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㎎/ℓ L-cysteine, 1mM sodium thiosulfate, 1mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (5㎎/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.

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㎎/ℓ L-cysteine, 1mM sodium thiosulfate, 1mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (5㎎/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.

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

A stable, efficient, and reliable selection system using bar and hpt as selectable markers for Agrobacterium mediatedco-transformation of rice with two separate vectors was developed. Two plasmids, pMJC-GB-IFS2 and pMJC-GH-CHR carryingbar or hpt gene as s

보리(Hordeum vulgare)의 유묘이 14 co2 를 10분 및 30분간 처리한 후 잎을 채취하여 아미노산, 당, 인산염 및 유기산과 같은 수용성 대사물질을 분석하였다. 배양기간에 따른 label 정도는 비슷하였으나 종류와 양에 있어서는 차이가 있었다. 10분 처리시에는 아미노산 분획보다 중성분회(당, 인산염에스퇴르 및 유기산)에서 label 양이 4.5배 증가하였으나 30분 처리시에는 1.7배로 감소하였다. 당분획에 있어서는 처리 시간이 길어짐에 따라 label 양이 약간 증가 하였으나 아미노산 분획에 있어서는 양과 질적으로 label 양이 현저히 증가하였다. 탄소 동화율은 10분에서 30분으로 길어질수록 현저히 감소하였다(0.125대 0.034와 0.042 mole CO2 /mg 엽록소/분).