한국콩연구회

국내 콩 품종 형질전환기술개발

Development of Agrohacterium-mediated transformation of Korean soybean cultivars

주관연구기관 : 동아대학교

연구책임자 : 정영수

발행년월 : 2004-08

주관부처 : 과학기술부

원문

http://www.ndsl.kr/ndsl/commons/util/ndslOriginalView.do?dbt=TRKO&cn=TRKO200400001565&rn=&url=&pageCode=PG18

초록

In order to achieve highly efficient soybean transformation method, genotype screen of Korean soybean cultivars for amenability to Agro-infection and diverse experimental trials have been introduced in the current study such as hormone pre-culture and various selection techniques. The active genome projects from several academically and economically important plants have provided sequence data of many genes and accumulated in Gene Bank data base promptly. Some of those genes reported have high potentials to improve plant quality in agronomical and industrial uses. However, difficulties to achieve the successful introduction of those valuable genes into plant can be found in recalcitrant genetic transformation procedure of some agriculturally important crops. Especially none of research group in Korea have succeeded to report any formal results on the production of genetically engineered soybean variety or research publication, even though there have been several GMO soybean varieties released from abroad soybean seed companies. To achieve long term goal for producing high-value soybean variety by genetic transformation, there are couple of procedures to be improved. First, genotype screen of Korean soybean varieties whether they are amenable to Agro-infection and tissue culture responsive has to be done. Second, efficiency for genetic transformation has to be increased. In this study various experiments have been carried to achieve both proposed goals.

To determine the amenability of Korean soybean cultivars to Agrobacterium infection most commonly grown 31 Korean cultivars were used for genetic transformation. Cotyledonary node was used as a explant and inoculated with Agrobacterium strain LBA4404 harboring pTOK233, super binary vector. After placement on selection media containing 10ppm of hygromycin, 4 to 6 week old shoots(0.5 - 1cm) were stained with GUS solution. Shoot formation rates were high in Muhankong(12%), Manrikong(10%), Eunhakomg(19%), Daewonkomg(23%), and high frequency of GUS positives were observed in Ilpumkumjeongkong, Muhankong, Eunhakomg, Daewonkomg. In addition that, strong GUS expression was found in Jangmikong and Baekwunkong.

- 국내 콩 품종에 대한 효율적인 형질전환체계를 확립하기 위하여 실험을 수행하였다.

- 아그로박테리움 감염에 순응적인 품종을 선발하기 위하여 31개의 국내 콩 장려품종을 스크린 하여 일품검정콩, 만리콩, 은하콩, 대원콩등 14개의 순응형 품종을 스크린 하였다.

- 효율적인 형질전환을 위하여 제균제로는 timentin이 선택되었고, 효율적인 선발을 위하여 선발 항생제로서 하이그로마이신이 선택되었다.

- 하이그로마이신 농도는 10ppm에서 15ppm이 효율적이었고, 절편체의 치상방향은 adaxial side를 down하여 치상하였을 때가 높은 GUS positive 빈도를 나타냈고, 상처방법은 매스보다 침을 사용하였을 때, 훨씬 높게 나타났다.

- 고효율 형질전환 체계 확립을 위하여 호르몬 전처리를 한 결과, BA 5ppm과 10ppm을 처리하였을 때, 후기 선발에서 높은 생존율을 보였고, 전처리후에 액체배지에서 선발을 하였을 때 escape의 빈도를 크게 낮출 수 있었다.

- 공배양중에 황화합물을 첨가하였을 때, 신초발생이 크게 증가하였으며, 하이그로마이신 선발배지에서 왕성한 생육을 나타내었다.

- GUS분석과 PCR을 통하여 도입유전자를 확인하였으며, 현재 종자확보를 위하여 뿌리형성이 유도되고 있다.