Establishment Of Agrobacterium Tumefaciens Mediated Soybean Transformation System And Developing Of Transgenic Soybean With Phy Gene

Soybean is an important oil crop, grain and forage crop in China. In recent years, the strategy of genetic engineering has been applied in improving soybean quality and enhancing soybean production. In this study, an efficient regeneration system of soybean cotyledonaiy node was established and optimized. The Phytase (Phy) gene was transferred into soybean with Agrobacterium-mediated method. The main factors influencing transformation rate were discussed and the transformation system was established preliminarily. The positive plants were obtained and confirmed by PCR. The main results were as follows:1. Establishment of the regeneration system of soybean cotyledonary node(1) Selection of the method for soybean seeds sterilization:The best sterilization method was using Cl2for its low pollution rate and less damage of soybean seeds.(2) Selection of seedling ages:There were large differences of shoot induction frequency among different ages of germination seedlings.4d germination seedlings were chosen to the transformation for the higest shoot induction frequency, up to88.9%.(3) Selection of genotypes:There were large differences of regeneration frequency between soybean genotypes. Regeneration frequency ranged from24%to84%in5soybean genotypes. Xiaolidou was chosen to be the research materials in this study for the highest regeneration frequency.(4) Selection of the concentration of6-BA:The concentration of6-BA directly affected the germination of seedlings and the shoot induction. The results indicated that germinated seedlings which addition of1mg/L6-BA to germination medium were suitable as explants. The optimal concentration of6-BA in shoots induction medium was1.67mg/L.(5) Selection of the concentration of IBA or NAA:The frequency of root regeneration was remarkably increased by the addition of IBA or NAA at the root regeneration stage. The optimal concentration of IBA in root medium was1mg/L.2. Introduction of Phy gene into soybean (1) Optimization of transformation condition of soybean:The concentration of Agrobacterium0.8(OD600) and co-infected30min, the3d dark co-cultivation in the co-cultivation medium which added25mg/L LA.(2) Determination of the best herbicide concentration:The suitable concentration of Glufosinate used in resistant shoots selection was5mg/L, and the late selection was used to overcome the difficulty of adventitious shoot differentiation.(3)345regenerated plants were tested by PCR,5positive plants with Phy gene were detected, the transformation frequency was1.45%.