• Molecules and Cells
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• 제39권9호
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• pp.705-713
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• 2016

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.

• Journal of Forest and Environmental Science
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• 제25권3호
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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.

• 한국초지조사료학회지
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• 제20권1호
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• pp.13-18
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• 2000

An efficient method for Agrobacterium-mediated transformation of forage crop alfalfa (Medicago sativa L.) was established using secondary somatic embryogenic calli. Agrobacterium tumefaciens strain EHAlOl and a binary vector pIG121-Hm which has selection markers for kanamycin and hygromycin have been shown to be an efticient materials for alfalfa transformation. The secondary somatic embryogenic calli originated from hypocotyl explants of alfalfa were efficient infection materials for Agrobacterium EHAlOl and normally germinated into plantlets. The introduced gene (GUS) was constitutively expressed in all tissues of transgenic alfalfa with different expression levels. These results indicate that the use of pIG121-Hm vector, Agrobacterium EHAlOl and improved culture system of callus facilitate the transformation of alfalfa. (Key words : Agrobacterium, Alfalfa, Gene transfer, Transformation)

• 식물조직배양학회지
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• 제27권5호
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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.

• 한국해양바이오학회지
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• 제16권1호
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• pp.26-35
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• 2024

Chlorella has various biotechnological applications, including in the biomedical and pharmaceutical industries, because of its advantages, including rich nutrients, fast growth rate, easy cultivation, and high biomass. We used the Agrobacterium-mediated transformation method to express human GM-CSF and EGF proteins, which are widely used in regenerative medicine, cosmetics, and pharmaceutical materials in Chlorella. The codon-optimized hGM-CSF and hEGF genes were cloned into plant binary vectors and transformed into Chlorella vulgaris using the Agrobacterium-mediated coculture transformation method. After transformation, genomic DNA PCR was performed for each C. vulgaris line that was stably subcultured on an antibiotic-resistant solid medium to confirm the insertion of hGM-CSF and hEGF into the chromosome. Furthermore, PT-PCR and protein expression of hGM-CSF and hEGF in each transformed C. vulgaris were significantly increased compared to the untransformed Chlorella. This study suggests that high-value proteins, including hGM-CSF and hEGF, which are foreign genes of C. vulgaris, can be stably expressed through the Agrobacterium-mediated Chlorella transformation system.

• 농업생명과학연구
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• 제44권6호
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• pp.141-146
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• 2010

본 연구에서는 국내에서 식용으로 재배되고 있는 팽이버섯의 균사체에 대하여 Agrobacterium을 이용한 형질전환을 시도하였다. 특히 물리적 연마제인 미세 aluminum oxide 입자를 팽이 균사체와 함께 강하게 교반함으로써 물리적 상해를 지니는 균사체를 제조하였으며 감압침윤에 의한 Agrobacterium 이용 형질전환을 시도하였다. Hygromycin 저항성을 이용한 선발 결과, 대조군에서는 균사체 생육이 전혀 관찰되지 않은 반면 물리적 상해군에서는 형질전환균사체 생육이 확인되었다. Genomic DNA PCR에 의한 유전자 도입을 확인함으로써 팽이균사체에 대한 매우 간편한 형질전환법을 제시할 수 있었다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 추계학술대회
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• pp.52-52
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• 2018

Development of herbicide resistant transgenic legume plants through Agrobacterium-mediated transformation has been worked in many previous studied. Plant regeneration after infection is the important step to obtain successful transgenic plants. Many attempts try to find the optimum media condition for plant regeneration after infection. However, the transformation efficiency of legume plants is still low. In this study, regeneration of some Korean legume species including two soybean cultivars (Dawon and Pungsan) and pea have been done with organogenesis which is used various kind of explants such as cotyledonary-nodes in soybean and bud-containing tissue in pea. We developed the optimum media condition for plant regeneration regulators under Agrobacterium-mediated transformation using different kind and various concentration of plant growth. As the results, B5 medium containing 2 mg/L of 6-benzylaminopurine was selected in this study for the optimum plant regeneration media. The segments were inoculated with Agrobacterium suspension harbored an IG2 vector containing bar gene which confers resistance to phosphinotricin (PPT) in 3, 5 and 7 days. The transformation efficiency was achieved in Dawon 3.03 % and pea 1.46 % with co-cultivation period of 7 days which is showed a high number of GUS positive expression period.

• Plant Biotechnology Reports
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• 제3권4호
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• pp.277-283
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• 2009

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for $4{\times}14$ days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter-bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with $4mg\;1^{-1}$ phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the $R_1$ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.

• Plant Biotechnology Reports
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• 제5권2호
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• pp.147-156
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• 2011

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with ${\beta}$-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with $1mg\;l^{-1}$ indole-3-butyric acid and $15mg\;l^{-1}$ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for ${\beta}$-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of $3.9{\pm}0.39$ transgenic plantlets per explant was achieved in the present transformation system. It took only 2-3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

• 농업생명과학연구
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• 제45권1호
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• pp.119-124
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• 2011

Trichoderma spp.는 white biotechnology에서 이용되는 대표적인 미생물로서 이들이 강력하게 분비 생산하는 효소들은 산업적으로 매우 중요하다. 본 연구에서는 amylase, pectinase, cellobiohydrolase 및 xylanase 분비활성이 높은 것으로 밝혀진 Trichoderma sp. KACC 40541균주에 대한 Agrobacterium이용 형질전환을 수행하였으며 균주개량을 위한 효율적인 유전자도입 방법을 제시하였다. 특히 형질전환을 위하여서는 균사체에 대한 적정 농도의 NaOH처리가 매우 효과적임을 보여주었다.