have long juvenile periods that drastically prolong the time required to analyze mature traits. Evaluation of characteristics related to fruits is a requisite to release any new variety into the market. Because of a decline in regenerative and transformation potential, genetic transformation procedures usually employ juvenile material as the source of plant tissue, therefore resulting in the production of juvenile plants. Direct transformation of mature material could ensure the production of adult transgenic plants, bypassing in this way the juvenile phase. Invigoration of the source adult material, establishment of adequate transformation and regeneration conditions, and acceleration of plant development through grafting allowed us to produce transgenic mature sweet orange trees flowering and bearing fruits in a short time period.

Simple screening strategy with only water bath needed for the identification of insect-resistant genetically modified rice.

Science.gov (United States)

Zhang, Fang; Wang, Liu; Wang, Rui; Ying, Yibin; Wu, Jian

2015-02-03

An informative, with simple instrument needed, rapid and easily updated strategy for the identification of insect-resistant genetically modified (GM) rice has been described. Such strategy is based on a parallel series of loop-mediated isothermal amplification (LAMP) reactions targeting the rice endogenous gene sucrose phosphate synthase (Sps), the top two most frequently used genetic elements (Agrobacterium tumefaciens nopaline synthase terminator (Nos) and Cauliflower mosaic virus 35S promoter (CaMV35S)), and an insect-resistant specific gene (Cry1Ac) and detected visually by phosphate ion (Pi)-induced coloration reaction. After a logical judgment of visible readouts has been obtained, three popular insect-resistant GM rice events in China can be successfully identified within 35 min, using either microwell strips or paper bases.

Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system

DEFF Research Database (Denmark)

Danhorn, T.; Hentzer, Morten; Givskov, Michael Christian

2004-01-01

The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces. Adherence of A. tumefaciens C58 was significantly enhanced under phosphate limitation compared to phosphate-replete conditions, despite slower overall growth under low-phosphate conditions...

Cotton transformation via pollen tube pathway.

Science.gov (United States)

Wang, Min; Zhang, Baohong; Wang, Qinglian

2013-01-01

Although many gene transfer methods have been employed for successfully obtaining transgenic cotton, the major constraint in cotton improvement is the limitation of genotype because the majority of transgenic methods require plant regeneration from a single transformed cell which is limited by cotton tissue culture. Comparing with other plant species, it is difficult to induce plant regeneration from cotton; currently, only a limited number of cotton cultivars can be cultured for obtaining regenerated plants. Thus, development of a simple and genotype-independent genetic transformation method is particularly important for cotton community. In this chapter, we present a simple, cost-efficient, and genotype-independent cotton transformation method-pollen tube pathway-mediated transformation. This method uses pollen tube pathway to deliver transgene into cotton embryo sacs and then insert foreign genes into cotton genome. There are three major steps for pollen tube pathway-mediated genetic transformation, which include injection of -foreign genes into pollen tube, integration of foreign genes into plant genome, and selection of transgenic plants.

Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

Science.gov (United States)

Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

2015-07-27

VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.

Science.gov (United States)

Yao, Jia-Long; Tomes, Sumathi; Gleave, Andrew P

2013-05-01

Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 μg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

Evaluations and modifications of semi-selective media for improved isolation of Agrobacterium tumefaciens biovar 1 from cultivated walnut

Science.gov (United States)

Agrobacterium tumefaciens, the causal agent of crown gall of walnut, is an aerobic, Gram negative bacterium belonging to the family Rhizobiaceae. Like many in this group, A. tumefaciens is a common inhabitant of soil and plant host tissue. Isolation from these complex environments is difficult even ...

Application of Carbon Nanotubes for Plant Genetic Transformation

Science.gov (United States)

Burlaka, Olga M.; Pirko, Yaroslav V.; Yemets, Alla I.; Blume, Yaroslav B.

In this chapter, the current state of using carbon nanotubes (CNTs; single- and multi-walled) that have attracted great interdisciplinary interest in recent decades due to their peculiar properties for genetic transformation of prokaryotic and eukaryotic cells will be enlightened. The covalent and non-covalent surface chemistry for the CNT functionalization with focus on the potential applications of surface modifications in design of biocompatible CNTs will be discussed. The properties of CNTs that are favorable for biotechnological use and current status of technical approaches that allow the increase in biocompatibility and lower nanotoxicity of engineered CNTs will be described. Decisions proposed by non-covalent surface modification of CNTs will be discussed. Existing data concerning mechanisms of CNT cell entry and factors governing toxicity, cellular uptake, intracellular traffic, and biodegradation of CNTs along with bioavailability of molecular cargoes of loaded CNTs will be discussed. Eco-friendly production of water dispersions of biologically functionalized multi-walled and single-walled CNTs for use as nano-vehicles for the DNA delivery in plant genetic transformation of plants will be described. The background, advantages, and problems of using CNTs in developing of novel methods of genetic transformation, including plant genetic transformation, will be highlighted. Special attention will be paid to the limitations of conventional gene transfer techniques and promising features of CNT-based strategies having improved efficacy, reproducibility, and accuracy along with less time consumption. Issues impeding manipulation of CNTs such as entangled bundle formation, low water solubility, inert properties of pristine CNTs, etc., and ways to solve arising tasks will be overviewed.

Recent Advances in the Genetic Transformation of Coffee

Science.gov (United States)

Mishra, M. K.; Slater, A.

2012-01-01

Coffee is one of the most important plantation crops, grown in about 80 countries across the world. The genus Coffea comprises approximately 100 species of which only two species, that is, Coffea arabica (commonly known as arabica coffee) and Coffea canephora (known as robusta coffee), are commercially cultivated. Genetic improvement of coffee through traditional breeding is slow due to the perennial nature of the plant. Genetic transformation has tremendous potential in developing improved coffee varieties with desired agronomic traits, which are otherwise difficult to achieve through traditional breeding. During the last twenty years, significant progress has been made in coffee biotechnology, particularly in the area of transgenic technology. This paper provides a detailed account of the advances made in the genetic transformation of coffee and their potential applications. PMID:22970380

Biophysical control of the growth of Agrobacterium tumefaciens using extremely low frequency electromagnetic waves at resonance frequency.

Science.gov (United States)

Fadel, M Ali; El-Gebaly, Reem H; Mohamed, Shaimaa A; Abdelbacki, Ashraf M M

2017-12-09

Isolated Agrobacterium tumefaciens was exposed to different extremely low frequencies of square amplitude modulated waves (QAMW) from two generators to determine the resonance frequency that causes growth inhibition. The carrier was 10 MHz sine wave with amplitude ±10 Vpp which was modulated by a second wave generator with a modulation depth of ± 2Vpp and constant field strength of 200 V/m at 28 °C. The exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min inhibited the bacterial growth by 49.2%. In addition, the tested antibiotics became more effective against A. tumefaciens after the exposure. Furthermore, results of DNA, dielectric relaxation and TEM showed highly significant molecular and morphological changes due to the exposure to 1.0 Hz QAMW for 90 min. An in-vivo study has been carried out on healthy tomato plants to test the pathogenicity of A. tumefaciens before and after the exposure to QAMW at the inhibiting frequency. Symptoms of crown gall and all pathological symptoms were more aggressive in tomato plants treated with non-exposed bacteria, comparing with those treated with exposed bacteria. We concluded that, the exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min modified its cellular activity and DNA structure, which inhibited the growth and affected the microbe pathogenicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of a rapid and simple Agrobacterium tumefaciens mediated transformation system for the fungal pathogen Heterobasidion annosum

Science.gov (United States)

Nicklas Samils; Malin Elfstrand; Daniel L. Lindner Czederpiltz; Jan Fahleson; Ake Olson; Christina Dixelius; Jan Stenlid

2006-01-01

Heterobasidion annosum causes root and butt-rot in trees and is the most serious forest pathogen in the northern hemisphere. We developed a rapid and simple Agrobacterium-mediated method of gene delivery into H. annosum to be used in functional studies of candidate genes and for visualization of mycelial interactions. Heterobasidion annosum TC 32-1 was cocultivated at...

Global analysis of differentially expressed genes and proteins in the wheat callus infected by Agrobacterium tumefaciens.

Directory of Open Access Journals (Sweden)

Xiaohong Zhou

Full Text Available Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs and differentially expressed proteins (DEPs were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq and two-dimensional electrophoresis (2-DE in conjunction with mass spectrometry (MS. A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.

Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens

Science.gov (United States)

Zhou, Xiaohong; Wang, Ke; Lv, Dongwen; Wu, Chengjun; Li, Jiarui; Zhao, Pei; Lin, Zhishan; Du, Lipu; Yan, Yueming; Ye, Xingguo

2013-01-01

Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops. PMID:24278131

NUTRITIONAL ENHANCEMENT OF ALFALFA THROUGH GENETIC ENGINEERING

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J. Faragó

2008-09-01

Full Text Available Alfalfa (Medicago sativa L. is a pasture legume crop of primary importance to animal production throughout the world. The nutritional quality of alfalfa, as of other leguminous forage crops, is mainly determined by their content in selected essential amino acids (EAAs, such as methionine (Met and cysteine (Cys. In alfalfa, however, these S-containing amino acids constitute only about 1% or less of crude proteins (Frame et al., 1998. This is significantly less than the 3.5% Met+Cys content in the recommended FAO reference protein (FAO, 1973. Recent advances in genetic engineering allow to use the transgenic approach to increase the content of specific essential amino acids in target plant species. A number of different molecular approaches have been developed to address this issue, such as over-expression of a heterologous or homologous Met-rich protein, expression of a synthetic protein, modification of protein sequence, and metabolic engineering of the free amino acid pool and protein sink. To study the possibility of transgenic enhancement of nutritional quality of alfalfa, we used the approach of expression of a heterologous protein rich in Met+Cys in cells of alfalfa. The T-DNA introduced into the genome of alfalfa, using Agrobacterium tumefaciens-mediated genetic transformation, contained the selectable merker gene nptII for kanamycin (Kn resistance, and a cDNA of Ov gene from Japanese quail (Coturnix coturnix coding for a high Met+Cys containing ovalbumine (Mucha et al., 1991, both under constitutive promoters. After cocultivation of petiole segment- and leaf blade-explants of two highly embryogenic alfalfa genotypes Rg9/I-14-22 and Rg11/I-10-68 (Faragó et al., 1997 with cells of A. tumefaciens strain AGL1 carrying the nptII and Ov genes, and selection of transgenic cells on Kn containing selective media, more than one hundred putatively transgenic regenerants were obtained through somatic embryogenesis. Biological (Kn rooting assay

Susceptibility of Juglans Species andInterspecific Hybrids to Agrobacterium tumefaciens

Science.gov (United States)

James R. McKenna; Lynn Epstein

2003-01-01

Crown gall, caused by the common soil-borne bacterium Agrobacterium tumefaciens, can be an economic problem in walnut nurseries and production orchards in Caliiornia. The principal rootstocks used for commercial walnut production in California are the native Northern California black walnut, Juglans hindsii, and "Paradox,...

Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P_SAG12-ipt gene delays leaf senescence and enhances resistance to exogenous ethylene

DEFF Research Database (Denmark)

Zakizadeh, Hedayatollah; Lütken, Henrik Vlk; Sriskandarajah, Sridevy

2013-01-01

Transgenic plants of Rosa hybrida ‘Linda’ were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the PSAG12-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P35S-INTGUS gene) were used for transformation...... of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1–6 copies of the nptII gene...

Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid

NARCIS (Netherlands)

Okker, Robert J.H.; Spaink, Herman; Hille, Jacques; Brussel, Ton A.N. van; Lugtenberg, Ben; Schilperoort, Rob A.

1984-01-01

Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for

Morphogenetic and chemical stability of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants.

Science.gov (United States)

Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Mathur, Ajay Kumar; Shanker, Karuna

2015-01-01

Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacterium rhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant.

A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae.

Directory of Open Access Journals (Sweden)

Raphaël Laurenceau

Full Text Available Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

DEFF Research Database (Denmark)

Sorensen, Lisette Quaade; Lysøe, Erik; Larsen, Jesper Erup

2014-01-01

The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infe...

Regeneration ability and genetic transformation of root type chicory ...

African Journals Online (AJOL)

This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes. Keywords: Chicory, regeneration, transformation, 1-Naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), ...

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Science.gov (United States)

Sarrion-Perdigones, Alejandro; Falconi, Erica Elvira; Zandalinas, Sara I.; Juárez, Paloma; Fernández-del-Carmen, Asun; Granell, Antonio; Orzaez, Diego

2011-01-01

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described. PMID:21750718

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

Science.gov (United States)

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

2013-11-01

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

Natural Genetic Transformation Generates a Population of Merodiploids in Streptococcus pneumoniae

Science.gov (United States)

Zomer, Aldert; Bootsma, Hester J.; Prudhomme, Marc; Granadel, Chantal; Hermans, Peter W. M.; Polard, Patrice; Martin, Bernard; Claverys, Jean-Pierre

2013-01-01

Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R) corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ∼3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD) formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ∼100 to ∼900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA). We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of merodiploids by

Natural genetic transformation generates a population of merodiploids in Streptococcus pneumoniae.

Directory of Open Access Journals (Sweden)

Calum Johnston

Full Text Available Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ~3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ~100 to ~900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA. We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of

Effects of X-ray irradiation to genetic transformation of Eruca sativa Mill mediated by agrobacterium rhizogenes

International Nuclear Information System (INIS)

Wu Zhenhua; Zhang Hong; Liu Bin; Li Ning; Niu Bingtao; Wang Genxuan; Li Sha; Sun Peng

2008-01-01

To investigate the effects of X-ray irradiation to the hairy root induction of Eruca sativa Mill, and to provide basic experimental data for concerted reaction of irradiation to the Genetic transformation technology. The aseptic cotyledons of Eruca sativa Mill were jointly treated with the X-ray at dose of 5, 10, 15, 20 Gy respectively and the different infected time, then the influence of explant age, pre-culturing time, dose of X-ray and infected time were studied. Furthermore, the hairy roots were testified in molecular level by the polymerase chain reaction(PCR). The irradiation of X-ray at dose of 15 Gy can improve the frequency of Eruca sativa Mill hairy root induction in a dose-dependent manner. Moreover, the transformation frequency in pre-infection plus irradiation group is higher than that in pre-irradiation plus infection group at the same irradiation dose. In addition, the PCR analysis also demonstrated that rolB gene of T-DNA from Ri plasmid had been integrated into the genome of the transformed roots. The irradiation of X-ray has some positive effects on the hairy root induction of Eruca sativa Mill, and the optimal revulsive dose is 15 Gy ora little bit more. (authors)

Transformational leadership and task cohesion in sport: the mediating role of inside sacrifice.

Science.gov (United States)

Cronin, Lorcan Donal; Arthur, Calum Alexander; Hardy, James; Callow, Nichola

2015-02-01

In this cross-sectional study, we examined a mediational model whereby transformational leadership is related to task cohesion via sacrifice. Participants were 381 American (Mage = 19.87 years, SD = 1.41) Division I university athletes (188 males, 193 females) who competed in a variety of sports. Participants completed measures of coach transformational leadership, personal and teammate inside sacrifice, and task cohesion. After conducting multilevel mediation analysis, we found that both personal and teammate inside sacrifice significantly mediated the relationships between transformational leadership behaviors and task cohesion. However, there were differential patterns of these relationships for male and female athletes. Interpretation of the results highlights that coaches should endeavor to display transformational leadership behaviors as they are related to personal and teammate inside sacrifices and task cohesion.

EXPRESIÓN GUS EN EXPLANTES DE Solanum phureja (Juz. et. Buk Var. Criolla Colombia, TRANSFORMADOS CON Agrobacterium tumefaciens

Directory of Open Access Journals (Sweden)

IVÁN DARÍO BARRERO-FARFÁN

2008-01-01

Full Text Available La expresión transitoria y estable del gen gusA-intron en explantes internodales de papa criolla variedad Criolla Colombia cocultivados con Agrobacterium tumefaciens es reportada. Con el fin de determinar la susceptibilidad de esta variedad a la transformación mediada por A. tumefaciens, explantes internodales de Solanum phureja fueron infectados con la cepa LBA4404 de A. tumefaciens que contiene el plásmido pCAMBIA2301. Este plásmido contiene el gen ntpII que confiere resistencia a kanamicina y el gen reportero gusA-intron. La selección de los explantes potencialmente transgénicos fue realizada en medios con kanamicina. La eficiencia de transformación estable y transitoria fue calculada con base en la actividad GUS (ß-glucuronidasa, detectada por el ensayo histoquímico X-gluc. La expresión transitoria y estable del gen gusA-intron fue observada en células del explante más bien que en tejidos completos. Estos resultados demuestran que la papa criolla (S. phureja Juz. et. Buk variedad Criolla Colombia es susceptible a la infección por A. tumefaciens.

Host genes involved in Agrobacterium-mediated transformation

NARCIS (Netherlands)

Soltani, Jalal

2009-01-01

Agrobacterium is the nature’s genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the

Metabolic Engineering Strategies for the Optimization of Medicinal and Aromatic Plants : Expectations and Realities

NARCIS (Netherlands)

Kayser, O.; Baricevic, D; Novak, J; Pank, F

2010-01-01

In recent years classic genetic and molecular biology strategies (Bioballistics, Agrobacterium tumefaciens transformation, recombinant enzymes) for production of natural compounds or even breeding of medicinal and aromatic plants have expanded and improved productivity of plant-derived fine

Natural Genetic Transformation Generates a Population of Merodiploids in Streptococcus pneumoniae.

OpenAIRE

Johnston, Calum; Caymaris, Stéphanie; Zomer, Aldert; Bootsma, Hester J.; Prudhomme, Marc; Granadel, Chantal; Hermans, Peter W. M.; Polard, Patrice; Martin, Bernard; Claverys, Jean-Pierre

2013-01-01

Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exog...

Biolistic- and Agrobacterium-mediated transformation protocols for wheat.

Science.gov (United States)

Tamás-Nyitrai, Cecília; Jones, Huw D; Tamás, László

2012-01-01

After rice, wheat is considered to be the most important world food crop, and the demand for high-quality wheat flour is increasing. Although there are no GM varieties currently grown, wheat is an important target for biotechnology, and we anticipate that GM wheat will be commercially available in 10-15 years. In this chapter, we summarize the main features and challenges of wheat transformation and then describe detailed protocols for the production of transgenic wheat plants both by biolistic and Agrobacterium-mediated DNA-delivery. Although these methods are used mainly for bread wheat (Triticum aestivum L.), they can also be successfully applied, with slight modifications, to tetraploid durum wheat (T. turgidum L. var. durum). The appropriate size and developmental stage of explants (immature embryo-derived scutella), the conditions to produce embryogenic callus tissues, and the methods to regenerate transgenic plants under increasing selection pressure are provided in the protocol. To illustrate the application of herbicide selection system, we have chosen to describe the use of the plasmid pAHC25 for biolistic transformation, while for Agrobacterium-mediated transformation the binary vector pAL156 (incorporating both the bar gene and the uidA gene) has been chosen. Beside the step-by-step methodology for obtaining stably transformed and normal fertile plants, procedures for screening and testing transgenic wheat plants are also discussed.

Transformational leadership, empowerment, and job satisfaction: the mediating role of employee empowerment.

Science.gov (United States)

Choi, Sang Long; Goh, Chin Fei; Adam, Muhammad Badrull Hisyam; Tan, Owee Kowang

2016-12-01

Recent studies have revealed that nursing staff turnover remains a major problem in emerging economies. In particular, nursing staff turnover in Malaysia remains high due to a lack of job satisfaction. Despite a shortage of healthcare staff, the Malaysian government plans to create 181 000 new healthcare jobs by 2020 through the Economic Transformation Programme (ETP). This study investigated the causal relationships among perceived transformational leadership, empowerment, and job satisfaction among nurses and medical assistants in two selected large private and public hospitals in Malaysia. This study also explored the mediating effect of empowerment between transformational leadership and job satisfaction. This study used a survey to collect data from 200 nursing staff, i.e., nurses and medical assistants, employed by a large private hospital and a public hospital in Malaysia. Respondents were asked to answer 5-point Likert scale questions regarding transformational leadership, employee empowerment, and job satisfaction. Partial least squares-structural equation modeling (PLS-SEM) was used to analyze the measurement models and to estimate parameters in a path model. Statistical analysis was performed to examine whether empowerment mediated the relationship between transformational leadership and job satisfaction. This analysis showed that empowerment mediated the effect of transformational leadership on the job satisfaction in nursing staff. Employee empowerment not only is indispensable for enhancing job satisfaction but also mediates the relationship between transformational leadership and job satisfaction among nursing staff. The results of this research contribute to the literature on job satisfaction in healthcare industries by enhancing the understanding of the influences of empowerment and transformational leadership on job satisfaction among nursing staff. This study offers important policy insight for healthcare managers who seek to increase job

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

Science.gov (United States)

Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji

2013-12-01

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.

Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments

OpenAIRE

Almeida,Weliton Antonio Bastos de; Mourão Filho,Francisco de Assis Alves; Mendes,Beatriz Madalena Januzzi; Pavan,Alexandra; Rodriguez,Adriana Pinheiro Martinelli

2003-01-01

Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck). Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod) were used...

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 ...

Regulatory focus and burnout in nurses: The mediating effect of perception of transformational leadership.

Science.gov (United States)

Shi, Rui; Zhang, Shilei; Xu, Hang; Liu, Xufeng; Miao, Danmin

2015-12-01

This correlation study investigated the relationship between nurses' regulatory focus and burnout, as mediated by their perceptions of transformational leadership, using a cross-sectional research design with anonymous questionnaires. In July-August 2012, data were collected from 378 nurses from three hospitals in Shaanxi Province, China, using self-report questionnaires for measuring the nurses' regulatory focus, their level of burnout and their perception of whether the leadership of their supervisor was transformational. Structural equation modelling and bootstrapping procedures were used to identify the mediating effect of their perceptions of transformational leadership. The results supported our hypothesized model. The type of regulatory focus emerged as a significant predictor of burnout. Having a perception of transformational leadership partially mediated the relationship between regulatory focus and burnout. Having a promotion focus reduced burnout when the participants perceived transformational leadership, whereas having a prevention focus exhibited the opposite pattern. The mediating effect of the perception of transformational leadership suggests that a promotion focus may help diminish burnout, directly and indirectly. Nurse managers must be aware of the role of a regulatory focus and cultivate promotion focus in their followers. © 2014 Wiley Publishing Asia Pty Ltd.

Computational prediction of over-annotated protein-coding genes in the genome of Agrobacterium tumefaciens strain C58

International Nuclear Information System (INIS)

Yu Jia-Feng; Sui Tian-Xiang; Wang Ji-Hua; Wang Hong-Mei; Wang Chun-Ling; Jing Li

2015-01-01

Agrobacterium tumefaciens strain C58 is a type of pathogen that can cause tumors in some dicotyledonous plants. Ever since the genome of A. tumefaciens strain C58 was sequenced, the quality of annotation of its protein-coding genes has been queried continually, because the annotation varies greatly among different databases. In this paper, the questionable hypothetical genes were re-predicted by integrating the TN curve and Z curve methods. As a result, 30 genes originally annotated as “hypothetical” were discriminated as being non-coding sequences. By testing the re-prediction program 10 times on data sets composed of the function-known genes, the mean accuracy of 99.99% and mean Matthews correlation coefficient value of 0.9999 were obtained. Further sequence analysis and COG analysis showed that the re-annotation results were very reliable. This work can provide an efficient tool and data resources for future studies of A. tumefaciens strain C58. (special topic)

Transformational leadership and project success : The mediating role of team-building

NARCIS (Netherlands)

Aga, Deribe Assefa; Noorderhaven, Niels; Vallejo, Bertha

2016-01-01

Although the effect of transformational leadership on project success is empirically supported, less is known about the mechanisms that explain this effect. To address this issue, we propose the mediating role of team-building as a possible explanation of the relationship between transformational

Agrobacterium rhizogenes-induced cotton hairy root culture as an alternative tool for cotton functional genomics

Science.gov (United States)

Although well-accepted as the ultimate method for cotton functional genomics, Agrobacterium tumefaciens-mediated cotton transformation is not widely used for functional analyses of cotton genes and their promoters since regeneration of cotton in tissue culture is lengthy and labor intensive. In cer...

Optimized integration of T-DNA in the taxol-producing fungus ...

African Journals Online (AJOL)

We previously reported a taxol-producing fungus Pestalotiopsis malicola. There, we described the transformation of the fungus mediated by Agrobacterium tumefaciens. T-DNA carrying the selection marker was transferred into the fungus and randomly integrated into the genome as shown by Southern blotting.

Genetic transformation of Neisseria gonorrhoeae shows a strand preference

OpenAIRE

Duffin, Paul M.; Seifert, H. Steven

2012-01-01

Natural transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Neisseria spp. have been shown to preferentially take up and transform their own DNA by recognizing a non-palindromic 10 or 12 nucleotide DNA uptake sequence (DUS10 or DUS12). We investigated the ability of the DUS12 to enhance single-stranded DNA (ssDNA) transformation. Given the non-palindromic nature of the DUS12, we tested whether both strands of the DUS equally en...

Does organizational culture mediate the relationship between transformational leadership and organizational commitment

Directory of Open Access Journals (Sweden)

Nor Hazana Abdullah

2015-02-01

Full Text Available To date, the relationships among organizational culture, transformational leadership and organizational commitment have been empirically investigated. However, majority of these studies have been focusing on direct effects of either transformational leadership or organizational culture on organizational commitment in large organizations. This approach might not only hinder our understanding on real predictors of organizational commitment but also obscure the presence of spurious relationships. Therefore, this study aims to determine the mediating effect of organizational culture on the relationship between transformational leadership and organizational commitment among small business employees. An explanatory research design was used with cross-sectional survey as data collection technique. Once the composite reliability, construct, and convergent and discriminant validity of the measurement constructs were established, a Partial Least Square Structural Equation Modeling (PLS-SEM was run to analyze the structural model and the mediating effect of organizational culture. The results showed that organizational culture mediates the effect of transformational leadership on organizational commitment among small business. This study cautions the generalization of findings obtained from large organizations to be extended to small organizations.

Comparison of agrobacterium mediated wheat and barley transformation with nucleoside diphosphate kinase 2 (NDPK2) gene

International Nuclear Information System (INIS)

Waheed, U.; Shah, M.M.; Smedley, M.; Harwood, W.

2016-01-01

An efficient and reliable transformation system is imperative for improvement of important crop species like barley and wheat. Wheat transformation is complex due to larger genome size and polyploidy while barley has a limitation of genotypic dependency. The objective of current study was to compare the relative transformation efficiency of wheat and barley using specific expression vector pBRACT 214-NDPK2 constructed through gateway cloning carrying Nucleoside Diphosphate Kinase 2 (NDPK2) gene. The vector was used to compare the transformation response in both crops using immature embryos through Agrobacterium mediated transformation. Both wheat and barley showed different responses towards callus induction and regeneration. Immature embryos of 1.5 to 2 mm in diameter was found optimum for wheat callus induction while 1 to 1.5 mm for barley. Both embryogenic and non-embryogenic calli were found in wheat with significantly greater tendency for embryogenecity in barley. The overall regeneration response was found different for all transformed wheat and barley cultivars. Wheat cultivars showed good response initially that drastically slowed down in later stages with the exception of Fielder that reached to the green shoots with good roots. The barley transformed lines showed good regeneration response as compared to wheat. PCR analysis of putative transformants using genomic DNA showed a maximum of 27% transformation efficiency in barely. No true transformation response was obtained in all cultivars of wheat used in this study. The protocol developed for wheat and barley transformation will greatly be helpful in crop improvement programme through genetic engineering especially in diploid relatives of cereals. (author)

Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla

Science.gov (United States)

Phomopsis seed decay (PSD) of soybean is caused primarily by the fungal pathogen Phomopsis longicolla. PSD impairs seed germination, reduces seedling vigor, and can substantially reduce stand establishment. In hot and humid conditions, PSD can cause significant yield losses. Few studies have explore...

Blueberry (Vaccinium corymbosum L.).

Science.gov (United States)

Song, Guo-Qing

2015-01-01

Vaccinium consists of approximately 450 species, of which highbush blueberry (Vaccinium corymbosum) is one of the three major Vaccinium fruit crops (i.e., blueberry, cranberry, and lingonberry) domesticated in the twentieth century. In blueberry the adventitious shoot regeneration using leaf explants has been the most desirable regeneration system to date; Agrobacterium tumefaciens-mediated transformation is the major gene delivery method and effective selection has been reported using either the neomycin phosphotransferase II gene (nptII) or the bialaphos resistance (bar) gene as selectable markers. The A. tumefaciens-mediated transformation protocol described in this chapter is based on combining the optimal conditions for efficient plant regeneration, reliable gene delivery, and effective selection. The protocol has led to successful regeneration of transgenic plants from leaf explants of four commercially important highbush blueberry cultivars for multiple purposes, providing a powerful approach to supplement conventional breeding methods for blueberry by introducing genes of interest.

Incidence of Agrobacterium tumefaciens biovar 1 in and on ‘Paradox’ (Juglans hindsii x Juglans regia) walnut seed collected from commercial nurseries

Science.gov (United States)

The walnut rootstock Paradox (Juglans hindsii (Jeps) Rehder x J. regia L.) is susceptible to Agrobacterium tumefaciens (7) which often results in a high incidence of crown gall in nursery or walnut production orchards. Though A. tumefaciens is susceptible to the commonly used preplant soil fumigant...

Plant regeneration and genetic transformation in Jatropha

KAUST Repository

Sujatha, M.

2012-07-01

Jatropha curcas, a non-edible oil bearing species with multiple uses, and considerable economic potential is emerging as a potential biofuel plant. The limited knowledge of this species, low and inconsistent yields, the narrow genetic variability, and vulnerability to insects and diseases are major constraints in successful cultivation of Jatropha as a biofuel crop. Hence, genetic improvement of Jatropha is essential by conventional and modern biotechnological tools to use as a viable alternative source of bio-diesel. Realising its potential as a bio-energy crop, in vitro regeneration methods have been established to meet the demand of large scale supply of superior clones, and also as a prelude for genetic improvement of the species through transgenic approaches. In this chapter, an overview of in vitro tissue culture and genetic transformation of Jatropha is discussed. © 2013 Springer Science+Business Media Dordrecht. All rights are reserved.

Particle bombardment and the genetic enhancement of crops: myths and realities

NARCIS (Netherlands)

Altpeter, F.; Baisakh, N.; Beachy, R.; Bock, R.; Capell, T.; Christou, P.; Daniell, H.; Datta, K.; Datta, S.; Dix, P.J.; Fauquet, C.; Huang, N.; Kohli, A.; Mooibroek, H.; Nicholson, L.; Nguyen, T.T.; Nugent, G.; Raemakers, C.J.J.M.; Romano, A.; Somers, D.A.; Stoger, E.; Taylor, N.; Visser, R.G.F.

2005-01-01

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA

Agrobacterium rhizogenes-Mediated Transformation – a Non-GMO Platform For Developing Compact Ornamentals

DEFF Research Database (Denmark)

Lütken, Henrik Vlk; Hegelund, Josefine Nymark; Lauridsen, Uffe Bjerre

of these compounds are potentially harmful to both the environment and human health. A new non-GMO molecular breeding strategy, as opposed to both the application of chemical growth retardants and conventional molecular breeding is Agrobacterium rhizogenes-mediated transformation. In this method, the soil borne...... for transformations, plants produced via this approach are not considered as GMOs in the European Union and Japan. We have developed an optimised Agrobacterium rhizogenes-mediated transformation platform useful for a wide range of ornamentals. Kalanchoë was the starting point and the effect of the rol-genes has now...

Optimization of genetic transformation with Agrobacterium rhizogenes

International Nuclear Information System (INIS)

Blanco, M.; Valverde, R.; Gomez, L.

2002-01-01

To optimize the genetic transformation efficiency using Agrobacterium rhizogenes, carrot sections inoculated with the Agrobacterium strain A4TC were co-cultivated with acetosyringone, phloroglucinol, and a mix of both. Acetosyringone is one of the phenolic compounds produced by plant tissues in response to wounding, which induces the transfer of T-DNA from the agrobacteria to the plant. Phloroglucinol is also a phenolic compound; however, it has a synergistic action with auxins by partially inhibiting cytokinin activity. The highest transformation efficiency (75%) was obtained with acetosyringone (100 mM) in combination with phloroglucinol (25 mg l - 1 ). In general, a 6-day co-cultivation, independently of treatments, induced the best transformation rate. Inclusion of 100 mg l - 1 kanamycin efficiently discriminated transformed roots from non-transgenic ones. This paper also presents a novel bacterial elimination method, by which Agrobacterium can be completely eliminated in 48 h with Cefotaxime at a dosage of 500 mg l - 1 . Author [es

Genetic Transformation of Artemisia carvifolia Buch with rol Genes Enhances Artemisinin Accumulation.

Directory of Open Access Journals (Sweden)

Erum Dilshad

Full Text Available The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01-0.8% DW. There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g, artesunate (2.24μg/g, dihydroartemisinin (13.6μg/g and artemether (12.8μg/g. Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS, cytochrome P450 (CYP71AV1, and aldehyde dehydrogenase 1 (ALDH1, with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1 showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism.

A quorum sensing-mediated switch contributes to natural transformation of Vibrio cholerae

OpenAIRE

Blokesch Melanie

2013-01-01

There is a fundamental gap in our understanding of how horizontal gene transfer contributes to the enormous range of genetic variations that are observed among bacteria. The objective of our study was to better understand how the acquisition of genetic material by natural transformation is regulated within a population of Vibrio cholerae cells. V. cholerae is an aquatic bacterium and a facultative human pathogen. It acquires natural competence for transformation in response to changing enviro...

Catalytic Organic Transformations Mediated by Actinide Complexes

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Isabell S. R. Karmel

2015-10-01

Full Text Available This review article presents the development of organoactinides and actinide coordination complexes as catalysts for homogeneous organic transformations. This chapter introduces the basic principles of actinide catalysis and deals with the historic development of actinide complexes in catalytic processes. The application of organoactinides in homogeneous catalysis is exemplified in the hydroelementation reactions, such as the hydroamination, hydrosilylation, hydroalkoxylation and hydrothiolation of alkynes. Additionally, the use of actinide coordination complexes for the catalytic polymerization of α-olefins and the ring opening polymerization of cyclic esters is presented. The last part of this review article highlights novel catalytic transformations mediated by actinide compounds and gives an outlook to the further potential of this field.

Unlocking the potential of tropical root crop biotechnology in east Africa by establishing a genetic transformation platform for local farmer-preferred cassava cultivars.

Science.gov (United States)

Nyaboga, Evans; Njiru, Joshua; Nguu, Edward; Gruissem, Wilhelm; Vanderschuren, Herve; Tripathi, Leena

2013-01-01

Cassava genetic transformation capacity is still mostly restricted to advanced laboratories in the USA, Europe and China; and its implementation and maintenance in African laboratories has remained scarce. The impact of transgenic technologies for genetic improvement of cassava will depend largely on the transfer of such capabilities to researchers in Africa, where cassava has an important socioeconomic niche. A major constraint to the development of genetic transformation technologies for cassava improvement has been the lack of an efficient and robust transformation and regeneration system. Despite the success achieved in genetic modification of few cassava cultivars, including the model cultivar 60444, transgenic cassava production remains difficult for farmer-preferred cultivars. In this study, a protocol for cultivar 60444 developed at ETH Zurich was successfully implemented and optimized to establish transformation of farmer-preferred cassava cultivars popular in east Africa. The conditions for production and proliferation of friable embryogenic calli (FEC) and Agrobacterium-mediated transformation were optimized for three east African farmer-preferred cultivars (Ebwanatereka, Kibandameno and Serere). Our results demonstrated transformation efficiencies of about 14-22 independent transgenic lines per 100 mg of FEC for farmer-preferred cultivars in comparison to 28 lines per 100 mg of the model cultivar 60444. The presence, integration and expression of the transgenes were confirmed by PCR, Southern blot analysis and histochemical GUS assay. This study reports the establishment of a cassava transformation platform at International Institute of Tropical Agriculture (IITA) hosted by Biosciences eastern and central Africa (BecA) hub in Kenya and provides the basis for transferring important traits such as virus resistance and prolonged shelf-life to farmer-preferred cultivars in east Africa. We anticipate that such platform will also be instrumental to transfer

Unlocking the potential of tropical root crop biotechnology in east Africa by establishing a genetic transformation platform for local farmer-preferred cassava cultivars

Directory of Open Access Journals (Sweden)

Evans eNyaboga

2013-12-01

Full Text Available Cassava genetic transformation capacity is still mostly restricted to advanced laboratories in the USA, Europe and China; and its implementation and maintainance in African laboratories has remained scarce. The impact of transgenic technologies for genetic improvement of cassava will depend largely on the transfer of such capabilities to researchers in Africa, where cassava has an important socioeconomic niche. A major constraint to the development of genetic transformation technologies for cassava improvement has been the lack of an efficient and robust transformation and regeneration system. Despite the success achieved in genetic modification of few cassava cultivars, including the model cultivar 60444, transgenic cassava production remains difficult for farmer-preferred cultivars. In this study, a protocol for cultivar 60444 developed at ETH Zurich was successfully implemented and optimized to establish transformation of farmer-preferred cassava cultivars popular in east Africa. The conditions for production and proliferation of friable embryogenic calli (FEC and Agrobacterium-mediated transformation were optimized for three east African farmer-preferred cultivars (Ebwanatereka, Kibandameno and Serere. Our results demonstrated transformation efficiencies of about 14-22 independent transgenic lines per 100 mg of FEC for farmer-preferred cultivars in comparison to 28 lines per 100 mg of the model cultivar 60444. The presence, integration and expression of the transgenes were confirmed by PCR, Southern blot analysis and histochemical GUS assay. This study reports the establishment of a cassava transformation platform at International Institute of Tropical Agriculture (IITA hosted by Biosciences eastern and central Africa (BecA hub in Kenya and provides the basis for transferring important traits such as virus resistance and prolonged shelf-life to farmer-preferred cultivars in east Africa. We anticipate that such platform will also be

Agrobacterium-mediated gene transfer in plants and biosafety considerations.

Science.gov (United States)

Mehrotra, Shweta; Goyal, Vinod

2012-12-01

Agrobacterium, the natures' genetic engineer, has been used as a vector to create transgenic plants. Agrobacterium-mediated gene transfer in plants is a highly efficient transformation process which is governed by various factors including genotype of the host plant, explant, vector, plasmid, bacterial strain, composition of culture medium, tissue damage, and temperature of co-cultivation. Agrobacterium has been successfully used to transform various economically and horticulturally important monocot and dicot species by standard tissue culture and in planta transformation techniques like floral or seedling infilteration, apical meristem transformation, and the pistil drip methods. Monocots have been comparatively difficult to transform by Agrobacterium. However, successful transformations have been reported in the last few years based on the adjustment of the parameters that govern the responses of monocots to Agrobacterium. A novel Agrobacterium transferred DNA-derived nanocomplex method has been developed which will be highly valuable for plant biology and biotechnology. Agrobacterium-mediated genetic transformation is known to be the preferred method of creating transgenic plants from a commercial and biosafety perspective. Agrobacterium-mediated gene transfer predominantly results in the integration of foreign genes at a single locus in the host plant, without associated vector backbone and is also known to produce marker free plants, which are the prerequisites for commercialization of transgenic crops. Research in Agrobacterium-mediated transformation can provide new and novel insights into the understanding of the regulatory process controlling molecular, cellular, biochemical, physiological, and developmental processes occurring during Agrobacterium-mediated transformation and also into a wide range of aspects on biological safety of transgenic crops to improve crop production to meet the demands of ever-growing world's population.

Genetic Algorithms Evolve Optimized Transforms for Signal Processing Applications

National Research Council Canada - National Science Library

Moore, Frank; Babb, Brendan; Becke, Steven; Koyuk, Heather; Lamson, Earl, III; Wedge, Christopher

2005-01-01

.... The primary goal of the research described in this final report was to establish a methodology for using genetic algorithms to evolve coefficient sets describing inverse transforms and matched...

Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

Energy Technology Data Exchange (ETDEWEB)

Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.

2009-05-14

ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens

International Nuclear Information System (INIS)

Atkinson, Sarah C.; Dogovski, Con; Dobson, Renwick C. J.; Perugini, Matthew A.

2012-01-01

Dihydrodipicolinate synthase from the plant pathogen A. tumefaciens has been cloned, expressed, purified and crystallized in its unliganded form, in the presence of its substrate pyruvate and in the presence of pyruvate and the allosteric inhibitor lysine. Diffraction data for the crystals were collected to a maximum resolution of 1.40 Å. Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP-354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS). Enzyme-kinetics studies demonstrate that AgT-DHDPS possesses DHDPS activity in vitro. Crystals of AgT-DHDPS were grown in the unliganded form and in forms with substrate bound and with substrate plus allosteric inhibitor (lysine) bound. X-ray diffraction data sets were subsequently collected to a maximum resolution of 1.40 Å. Determination of the structure with and without substrate and inhibitor will offer insight into the design of novel pesticide agents

Promoter deletions are essential for transformation of lettuce by the T-cyt gene: the phenotypes of transgenic plants

NARCIS (Netherlands)

Curtis, H.E.C.; Jordi, W.; Davelaar, E.; Power, J.B.; Laat, de A.M.M.; Davey, M.R.

1999-01-01

The Agrobacterium T- cyt gene was transferred into lettuce, Latuca sativa‘Saladin’ using a genotype-independent transformation procedure employing a supervirulent Agrobacterium tumefaciens strain carrying the binary vector pMOG23. Kanamycin-resistant shoots were initiated from inoculated explants

Computational prediction of over-annotated protein-coding genes in the genome of Agrobacterium tumefaciens strain C58

Science.gov (United States)

Yu, Jia-Feng; Sui, Tian-Xiang; Wang, Hong-Mei; Wang, Chun-Ling; Jing, Li; Wang, Ji-Hua

2015-12-01

Agrobacterium tumefaciens strain C58 is a type of pathogen that can cause tumors in some dicotyledonous plants. Ever since the genome of A. tumefaciens strain C58 was sequenced, the quality of annotation of its protein-coding genes has been queried continually, because the annotation varies greatly among different databases. In this paper, the questionable hypothetical genes were re-predicted by integrating the TN curve and Z curve methods. As a result, 30 genes originally annotated as “hypothetical” were discriminated as being non-coding sequences. By testing the re-prediction program 10 times on data sets composed of the function-known genes, the mean accuracy of 99.99% and mean Matthews correlation coefficient value of 0.9999 were obtained. Further sequence analysis and COG analysis showed that the re-annotation results were very reliable. This work can provide an efficient tool and data resources for future studies of A. tumefaciens strain C58. Project supported by the National Natural Science Foundation of China (Grant Nos. 61302186 and 61271378) and the Funding from the State Key Laboratory of Bioelectronics of Southeast University.

Inhibition and dispersal of Agrobacterium tumefaciens biofilms by a small diffusible Pseudomonas aeruginosa exoproduct(s).

Science.gov (United States)

Hibbing, Michael E; Fuqua, Clay

2012-06-01

Environmental biofilms often contain mixed populations of different species. In these dense communities, competition between biofilm residents for limited nutrients such as iron can be fierce, leading to the evolution of competitive factors that affect the ability of competitors to grow or form biofilms. We have discovered a compound(s) present in the conditioned culture fluids of Pseudomonas aeruginosa that disperses and inhibits the formation of biofilms produced by the facultative plant pathogen Agrobacterium tumefaciens. The inhibitory activity is strongly induced when P. aeruginosa is cultivated in iron-limited conditions, but it does not function through iron sequestration. In addition, the production of the biofilm inhibitory activity is not regulated by the global iron regulatory protein Fur, the iron-responsive extracytoplasmic function σ factor PvdS, or three of the recognized P. aeruginosa quorum-sensing systems. In addition, the compound(s) responsible for the inhibition and dispersal of A. tumefaciens biofilm formation is likely distinct from the recently identified P. aeruginosa dispersal factor, cis-2-decenoic acid (CDA), as dialysis of the culture fluids showed that the inhibitory compound was larger than CDA and culture fluids that dispersed and inhibited biofilm formation by A. tumefaciens had no effect on biofilm formation by P. aeruginosa.

Genetic transformation of fruit trees: current status and remaining challenges.

Science.gov (United States)

Gambino, Giorgio; Gribaudo, Ivana

2012-12-01

Genetic transformation has emerged as a powerful tool for genetic improvement of fruit trees hindered by their reproductive biology and their high levels of heterozygosity. For years, genetic engineering of fruit trees has focussed principally on enhancing disease resistance (against viruses, fungi, and bacteria), although there are few examples of field cultivation and commercial application of these transgenic plants. In addition, over the years much work has been performed to enhance abiotic stress tolerance, to induce modifications of plant growth and habit, to produce marker-free transgenic plants and to improve fruit quality by modification of genes that are crucially important in the production of specific plant components. Recently, with the release of several genome sequences, studies of functional genomics are becoming increasingly important: by modification (overexpression or silencing) of genes involved in the production of specific plant components is possible to uncover regulatory mechanisms associated with the biosynthesis and catabolism of metabolites in plants. This review focuses on the main advances, in recent years, in genetic transformation of the most important species of fruit trees, devoting particular attention to functional genomics approaches and possible future challenges of genetic engineering for these species in the post-genomic era.

Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi.

Science.gov (United States)

Lin, Xinping; Liu, Sasa; Bao, Ruiqi; Gao, Ning; Zhang, Sufang; Zhu, Rongqian; Zhao, Zongbao Kent

2017-11-01

Oleaginous yeast Lipomyces starkeyi, a promising strain of great biotechnical importance, is able to accumulate over 60% of its cell biomass as triacylglycerols (TAGs). It is promising to directly produce the derivatives of TAGs, such as long-chain fatty acid methyl esters and alkanes, in L. starkeyi. However, techniques for genetic modification of this oleaginous yeast are lacking, thus, further research is needed to develop genetic tools and functional elements. Here, we used two exogenous promoters (pGPD and pPGK) from oleaginous yeast Rhodosporidium toruloides to establish a simpler Agrobacterium-mediated transformation (AMT) method for L. starkeyi. Hygromycin-resistant transformants were obtained on antibiotic-contained plate. Mitotic stability test, genotype verification by PCR, and protein expression confirmation all demonstrated the success of this method. Furthermore, the strength of these two promoters was evaluated at the phenotypic level on a hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. The PGK promoter strength was 2.2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. starkeyi for constructing superior strains to produce advanced biofuels.

Radiation-induced irreparable heritable changes in cells promoting their tumoral transformation

International Nuclear Information System (INIS)

Kuzin, A.M.; Vagabova, M.Eh.; Yurov, S.S.

1988-01-01

In experiments with model plant tumors (Kalanchoe-ti plasmid Agrobat. tumefaciens C-58D) it was shown that exposure of the recepient plant to low-level γ-radiation of Gy induced changes in cells that were not repaired over two months promoting tumoral transformations in them. Those changes were shown to persist in the offspring of the exposed somatic cells

Generation of doubled haploid transgenic wheat lines by microspore transformation.

Directory of Open Access Journals (Sweden)

Rhoda A T Brew-Appiah

Full Text Available Microspores can be induced to develop homozygous doubled haploid plants in a single generation. In the present experiments androgenic microspores of wheat have been genetically transformed and developed into mature homozygous transgenic plants. Two different transformation techniques were investigated, one employing electroporation and the other co-cultivation with Agrobacterium tumefaciens. Different tissue culture and transfection conditions were tested on nine different wheat cultivars using four different constructs. A total of 19 fertile transformants in five genotypes from four market classes of common wheat were recovered by the two procedures. PCR followed by DNA sequencing of the products, Southern blot analyses and bio/histo-chemical and histological assays of the recombinant enzymes confirmed the presence of the transgenes in the T0 transformants and their stable inheritance in homozygous T1∶2 doubled haploid progenies. Several decisive factors determining the transformation and regeneration efficiency with the two procedures were determined: (i pretreatment of immature spikes with CuSO4 solution (500 mg/L at 4°C for 10 days; (ii electroporation of plasmid DNA in enlarged microspores by a single pulse of ∼375 V; (iii induction of microspores after transfection at 28°C in NPB-99 medium and regeneration at 26°C in MMS5 medium; (iv co-cultivation with Agrobacterium AGL-1 cells for transfer of plasmid T-DNA into microspores at day 0 for <24 hours; and (v elimination of AGL-1 cells after co-cultivation with timentin (200-400 mg/L.

Transformation of Inhibitor of Meristem Activity (IMA Gene into Jatropha curcas L.

Directory of Open Access Journals (Sweden)

Asri Pirade Paserang

2015-09-01

Full Text Available Jatropha is one of the many biodiesel plants developed in tropical countries. Efforts to increase its productivity can be done using various methods of breeding. One of the breeding methods is the introduction of genes into the Jatropha plant. The aim of this study is to assess the success of genetic transformation using the Inhibitor of Meristem Activity (IMA gene in Jatropha curcas. The research procedures included inoculation of explants with Agrobacterium tumefaciens, callus induction, screening test of selection media, regeneration, and gene expression analysis using Polymerase Chain Reaction (PCR. IMA is one of the genes that controls flowering genes and ovule development. It was first isolated from tomato plants and has been successfully overexpressed in these plants using the Cauliflower Mosaic Virus (CaMV 35S promoter. In this experiment, plant transformation was performed on J. curcas as the target. Explant callus formation in both the control and treated samples was good, but shoot formation decreased dramatically in the treated explants. PCR analysis indicated that IMA genes can be inserted into J. curcas with the size of the IMA gene is 500 bp.

The Impact of Transformational Leadership on Employee Sustainable Performance: The Mediating Role of Organizational Citizenship Behavior

Directory of Open Access Journals (Sweden)

Weiping Jiang

2017-09-01

Full Text Available Transformational leadership has drawn extensive attention in management research. In this field, the influence of transformational leadership on employee performance is an important branch. Recent research indicates that organizational citizenship behavior plays a mediating role between transformational leadership and employee performance. However, some of these findings contradict each other. Given the background where greater attention is being paid to transformational leadership in the construction industry, this research aims to find the degree of the influence of transformational leadership on employee sustainable performance, as well as the mediating role of organizational citizenship behavior. A total of 389 questionnaires were collected from contractors and analyzed via structural equation modeling. The findings reveal that employee sustainable performance is positively influenced by transformational leadership. In addition, more than half of that influence is mediated by their organizational citizenship behavior. These findings remind project managers of the need to pay close attention to transformational leadership, to cultivate organizational citizenship behavior, and thereby to eventually improve employee’s sustainable performance.

Better Rooting Procedure to Enhance Survival Rate of Field Grown Malaysian Eksotika Papaya Transformed with 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Gene

Science.gov (United States)

Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom

2013-01-01

A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets. PMID:25969786

Better rooting procedure to enhance survival rate of field grown malaysian eksotika papaya transformed with 1-aminocyclopropane-1-carboxylic Acid oxidase gene.

Science.gov (United States)

Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom

2013-01-01

A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets.

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

NARCIS (Netherlands)

Wang, Yue; Kronenburg-van de Ven, van B.C.E.; Menzel, T.R.; Maliepaard, C.A.; Shen, X.; Krens, F.A.

2012-01-01

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in

Abundant genetic overlap between blood lipids and immune-mediated diseases indicates shared molecular genetic mechanisms.

Directory of Open Access Journals (Sweden)

Ole A Andreassen

Full Text Available Epidemiological studies suggest a relationship between blood lipids and immune-mediated diseases, but the nature of these associations is not well understood. We used genome-wide association studies (GWAS to investigate shared single nucleotide polymorphisms (SNPs between blood lipids and immune-mediated diseases. We analyzed data from GWAS (n~200,000 individuals, applying new False Discovery Rate (FDR methods, to investigate genetic overlap between blood lipid levels [triglycerides (TG, low density lipoproteins (LDL, high density lipoproteins (HDL] and a selection of archetypal immune-mediated diseases (Crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, celiac disease, psoriasis and sarcoidosis. We found significant polygenic pleiotropy between the blood lipids and all the investigated immune-mediated diseases. We discovered several shared risk loci between the immune-mediated diseases and TG (n = 88, LDL (n = 87 and HDL (n = 52. Three-way analyses differentiated the pattern of pleiotropy among the immune-mediated diseases. The new pleiotropic loci increased the number of functional gene network nodes representing blood lipid loci by 40%. Pathway analyses implicated several novel shared mechanisms for immune pathogenesis and lipid biology, including glycosphingolipid synthesis (e.g. FUT2 and intestinal host-microbe interactions (e.g. ATG16L1. We demonstrate a shared genetic basis for blood lipids and immune-mediated diseases independent of environmental factors. Our findings provide novel mechanistic insights into dyslipidemia and immune-mediated diseases and may have implications for therapeutic trials involving lipid-lowering and anti-inflammatory agents.

Effects of a petunia scaffold/matrix attachment region on copy number dependency and stability of transgene expression in Nicotiana tabacum.

Science.gov (United States)

Dietz-Pfeilstetter, Antje; Arndt, Nicola; Manske, Ulrike

2016-04-01

Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region.

Green Transformational Leadership and Green Performance: The Mediation Effects of Green Mindfulness and Green Self-Efficacy

Directory of Open Access Journals (Sweden)

Yu-Shan Chen

2014-09-01

Full Text Available No prior literature explores the influence of green transformational leadership on green performance, thus, this study develops a novel research framework to fill the research gap. This study investigates the influence of green transformational leadership on green performance and discusses the mediation effects of green mindfulness and green self-efficacy by means of structural equation modeling (SEM. The results indicate that green transformational leadership positively influences green mindfulness, green self-efficacy, and green performance. Moreover, this study demonstrates that the positive relationship between green transformational leadership and green performance is partially mediated by the two mediators: green mindfulness and green self-efficacy. It means that green transformational leadership can not only directly affect green performance positively but also indirectly affect it positively through green mindfulness and green self-efficacy. Therefore, firms need to raise their green transformational leadership, green mindfulness, and green self-efficacy to increase their green performance.

Agrobacterium-mediated transformation of Mexican lime (Citrus aurantifolia Swingle) using optimized systems for epicotyls and cotelydons

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Epicotyl and internodal stem segments provide the predominantly used explants for regeneration of transgenic citrus plants following co-cultivation with Agrobacterium. Previous reports using epicotyls segments from Mexican lime have shown low affinity for Agrobacterium tumefaciens infection which re...

Growth kinetics and scale-up of Agrobacterium tumefaciens.

Science.gov (United States)

Leth, Ingrid K; McDonald, Karen A

2017-06-01

Production of recombinant proteins in plants through Agrobacterium-mediated transient expression is a promising method of producing human therapeutic proteins, vaccines, and commercial enzymes. This process has been shown to be viable at a large scale and involves growing large quantities of wild-type plants and infiltrating the leaf tissue with a suspension of Agrobacterium tumefaciens bearing the genes of interest. This study examined one of the steps in this process that had not yet been optimized: the scale-up of Agrobacterium production to sufficient volumes for large-scale plant infiltration. Production of Agrobacterium strain C58C1 pTFS40 was scaled up from shake flasks (50-100 mL) to benchtop (5 L) scale with three types of media: Lysogeny broth (LB), yeast extract peptone (YEP) media, and a sucrose-based defined media. The maximum specific growth rate (μ max ) of the strain in the three types of media was 0.46 ± 0.04 h -1 in LB media, 0.43 ± 0.03 h -1 in YEP media, and 0.27 ± 0.01 h -1 in defined media. The maximum biomass concentration reached at this scale was 2.0 ± 0.1, 2.8 ± 0.1, and 2.6 ± 0.1 g dry cell weight (DCW)/L for the three media types. Production was successfully scaled up to a 100-L working volume reactor with YEP media, using k L a as the scale-up parameter.

Genetic transformation of rare Verbascum eriophorum Godr. plants and metabolic alterations revealed by NMR-based metabolomics.

Science.gov (United States)

Marchev, Andrey; Yordanova, Zhenya; Alipieva, Kalina; Zahmanov, Georgi; Rusinova-Videva, Snezhana; Kapchina-Toteva, Veneta; Simova, Svetlana; Popova, Milena; Georgiev, Milen I

2016-09-01

To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.

Transformational Leaders and Work Performance: The Mediating Roles of Identification and Self-efficacy

Directory of Open Access Journals (Sweden)

Flávia Cavazotte

2013-10-01

Full Text Available In this study we investigate the connections between transformational leadership and subordinate formal and contextual performance among Brazilian employees. We also proposed and tested two mediating processesthrough which transformational leaders would enhance the performance of their staff: stronger follower identification with the leader and efficacy beliefs. These relations were tested with a sample of 107 managers from a multinational company that operates in the financial sector. The proposed structural equation model was assessed with Partial Least Squares (PLS techniques. The results suggest that perceived transformational leadership is associated with higher levels of task performance and helping behaviors. Moreover, the proposed mediating processes were empirically supported. We discuss implications for theory and practice.

Overexpression of Arabidopsis cytokinin oxidase/dehydrogenase genes AtCKX1 and AtCKX2 in transgenic Centaurium erythraea Rafn

Czech Academy of Sciences Publication Activity Database

Trifunovic, M.; Cingel, A.; Simonovic, A.; Jevremovic, S.; Petric, M.; Dragicevic, I.; Motyka, Václav; Dobrev, Petre; Zahajská, Lenka; Subotic, A.

2013-01-01

Roč. 115, č. 2 (2013), s. 139-150 ISSN 0167-6857 R&D Projects: GA ČR(CZ) GAP506/11/0774 Institutional research plan: CEZ:AV0Z50380511 Keywords : Centaurium erythraea Rafn. * Agrobacterium tumefaciens * Genetic transformation Subject RIV: EF - Botanics Impact factor: 2.612, year: 2013

Mediation and modification of genetic susceptibility to obesity by eating behaviors.

Science.gov (United States)

de Lauzon-Guillain, Blandine; Clifton, Emma Ad; Day, Felix R; Clément, Karine; Brage, Soren; Forouhi, Nita G; Griffin, Simon J; Koudou, Yves Akoli; Pelloux, Véronique; Wareham, Nicholas J; Charles, Marie-Aline; Heude, Barbara; Ong, Ken K

2017-10-01

Background: Many genetic variants show highly robust associations with body mass index (BMI). However, the mechanisms through which genetic susceptibility to obesity operates are not well understood. Potentially modifiable mechanisms, including eating behaviors, are of particular interest to public health. Objective: Here we explore whether eating behaviors mediate or modify genetic susceptibility to obesity. Design: Genetic risk scores for BMI (BMI-GRSs) were calculated for 3515 and 2154 adults in the Fenland and EDEN (Etude des déterminants pré et postnatals de la santé et du développement de l'enfant) population-based cohort studies, respectively. The eating behaviors-emotional eating, uncontrolled eating, and cognitive restraint-were measured through the use of a validated questionnaire. The mediating effect of each eating behavior on the association between the BMI-GRS and measured BMI was assessed by using the Sobel test. In addition, we tested for interactions between each eating behavior and the BMI-GRS on BMI. Results: The association between the BMI-GRS and BMI was mediated by both emotional eating (EDEN: P- Sobel = 0.01; Fenland: P- Sobel = 0.02) and uncontrolled eating (EDEN: P- Sobel = 0.04; Fenland: P -Sobel = 0.0006) in both sexes combined. Cognitive restraint did not mediate this association ( P -Sobel > 0.10), except among EDEN women ( P -Sobel = 0.0009). Cognitive restraint modified the relation between the BMI-GRS and BMI among men (EDEN: P -interaction = 0.0001; Fenland: P -interaction = 0.04) and Fenland women ( P -interaction = 0.0004). By tertiles of cognitive restraint, the association between the BMI-GRS and BMI was strongest in the lowest tertile of cognitive restraint, and weakest in the highest tertile. Conclusions: Genetic susceptibility to obesity was partially mediated by the "appetitive" eating behavior traits (uncontrolled and emotional eating) and, in 3 of the 4 population groups studied, was modified by cognitive restraint

Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles.

Science.gov (United States)

Rasmussen, J L; Kikkert, J R; Roy, M K; Sanford, J C

1994-01-01

We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.

Improvements in the transformation of Arabidopsis thaliana C24 leaf-discs by Agrobacterium tumefaciens

DEFF Research Database (Denmark)

van der Graaff, Eric; Hooykaas, P J

1996-01-01

We report here an efficient Arabidopsis leafdisc transformation protocol yielding an average transformation frequency of 1.6 transgenic shoots per leaf explant 4 weeks after the bacterial infection period. Subsequent cultivation in vitro is such that a high percentage (85-90%) of the primary...... transformants produces seeds with an average seed yield of 100-300 seeds per plant. This improved transformation protocol yields mainly (70%) transformants segregating for a single T-DNA locus of which 68% actually contain one T-DNA insert. The objective is to generate a pool of independent transformants...

Convergent evolution of Amadori opine catabolic systems in plasmids of Agrobacterium tumefaciens.

Science.gov (United States)

Baek, Chang-Ho; Farrand, Stephen K; Lee, Ko-Eun; Park, Dae-Kyun; Lee, Jeong Kug; Kim, Kun-Soo

2003-01-01

Deoxyfructosyl glutamine (DFG, referred to elsewhere as dfg) is a naturally occurring Amadori compound found in rotting fruits and vegetables. DFG also is an opine and is found in tumors induced by chrysopine-type strains of Agrobacterium tumefaciens. Such strains catabolize this opine via a pathway coded for by their plasmids. NT1, a derivative of the nopaline-type A. tumefaciens strain C58 lacking pTiC58, can utilize DFG as the sole carbon source. Genes for utilization of DFG were mapped to the 543-kb accessory plasmid pAtC58. Two cosmid clones of pAtC58 allowed UIA5, a plasmid-free derivative of C58, harboring pSa-C that expresses MocC (mannopine [MOP] oxidoreductase that oxidizes MOP to DFG), to grow by using MOP as the sole carbon source. Genetic analysis of subclones indicated that the genes for utilization of DFG are located in a 6.2-kb BglII (Bg2) region adjacent to repABC-type genes probably responsible for the replication of pAtC58. This region contains five open reading frames organized into at least two transcriptional soc (santhopine catabolism) groups: socR and socABCD. Nucleotide sequence analysis and analyses of transposon-insertion mutations in the region showed that SocR negatively regulates the expression of socR itself and socABCD. SocA and SocB are responsible for transport of DFG and MOP. SocA is a homolog of known periplasmic amino acid binding proteins. The N-terminal half of SocB is a homolog of the transmembrane transporter proteins for several amino acids, and the C-terminal half is a homolog of the transporter-associated ATP-binding proteins. SocC and SocD could be responsible for the enzymatic degradation of DFG, being homologs of sugar oxidoreductases and an amadoriase from Corynebacterium sp., respectively. The protein products of socABCD are not related at the amino acid sequence level to those of the moc and mot genes of Ti plasmids responsible for utilization of DFG and MOP, indicating that these two sets of genes and their

Silently transformable: the many ways bacteria conceal their built-in capacity of genetic exchange.

Science.gov (United States)

Attaiech, Laetitia; Charpentier, Xavier

2017-06-01

Bacteria can undergo genetic transformation by actively integrating genetic information from phylogenetically related or unrelated organisms. The original function of natural transformation remains a subject of debate, but it is well established as a major player in genome evolution. Naturally transformable bacteria use a highly conserved DNA uptake system to internalize DNA and integrate it in their chromosome by homologous recombination. Expression of the DNA uptake system, often referred to as competence, is tightly controlled and induced by signals that are often elusive. Initially thought to be restricted to a few bacterial species, natural transformation increasingly seems widespread in bacteria. Yet, the triggering signals and regulatory mechanisms involved appear diverse and are understood only in a limited set of species. As a result, natural transformation in most bacterial species remains poorly documented and the potential impact of this mechanism on global genetic mobilization is likely underappreciated. Indeed, even when a conserved activator can be identified to artificially induce the expression of the DNA uptake system, the considered species may still remain non-transformable. Recent works indicate that the DNA uptake system is directly subjected to silencing. At least in Legionella pneumophila and possibly in other species, a small non-coding RNA prevents expression of the DNA uptake system. Silencing constitutes one more way bacteria control expression of their engine of genetic exchange. It may also be the underlying reason of the undetectable natural transformation of many bacterial species grown under laboratory conditions even though they possess a DNA uptake system.

Agrobacterium-mediated transformation: state of the art and future prospect

Institute of Scientific and Technical Information of China (English)

无

2000-01-01

Great progress has been made in recent years in studies on the mechanism of Agrobacterium-mediated transformation and its application. Many details of the key molecular events within the bacterial cells involved in T-DNA transfer have been elucidated, and it is notable that some plant factors which were elusive before are purified and characterized. Vast kinds of species, which were either recalcitrant to or not included in the host range of Agrobacterium, can now be transformed by this bacterium, and they include the very important cereal species, gymnosperms, yeast and many filamentous fungi. The simple in vivo transformation of tissue in intact plants and the "agrolistic" methods to transform recalcitrant plants are the two novel technical achievements. Combined with other powerful techniques such as bacterial artificial chromosome, very large DNA fragment can be transformed into the plant genome by Agrobacterium. Further studies will elucidate more plant-encoded factors involved in T-DNA transformation and there is a need to develop more powerful Agrobacterium-based transformation systems to meet different needs in basic research and crop improvement practice.

Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants.

Science.gov (United States)

Ceasar, S Antony; Ignacimuthu, S

2011-09-01

A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM L: -cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R(1) progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R(1) progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.

AgarTrap: a simplified Agrobacterium-mediated transformation method for sporelings of the liverwort Marchantia polymorpha L.

Science.gov (United States)

Tsuboyama, Shoko; Kodama, Yutaka

2014-01-01

The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha.

Transformative Mediation and Human Rights: the Possibility of Insertion of Alterity in the Conflict

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Romulo Rhemo Palitot Braga

2015-12-01

Full Text Available This paper intends to present the transformative mediation, from the theoretical perspective of Luis Alberto Warat, as a proposal for promoting a culture of human rights, seen articular in your exercise the individuals's autonomy and the inclusion of alterity in the conflict. It is from this last element that will be further articulated discussion between human rights and mediation through the debate on the recognition of equality and difference, currently so expensive for human rights. The transformative mediation aims through a dialogic relationship building with the other, a participatory approach and shared the problems and dilemmas common to human and social life.

Overlapping protective roles for glutathione transferase gene family members in chemical and oxidative stress response in Agrobacterium tumefaciens.

Science.gov (United States)

Skopelitou, Katholiki; Muleta, Abdi W; Pavli, Ourania; Skaracis, Georgios N; Flemetakis, Emmanouil; Papageorgiou, Anastassios C; Labrou, Nikolaos E

2012-03-01

In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.

In vitro organogenesis of potato (Solanum tuberosum L. cultivar Atlantic for the genetic transformationOrganogênese in vitro de batata (Solanum tuberosum L. cultivar Atlantic visando transformação genética

Directory of Open Access Journals (Sweden)

Rodrigo Kelson Silva Rezende

2013-06-01

Full Text Available The obtaining of commercial cultivars of potato with resistance to the phytopatogen is a very promising alternative that the genetic engineering can provide, through the introduction of exogenous genes for the genetic transformation. In order to establish an efficient system of transformation, it is crucial first to optimize in vitro organogenesis protocol. This work aimed to establish an efficient protocol for the genetic transformation of potato cv. Atlantic. The efficiency of the in vitro organogenesis is influenced by the explant type and by the kind and concentrations of growth regulators used. Shoot segments of potato cv. Atlantic present better organogenesis capacity than leaf explants. It is recommended to use the woody plant medium (WPM supplied with 1,0 mg L-1 of naphthalene acetic acid (NAA + 5,0 mg L-1 of zeatin riboside (ZEA, to obtain shoots from shoot segments, and the carbenicilin (Cb added to this medium increased this formation. For the process to enlarge shoots, this same antibiotic when added to the WPM medium in growing concentrations (100; 250; 500 mg L-1 promoted a decrease in the height of plants, number of shoots and length of roots. After the co-cultivation of shoots segments with Agrobacterium tumefaciens these concentrations of Cb are not efficient in the elimination of the bacteria, which commits the organogenesis. However, it is possible to establish a protocol of in vitro organogenesis of potato cv. Atlantic starting from shoots segments not co-cultivated. These results will be useful for futures experiments of genetic transformation with this and another commercials cultivars of potato.A obtenção de cultivares comerciais de batata com resistência a fitopatógenos é uma alternativa muito promissora que a engenharia genética pode proporcionar, através da introdução de genes exógenos visando transformação genética. Para tal, torna-se imprescindível estabelecer um sistema eficiente de transformação, e

In situ measurement of solvent-mediated phase transformations during dissolution testing

DEFF Research Database (Denmark)

Aaltonen, Jaakko; Heinänen, Paula; Peltonen, Leena

2006-01-01

In this study, solvent-mediated phase transformations of theophylline (TP) and nitrofurantoin (NF) were measured in a channel flow intrinsic dissolution test system. The test set-up comprised simultaneous measurement of drug concentration in the dissolution medium (with UV-Vis spectrophotometry......) and measurement of the solid-state form of the dissolving solid (in situ with Raman spectroscopy). The solid phase transformations were also investigated off-line with scanning electron microscopy. TP anhydrate underwent a transformation to TP monohydrate, and NF anhydrate (form beta) to NF monohydrate (form II......). Transformation of TP anhydrate to TP monohydrate resulted in a clear decrease in the dissolution rate, while the transformation of NF anhydrate (form beta) to NF monohydrate (form II) could not be linked as clearly to changes in the dissolution rate. The transformation of TP was an order of magnitude faster than...

Translating genetic research into preventive intervention: The baseline target moderated mediator design

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George W. Howe

2016-01-01

Full Text Available In this paper we present and discuss a novel research approach, the baseline target moderated mediation (BTMM design, that holds substantial promise for advancing our understanding of how genetic research can inform prevention research. We first discuss how genetically informed research on developmental psychopathology can be used to identify potential intervention targets. We then describe the BTMM design, which employs moderated mediation within a longitudinal study to test whether baseline levels of intervention targets moderate the impact of the intervention on change in that target, and whether change in those targets mediates causal impact of preventive or treatment interventions on distal health outcomes. We next discuss how genetically informed BTMM designs can be applied to both microtrials and full-scale prevention trials. We end with a discussion of some of the advantages and limitations of this approach.

Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii.

Science.gov (United States)

Douradinha, Bruno; Reis, Viviane C B; Rogers, Matthew B; Torres, Fernando A G; Evans, Jared D; Marques, Ernesto T A

2014-01-01

Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters and resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (> 96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest.

Genetic transformation of lettuce ( Lactuca sativa ): A review | Dan ...

African Journals Online (AJOL)

Lettuce (Lactuca sativa L.) is a globally important leafy vegetable that can be grown worldwide. Due to the rapid growth of population and the human desire to progress, there have been a lot of studies made by researchers, especially in genetic engineering. Improvements in regeneration system and transformation ...

Transformational leadership, intrinsic motivation, and trust: a moderated-mediated model of workplace safety.

Science.gov (United States)

Conchie, Stacey M

2013-04-01

Two studies examine the role of motivation and trust in the relationship between safety-specific transformational leadership and employees' safety behavior. Study 1 tested the prediction that intrinsic and identified regulation motivations mediate the relationship between safety-specific transformational leadership and employees' safety behaviors. Study 2 further explored this relationship by testing the prediction that the mediating role of intrinsic motivation is dependent on employees' level of trust in their leader. Survey data from the U.K. construction industry supported both predictions. However, the mediating role of intrinsic motivation was found only for challenge safety citizenship behaviors (i.e., voice) and not for affiliative safety citizenship behaviors (i.e., helping). These findings suggest that employees' intrinsic motivation is important to the effectiveness of leaders' efforts to promote some but not all forms of safety behavior.

Genetic transformation of cry1EC gene into cotton (Gossypium ...

African Journals Online (AJOL)

welcome

2013-04-10

Apr 10, 2013 ... Full Length Research Paper. Genetic ... This research work was carried out to transform ... were maintained on the same medium till somatic embryos matured. ... of secondary pests, as well as risk to human health and.

Generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation

Directory of Open Access Journals (Sweden)

Wang Genping

2016-09-01

Full Text Available Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154 and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154 were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of ‘clean’ GM wheat containing only the foreign genes of agronomic importance.

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Science.gov (United States)

Wang, Gen-Ping; Yu, Xiu-Dao; Sun, Yong-Wei; Jones, Huw D; Xia, Lan-Qin

2016-01-01

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium -mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

Translating Genetic Research into Preventive Intervention: The Baseline Target Moderated Mediator Design.

Science.gov (United States)

Howe, George W; Beach, Steven R H; Brody, Gene H; Wyman, Peter A

2015-01-01

In this paper we present and discuss a novel research approach, the baseline target moderated mediation (BTMM) design, that holds substantial promise for advancing our understanding of how genetic research can inform prevention research. We first discuss how genetically informed research on developmental psychopathology can be used to identify potential intervention targets. We then describe the BTMM design, which employs moderated mediation within a longitudinal study to test whether baseline levels of intervention targets moderate the impact of the intervention on change in that target, and whether change in those targets mediates causal impact of preventive or treatment interventions on distal health outcomes. We next discuss how genetically informed BTMM designs can be applied to both microtrials and full-scale prevention trials. We use simulated data to illustrate a BTMM, and end with a discussion of some of the advantages and limitations of this approach.

Speeding-up exchange-mediated saturation transfer experiments by Fourier transform

Energy Technology Data Exchange (ETDEWEB)

Carneiro, Marta G.; Reddy, Jithender G.; Griesinger, Christian; Lee, Donghan, E-mail: dole@nmr.mpibpc.mpg.de [Max-Planck Institute for Biophysical chemistry, Department of NMR-based Structural Biology (Germany)

2015-11-15

Protein motions over various time scales are crucial for protein function. NMR relaxation dispersion experiments play a key role in explaining these motions. However, the study of slow conformational changes with lowly populated states remained elusive. The recently developed exchange-mediated saturation transfer experiments allow the detection and characterization of such motions, but require extensive measurement time. Here we show that, by making use of Fourier transform, the total acquisition time required to measure an exchange-mediated saturation transfer profile can be reduced by twofold in case that one applies linear prediction. In addition, we demonstrate that the analytical solution for R{sub 1}ρ experiments can be used for fitting the exchange-mediated saturation transfer profile. Furthermore, we show that simultaneous analysis of exchange-mediated saturation transfer profiles with two different radio-frequency field strengths is required for accurate and precise characterization of the exchange process and the exchanging states.

Speeding-up exchange-mediated saturation transfer experiments by Fourier transform

International Nuclear Information System (INIS)

Carneiro, Marta G.; Reddy, Jithender G.; Griesinger, Christian; Lee, Donghan

2015-01-01

Protein motions over various time scales are crucial for protein function. NMR relaxation dispersion experiments play a key role in explaining these motions. However, the study of slow conformational changes with lowly populated states remained elusive. The recently developed exchange-mediated saturation transfer experiments allow the detection and characterization of such motions, but require extensive measurement time. Here we show that, by making use of Fourier transform, the total acquisition time required to measure an exchange-mediated saturation transfer profile can be reduced by twofold in case that one applies linear prediction. In addition, we demonstrate that the analytical solution for R 1 ρ experiments can be used for fitting the exchange-mediated saturation transfer profile. Furthermore, we show that simultaneous analysis of exchange-mediated saturation transfer profiles with two different radio-frequency field strengths is required for accurate and precise characterization of the exchange process and the exchanging states

Transformational leadership and organizational citizenship behavior: Modeling emotional intelligence as mediator

Directory of Open Access Journals (Sweden)

Majeed Nauman

2017-12-01

Full Text Available Leadership and organizational citizenship behavior (OCB stayed at pinnacle in the arena of organizational behavior research since decades and has attained significant consideration of scholars pursuing to define multifaceted dynamics of leadership and their influence on follower’s behavior at work. The voluntary behavior of Organizational citizenship improves organizational effectiveness, and it goes beyond formal job duties. This study attempts to explore the association amongst transformational leadership and organizational citizenship behavior of teachers in public sector higher education institutions in Pakistan. Study of organizational citizenship behavior in educational organizations and academicians is of high value that definitely requires attention. This study examines the direct and indirect influence of transformational leadership through exploring the mediating role of emotional intelligence. The model was tested by employing structural equation modelling technique on survey responses collected from academicians. Results from 220 responses indicated that relationship between transformational leadership and Organizational Citizenship Behavior is statistically significant where Emotional Intelligence plays an important role as a mediator. The results support and add to the positive effects of transformational leadership style interconnected with extra role behavior at work making it more meaningful. The findings make a significant contribution to leadership and organizational behavior literature in higher education sector and propose that organizations should implement practices that help in enhancing the level of organizational citizenship behavior in organizations.

Development of salt tolerant plants through genetic engineering (abstract)

International Nuclear Information System (INIS)

Mukhtar, Z.; Khan, S.A.; Zafar, Y.

2005-01-01

Salinity stress is one of the most serious factors limiting the productivity of agricultural crops. Genetic engineering provides a useful tool for tailoring plants with enhanced salt tolerance characteristics. Many organisms have evolved mechanisms to survive and grow under such extreme environments. These organisms provide us with a useful source of genes which can be used to improve salt tolerance in plants. The present study aims at identification and cloning of useful halo tolerance conferring genes from fungi and plants and to develop salt tolerant transgenic plants. Here we describe the cloning and use of HSR1 gene (a yeast transcription factor known to confer salt tolerance) and Na/sup +//H/sup +/ antiporter gene AtNHX1 (3016 bp) from Arabidopsis thaliana, and transformation of tobacco with HSR1 and AtNHX1 genes through Agrobacterium method. A number of transgenic tobacco plants were regenerated from leaf explants transformed with Agrobacterium tumefaciens (LBA4404) having HSR1 and AtNHX1 genes by leaf disc method. The putative transgenic plants were analyzed by PCR and dot blot analysis. Screening of these transgenic plants at different salinity levels is in progress which will help identify the suitable plant lines and thus the promising genes which can be further exploited to engineer salt tolerant crop plants. (author)

Petunia (Petunia hybrida).

Science.gov (United States)

Lutke, W Kevin

2006-01-01

Petunia hybrida genetic transformation continues to be a valuable tool for genetic research into biochemical pathways and gene expression, as well as generating commercial products with varying floral colors. In this chapter, we describe a simple and reproducible genetic transformation protocol for generating transgenic petunia plants harboring a gene of interest and selectable marker. The system utilizes Agrobacterium tumefaciens for transgene integration with plant recovery via shoot organogenesis from leaf explant material. Selection for transgenic plants is achieved using the bar gene conferring resistance to glufosinate or nptII gene for resistance to kanamycin. Transformation efficiencies of around 10% are achievable with shoots being recovered about 8 wk after transgene insertion and rooted plants transferred to the greenhouse about twelve weeks after inoculation.

Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani

DEFF Research Database (Denmark)

Romans-Fuertes, Patricia; Sondergaard, Teis Esben; Sandmann, Manuela Ilse Helga

2016-01-01

Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate kno...... and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread....

Assessment of ptxD gene as an alternative selectable marker for Agrobacterium-mediated maize transformation.

Science.gov (United States)

Nahampun, Hartinio N; López-Arredondo, Damar; Xu, Xing; Herrera-Estrella, Luis; Wang, Kan

2016-05-01

Bacterial phosphite oxidoreductase gene and chemical phosphite can be used as a selection system for Agrobacterium -mediated maize transformation. Application of phosphite (Phi) on plants can interfere the plant metabolic system leading to stunted growth and lethality. On the other hand, ectopic expression of the ptxD gene in tobacco and Arabidopsis allowed plants to grow in media with Phi as the sole phosphorous source. The phosphite oxidoreductase (PTXD) enzyme catalyzes the conversion of Phi into phosphate (Pi) that can then be metabolized by plants and utilized as their essential phosphorous source. Here we assess an alternative selectable marker based on a bacterial ptxD gene for Agrobacterium-mediated maize transformation. We compared the transformation frequencies of maize using either the ptxD/Phi selection system or a standard herbicide bar/bialaphos selection system. Two maize genotypes, a transformation amenable hybrid Hi II and an inbred B104, were tested. Transgene presence, insertion copy numbers, and ptxD transcript levels were analyzed and compared. This work demonstrates that the ptxD/Phi selection system can be used for Agrobacterium-mediated maize transformation of both type I and type II callus culture and achieve a comparable frequency as that of the herbicide bar/bialaphos selection system.

Relationship between transformational leadership style and organizational commitment: Mediating effect of psychological empowerment

Science.gov (United States)

Asif, Muhammad; Ayyub, Samia; Bashir, Muhammad Khawar

2014-12-01

This study explores the relationship between style of transformational leadership and organizational commitment of employees with mediating role of psychological empowerment in the textile sector Punjab Pakistan. Data was collected using tools from 250 employees. The transformational leadership questionnaire, MLQ-Multifactor leadership Questionnaire [1] was used to verify the perception of the employees towards transformational leadership style in two dimensions i.e. idealized influence and inspirational motivation. The organizational commitment questionnaire designed by [2] was used to verify the affective organizational commitment. Further, psychological empowerment questionnaire was developed by [3] which was used to examine the state of psychological empowerment of textile sector employees. Pearson Correlation revealed that there exists a positive significant relationship between idealized influence and affective organizational commitment, Inspirational motivation and affective organizational commitment, affective organizational commitment and psychological empowerment. The results from the study put forward that there is a significant relationship between style of transformational leadership and organizational commitment. The mediating variable which one is suitable in the model i.e. psychological empowerment and the model is good fit as the F value is significant.

Optimization design for the stepped impedance transformer based on the genetic algorithm

International Nuclear Information System (INIS)

Zou Dehui; Lai Wanchang; Qiu Dong

2007-01-01

This paper introduces the basic principium and mathematic model of the stepped impedance transformer, then puts the emphasis on comparing two kinds of design methods of the stepped impedance transformer. The design results are simulated by EDA, which indicates that genetic algorithm design is better than Chebyshev integrated design in the term of the most reflect coefficient's module. (authors)

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici.

Science.gov (United States)

Wang, Mei; Zhang, Jie; Wang, Lanying; Han, Lirong; Zhang, Xing; Feng, Juntao

2018-05-24

Take-all, caused by Gaeumannomyces tritici , is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors-temperature, enzyme lysis time, and concentration of the lysing enzyme-were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 10⁷ protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46⁻54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici . An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

Non-oncogenic T-region mutants of Agrobacterium tumefaciens do transfer T-DNA into plant cells

NARCIS (Netherlands)

Hille, Jacques; Wullems, George; Schilperoort, Rob

1983-01-01

A new procedure for site-directed mutagenesis has been applied to the shooting and rooting loci of T-DNA of an octopine Ti-plasmid of Agrobacterium tumefaciens. Mutants have been obtained which induced tumours that either developed shoots or produced more roots than normally observed. Double

Development of glyphosate-resistant alfalfa (Medicago sativa L.) upon transformation with the GR79Ms gene encoding 5-enolpyruvylshikimate-3-phosphate synthase.

Science.gov (United States)

Yi, Dengxia; Ma, Lin; Lin, Min; Li, Cong

2018-07-01

The glyphosate-resistant gene, GR79Ms, was successfully introduced into the genome of alfalfa. The transgenic events may serve as novel germplasm resources in alfalfa breeding. Weed competition can reduce the alfalfa yield, generating new alfalfa germplasm with herbicide resistance is essential. To obtain transgenic alfalfa lines with glyphosate resistance, a new synthetic glyphosate-resistant gene GR79Ms encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was introduced into alfalfa germplasm by Agrobacterium tumefaciens-mediated transformation. In total, 67 transformants were obtained. PCR and Southern blot analyses confirmed that GR79Ms was successfully inserted into the genome of alfalfa. Reverse transcription-PCR and western blot analyses further demonstrated the expression of GR79Ms and its product, GR79Ms EPSPS. Moreover, two homozygous transgenic lines were developed in the T 2 generation by means of molecular-assisted selection. Herbicide tolerance spray tests showed that the transgenic plants T 0 -GR1, T 0 -GR2, T 0 -GR3 and two homozygous lines were able to tolerate fourfold higher commercial usage of glyphosate than non-transgenic plants.

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana.

Science.gov (United States)

Radakovits, Randor; Jinkerson, Robert E; Fuerstenberg, Susan I; Tae, Hongseok; Settlage, Robert E; Boore, Jeffrey L; Posewitz, Matthew C

2012-02-21

The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga.

Sweet Potato [Ipomoea batatas (L.) Lam].

Science.gov (United States)

Song, Guo-qing; Yamaguchi, Ken-ichi

2006-01-01

Among the available transformation methods reported on sweet potato, Agrobacterium tumefaciens-mediated transformation is more successful and desirable. Stem explants have shown to be ideal for the transformation of sweet potato because of their ready availability as explants, the simple transformation process, and high-frequency-regeneration via somatic embryogenesis. Under the two-step kanamycin-hygromycin selection method and using the appropriate explants type (stem explants), the efficiency of transformation can be considerably improved in cv. Beniazuma. The high efficiency in the transformation of stem explants suggests that the transformation protocol described in this chapter warrants testing for routine stable transformation of diverse varieties of sweet potato.

Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens

International Nuclear Information System (INIS)

Hou, Feng; Miyakawa, Takuya; Takeshita, Daijiro; Kataoka, Michihiko; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2012-01-01

The purification and crystallization of 3-quinuclidinone reductase from A. tumefaciens allowed the collection of a diffraction data set to 1.72 Å resolution. (R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2 1 , with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V M = 2.08 Å 3 Da −1 )

INTRODUKSI GEN METALLOTHIONEIN TIPE II KE DALAM RUMPUT LAUT Kappaphycus alvarezii MENGGUNAKAN Agrobacterium tumefaciens

Directory of Open Access Journals (Sweden)

Ulia Fajriah

2014-12-01

Full Text Available Kappaphycus alvarezii adalah jenis alga merah yang memproduksi kappa karagenan yang sangat penting untuk industri makanan, farmasi, dan kosmetik. Untuk meningkatkan produksi, diperlukan ketersediaan bahan baku yang baik. Salah satu yang memengaruhi ketersediaan bahan baku adalah kondisi ingkungan perairan untuk budidaya. Metallothionein (MT adalah protein yang memiliki kemampuan untuk mengikat ion logam seperti Cd, Zn, dan Cu. Tujuan penelitian ini adalah untuk mengintroduksi gen Metallothionein Tipe II (MaMt2 ke dalam genom K. alvarezii menggunakan Agrobacterium tumefaciens. Talus rumput laut diinokulasi dengan A. tumefaciens mengandung plasmid pIG6-SMt2 yang membawa gen MaMt2, selanjutnya dilakukan seleksi bertingkat menggunakan higromisin 10 mg/L dan 20 mg/L. Hasil efisiensi transformasi yang diperoleh adalah 27,4%, efisiensi regenerasi tunas transgenik adalah 27,6%. Analisis molekuler dengan PCR menunjukkan bahwa 13 tunas transgenik mengandung gen MaMt2. Tunas transgenik putatif ditumbuhkan hingga menjadi talus baru dan dapat dilakukan uji tantang pada penelitian selanjutnya.

Transformation of natural genetic variation into Haemophilus influenzae genomes.

Directory of Open Access Journals (Sweden)

Joshua Chang Mell

2011-07-01

Full Text Available Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ~40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ~1000 donor polymorphisms in 3-6 contiguous runs (8.1±4.5 kb in length that collectively comprised ~1-3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.

Genetics of immune-mediated disorders : from genome-wide association to molecular mechanism

NARCIS (Netherlands)

Kumar, Vinod; Wijmenga, Cisca; Xavier, Ramnik J.

2014-01-01

Genetic association studies have identified not only hundreds of susceptibility loci to immune-mediated diseases but also pinpointed causal amino-acid variants of HLA genes that contribute to many autoimmune reactions. Majority of non-HLA genetic variants are located within non-coding regulatory

Plant biotechnology for food security and bioeconomy.

Science.gov (United States)

Clarke, Jihong Liu; Zhang, Peng

2013-09-01

This year is a special year for plant biotechnology. It was 30 years ago, on January 18 1983, one of the most important dates in the history of plant biotechnology, that three independent groups described Agrobacterium tumefaciens-mediated genetic transformation at the Miami Winter Symposium, leading to the production of normal, fertile transgenic plants (Bevan et al. in Nature 304:184-187, 1983; Fraley et al. in Proc Natl Acad Sci USA 80:4803-4807, 1983; Herrera-Estrella et al. in EMBO J 2:987-995, 1983; Vasil in Plant Cell Rep 27:1432-1440, 2008). Since then, plant biotechnology has rapidly advanced into a useful and valuable tool and has made a significant impact on crop production, development of a biotech industry and the bio-based economy worldwide.

Advances in improvement of stress tolerance by induced mutation and genetic transformation in alfalfa

International Nuclear Information System (INIS)

Huang Xin; Ye Hongxia; Shu Xiaoli; Wu Dianxing

2008-01-01

In order to provide references for stress-tolerant breeding of alfalfa, genetic basis of stress-tolerant traits was briefly introduced and advanced in improvement of stress-tolerance by induced mutation and genetic transformation in alfalfa were reviewed. (authors)

Plant genetic variation mediates an indirect ecological effect between belowground earthworms and aboveground aphids.

Science.gov (United States)

Singh, Akanksha; Braun, Julia; Decker, Emilia; Hans, Sarah; Wagner, Agnes; Weisser, Wolfgang W; Zytynska, Sharon E

2014-10-21

Interactions between aboveground and belowground terrestrial communities are often mediated by plants, with soil organisms interacting via the roots and aboveground organisms via the shoots and leaves. Many studies now show that plant genetics can drive changes in the structure of both above and belowground communities; however, the role of plant genetic variation in mediating aboveground-belowground interactions is still unclear. We used an earthworm-plant-aphid model system with two aphid species (Aphis fabae and Acyrthosiphon pisum) to test the effect of host-plant (Vicia faba) genetic variation on the indirect interaction between the belowground earthworms (Eisenia veneta) on the aboveground aphid populations. Our data shows that host-plant variety mediated an indirect ecological effect of earthworms on generalist black bean aphids (A. fabae), with earthworms increasing aphid growth rate in three plant varieties but decreasing it in another variety. We found no effect of earthworms on the second aphid species, the pea aphid (A. pisum), and no effect of competition between the aphid species. Plant biomass was increased when earthworms were present, and decreased when A. pisum was feeding on the plant (mediated by plant variety). Although A. fabae aphids were influenced by the plants and worms, they did not, in turn, alter plant biomass. Previous work has shown inconsistent effects of earthworms on aphids, but we suggest these differences could be explained by plant genetic variation and variation among aphid species. This study demonstrates that the outcome of belowground-aboveground interactions can be mediated by genetic variation in the host-plant, but depends on the identity of the species involved.

Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens

Energy Technology Data Exchange (ETDEWEB)

Liu, D.; Thomas, P.W.; Momb, J.; Hoang, Q.Q.; Petsko, G.A.; Ringe, D.; Fast, W.

2009-06-03

N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.

High-Throughput Agrobacterium-mediated Transformation of Medicago Truncatula in Comparison to Two Expression Vectors

International Nuclear Information System (INIS)

Sultana, T.; Deeba, F.; Naqvi, S. M. S.

2016-01-01

Legumes have been turbulent to efficient Agrobacterium-mediated transformation for a long time. The selection of Medicago truncatula as a model legume plant for molecular analysis resulted in the development of efficient Agrobacterium-mediated transformation protocols. In current study, M. truncatula transformed plants expressing OsRGLP1 were obtained through GATEWAY technology using pGOsRGLP1 (pH7WG2.0=OsRGLP1). The transformation efficiency of this vector was compared with expression vector from pCAMBIA series over-expressing same gene (pCOsRGLP1). A lower number of explants generated hygromycin resistant plantlet for instance, 18.3 with pGOsRGLP1 vector as compared to 35.5 percent with pCOsRGLP1 vector. Transformation efficiency of PCR positive plants generated was 9.4 percent for pGOsRGLP1 while 21.6 percent for pCOsRGLP1. Furthermore 24.4 percent of explants generated antibiotic resistant plantlet on 20 mgl/sup -1/ of hygromycin which was higher than on 15 mgl/sup -1/ of hygromycin such as 12.2 percent. T/sub 1/ progeny analysis indicated that the transgene was inherited in Mendelian manner. The functionally active status of transgene was monitored by high level of Superoxide dismutase (SOD) activity in transformed progeny. (author)

Testing Transgenic Aspen Plants with bar Gene for Herbicide Resistance under Semi-natural Conditions.

Science.gov (United States)

Lebedev, V G; Faskhiev, V N; Kovalenko, N P; Shestibratov, K A; Miroshnikov, A I

2016-01-01

Obtaining herbicide resistant plants is an important task in the genetic engineering of forest trees. Transgenic European aspen plants (Populus tremula L.) expressing the bar gene for phosphinothricin resistance have been produced using Agrobacterium tumefaciens-mediated transformation. Successful genetic transformation was confirmed by PCR analysis for thirteen lines derived from two elite genotypes. In 2014-2015, six lines were evaluated for resistance to herbicide treatment under semi-natural conditions. All selected transgenic lines were resistant to the herbicide Basta at doses equivalent to 10 l/ha (twofold normal field dosage) whereas the control plants died at 2.5 l/ha. Foliar NH4-N concentrations in transgenic plants did not change after treatment. Extremely low temperatures in the third ten-day period of October 2014 revealed differences in freeze tolerance between the lines obtained from Pt of f2 aspen genotypes. Stable expression of the bar gene after overwintering outdoors was confirmed by RT-PCR. On the basis of the tests, four transgenic aspen lines were selected. The bar gene could be used for retransformation of transgenic forest trees expressing valuable traits, such as increased productivity.

Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

Science.gov (United States)

The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brac...

Abdomen disease diagnosis in CT images using flexiscale curvelet transform and improved genetic algorithm.

Science.gov (United States)

Sethi, Gaurav; Saini, B S

2015-12-01

This paper presents an abdomen disease diagnostic system based on the flexi-scale curvelet transform, which uses different optimal scales for extracting features from computed tomography (CT) images. To optimize the scale of the flexi-scale curvelet transform, we propose an improved genetic algorithm. The conventional genetic algorithm assumes that fit parents will likely produce the healthiest offspring that leads to the least fit parents accumulating at the bottom of the population, reducing the fitness of subsequent populations and delaying the optimal solution search. In our improved genetic algorithm, combining the chromosomes of a low-fitness and a high-fitness individual increases the probability of producing high-fitness offspring. Thereby, all of the least fit parent chromosomes are combined with high fit parent to produce offspring for the next population. In this way, the leftover weak chromosomes cannot damage the fitness of subsequent populations. To further facilitate the search for the optimal solution, our improved genetic algorithm adopts modified elitism. The proposed method was applied to 120 CT abdominal images; 30 images each of normal subjects, cysts, tumors and stones. The features extracted by the flexi-scale curvelet transform were more discriminative than conventional methods, demonstrating the potential of our method as a diagnostic tool for abdomen diseases.

The molecular genetics of crown gall tumorigenesis

International Nuclear Information System (INIS)

Hooykaas, P.J.J.; Schilperoort, R.A.

1984-01-01

The phytopathogenic bacteria Agrobacterium tumefaciens and A. rhizogenes are the causative agents of the widespread plant diseases ''crown gall'' and ''hairy root'' respectively. It is now well established that virulent strains of these bacterial species transfer a piece of bacterial DNA into plant cells, thereby transforming these into tumor cells. In research much attention has been paid to the agrobacteria for several reasons. First is the desire to develop a system for the genetic engineering of plant cells based on the natural system for gene transfer between Agrobacterium species and plant cells. Second, there is a striking resemblance between the etiology of animal cancers and the plant cancer crown gall that was recognized as early as in 1927. This led to basic studies on the process of plant tumor induction and on the recovery of plant cells from the tumorous state. A third important interest lies in crown gall as a disease that is the cause of economically important losses in agriculture an horticulture in Europe, North America, and Austrailia. Research has been aimed at finding means to prevent crown gall and to cure plants of this disease

Shared genetics in coeliac disease and other immune-mediated diseases

NARCIS (Netherlands)

Gutierrez-Achury, J.; Coutinho de Almeida, R.; Wijmenga, C.

Gutierrez-Achury J, Coutinho de Almeida R, Wijmenga C (University Medical Centre Groningen and University of Groningen, Groningen, the Netherlands; University of Brasilia School of Health Sciences, Brasilia, DF, Brazil). Shared genetics in coeliac disease and other immune-mediated diseases

[Construction of plant expression vectors with PMI gene as selection marker and their utilization in transformation of Salvia miltiorrhiza f. alba].

Science.gov (United States)

Tao, Ru; Zhang, You-Can; Fang, Qian; Shi, Ren-Jiu; Li, Yan-Ling; Huang, Lu-Qi; Hao, Gang-Ping

2014-04-01

To construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system. The 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI. Plant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%. Genetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

Genetic Associations in Acquired Immune-Mediated Bone Marrow Failure Syndromes: Insights in Aplastic Anemia and Chronic Idiopathic Neutropenia

Science.gov (United States)

Mavroudi, Irene; Papadaki, Helen A.

2012-01-01

Increasing interest on the field of autoimmune diseases has unveiled a plethora of genetic factors that predispose to these diseases. However, in immune-mediated bone marrow failure syndromes, such as acquired aplastic anemia and chronic idiopathic neutropenia, in which the pathophysiology results from a myelosuppressive bone marrow microenvironment mainly due to the presence of activated T lymphocytes, leading to the accelerated apoptotic death of the hematopoietic stem and progenitor cells, such genetic associations have been very limited. Various alleles and haplotypes of human leucocyte antigen (HLA) molecules have been implicated in the predisposition of developing the above diseases, as well as polymorphisms of inhibitory cytokines such as interferon-γ, tumor necrosis factor-α, and transforming growth factor-β1 along with polymorphisms on molecules of the immune system including the T-bet transcription factor and signal transducers and activators of transcription. In some cases, specific polymorphisms have been implicated in the outcome of treatment on those patients. PMID:22956967

An efficient in planta transformation of Jatropha curcas (L.) and multiplication of transformed plants through in vivo grafting.

Science.gov (United States)

Jaganath, Balusamy; Subramanyam, Kondeti; Mayavan, Subramanian; Karthik, Sivabalan; Elayaraja, Dhandapani; Udayakumar, Rajangam; Manickavasagam, Markandan; Ganapathi, Andy

2014-05-01

An efficient and reproducible Agrobacterium-mediated in planta transformation was developed in Jatropha curcas. The various factors affecting J. curcas in planta transformation were optimized, including decapitation, Agrobacterium strain, pin-pricking, vacuum infiltration duration and vacuum pressure. Simple vegetative in vivo cleft grafting method was adopted in the multiplication of transformants without the aid of tissue culture. Among the various parameters evaluated, decapitated plants on pin-pricking and vacuum infiltrated at 250 mmHg for 3 min with the Agrobacterium strain EHA 105 harbouring the binary vector pGA 492 was proved to be efficient in all terms with a transformation efficiency of 62.66%. Transgene integration was evinced by the GUS histochemical analysis, and the GUS positive plants were subjected to grafting. Putatively transformed J. curcas served as "Scion" and the wild type J. curcas plant severed as "Stock". There was no occurrence of graft rejection and the plants were then confirmed by GUS histochemical analysis, polymerase chain reaction (PCR) and Southern hybridization. Genetic stability of the grafted plants was evaluated by using randomly amplified polymorphic DNA (RAPD), marker which showed 100% genetic stability between mother and grafted plants. Thus, an efficient in planta transformation and grafting based multiplication of J. curcas was established.

An Efficient Agrobacterium-Mediated Transformation of Strawberry cv. Camarosa by a Dual Plasmid System

Directory of Open Access Journals (Sweden)

Fatemeh Haddadi

2015-02-01

Full Text Available An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100% of shoot formation and the highest mean number of shoots (24 produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86% in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

A genetic-algorithm-based method to find unitary transformations for any desired quantum computation and application to a one-bit oracle decision problem

Energy Technology Data Exchange (ETDEWEB)

Bang, Jeongho [Seoul National University, Seoul (Korea, Republic of); Hanyang University, Seoul (Korea, Republic of); Yoo, Seokwon [Hanyang University, Seoul (Korea, Republic of)

2014-12-15

We propose a genetic-algorithm-based method to find the unitary transformations for any desired quantum computation. We formulate a simple genetic algorithm by introducing the 'genetic parameter vector' of the unitary transformations to be found. In the genetic algorithm process, all components of the genetic parameter vectors are supposed to evolve to the solution parameters of the unitary transformations. We apply our method to find the optimal unitary transformations and to generalize the corresponding quantum algorithms for a realistic problem, the one-bit oracle decision problem, or the often-called Deutsch problem. By numerical simulations, we can faithfully find the appropriate unitary transformations to solve the problem by using our method. We analyze the quantum algorithms identified by the found unitary transformations and generalize the variant models of the original Deutsch's algorithm.

Are Associations between Parental Divorce and Children's Adjustment Genetically Mediated? An Adoption Study.

Science.gov (United States)

O'Connor, Thomas G.; Caspi, Avshalom; DeFries, John C.; Plomin, Robert

2000-01-01

Data from Colorado Adoption Project were used to examine hypothesis that association between parental divorce and children's adjustment is mediated by genetic factors. Findings for psychopathology were consistent with an environmentally mediated explanation for the association. Findings for achievement and social adjustment were consistent with a…

Transformational leadership and group potency in small military units: The mediating role of group identification and cohesion

Directory of Open Access Journals (Sweden)

Carlos García-Guiu

2016-12-01

Full Text Available In the present study, we examined an exploratory model to assess the relationship between transformational leadership and group potency and analyze the mediating role of group identification and cohesion. The research was conducted with squads of the Spanish Army. The sample was composed of 243 members of 51 squads of operational units. Our findings highlighted the importance of the transformational leadership style of command of non-commissioned officers (NCOs due to its positive relationship with the group potency of the squad. We also analyzed the indirect relationships between transformational leadership and group identification and group cohesion and found that the latter variables played a mediating role between transformational leadership and group potency. The conclusions of this study are relevant due to the growing importance of transformational leadership and actions implemented at lower levels of the command chain for the success of missions of security organizations and defense.

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

Science.gov (United States)

Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

1999-01-01

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

Science.gov (United States)

Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

2014-01-01

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

Genetic transformation of Bacillus strains close to bacillus subtilis and isolated from the soil

International Nuclear Information System (INIS)

Van, C.K.; Kuzin, Yu.Yu.; Kozlovskii, Yu.E.; Prozorov, A.A.

1986-01-01

Chromosomal and plasmid transformation was found in five out of 118 Bacillus strains, close or identical to Bacillus subtilis, and isolated from soil in Moscow or in the Moscow district. The efficiency of transformation in these strains was lower than that in derivatives of Bac. subtilis strain 168. In these strains the ability to undergo transformation was dependent on the rate of sporulation and the presence of restrictases. As in the case of Bac. subtilis 168 the strains isolated may be used as models in genetic transformation studies on Bac. subtilis

Cyanuric acid biodegradation by a mixed bacterial culture of Agrobacterium tumefaciens and Acinetobacter sp. in a packed bed biofilm reactor.

Science.gov (United States)

Galíndez-Nájera, S P; Llamas-Martínez, M A; Ruiz-Ordaz, N; Juárez-Ramírez, C; Mondragón-Parada, M E; Ahuatzi-Chacón, D; Galíndez-Mayer, J

2009-02-01

Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L(-1)), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L(-1)) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support.

X-ray structure of imidazolonepropionase from Agrobacterium tumefaciens at 1.87 Å resolution

Energy Technology Data Exchange (ETDEWEB)

Tyagi, Rajiv; Kumaran, Desigan; Burley, Stephen K.; Swaminathan, Subramanyam (SGX); (BNL)

2010-01-12

Histidine degradation in Agrobacterium tumefaciens involves four enzymes, including histidase (EC 4.3.1.3), urocanase (EC 4.2.1.49), imidazolonepropionase (EC 3.5.2.7), and N-formylglutamate amidohydrolase (EC 3.5.3.8). The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-L-glutamate. Initial studies of the role of imidazolonepropionase in histidine degradation were published in 1953. Subsequent publications have been limited to enzyme kinetics, crystallization, and a recently reported structure determination. The imidazolonepropionases are members of metallodepenent-hydrolases (or amidohydroase) superfamily, which includs ureases, adenosine deaminases, phosphotriesterases, dihydroorotases, allantoinases, hydantoinases, adenine and cytosine deaminases, imidazolonepropionases, aryldial-kylphosphatases, chlorohydrolases, and formylmethanofuran dehydroases. Proteins belonging to this large group share a common three-dimensional structural motif (an eightfold {alpha}/{beta} or TIM barrel) with similar active sites. Most superfamily members also share a conserved metal binding site, involving four histidine residues and one aspartic acid. Imidazolonepropionase is one of the targets selected for X-ray crystallpgrahpic structure determination by the New York Structural GenomiX Research Consortium (NYSGXRC) Target ID: 9252b to correlate the structure function relationship of poorly studied by important enzyme. Here they report the crystal structure of imidazolonepropionase from Agrobacterium tumefaciens determined at 1.87 {angstrom} resolution.

Regulation of hydantoin-hydrolyzing enzyme expression in Agrobacterium tumefaciens strain RU-AE01.

Science.gov (United States)

Jiwaji, Meesbah; Dorrington, Rosemary Ann

2009-10-01

Optically pure D-: amino acids, like D-: hydroxyphenylglycine, are used in the semi-synthetic production of pharmaceuticals. They are synthesized industrially via the biocatalytic hydrolysis of p-hydroxyphenylhydantoin using enzymes derived from Agrobacterium tumefaciens strains. The reaction proceeds via a three-step pathway: (a) the ring-opening cleavage of the hydantoin ring by a D-: hydantoinase (encoded by hyuH), (b) conversion of the resultant D-: N-carbamylamino acid to the corresponding amino acid by a D-: N-carbamoylase (encoded by hyuC), and (c) chemical or enzymatic racemization of the un-reacted hydantoin substrate. While the structure and biochemical properties of these enzymes are well understood, little is known about their origin, their function, and their regulation in the native host. We investigated the mechanisms involved in the regulation of expression of the hydantoinase and N-carbamoylase enzyme activity in A. tumefaciens strain RU-AE01. We present evidence for a complex regulatory network that responds to the growth status of the cells, the presence of inducer, and nitrogen catabolite repression. Deletion analysis and site-directed mutagenesis were used to identify regulatory elements involved in transcriptional regulation of hyuH and hyuC expression. Finally, a comparison between the hyu gene clusters in several Agrobacterium strains provides insight into the function of D-: selective hydantoin-hydrolyzing enzyme systems in Agrobacterium species.

Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

International Nuclear Information System (INIS)

Bäuerle, Bettina; Sandalova, Tatyana; Schneider, Gunter; Rieger, Paul-Gerhard

2006-01-01

This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH 4 ) 2 SO 4 and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na 2 SO 4 and 0.1 M bis-Tris propane pH 8.5

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

Science.gov (United States)

Nanjareddy, Kalpana; Arthikala, Manoj-Kumar; Blanco, Lourdes; Arellano, Elizabeth S; Lara, Miguel

2016-06-24

Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for protoplast isolation from multiple P

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line.

Science.gov (United States)

Cho, Myeong-Je; Wu, Emily; Kwan, Jackie; Yu, Maryanne; Banh, Jenny; Linn, Wutt; Anand, Ajith; Li, Zhi; TeRonde, Susan; Register, James C; Jones, Todd J; Zhao, Zuo-Yu

2014-10-01

An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds. This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.

Genetic transformation of deciduous fruit trees conferring resistance against diseases

International Nuclear Information System (INIS)

Mansvelt, E.L.; Glyn-Woods, T.; Watts, L.; Rabie, A.; Appel, M.; Bellstedt, D.U.

1998-01-01

Long breeding cycles make cultivar development a lengthy process in deciduous fruit species. Gene transfer is, accordingly, a goal with significant commercial value. In many plant species, especially in woody plants, a prerequisite for genetic engineering is the ability to regenerate plants from transformed cells. Development of single cell regeneration is the first step towards exploration of gene transfer techniques. In this investigation media for plum and apple leaf disk regeneration were developed. Transformation experiments were performed. The vector EHA105 containing the gus-intron gene was found to be effective for gene transfer. Induction of the virG genes with aceto-syringone did not enhance transformation. Cefotaxime that was supplemented in the plum selection medium to suppress the Agrobacterium vector seriously inhibited leaf disk regeneration. However, in applies it was not detrimental. With further apple transformation experiments, factors such as preculturing, age of leaves, sucrose and cefotaxime concentrations did not increase the transformation efficiency of the marker gene. The harpin protein, essential for the pathogenicity of Pseudomonas syringae pv. syringae which incites bacterial canker of stone fruit, ws amplified and cloned into an expression vector. The fusion protein was purified. This will be used in future studies to elucidate the host-pathogen interaction, and to identify antibacterial genes. (author)

Green Transformational Leadership and Green Performance: The Mediation Effects of Green Mindfulness and Green Self-Efficacy

OpenAIRE

Yu-Shan Chen; Ching-Hsun Chang; Yu-Hsien Lin

2014-01-01

No prior literature explores the influence of green transformational leadership on green performance, thus, this study develops a novel research framework to fill the research gap. This study investigates the influence of green transformational leadership on green performance and discusses the mediation effects of green mindfulness and green self-efficacy by means of structural equation modeling (SEM). The results indicate that green transformational leadership positively influences green min...

Mediating the effects of work-life conflict between transformational leadership and health-care workers' job satisfaction and psychological wellbeing.

Science.gov (United States)

Munir, Fehmidah; Nielsen, Karina; Garde, Anne H; Albertsen, Karen; Carneiro, Isabella G

2012-05-01

To explore the mediating effects of work-life conflict between transformational leadership and job satisfaction and psychological wellbeing. The importance of work-life balance for job satisfaction and wellbeing among health-care employees is well-recognized. Evidence shows that transformational leadership style is linked to psychological wellbeing. It is possible that transformational leadership is also associated with employees' perceptions of work-life conflict, thereby influencing their job satisfaction and wellbeing. A longitudinal design was used where staff working within Danish elderly care completed a questionnaire at baseline and 18-month follow-up (N=188). Regression analyses showed that transformational leadership style was directly associated with perceptions of work-life conflict, job satisfaction and psychological wellbeing. Work-life conflict mediated between transformational leadership and wellbeing, but not job satisfaction. The findings suggest transformational leadership style may improve perceptions of work-life balance and employee wellbeing. Managers should adopt transformational leadership styles to reduce work-life conflict and enhance the wellbeing of their staff. © 2011 Blackwell Publishing Ltd.

Promoting safety voice with safety-specific transformational leadership: the mediating role of two dimensions of trust.

Science.gov (United States)

Conchie, Stacey M; Taylor, Paul J; Donald, Ian J

2012-01-01

Although safety-specific transformational leadership is known to encourage employee safety voice behaviors, less is known about what makes this style of leadership effective. We tested a model that links safety-specific transformational leadership to safety voice through various dimensions of trust. Data from 150 supervisor-employee dyads from the United Kingdom oil industry supported our predictions that the effects of safety-specific transformational leadership are sequentially mediated by affect-based trust beliefs and disclosure trust intentions. Moreover, we found that reliance trust intentions moderated the effect of disclosure: employees' disclosure intentions mediated the effects of affect-based trust on safety voice behaviors only when employees' intention to rely on their leader was moderate to high. These findings suggest that leaders seeking to encourage safety voice behaviors should go beyond "good reason" arguments and develop affective bonds with their employees.

Theoretical Study of Molecular Determinants Involved in Signal Binding to the TraR Protein of Agrobacterium tumefaciens

Directory of Open Access Journals (Sweden)

N. Kumar

2005-10-01

Full Text Available N-acylated homoserine lactone (AHL mediated cell-cell communication in bacteria is dependent on the recognition of the cognate signal by its receptor. This interaction allows the receptor-ligand complex to act as a transcriptional activator, controlling the expression of a range of bacterial phenotypes, including virulence factor expression and biofilm formation. One approach to determine the key features of signal- binding is to model the intermolecular interactions between the receptor and ligand using computational-based modeling software (LigandFit. In this communication, we have modeled the crystal structure of the AHL receptor protein TraR and its AHL signal N-(3- oxooctanoyl-homoserine lactone from Agrobacterium tumefaciens and compared it to the previously reported antagonist behaviour of a number of AHL analogues, in an attempt to determine structural constraints for ligand binding. We conclude that (i a common conformation of the AHL in the hydrophobic and hydrophilic region exists for ligand-binding, (ii a tail chain length threshold of 8 carbons is most favourable for ligand-binding affinity, (iii the positive correlation in the docking studies could be used a virtual screening tool.

Agrobacterium-Mediated Transformation of Bread and Durum Wheat Using Freshly Isolated Immature Embryos

Science.gov (United States)

Wu, Huixia; Doherty, Angela; Jones, Huw D.

Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.

The biotechnology (genetic transformation and molecular biology) of Bixa orellana L. (achiote).

Science.gov (United States)

Teixeira da Silva, Jaime A; Dobránszki, Judit; Rivera-Madrid, Renata

2018-05-10

Genetic transformation allows for greater bixin or norbixin production in achiote. Knowledge of genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants. Annatto is a natural dye or coloring agent derived from the seeds, or their arils, of achiote (Bixa orellana L.), and is commercially known as E160b. The main active component of annatto dye is water-insoluble bixin, although water-soluble norbixin also has commercial applications. Relative to other antioxidants, bixin is light- and temperature stable and is thus safe for human consumption. Bixin is, therefore, widely applied as a dye and as an antioxidant in the medico-pharmaceutical, food, cosmetic, and dye industries. Even though bixin has also been isolated from leaves and bark, yield is lower than from seeds. More biotechnology-based research of this industrial and medicinal plant is needed. Building on provisional genetic transformation studies, it would be advantageous to transform genes that could result in greater bixin or norbixin production. Reliable protocols for the extraction of bixin and norbixin, as well as deeper knowledge of the genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants.

IMPROVING PLANT GENETIC ENGINEERING BY MANIPULATING THE HOST. (R829479C001)

Science.gov (United States)

Agrobacterium-mediated transformation is a major technique for the genetic engineering of plants. However, there are many economically important crop and tree species that remain highly recalcitrant to Agrobacterium infection. Although attempts have been made to ...

Relationship of transformational leadership style with employee health and well-being: The mediating role of trust in the leader

Directory of Open Access Journals (Sweden)

Lyria Esperanza Perilla-Toro

2017-07-01

Full Text Available This study examined the relationship between transformational leadership and employee well-being indicators in developing countries, as well as the mediation role of trust in the leader. Five hundred ninety-seven employees of Colombian and Mexican organizations answered a questionnaire. Results indicated that transformational leadership relates positively with job satisfaction and negatively with distress symptomatology. No relationship was established between transformational leadership and psychological well-being. Trust in the leader mediated totally the relationship between transformational leadership and job satisfaction and symptoms of distress. These results confirm the previously described relationship between transformational leadership, less distress, and higher job satisfaction. However, it suggests too that the possible effect of transformational leadership on employees health and well-being would be limited to promoting affective aspects of well-being, but not psychological well-being. This result invites to a thorough review of the meaning and use of the concepts affective and psychological well-being and the differences between them.

Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

Energy Technology Data Exchange (ETDEWEB)

Bäuerle, Bettina [Institute of Microbiology, University of Stuttgart, 70569 Stuttgart (Germany); Sandalova, Tatyana; Schneider, Gunter [Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm (Sweden); Rieger, Paul-Gerhard, E-mail: pg.rieger@imb.uni-stuttgart.de [Institute of Microbiology, University of Stuttgart, 70569 Stuttgart (Germany)

2006-08-01

This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH{sub 4}){sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na{sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 8.5.

Nano-MnO2-mediated transformation of triclosan with humic molecules present: kinetics, products, and pathways.

Science.gov (United States)

Sun, Kai; Li, Shunyao; Waigi, Michael Gatheru; Huang, Qingguo

2018-05-01

It has been shown that manganese dioxide (MnO 2 ) can mediate transformation of phenolic contaminants to form phenoxyl radical intermediates, and subsequently, these intermediates intercouple to form oligomers via covalent binding. However, the reaction kinetics and transformation mechanisms of phenolic contaminants with humic molecules present in nano-MnO 2 -mediated systems were still unclear. In this study, it was proven that nano-MnO 2 were effective in transforming triclosan under acidic conditions (pH 3.5-5.0) during manganese reduction, and the apparent pseudo first-order kinetics rate constants (k = 0.0599-1.5314 h -1 ) increased as the pH decreased. In particular, the transformation of triclosan by nano-MnO 2 was enhanced in the presence of low-concentration humic acid (1-10 mg L -1 ). The variation in the absorption of humic molecules at 275 nm supported possible covalent binding between humic molecules and triclosan in the nano-MnO 2 -mediated systems. A total of four main intermediate products were identified by high-resolution mass spectrometry (HRMS), regardless of humic molecules present in the systems or not. These products correspond to a suite of radical intercoupling reactions (dimers and trimers), ether cleavage (2,4-dichlorophenol), and oxidation to quinone-like products, triggered by electron transfer from triclosan molecules to nano-MnO 2 . A possible reaction pathway in humic acid solutions, including homo-coupling, decomposition, oxidation, and cross-coupling, was proposed. Our findings provide valuable information regarding the environmental fate and transformation mechanism of triclosan by nano-MnO 2 in complex water matrices.

TRANSFORMATION

Energy Technology Data Exchange (ETDEWEB)

LACKS,S.A.

2003-10-09

Transformation, which alters the genetic makeup of an individual, is a concept that intrigues the human imagination. In Streptococcus pneumoniae such transformation was first demonstrated. Perhaps our fascination with genetics derived from our ancestors observing their own progeny, with its retention and assortment of parental traits, but such interest must have been accelerated after the dawn of agriculture. It was in pea plants that Gregor Mendel in the late 1800s examined inherited traits and found them to be determined by physical elements, or genes, passed from parents to progeny. In our day, the material basis of these genetic determinants was revealed to be DNA by the lowly bacteria, in particular, the pneumococcus. For this species, transformation by free DNA is a sexual process that enables cells to sport new combinations of genes and traits. Genetic transformation of the type found in S. pneumoniae occurs naturally in many species of bacteria (70), but, initially only a few other transformable species were found, namely, Haemophilus influenzae, Neisseria meningitides, Neisseria gonorrheae, and Bacillus subtilis (96). Natural transformation, which requires a set of genes evolved for the purpose, contrasts with artificial transformation, which is accomplished by shocking cells either electrically, as in electroporation, or by ionic and temperature shifts. Although such artificial treatments can introduce very small amounts of DNA into virtually any type of cell, the amounts introduced by natural transformation are a million-fold greater, and S. pneumoniae can take up as much as 10% of its cellular DNA content (40).

Transformational Leadership and Knowledge Sharing: Mediating Roles of Employee's Empowerment, Commitment, and Citizenship Behaviors

Science.gov (United States)

Han, Seung Hyun; Seo, Gaeun; Yoon, Seung Won; Yoon, Dong-Yeol

2016-01-01

Purpose: The purpose of this paper is to empirically examine the fundamental process through which transformational leaders play a significant role in employees' knowledge sharing by investigating mediating roles of individual affects, particularly psychological empowerment, organizational commitment and organizational citizenship behavior (OCB).…

Studies of transformational leadership in consumer service: leadership trust and the mediating-moderating role of cooperative conflict management.

Science.gov (United States)

Yang, Yi-Feng

2012-02-01

This is the third in a series of studies evaluating how transformational leadership is associated with related variables such as job satisfaction, change commitment, leadership trust, cooperative conflict management, and market orientation. The present paper evaluates the effects of transformational leadership and cooperative conflict management along with their mediating and moderating of leadership trust in the life insurance industry for two sample groups, sales managers and sales employees. The main effect of leadership trust was mediated and moderated by cooperative conflict management. Cooperative conflict management made a more important contribution than transformational leadership or the moderating effect (interaction), but these three together were the most important variables predicting highest leadership trust. Transformational leadership has an indirect influence on leadership trust. This work summarizes the specific contribution and importance of building successful leadership trust associations with employees in relation to leadership and satisfaction with change commitment.

Regeneration and genetic transformation of cowpea (Vigna unguiculata Walp.)

Energy Technology Data Exchange (ETDEWEB)

Filippone, E; Colucci, G; Ciardi, F; Monti, L [Department of Agronomy and Plant Genetics, Univ. of Naples Federico 11, Portici (Italy)

1997-07-01

Regeneration of cowpea (Vigna unguiculata Walp.) was achieved through massive bud formation induced in apical and lateral meristems by the herbicide Thidiazuron (TDZ). The effect of TDZ (5, 10, or 20 {mu}M) was tested in vitro on four different cowpea genotypes. Thidiazuron, even at the highest concentration, had no effect on seed germination. After one month of culture, multiple bud cluster formation was observed in all genotypes tested; about 80% of shoot apices regenerated multiple buds, whilst only 34% of cotyledonary nodes behaved in the same way. Histology of regenerating multiple bud clusters revealed that regeneration initiated from pre-existing meristems in the apex and cotyledonary node. Thidiazuron at 10 {mu}M appeared to be the best concentration to produce clusters with high number of buds, ranging from 5 to 10. Shoot elongation occurred only on MS medium without TDZ. On the same medium, 75% of elongated shoots rooted. For genetic transformation of cowpea, a direct DNA transfer methods in plants under in vivo conditions was tested by electroporation of plasmid DNA into the nodal meristematic cells. Some transformed plants were obtained, and produced T{sub 1} transformed progenies; their transgenic nature was confirmed by Southern analysis. (author). 21 refs, 2 figs, 3 tabs.

Regeneration and genetic transformation of cowpea (Vigna unguiculata Walp.)

International Nuclear Information System (INIS)

Filippone, E.; Colucci, G.; Ciardi, F.; Monti, L.

1997-01-01

Regeneration of cowpea (Vigna unguiculata Walp.) was achieved through massive bud formation induced in apical and lateral meristems by the herbicide Thidiazuron (TDZ). The effect of TDZ (5, 10, or 20 μM) was tested in vitro on four different cowpea genotypes. Thidiazuron, even at the highest concentration, had no effect on seed germination. After one month of culture, multiple bud cluster formation was observed in all genotypes tested; about 80% of shoot apices regenerated multiple buds, whilst only 34% of cotyledonary nodes behaved in the same way. Histology of regenerating multiple bud clusters revealed that regeneration initiated from pre-existing meristems in the apex and cotyledonary node. Thidiazuron at 10 μM appeared to be the best concentration to produce clusters with high number of buds, ranging from 5 to 10. Shoot elongation occurred only on MS medium without TDZ. On the same medium, 75% of elongated shoots rooted. For genetic transformation of cowpea, a direct DNA transfer methods in plants under in vivo conditions was tested by electroporation of plasmid DNA into the nodal meristematic cells. Some transformed plants were obtained, and produced T 1 transformed progenies; their transgenic nature was confirmed by Southern analysis. (author). 21 refs, 2 figs, 3 tabs

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

Science.gov (United States)

Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

2014-07-01

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

The experimental study of genetic engineering human neural stem cells mediated by lentivirus to express multigene.

Science.gov (United States)

Cai, Pei-qiang; Tang, Xun; Lin, Yue-qiu; Martin, Oudega; Sun, Guang-yun; Xu, Lin; Yang, Yun-kang; Zhou, Tian-hua

2006-02-01

To explore the feasibility to construct genetic engineering human neural stem cells (hNSCs) mediated by lentivirus to express multigene in order to provide a graft source for further studies of spinal cord injury (SCI). Human neural stem cells from the brain cortex of human abortus were isolated and cultured, then gene was modified by lentivirus to express both green fluorescence protein (GFP) and rat neurotrophin-3 (NT-3); the transgenic expression was detected by the methods of fluorescence microscope, dorsal root ganglion of fetal rats and slot blot. Genetic engineering hNSCs were successfully constructed. All of the genetic engineering hNSCs which expressed bright green fluorescence were observed under the fluorescence microscope. The conditioned medium of transgenic hNSCs could induce neurite flourishing outgrowth from dorsal root ganglion (DRG). The genetic engineering hNSCs expressed high level NT-3 which could be detected by using slot blot. Genetic engineering hNSCs mediated by lentivirus can be constructed to express multigene successfully.

Transformational leadership and safety performance among nurses: the mediating role of knowledge-related job characteristics.

Science.gov (United States)

Lievens, Ilse; Vlerick, Peter

2014-03-01

To report the impact of transformational leadership on two dimensions of nurses' safety performance (i.e. safety compliance and safety participation) and to study the mediating role of knowledge-related job characteristics in this relationship. Safety performance refers to the behaviours that employees exhibit to adhere to safety guidelines and to promote health and safety at their workplace. Nurses' safety performance is a major challenge for healthcare settings, urging the need to identify the key determinants and psychological mechanisms that influence it. A cross-sectional survey study. The study was carried out in September 2010 in a large Belgian hospital. We used self-administered questionnaires; 152 nurses participated. The hypotheses were tested using hierarchical regression analyses. In line with our first hypothesis, the results show that transformational leadership exerted a significant positive impact on both dimensions of nurses' safety performance. This positive relation was mediated by knowledge-related job characteristics, supporting our second hypothesis. Head nurses' transformational leadership can enhance nurses' compliance with and participation in safety. Furthermore, transformational head nurses are able to influence the perception that their nurses have about the kind and amount of knowledge in their job, which can also lead to increases in both dimensions of nurses' safety performance. This study therefore demonstrates the key impact that transformational head nurses have, both directly and indirectly, on the safety performance of their nurses. © 2013 John Wiley & Sons Ltd.

Future strategy and puzzles of heavy ion beam mediated technique in genetic improvement of biological bodies

International Nuclear Information System (INIS)

Huang Qunce

2007-01-01

The 7 research puzzles in the genetic improvement of biological bodies made by ion beam mediated technique, are worth noticed. The technical ideas, including one mediated technique in physics, 2 significant subjects, 3 effective changes, the mediated evidences of 4 aspects and 5 biological characteristics, were particularly put forward according to the existing states in the field. The 2 significant subjects consist of the mechanics of the allogenetic materials entering into the acceptor and they being to be recombined. The 3 effective changes include from studying morphology to genetic laws, from researching M1 generation to the next generations, from determining the single character to the synthetic traits. The mediated evidences of 4 aspects come from morphology, physiology and biochemistry, molecule biology. The 5 biological characteristics are mainly reproduction, development, photosynthesis, bad condition-resistant and quality. (authors)

EGG transformation through the use of irradiated pollen: 'Poor man's genetic engineering'

International Nuclear Information System (INIS)

Pandey, K.K.

1981-01-01

There is no way that the 'fertilization cummutation hypothesis' can be considered to be an alternative to transformation. 'Poor man's genetic engineering' as a tool for plant breeders should be the development and application of the knowledge about growth-promothing genes which are thought to occur in self-compatible as well as in self-incompatible species. (AJ)

In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance

OpenAIRE

Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath

2016-01-01

Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbN...

Transformational Leadership, Transactional Contingent Reward, and Organizational Identification: The Mediating Effect of Perceived Innovation and Goal Culture Orientations.

Science.gov (United States)

Xenikou, Athena

2017-01-01

Purpose: The aim of this research was to investigate the effect of transformational leadership and transactional contingent reward as complementary, but distinct, forms of leadership on facets of organizational identification via the perception of innovation and goal organizational values. Design/Methodology/Approach: Three studies were carried out implementing either a measurement of mediation or experimental-causal-chain design to test for the hypothesized effects. Findings: The measurement of mediation study showed that transformational leadership had a positive direct and indirect effect, via innovation value orientation, on cognitive identification, whereas transactional contingent reward was more strongly related to affective, rather than cognitive, identification, and goal orientation was a mediator of their link. The findings of the two experimental-causal-chain studies further supported the hypothesized effects. Transformational leadership was found to lead subordinates to perceive the culture as more innovative compared to transactional contingent reward, whereas transactional contingent reward led employees to perceive the culture as more goal, than innovation, oriented. Finally, innovation, compared to goal, value orientation increased cognitive identification, while goal orientation facilitated affective, rather than cognitive, identification. Implications: The practical implications involve the development of strategies organizations can apply, such as leadership training programs, to strengthen their ties with their employees, which, in turn, may have a positive impact on in-role, as well as extra-role, behaviors. Originality/Value: The originality of this research concerns the identification of distinct mechanisms explaining the effect of transformational leadership and transactional contingent reward on cognitive and affective identification applying an organizational culture perspective and a combination of measurement and causal mediation designs.

Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

International Nuclear Information System (INIS)

Mase, Tomoko; Yabuki, Hideya; Okai, Masahiko; Ohtsuka, Jun; Imai, Fabiana Lica; Nagata, Yuji; Tanokura, Masaru

2012-01-01

The haloalkane dehalogenase DatA from A. tumefaciens C58 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.70 Å resolution. Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit

Microbe and Mineral Mediated Transformation of Heavy Metals, Radionuclides, and Organic Contaminants

Science.gov (United States)

Gerlach, R.

2011-12-01

Microorganisms influence their surroundings in many ways and humans have utilized microbially catalyzed reactions for benefit for centuries. Over the past few decades, microorganisms have been used for the control of contaminant transport in subsurface environments where many microbe mineral interactions occur. This presentation will discuss microbially influenced mineral formation and transformation as well as their influence on the fate of organic contaminants such as chlorinated aliphatics & 2,4,6-trinitrotoluene (TNT), heavy metals such as chromium, and radionuclides such as uranium & strontium. Both, batch and flow experiments have been performed, which monitor the net effect of microbe mineral interactions on the fate of these contaminants. This invited presentation will place an emphasis on the relative importance of direct microbial (i.e. biotic) transformations, mineral-mediated transformations as well as other abiotic reactions influencing the fate of environmental contaminants. Experiments will be summarized and placed in context of past and future engineered applications for the control of subsurface contaminants.

Exosome-Mediated Genetic Information Transfer, a Missing Piece of Osteoblast-Osteoclast Communication Puzzle.

Science.gov (United States)

Yin, Pengbin; Lv, Houchen; Li, Yi; Deng, Yuan; Zhang, Licheng; Tang, Peifu

2017-01-01

The skeletal system functions and maintains itself based on communication between cells of diverse origins, especially between osteoblasts (OBs) and osteoclasts (OCs), accounting for bone formation and resorption, respectively. Previously, protein-level information exchange has been the research focus, and this has been discussed in detail. The regulative effects of microRNAs (miRNAs) on OB and OC ignite the question as to whether genetic information could be transferred between bone cells. Exosomes, extracellular membrane vesicles 30-100 nm in diameter, have recently been demonstrated to transfer functional proteins, mRNAs, and miRNAs, and serve as mediators of intercellular communication. By reviewing the distinguishing features of exosomes, a hypothesis was formulated and evaluated in this article that exosome-mediated genetic information transfer may represent a novel strategy for OB-OC communication. The exosomes may coordinately regulate these two cells under certain physiological conditions by transferring genetic information. Further research in exosome-shuttered miRNAs in OB-OC communication may add a missing piece to the bone cells communication "puzzle."

Genetic Transformation of Bacteria.

Science.gov (United States)

Moss, Robert.

1991-01-01

An activity in which students transform an ampicillin-sensitive strain of E. coli with a plasmid containing a gene for ampicillin resistance is described. The procedure for the preparation of competent cells and the transformation of competent E. coli is provided. (KR)

Isolation of a conjugative F-like plasmid from a multidrug-resistant Escherichia coli strain CM6 using tandem shock wave-mediated transformation.

Science.gov (United States)

Soto-Alonso, G; Cruz-Medina, J A; Caballero-Pérez, J; Arvizu-Hernández, I; Ávalos-Esparza, L M; Cruz-Hernández, A; Romero-Gómez, S; Rodríguez, A L; Pastrana-Martínez, X; Fernández, F; Loske, A M; Campos-Guillén, J

2015-07-01

Genetic characterization of plasmids from bacterial strains provides insight about multidrug resistance. Ten wild type Escherichia coli (E. coli) strains isolated from cow fecal samples were characterized by their antibiotic resistance profile, plasmid patterns and three different identification methods. From one of the strains, a fertility factor-like plasmid was replicated using tandem shock wave-mediated transformation. Underwater shock waves with a positive pressure peak of up to approximately 40 MPa, followed by a pressure trough of approximately -19 MPa were generated using an experimental piezoelectric shock wave source. Three different shock wave energies and a fixed delay of 750 μs were used to study the relationship between energy and transformation efficiency (TE), as well as the influence of shock wave energy on the integrity of the plasmid. Our results showed that the mean shock wave-mediated TE and the integrity of the large plasmid (~70 kb) were reduced significantly at the energy levels tested. The sequencing analysis of the plasmid revealed a high identity to the pHK17a plasmid, including the replication system, which was similar to the plasmid incompatibility group FII. It also showed that it carried an extended spectrum beta-lactamase gene, ctx-m-14. Furthermore, diverse genes for the conjugative mechanism were identified. Our results may be helpful in improving methodologies for conjugative plasmid transfer and directly selecting the most interesting plasmids from environmental samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Linking transformational leadership to nurses' extra-role performance: the mediating role of self-efficacy and work engagement.

Science.gov (United States)

Salanova, Marisa; Lorente, Laura; Chambel, Maria J; Martínez, Isabel M

2011-10-01

This paper is a report of a social cognitive theory-guided study about the link between supervisors' transformational leadership and staff nurses' extra-role performance as mediated by nurse self-efficacy and work engagement. Past research has acknowledged the positive influence that transformational leaders have on employee (extra-role) performance. However, less is known about the psychological mechanisms that may explain the links between transformational leaders and extra-role performance, which encompasses behaviours that are not considered formal job requirements, but which facilitate the smooth functioning of the organization as a social system. Seventeen supervisors evaluated nurses' extra-role performance, the data generating a sample consisting of 280 dyads. The nurses worked in different health services in a large Portuguese hospital and the participation rate was 76·9% for nurses and 100% for supervisors. Data were collected during 2009. A theory-driven model of the relationships between transformation leadership, self-efficacy, work engagement and nurses' extra-role performance was tested using Structural Equation Modelling. Data analysis revealed a full mediation model in which transformational leadership explained extra-role performance through self-efficacy and work engagement. A direct relationship between transformational leadership and work engagement was also found. Nurses' supervisors with a transformational leadership style enhance different 'extra-role' performance in nurses and this increases hospital efficacy. They do so by establishing a sense of self-efficacy but also by amplifying their levels of engagement in the workplace. © 2011 Blackwell Publishing Ltd.

Forest, water and people: The roles and limits of mediation in transforming watershed conflict in Northern Thailand

Directory of Open Access Journals (Sweden)

Ahmad Dhiaulhaq

2017-11-01

Full Text Available This study focuses on watershed management in Northern Thailand, where conflict over forest, land and water-use is a prevailing problem. A characteristic of watershed conflicts is that they are often multifaceted and involve multiple stakeholders with different interests and values, consequently requiring conflict management approaches that are sustainable in their outcomes, including addressing the underlying causes of the conflicts. Drawing from a case study in Mae Tia Mae Tae watershed in Northern Thailand, this study explores how mediation by external third party can contribute to the transformation of conflicts in the watershed and how the broader institutional contexts in which the conflict is embedded shapes the mediation outcomes. The study suggests that co-creation of mutual understanding and recognition of each party’s socio-cultural differences, including land-use practices, are critical in building trust and in how conflict transformation processes moved forward. Moreover, the ability of the mediator in facilitating the establishment of a deliberative institution (i.e. a watershed network committee and agreed rules on forest utilization were also critical in maintaining long-term collaboration in the watershed and potentially preventing other conflicts arising in the future. Some issues, however, may threaten the continuity of the cooperation and sustainability of peace in the watershed, including the lack of structural reform that formally recognizes local people’s rights, insecure land tenure, and the absence of legal recognition for the watershed network committee as a legitimate mechanism for watershed decision making. The paper discusses these findings by comparing it with those from our previous studies in other locations (Cambodia, Indonesia and Western Thailand to strengthen the insights from Northern Thailand. Finally, the research puts forward some recommendations for reforms and to strengthen the use of effective

Abiotic and biotic factors responsible for antimonite oxidation in Agrobacterium tumefaciens GW4

Science.gov (United States)

Li, Jingxin; Yang, Birong; Shi, Manman; Yuan, Kai; Guo, Wei; Wang, Qian; Wang, Gejiao

2017-03-01

Antimonite [Sb(III)]-oxidizing bacteria can transform the toxic Sb(III) into the less toxic antimonate [Sb(V)]. Recently, the cytoplasmic Sb(III)-oxidase AnoA and the periplasmic arsenite [As(III)] oxidase AioAB were shown to responsible for bacterial Sb(III) oxidation, however, disruption of each gene only partially decreased Sb(III) oxidation efficiency. This study showed that in Agrobacterium tumefaciens GW4, Sb(III) induced cellular H2O2 content and H2O2 degradation gene katA. Gene knock-out/complementation of katA, anoA, aioA and anoA/aioA and Sb(III) oxidation and growth experiments showed that katA, anoA and aioA were essential for Sb(III) oxidation and resistance and katA was also essential for H2O2 resistance. Furthermore, linear correlations were observed between cellular H2O2 and Sb(V) content in vivo and chemical H2O2 and Sb(V) content in vitro (R2 = 0.93 and 0.94, respectively). These results indicate that besides the biotic factors, the cellular H2O2 induced by Sb(III) also catalyzes bacterial Sb(III) oxidation as an abiotic oxidant. The data reveal a novel mechanism that bacterial Sb(III) oxidation is associated with abiotic (cellular H2O2) and biotic (AnoA and AioAB) factors and Sb(III) oxidation process consumes cellular H2O2 which contributes to microbial detoxification of both Sb(III) and cellular H2O2.

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

OpenAIRE

Metts, J; West, J; Doares, S H; Matthysse, A G

1991-01-01

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was d...

Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens

International Nuclear Information System (INIS)

Amikam, D.; Benziman, M.

1989-01-01

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32 P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [ 14 C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation[OPEN

Science.gov (United States)

Lowe, Keith; Wu, Emily; Cho, Myeong-Je; Lenderts, Brian; Chamberlin, Mark; Cushatt, Josh; Ryan, Larisa; Khan, Tanveer; Chow-Yiu, Julia; Hua, Wei; Banh, Jenny; Bao, Zhongmeng; Brink, Kent; Igo, Elizabeth; Rudrappa, Bhojaraja; Shamseer, PM; Shen, Bo; Zheng, Peizhong; Bidney, Dennis; Falco, Carl; Zhao, Zuo-Yu; Xu, Deping

2016-01-01

While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus. PMID:27600536

Natural transformation of Campylobacter jejuni occurs beyond limits of growth

DEFF Research Database (Denmark)

Vegge, Christina Skovgaard; Brøndsted, Lone; Ligowska, Małgorzata

2012-01-01

of transformation is correlated to the growth conditions, but more importantly that transformation occurs at growth-restrictive conditions as well as in the late stationary phase; hence revealing that growth per se is not required for C. jejuni to be competent. Yet, natural transformation of C. jejuni is an energy......Campylobacter jejuni is a human bacterial pathogen. While poultry is considered to be a major source of food borne campylobacteriosis, C. jejuni is frequently found in the external environment, and water is another well-known source of human infections. Natural transformation is considered...... to be one of the main mechanisms for mediating transfer of genetic material and evolution of the organism. Given the diverse habitats of C. jejuni we set out to examine how environmental conditions and physiological processes affect natural transformation of C. jejuni. We show that the efficiency...

CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

Science.gov (United States)

Yang, Fayu; Liu, Changbao; Chen, Ding; Tu, Mengjun; Xie, Haihua; Sun, Huihui; Ge, Xianglian; Tang, Lianchao; Li, Jin; Zheng, Jiayong; Song, Zongming; Qu, Jia; Gu, Feng

2017-06-16

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A Rigid Image Registration Based on the Nonsubsampled Contourlet Transform and Genetic Algorithms

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Nasreddine Taleb

2010-09-01

Full Text Available Image registration is a fundamental task used in image processing to match two or more images taken at different times, from different sensors or from different viewpoints. The objective is to find in a huge search space of geometric transformations, an acceptable accurate solution in a reasonable time to provide better registered images. Exhaustive search is computationally expensive and the computational cost increases exponentially with the number of transformation parameters and the size of the data set. In this work, we present an efficient image registration algorithm that uses genetic algorithms within a multi-resolution framework based on the Non-Subsampled Contourlet Transform (NSCT. An adaptable genetic algorithm for registration is adopted in order to minimize the search space. This approach is used within a hybrid scheme applying the two techniques fitness sharing and elitism. Two NSCT based methods are proposed for registration. A comparative study is established between these methods and a wavelet based one. Because the NSCT is a shift-invariant multidirectional transform, the second method is adopted for its search speeding up property. Simulation results clearly show that both proposed techniques are really promising methods for image registration compared to the wavelet approach, while the second technique has led to the best performance results of all. Moreover, to demonstrate the effectiveness of these methods, these registration techniques have been successfully applied to register SPOT, IKONOS and Synthetic Aperture Radar (SAR images. The algorithm has been shown to work perfectly well for multi-temporal satellite images as well, even in the presence of noise.

A rigid image registration based on the nonsubsampled contourlet transform and genetic algorithms.

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Meskine, Fatiha; Chikr El Mezouar, Miloud; Taleb, Nasreddine

2010-01-01

Image registration is a fundamental task used in image processing to match two or more images taken at different times, from different sensors or from different viewpoints. The objective is to find in a huge search space of geometric transformations, an acceptable accurate solution in a reasonable time to provide better registered images. Exhaustive search is computationally expensive and the computational cost increases exponentially with the number of transformation parameters and the size of the data set. In this work, we present an efficient image registration algorithm that uses genetic algorithms within a multi-resolution framework based on the Non-Subsampled Contourlet Transform (NSCT). An adaptable genetic algorithm for registration is adopted in order to minimize the search space. This approach is used within a hybrid scheme applying the two techniques fitness sharing and elitism. Two NSCT based methods are proposed for registration. A comparative study is established between these methods and a wavelet based one. Because the NSCT is a shift-invariant multidirectional transform, the second method is adopted for its search speeding up property. Simulation results clearly show that both proposed techniques are really promising methods for image registration compared to the wavelet approach, while the second technique has led to the best performance results of all. Moreover, to demonstrate the effectiveness of these methods, these registration techniques have been successfully applied to register SPOT, IKONOS and Synthetic Aperture Radar (SAR) images. The algorithm has been shown to work perfectly well for multi-temporal satellite images as well, even in the presence of noise.

Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination.

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Döhlemann, Johannes; Brennecke, Meike; Becker, Anke

2016-09-10

The soil-dwelling α-proteobacterium Sinorhizobium meliloti serves as model for studies of symbiotic nitrogen fixation, a highly important process in sustainable agriculture. Here, we report advancements of the genetic toolbox accelerating genome editing in S. meliloti. The hsdMSR operon encodes a type-I restriction-modification (R-M) system. Transformation of S. meliloti is counteracted by the restriction endonuclease HsdR degrading DNA which lacks the appropriate methylation pattern. We provide a stable S. meliloti hsdR deletion mutant showing enhanced transformation with Escherichia coli-derived plasmid DNA and demonstrate that using an E. coli plasmid donor, expressing S. meliloti methyl transferase genes, is an alternative strategy of increasing the transformation efficiency of S. meliloti. Furthermore, we devise a novel cloning-free genome editing (CFGE) method for S. meliloti, Agrobacterium tumefaciens and Xanthomonas campestris, and demonstrate the applicability of this method for intricate applications of the Cre/lox recombination system in S. meliloti. An enhanced Cre/lox system, allowing for serial deletions of large genomic regions, was established. An assay of lox spacer mutants identified a set of lox sites mediating specific recombination. The availability of several non-promiscuous Cre recognition sites enables simultaneous specific Cre/lox recombination events. CFGE combined with Cre/lox recombination is put forward as powerful approach for targeted genome editing, involving serial steps of manipulation to expedite the genetic accessibility of S. meliloti as chassis. Copyright © 2016 Elsevier B.V. All rights reserved.

Cannabis sativa L. genetically transformed root based culture via Agrobacterium rhizogenes

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Farnoush Berahmand

2016-09-01

Full Text Available It is an increased interest in the therapeutic potential of Cannabis sativa L. (marijuana for treatment of multiple sclerosis and HIV neuropathy. Because of limitation in cultivation of this plant, an efficient hairy root induction system for Cannabis sativa L. was developed in the present study. Agrobacterium rhizogenes mediated transformation performed by two different co-cultivation mediums and four different bacterial strains including A4, ATCC15834, MSU440, and A13 (MAFF-02-10266. Genomic DNA from putative transgenic hairy root lines and the control root was extracted using a modified CTAB protocol. Molecular analysis of transformed root lines was confirmed by polymerase chain reaction using specific primers of the rolB gene. The transformation frequency by Murashige and Skoog co-cultivation medium resulting in hairy root induction frequencies of 42.3%, 46.3%, 68.6% and 39.3% by A4, ATCC15834, MSU440, and A13 strains, respectively. There was no significant difference between MS or ½ MS co-cultivation mediums.  This study established a reliable protocol for induction of hairy roots of C. sativa. The best A. rhisogenes strain was MSU440. It was observed no significant difference between MS and ½ MS co-cultivation mediums on transformation frequency.

Transformational Leadership, Transactional Contingent Reward, and Organizational Identification: The Mediating Effect of Perceived Innovation and Goal Culture Orientations

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Xenikou, Athena

2017-01-01

Purpose: The aim of this research was to investigate the effect of transformational leadership and transactional contingent reward as complementary, but distinct, forms of leadership on facets of organizational identification via the perception of innovation and goal organizational values. Design/Methodology/Approach: Three studies were carried out implementing either a measurement of mediation or experimental-causal-chain design to test for the hypothesized effects. Findings: The measurement of mediation study showed that transformational leadership had a positive direct and indirect effect, via innovation value orientation, on cognitive identification, whereas transactional contingent reward was more strongly related to affective, rather than cognitive, identification, and goal orientation was a mediator of their link. The findings of the two experimental-causal-chain studies further supported the hypothesized effects. Transformational leadership was found to lead subordinates to perceive the culture as more innovative compared to transactional contingent reward, whereas transactional contingent reward led employees to perceive the culture as more goal, than innovation, oriented. Finally, innovation, compared to goal, value orientation increased cognitive identification, while goal orientation facilitated affective, rather than cognitive, identification. Implications: The practical implications involve the development of strategies organizations can apply, such as leadership training programs, to strengthen their ties with their employees, which, in turn, may have a positive impact on in-role, as well as extra-role, behaviors. Originality/Value: The originality of this research concerns the identification of distinct mechanisms explaining the effect of transformational leadership and transactional contingent reward on cognitive and affective identification applying an organizational culture perspective and a combination of measurement and causal mediation designs

Transport and transformation of genetic information in the critical zone: The case of antibiotic resistance genes

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Zhu, Y. G.

2015-12-01

In addition to material and energy flows, the dynamics and functions of the Earth's critical zone are intensively mediated by biological actions performed by diverse organisms. These biological actions are modulated by the expression of functional genes and their translation into enzymes that catalyze geochemical reactions, such as nutrient turnover and pollutant biodegradation. Although geobiology, as an interdisciplinary research area, is playing and vital role in linking biological and geochemical processes at different temporal and spatial scales, the distribution and transport of functional genes have rarely been investigated from the Earth's critical zone perspectives. To illustrate the framework of studies on the transport and transformation of genetic information in the critical zone, antibiotic resistance is taken as an example. Antibiotic resistance genes are considered as a group of emerging contaminants, and their emergence and spread within the critical zone on one hand are induced by anthropogenic activities, and on other hand are threatening human health worldwide. The transport and transformation of antibiotic resistance genes are controlled by both horizontal gene transfer between bacterial cells and the movement of bacteria harboring antibiotic resistance genes. In this paper, the fate and behavior of antibiotic resistance genes will be discussed in the following aspects: 1) general overview of environmental antibiotic resistance; 2) high through quantification of the resistome in various environmental media; 3) pathways of resistance gene flow within the critical zone; and 4) potential strategies in mitigating antibiotic resistance, particularly from the critical zone perspectives.

Genetic transformation of midgut bacteria from the red imported fire ant (Solenopsis invicta).

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Medina, Freder; Li, Haiwen; Vinson, S Bradleigh; Coates, Craig J

2009-05-01

In our previous study we isolated 10 bacterial species from fourth-instar larval midguts of the red imported fire ant, Solenopsis invicta. Here we report the genetic transformation and reintroduction of three species (Kluyvera cryocrescens, Serratia marcescens, and isolate 38) into the fire ant host. All three species were transformed with the plasmid vector, pZeoDsRed. High expression levels of DsRed were observed and the plasmid is maintained in these bacteria at 37 degrees C in the absence of antibiotic selection for at least 9 days of subculturing. The transformed bacteria were successfully reintroduced into fire ant larvae and survived in the fire ant gut for at least 7 days. Upon pupal emergence, 7 days after reintroduction, transformed bacteria can still be isolated, however, most were passed out in the meconium. We further demonstrated that the engineered bacteria could be spread within the colony by feeding this meconium to naive larvae with the aid of worker fire ants.

A study on ranking the effects of transformational leadership style on organizational agility and mediating role of organizational creativity

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Seidmehdi Veiseh

2014-09-01

Full Text Available The purpose of this study was to investigate the effects of the components of transformational leadership style on organizational agility and mediating role of organizational creativity in Ilam Gas Refinery located in province of Ilam, Iran. The method of the present study was descriptive and correlational-structural equation modeling. The population of this research included all 400 workers of Ilam gas refinery and the study chose a sample of196 employees. The questionnaire was standardized using Cronbach's alpha; the obtained reliability was 0.90, which indicated the reliability of the questionnaire. At the end, the data was analyzed by LISREL software and structural equation modeling analysis was conducted. The findings showed that transformational leadership style had an effect on organizational agility. In addition, organizational creativity maintained a mediator role on influencing the transformational leadership on organizational agility. The four dimensions of transformational leadership, hopeful influence, inspirational motivation, intellectual encouragement as well as personal considerations also influenced on the agility of organizations.

Unlocking the potential of tropical root crop biotechnology in east Africa by establishing a genetic transformation platform for local farmer-preferred cassava cultivars

OpenAIRE

Evans eNyaboga; Evans eNyaboga; Evans eNyaboga; Joshua eNjiru; Edward eNguu; Wilhelm eGruissem; Herve eVanderschuren; Leena eTripathi

2013-01-01

Cassava genetic transformation capacity is still mostly restricted to advanced laboratories in the USA, Europe and China; and its implementation and maintainance in African laboratories has remained scarce. The impact of transgenic technologies for genetic improvement of cassava will depend largely on the transfer of such capabilities to researchers in Africa, where cassava has an important socioeconomic niche. A major constraint to the development of genetic transformation technologies for c...

Biolistic mediated sorghum (Sorghum bicolor L. Moench) transformation via mannose and bialaphos based selection systems

CSIR Research Space (South Africa)

Grootboom, AW

2010-01-01

Full Text Available of transformation. In sorghum, concerns about flow of herbicide and antibiotic resistance gene into genetically related wild and weedy species have a direct bearing on the choice of suitable selectable markers in many tropical and subtropical regions. The authors...

Prefrontal gray matter volume mediates genetic risks for obesity.

Science.gov (United States)

Opel, N; Redlich, R; Kaehler, C; Grotegerd, D; Dohm, K; Heindel, W; Kugel, H; Thalamuthu, A; Koutsouleris, N; Arolt, V; Teuber, A; Wersching, H; Baune, B T; Berger, K; Dannlowski, U

2017-05-01

Genetic and neuroimaging research has identified neurobiological correlates of obesity. However, evidence for an integrated model of genetic risk and brain structural alterations in the pathophysiology of obesity is still absent. Here we investigated the relationship between polygenic risk for obesity, gray matter structure and body mass index (BMI) by the use of univariate and multivariate analyses in two large, independent cohorts (n=330 and n=347). Higher BMI and higher polygenic risk for obesity were significantly associated with medial prefrontal gray matter decrease, and prefrontal gray matter was further shown to significantly mediate the effect of polygenic risk for obesity on BMI in both samples. Building on this, the successful individualized prediction of BMI by means of multivariate pattern classification algorithms trained on whole-brain imaging data and external validations in the second cohort points to potential clinical applications of this imaging trait marker.

Genetic Transformation of Streptococcus mutans

OpenAIRE

Perry, Dennis; Kuramitsu, Howard K.

1981-01-01

Three strains of Streptococcus mutans belonging to serotypes a, c, and f were transformed to streptomycin resistance by deoxyribonucleic acids derived from homologous and heterologous streptomycin-resistant strains of S. mutans and Streptococcus sanguis strain Challis. Homologous transformation of S. mutans was less efficient than heterologous transformation by deoxyribonucleic acids from other strains of S. mutans.

EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.

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Leah A Owen

2008-04-01

Full Text Available EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.

Crystal Structure of AGR_C_4470p from Agrobacterium tumefaciens

Energy Technology Data Exchange (ETDEWEB)

Vorobiev,S.; Neely, H.; Seetharaman, J.; Ma, L.; Xiao, R.; Acton, T.; Montelione, G.; Tong, L.

2007-01-01

We report here the crystal structure at 2.0 {angstrom} resolution of the AGR{_}C{_}4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR{_}C{_}4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR{_}C{_}4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR{_}C{_}4470p in E. coli, in addition to the ChuS protein.

Biolistic transformation of Scoparia dulcis L.

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Srinivas, Kota; Muralikrishna, Narra; Kumar, Kalva Bharath; Raghu, Ellendula; Mahender, Aileni; Kiranmayee, Kasula; Yashodahara, Velivela; Sadanandam, Abbagani

2016-01-01

Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0 %) transient expression of GUS gene bombarded at a flight distance of 6 cm and rupture disc pressure/acceleration pressure of 650 psi. Highest stable expression of 52.0 % was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant.

Optimization of an Efficient Non-Tissue Culture Transformation Method for Brassica Juncea

International Nuclear Information System (INIS)

Naeem, I.; Munir, I.; Iqbal, A.; Ullah, F.

2016-01-01

The major hurdles in successful in vitro transformation of Brassica juncea through standard tissue culture (STC) method are: culture contamination, somaclonal variations, and lack of expertise. Moreover, the current STC method is time consuming and needs continuous electricity. In the present study, the in planta transformation method through floral dip with or without vacuum infiltration was optimized for successful transformation of B. juncea. The B. juncea CV RAYA Anmol was used for transformation through Agrobacterium tumefaciens strain GV3101 harboring the binary vector plasmid pBinGlyBar4-EADcT. Based on the resistance reaction to the herbicide Basta, 20 and 40 resistant seedlings were obtained from 2000 seed germinated from the plants transformed through floral dip and vacuum infiltration methods, respectively. The PCR analyses further confirmed the presence of transgene in 3 floral dipped plants without vacuum infiltration and 17 floral dipped plants with vacuum infiltration, giving the transformation frequencies of 1.5*10/sup -3/ and 8.5*10/sup -3/, respectively. This method, which avoids tissue culture, will reduce the somaclonal variation accompanying prolonged culture of cells in a dedifferentiated state, will facilitate functional genomics and improvement of Brassica juncea with novel desirable traits while reducing time and expense. (author)

GENETIC TRANSFORMATION AND ANALYSIS OF WHEAT TRANSGENIC CELL LINES BY IRAP-PCR

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Bavol A. V.

2013-12-01

Full Text Available The transgenic wheat cell lines were obtained via biolistic transformation of the callus cultures initiated from the 3-day-old sterile seedling shoot apexes. The pAHC25 vector construction used for 14- and 28-day-old callus cultures transformation carried the selective phosphinothricin-N-acetyltransferase (bar gene and reporter ?-glucuronidase gene. The cell line selection was carried out on the media with phosphinothricin by means of graduated cell selection. The transgenic status of the obtained forms was proved by PCR-analysis. The presence of new relatively high molecular (more than 1 000 bp amplicons were found out for three transformed lines by means of IRAP PCRanalysis with the primers coding for long termainal repeats sequences of SIRE 1 retrotransposon. This fact may prove transposition of this mobile genetic element. The new DNA fragments were detected for three of the seven analyzed lines but for the control callus. It is possible to assume at induction of SIRE 1 transposition bis probably caused by the genomic stress of foreign DNA inserting or associated with the transformation process (mechanical wounding, cultivation on selective media.

Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker.