Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Production of Transgenic Plants in Brassica napus Winter Cultivar 'Youngsan'

영산 유채를 이용한 형질전환체 생산

  • Roh, Kyung-Hee (Department of Agricultural Biotechnology, National Academy of Agricultural Science, RDA) ;
  • Kwak, Bo-Kyoung (Department of Agricultural Biotechnology, National Academy of Agricultural Science, RDA) ;
  • Kim, Hyun-Uk (Department of Agricultural Biotechnology, National Academy of Agricultural Science, RDA) ;
  • Lee, Kyeong-Ryeol (Department of Agricultural Biotechnology, National Academy of Agricultural Science, RDA) ;
  • Kim, Sun-Hee (Department of Agricultural Biotechnology, National Academy of Agricultural Science, RDA) ;
  • Suh, Mi-Chung (Department of Bioenergy Science & Technology, Chonnam National University) ;
  • Kim, Hyo-Jin (Department of Bioenergy Science & Technology, Chonnam National University) ;
  • Kim, Jong-Beom (Department of Agricultural Biotechnology, National Academy of Agricultural Science, RDA)
  • 노경희 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 곽보경 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 김현욱 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 이경렬 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 김순희 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 서미정 (전남대학교 바이오에너지공학과) ;
  • 김효진 (전남대학교 바이오에너지공학과) ;
  • 김종범 (농촌진흥청 국립농업과학원 농업생명자원부)
  • Received : 2011.01.27
  • Accepted : 2011.03.07
  • Published : 2011.03.31
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Abstract

To improve genetic transformation of Brassica napus winter cultivar 'Youngsan', factors influencing shoot regeneration and transformation from cotyledonary petioles were investigated. Shoot induction was enhanced in the combination of 0.5 mg/L NAA and 2~4 mg/L kinetin. Silver nitrate was essential for successful shoot regeneration, ranging from 5 to 9 mg/L. The addition of $GA_3$ promoted plant regeneration. Among the tested Agrobacterium strains, co-cultivation times, and antibiotic selection regimes, choice of appropriate Agrobacterium strain was the most critical factor for efficient transformation of B. napus cv. 'Youngsan'. The EHA105 succinamopine strain was the most efficient and the maximum transformation efficiency was 26.8%. Transgenic shoots were selected on 10 mg/L phosphinothricin (PPT) containing media. The transgenic plants expressing bar and gus genes were resistant for commercial herbicide "Basta" and stained with X-Gluc. Southern blot hybridization indicated that the presence of one to three gus gene copies per genome and inheritance of the gus gene into the $T_1$ generation.

국내 육성 추파 품종인 '영산' 유채의 자엽 부착 잎자루 부위를 이용하여 재분화와 형질전환의 효율 증진에 영향을 주는 요인을 살펴보았다. 줄기 형성율은 NAA 0.5 mg/L와 Kinetin 2~4mg/L가 혼용 첨가된 배지에서 61~71%로 가장 양호하였다. 또한 질산은(Silver nitrate) 5~9 mg/L 첨가는 재분화에 필수적이었고, $GA_3$ 0.01 mg/L 첨가는 재분화에 긍정적인 영향을 주는 것으로 관찰되었다. 아그로박테리움의 Strain 종류, 공동배양 시간, 그리고 형질전환체 초기 선발시 항생제 농도 조절(저${\rightarrow}$고농도)에 따른 '영산' 유채의 형질전환 효율을 살펴본 결과, 아그로박테리움의 Strain 선정이 가장 중요한 요인으로 조사되었다. 특히, EHA105 succinamopine strain을 사용하였을 때 형질전환 효율이 26.8%로 가장 효과적이었다. Bar 유전자와 GUS 유전자가 내재된 형질전환체는 제초제 '바스타' 살포시 생존하고, X-Gluc으로 염색되었다. 그리고 Southern 분석을 통해 후대로 유전자가 안정적으로 유전됨을 확인하였다.

Keywords

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