• Journal of Forest and Environmental Science
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• v.25 no.3
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• pp.195-204
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• 2009

Agrobacterium-mediated genetic transformation has been widely used for the production of genetically modified transgenic plants to obtain specific desired traits. Most of the molecular mechanisms that underlie the transformation steps have been well elucidated over the years. However, a few steps, such as nuclear targeting, T-DNA integration, and Agrobacterium-plant proteins involved remain largely obscure and are still under extensive studies. This review describes the major steps involved in the molecular mechanism of Agrobacterium-mediated transformation and provides insight in the recent developments in studies on the Agrobacterium-mediated genetic transformation system. Some factors affecting the transformation efficiency are also briefly discussed.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.683-688
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• 2005

A transformation system mediated by Agrobacterium tumefaciens is routinely used for the genetic engineering of plants. Here, we report an efficient and stable method for transformation of heterogeneous genes into an industrial Cephalosporium acremonium by using a similar transformation system established in plants. Both the phleomycin-resistant gene and vgb gene were used as screening markers to confirm the success of transformation by either Southern hybridization or PCR amplification. It was found that acetosyringone (AS) was necessary only for protoplast transformation and the heterogeneous genes transferred were integrated into the genome of C. acremonium. The transformation efficiency obtained with this system was much higher than the conventional techniques used for transformation of C. acremonium.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.387-393
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• 2000

The process by which Agrobacterium tumefaciens genetically transforms plants involves a complex series of reactions communicated between the pathogen and the plants. To identify plant factors involved in agrobacterium-mediated plant transformation, a large number of T-DNA inserted Arabidopsis thaliana mutant lines were investigated for susceptibility to Agrobacterium infection by using an in vitro root inoculation assay. Based on the phenotype of tumorigenesis, twelve T-DNA inserted Arabidopsis mutants(rat) that were resistant to Agrobacterium transformation were found. Three mutants, rat1, rat3, and rat4 were characterized in detail. They showed low transient GUS activity and very low stable transformation efficiency compared to the wild-type plant. The resistance phenotype of rat1 and rats resulted from decreased attachment of Agrobacterium tumefaciens to inoculated root explants. They may be deficient in plant actors that are necessary for bacterial attachment to plant cells. The disrupted genes in rat1, rat3, and rat4 mutants were coding a arabinogalactan protein, a likely cell wall protein and a cellulose synthase-like protein, respectively.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.3
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• pp.217-222
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• 2004

As Agrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast, Saccharomyces cerevisiae, a variety of fungi were subjected to the A. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. The A. tumefaciens-mediated transformation of chestnut blight fungus, Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1${\times}$10$\^$6/ conidia of C. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.754-758
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• 2008

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when $1{\times}10^6$ spores were used with the same volume of bacteria. The stability of transform ants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.66-75
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• 2016

Agrobacterium-mediated gene transfer has recently been developed to improve rice transformation. In this study, 3 different transformation methods were tested including soaking, co-cultivation, and vacuum infiltration. Agrobacterium tumefaciens GV3101 harboring the binary vector pGreen:: LeGSNOR was used in this experiment. This study aimed to identify the most appropriate method for transferring LeGSNOR into rice. Vacuum infiltration of the embryonic calli for 5 min in Ilpum resulted in high transformation efficiency based on confirmation by PCR, RT-PCR, and qRT-PCR analyses. In conclusion, we described the development of an efficient transformation protocol for the stable integration of foreign genes into rice; furthermore, the study results confirmed that PCR is suitable for efficient detection of the integrated gene. The vacuum infiltration system is a potentially useful tool for future studies focusing on transferring important genes into rice seed calli, and may help reduce time and effort.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1390-1394
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• 2004

We have achieved efficient transformation system for forage-type tall fescue plants by Agrobacterium tumefaciens. Mature seed-derived embryogenic calli were infected and co-cultivated with each of three A. tumefaciens strains, all of which harbored a standard binary vector pIG121Hm encoding the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing $\beta$-glucuronidase (intron-GUS) genes in the T-DNA region. Transformation efficiency was influenced by the A. tumefaciens strain, addition of the phenolic compound acetosyringone and duration of vacuum treatment. Of the three A. tumefaciens strains tested, EHA101/pIG121Hm was found to be most effective followed by GV3101/pIG121Hm and LBA4404/pIG121Hm for transient GUS expression after 3 days co-cultivation. Inclusion of 100 $\mu$M acetosyringone in both the inoculation and co-cultivation media lead to an improvement in transient GUS expression observed in targeted calli. Vacuum treatment during infection of calli with A. tumefaciens strains increased transformation efficiency. The highest stable transformation efficiency of transgenic plants was obtained when mature seed-derived calli infected with A. tumefaciens EHA101/pIG121Hm in the presence of 100 $\mu$M acetosyringone and vacuum treatment for 30 min. Southern blot analysis indicated integration of the transgene into the genome of tall fescue. The transformation system developed in this study would be useful for Agrobacterium-mediated genetic transformation of tall fescue plants with genes of agronomic importance.

• Journal of Plant Biotechnology
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• v.6 no.2
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• pp.69-75
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• 2004

Optimal protocol for efficient genetic transformation has been defined to aid future strategies of genetic engineering in pigeon pea with agronomically important genes. Transgenic pigeonpea plants were successfully produced through Agrobacterium tumefaciens-mediated genetic transformation method using cotyledonary node explants by employing defined culture media. The explants were co-cultivated with A. tumefaciens strain C-58 harboring the binary plasmid, pCAMBIA-1301 [con-ferring $\beta$-glucuronidase(GUS) activity and resistance to hygromycin] and cultured on selection medium (regeneration medium supplemented with hygromycin) to select putatively transformed shoots. The shoots were then rooted on root induction medium and transferred to pots containing sand and soil mixture in the ratio of 1:1. About 22 putative TO transgenic plants have been produced. Stable expression and integration of the transgenes in the putative transgenics were confirmed by GUS assay, PCR and Southern blot hybridization with a transformation efficiency of over 45%. Stable integration and expression of the marker gene has been confirmed in the TO and T1 transgenics through PCR, and Southern hybridization.

• Horticultural Science & Technology
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• v.18 no.6
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• pp.797-801
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• 2000

The present study was conducted to improve the efficiency of transformation mediated by Agrobacterium tumefaciens in hot pepper. Both regeneration ratio and transformation frequency after the cocultivation with A. tumefaciens were affected by inoculation time and artificial wounding. Transformation frequency was increased over 50% by combining artificial wounding with 120 s of inoculation treatment. Confirmation for the transformation of regenerated shoots was carried out by histochemical ${\beta}$-glucuronidase assay and polymerase chain reaction analysis using npt II primer.

• Mycobiology
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• v.45 no.2
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• pp.84-89
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• 2017

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.