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국내 콩(Glycine max) 품종 형질전환 초기조건 확립

Optimization of Genetic Transformation Conditions for Korean Soybean Cultivars

  • 이기정 (동아대학교 생명자원과학대학 유전공학과) ;
  • 서진경 (동아대학교 생명자원과학대학 유전공학과) ;
  • 이혜영 (동아대학교 생명자원과학대학 유전공학과) ;
  • 전은희 (동아대학교 생명자원과학대학 유전공학과) ;
  • 신상현 (동아대학교 생명자원과학대학 유전공학과) ;
  • 이재헌 (동아대학교 생명자원과학대학 유전공학과) ;
  • 김도훈 (동아대학교 생명자원과학대학 유전공학과) ;
  • 고종민 (영남농업연구소 전특작과 두류육종연구실) ;
  • 한원영 (영남농업연구소 전특작과 두류육종연구실) ;
  • 백인열 (영남농업연구소 전특작과 두류육종연구실) ;
  • 오병준 (전남생물산업연구센터) ;
  • 정영수 (동아대학교 생명자원과학대학 유전공학과)
  • 발행 : 2006.04.01

초록

경제적으로 중요한 식용작물인 콩(Glycine max)의 초기형 질전환 효율증대를 위하여 연구를 수행하였다. 높은 초기형 질전환 조건 확립을 위하여 기초실험으로 Agrobacterium 감 염에 순응적인 국내 콩 품종을 스크린 하였으며, 적정한 agar 농도와 선발항생제 hygromycin 농도 규명, 상처방법, explant의 치상방향, 호르몬 전처리, 액체배지 선발 등 형질 전환 초기효율에 미치는 영향을 transient GUS 분석을 통하여 알아보았다. Agrobacterium 감염에 순응적인 품종을 선발하기 위하여 32개의 국내 콩 장려품종을 스크린한 결과, 일품검정콩, 은하콩, 만리콩, 대원콩 등 14개의 순응형 품종을 선발하였으며, 효율적인 제균제로는 cefotaxime이, 효율적인 선발항생제로서는 hygromycin이 선택되었다. Hygromycin 농도는 10-15 ppm이 효율적이었고, agar 농도 0.6-0.8%, explant의 치상방향은 향배축(adaxial side)을 down하여 치상하였을 때가 높은 GUS ($\beta$-glucuronidase)발현빈도를 나타냈고 상처방법은 scalpel보다. 동양침을 사용하였을 때 높게 나타났다. 형질전환 체계확립을 위하여 호르몬 전처리를 한 결과, BA 5 ppm 과 10 ppm을 처리하였을 때 후기 선발에서 높은 생존율을 보였고, 전처리 후에 액체배지에서 선발을 하였을 때 비형질 전환체(escape)의 빈도를 크게 낮출 수 있었다. 또한 여러 조건의 결과를 비교하여 볼 때, 국내 콩 품종 중에서 형질전환을 위한 재료로써는 은하콩이 가장 적합한 것으로 생각된다.

In order to establish highly efficient gene transfer condition at early stage of soybean transformation, various experiments were performed and compared their efficiencies by transient GUS analysis; those conditions are genotype determination of Korean soybean cultivars for amenability to Agro-infection, appropriate agar and selective agent concentration, orientation of explant placement, hormone pre-culture, and liquid selection condition. In the genotype screen of Korean soybean varieties, 14 amenable genotypes were selected. For efficient Agrobacterium washing, cefotaxime was chosen and hygromycin at the concentration of 10 and 15 ppm was used as selection agent in the media. Agar concentration was slightly better in 0.6% and 0.8% for both shoot and callus formation, and explant placement with adaxial side down showed high frequency of GUS expression. For wounding treatment, oriental needle was efficient than scalpel for shoot formation and gene transfer. To increase the frequency of gene transfer, hormone pre-treatment was applied. BA at the concentration of 5 and 10 ppm resulted in better survival at the late stage of selection in shoot elongation media. Selection in liquid media after hormone pre-treatment seemed to be effective to remove the escaped non-transformants at early stage of procedure. Considering the results obtained, Eunhakong could be the first choice as a material for soybean transformation among Korean soybean genotypes.

키워드

참고문헌

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피인용 문헌

  1. Characterization of SMV resistance of soybean produced by genetic transformation of SMV-CP gene in RNAi vol.7, pp.4, 2013, https://doi.org/10.1007/s11816-013-0279-y
  2. Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression vol.7, pp.10, 2012, https://doi.org/10.1371/journal.pone.0048287
  3. RNAi-mediated Soybean mosaic virus (SMV) resistance of a Korean Soybean cultivar vol.10, pp.5, 2016, https://doi.org/10.1007/s11816-016-0402-y