Optimization Of Regeneration System Via Cotyledonary Node And Transformation Of EPSPS Gene In Soybean

EPSPS gene was transformed into soybean for boosting up herbicide resistant,having important economic significance. Glyphosate has been extensively used forbroad-spectrum control of weeds in the word, and much attention has been paid to thesearch for glyphosate-tolerant enzymes for transgenic crops. Soybean is one of thecrops whose transformation is difficult，the low transformation efficiency in soybeanis limited to soybean gene function and gene transformation research. So, it ismeaningful to establish a high efficiency transformation method.In this study, the EPSPS gene was transformed into soybean by Agrobactrium-mediated genetic transformation system with cotyledonary node as explants insoybean. Some major factors, which affect the regeneration efficiency andtransformation efficiency of soybean were assessed. The main conclusion of thisexperiment was as following:Four varieties of soybean were chosen and the cotyledonary nodes of Hefeng35and Heinong37were used to be the target explants. The best seed germinationcondition is to place soybean seeds on germination medium for five days. In addition,we found it could improve induction efficiency when the concentration of6-BA wasfrom1.0to2.0mg/L in germination medium. During the period of shoot induction, ahigher shoot induction rate were obtained with1.6mg/L6-BA in this experiment. Forrooting, the optimal concentration of IBA was from0.5to1.0mg/L.And the optimalconcentration of cefotaxime sodium in medium was500mg/L. The appropriateconcentration of glyphosate for selection culture was from7to9mg/L for Hefeng35and from5to7mg/L for Heinong37.We assessed factors affecting transformation efficiency, including the conditionsof co-cultivation, antioxidants, and concentration of ZT. We found400mg/L L-cysand154.2mg/L DTT could improve transformation effieiency greatly and otherfactors, such as light, the time of co-cultivation, also play important roles intransformation. In addition, the optimal mass concentration of ZT was0.5mg/L forHefeng35and1.0mg/L for Heinong37. After optimizing these factors, a stable andefficient genetic transformation system using soybean cotyledonary nodes as explantswas established, and some of herbicide resistant soybean plants were obtained.