공동배양과정의 배지조성과 배양조건이 벼 형질전환효율에 미치는 영향

• Published in: Korean journal of breeding Vol. 41; no. 3; pp. 252 - 260
• Main Authors: 김율호(Yul Ho Kim), 박향미(Hyang Mi Park), 최만수(Man Soo Choi), 윤홍태(Hong Tai Yun), 최임수(Im Soo Choi), 신동범(Dong Bum Shin), 김정곤(Chung Kon Kim), 이장용(Jang Yong Lee)
• Format: Journal Article
• Language: Korean
• Published: 한국육종학회 30.09.2009; The Korean Breeding Society
• Subjects: Rice transformation; Transformation rate; Co-cultivation medium; Culture conditions
• ISSN: 0250-3360; 2287-5174

1. 본 연구에서는 공동배양 배지에 Agrobacterium 성장 억제물질인 silver nitrate를 첨가하고 변온과 여과지처리를 추가하여 공동배양 기간을 7일로 늘였으며, 또한 항산화 물질 3종을 공동배양 배지에 첨가하여 세포의 oxidative burst를 최소화함으로써 벼 형질전환효율을 높일 수 있었다. 또한 이 방법을 적용하여 형질전환이 어려운 품종을 대상으로도 형질전환 식물체를 작성할 수 있었다. 2. 벼 형질전환체의 70%에서 도입유전자 Rice is the most important cereal crop not only in supplying the basic staple food for more than half of the world's population but also as a model plant for functional genomic studies of monocotyledons. Although rice transformation method using A. tumefaciens has already been widely used to generate transgenic plants, the transformation rate is still low in most Korean elite cultivars. We made several modifications of the standard protocol especially in the co-cultivation step to improve the efficiency of the rice transformation. The co-culture medium was modified by the addition of three antioxidant compounds (10.5 mg/L L-cysteine, 1 mM sodium thiosulfate, 1 mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (5 mg/L silver nitrate). Co-cultivation temperature (23.5^℃ for 1 day, 26.5^℃ for 6 days) and duration (7 days) were also changed. The plasmid of pMJC-GB-GUS carrying the GUS reporter gene and the bar gene as the selectable marker was used to evaluate the efficiency of the transformation. After co-cultivation, a high level of GUS gene expression was observed in calli treated with the modified method. It is likely that those newly added compounds helped to minimize the damage due to oxidative bursts during plant cell-Agrobacterium interaction and to prevent necrosis of rice cells. And the transformation rate under the modified method was also remarkably increased approximately 8-fold in Heungnambyeo and 2-fold in Ilmibyeo as compared to the corresponding standard method. Furthermore, we could produce the transgenic plants stably from Ilpumbyeo which is a high-quality rice but its transformation rate is extremely low. Transformation and the copy number of transgenes were confirmed by PCR, bar strip and Southern blot analysis. The improved method would attribute reducing the effort and the time required to produce a large number of transgenic rice plants.