Agrobacterium Tumefaciens-mediated Transformation Of TCS Gene Into Soybean

Soybean(Glycine max) is the most important source of grain and oil crops.It also has the main resource of plant oil and protein for human being.Soybean was injured seriously by pests during the period of duration,which resulted in great loss of production of soybean. Trichosanthin(TCS) is obtained from root tuber of Trichosanthes kirilowii.TCS is ribosome inactivating protein(RIP) of single chain,which can resist 7 kinds of virus(for example tobacco mosaic virus),9 kinds of pathogen fungi(for example rice blast) and 4 kinds of insect pest(for example aphides).TCS gene was transformed into soybean for boosting up antiadversity, having important economic significance.In this study,cotyledon nodes of general soybean were used as experiment material.TCS gene was introduced into soybean successfully with Agrobacterium-cotyledonary transformation system.The main conclusion of this experiment was as following:Ten varieties of soybean were chosen and the cotyledonary nodes of these varieties were used to be the target explants of TCS gene transformation.The results showed that regeneration frequency was high of Hefeng 35,Hefeng 25 and Heinong 35.The appropriate concentration of hygromycin for selection culture was determined.For cotyledonary node,the optimal mass concentration of hygromycin was 6mg/L for Heinong35,4mg/L for Hefeng35 and Hefeng25. For rooting,the optimal mass concentration of hygromycin was 2mg/L for Heinong35 and Hefeng35,1mg/L for Hefeng25.Using Hefeng35 cotyledon node for materials determined the suitable condition.The impact of pre-culture,concentration of liquid bacterium and infection time on genetic transformation frequency of soybean was studied,with the rate of transforming.When the cotyledon node pre-culture was 24 hours,infected 30 min by 0.4-0.6 of OD600,and the coculture time was 72 hours,the genetic transformation was the highest.Soybean cotyledons varieties cultured were inoculated with Agrobacterium tumefacines LBA4404.The resistant calli were induced and regenerated plantlets were obtained.Among the 44 transplanted survival plants,29 plants gave positive PCR reaction.19 plants were from Hefeng35 and the transformation frequence was 1.03%.7 plants were from Hefeng25 and the transformation frequence was 0.51%.3 plants were from Heinong35 and the transformation frequence was 0.23%.PCR and PCR-Southern analysis showed that TCS gene was integrated into soybean's genome.