• 제목/요약/키워드: Agrobacterium-mediated Transformation

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Agrobacterium을 이용한 내열성 유전자의 벼로의 형질전환 및 발현 (Introduction of Thermotolerant Gene into Rice Plant by Agrobacterium Mediated Transformation)

  • 이병현;이효신;원성혜;조진기
    • Current Research on Agriculture and Life Sciences
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    • 제17권
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    • pp.39-43
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    • 1999
  • 벼에 있어서 엽록체 small heat shock protein(small HSP)의 기능을 밝히기 위하여 Agrobacterium을 이용한 형질전환법을 이용하여 벼로부터 분리한 small HSP cDNA를 도입하였다. Agrobacterium의 감염에는 벼의 미성숙 배로부터 유도한 callus를 이용하였다. 형질전환 후의 재분화율은 약 30%였다. 형질전환을 통하여 얻어진 식물체의 genomic DNA로부터 PCR 분석과 Southern blot 분석으로 엽록체 small HSP 유전자의 도입을 확인하였다. 도입 유전자의 형질전환 벼에 있어서 유전자의 발현 양상을 northern blot 분석으로 조사하였다. 그 결과 도입된 유전자는 상온에서의 발현량이 서로 다르게 나타났으며 항상적으로 발현하고 있음을 확인하였다.

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Factors Affecting Agrobacterium tumefaciens-mediated Transformation of Panax ginseng C.A. Meyer

  • Kim, Ok-Tae;Jung, Su-Jin;Bang, Kyong-Hwan;Kim, Young-Chang;Shin, Yu-Su;Sung, Jung-Sook;Park, Chun-Geon;Seong, Nak-Sul;Cha, Seon-Woo;Park, Hee-Woon
    • 한국약용작물학회지
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    • 제15권2호
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    • pp.100-104
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    • 2007
  • A protocol for the production of transgenic Panax ginseng C.A. Meyer was established via Agrobacterium tumefaciens-mediated genetic transformation of direct somatic embryos. A number of conditions related to the co-cultivation were tested with respect to maximizing transformation efficiency. The results showed that pH of the co-cultivation medium (5.7), the bacterial growth phase (optical density; $OD_{600}$ = 0.8), co-cultivation period (3 days), and acetosyringone concentration $(100\;{\mu}M)$ had positive effects on transformation. Selected plantlets were cultured on the medium at an elevated hygromycin level(30 mg/l). Integration of the transgenes into the P. ginseng nuclear genome was confirmed by PCR analysis using hpt primers and by Southern hybridization using hpt-specific probe. The transgenic plantlets were obtained after 3-month cultivation and did not show any detectable variation in morphology or growth characteristics compared to wild-type plants.

Production of Transgenic Petunia hybrida cv. Rosanpion Using Agrobacterium-mediated Transformation

  • Ko, Jeong-Ae;Kim, Young-Sook;Kim, Myung-Jun;Kim, Hyun-Soon
    • Plant Resources
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    • 제4권1호
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    • pp.36-40
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    • 2001
  • Transgenic Petunia hybrida cv. Rosanpion was produced by Agrobactepium tumefaciens LBA4404 harboring a binary vector pBI 121 containing $\beta$-glucuronidase (gus) and neomycin phosphotransferase (nptII). For genetic transformation, leaf discs were precultured on MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L BA (MNB) for 2 days and cocultured for 15 mins with A. tumefaciens. For selection of transformant, leaf discs were transferred to fresh MNB containing 50 mg/L kanamycin and 500 mg/L cefotaxime. Eighteen plants were regenerated and four were confirmed by PCR for detection of gus and nptII gene integrated into the nuclear genome of petunia ‘Rosanpion’. Using this transformation system, we expect that transgenic petunia ‘Rosanpion’ incorporating a useful gene can be produced.

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Optimization of Agrobacterium-mediated transformation procedure for grapevine 'Kyoho' with carrot antifreeze protein gene

  • Shin, Hye Young;Kim, Gi Hoon;Kang, Sang Jae;Han, Jeung-Sul;Choi, Cheol
    • Journal of Plant Biotechnology
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    • 제44권4호
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    • pp.388-393
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    • 2017
  • We report an Agrobacterium-mediated transformation procedure optimized for 'Kyoho' that is a major table grapevine cultivar in Korea, and its transgenic plants with antifreeze protein gene of carrot (DcAFP). The full length of DcAFP coding region in accordance with the previous report was isolated from young leaves of carrot and recombined into a plant transformation vector. Ethylene inhibitors such as silver nitrate and aminoethoxyvinylglycin (AVG) supplemented in a co-cultivation medium distinctly increased frequency of shoot regeneration when explants were sub-cultured in a selection medium: particularly ten-fold higher in treatment with 0.1 mg/L AVG than one without ethylene inhibitor. Among various antibiotics and their concentrations, the combination of 150 mg/L cefotaxime plus 150 mg/L $Clavamox^{TM}$ was selected for elimination of Agrobacterium cells in addition to minimization of adverse effect on shoot regeneration, while 50 mg/L kanamycin monosulfate effectively suppressed regeneration of non-transgenic shoots. Applying the elucidated culture condition, we finally obtained a total of 5 transgenic 'Kyoho' plantlets with DcAFP, of which integration with the grapevine genome and transcription was confirmed by nucleic acid analyses.

Assessment of Factors Influencing Agrobacterium Mediated Transformation in Cucumber (Cucumis sativus L)

  • Sureshkumar P.;Selvaraj N.;Ganapathi A.;Kasthurirengan S.;Vasudevan A.;Anbazhagan V. Ramesh
    • Journal of Plant Biotechnology
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    • 제7권4호
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    • pp.225-231
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    • 2005
  • Five day old cotyledon explants of Cucumber (Cucumis sativus L) cv Poinsett 76 were cocultivated with two Agrobacterium strains (EHA105 and LBA 4404) each carrying GUS as the reporter gene and npt-II as the selection marker gene in the T-DNA region of the vector. Transformed shoots were selected at 150 mg/L kanamycin. A two day cocultivation coupled with $20\;{\mu}M$ acetosyringone increased the frequency (8.2 and 15.4 shoots) of GUS expression in the shoots of transformed plant. Among the two Agrobacterium strains, EHA 105 performed better than LBA 4404 in bringing two-fold increase in transformation efficiency (14%) than LBA 4404 (7.4%). PCR analysis was done to confirm the integration of T-DNA into cucumber genome.

Agrobacterium-mediated Transformation of Rice 'Ilmibyeo' using HPT Selection Maker Gene

  • Guo, Jia;Cho, Joon-Hyeong;Jo, Hye-Jeong;Seong, Eun-Soo;Wang, Myeong-Hyeon
    • 한국자원식물학회지
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    • 제20권3호
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    • pp.242-246
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    • 2007
  • This study was conducted to produce the transgenic plant of rice. We obtained Agrobacterium AGL1 harbaring pCambial 300 vector with HPT gene. We carried out PCR analysis of 22 ea putative transgenic rice to investigate transformed lines. The 3 ea transgenic lines were detected insertion of HPT gene. Transgenic lines selected from PCR analysis were performed by Southern blot. From Southern blot, we obtained that two transgenic lines detected single band. We are going to study the method improving of cotransformation as well as transformation efficiency in rice.