Establishment Of Soybean Regenerative System Of Cotyledonary Node And Study On GAFP Gene Transfer Mediated With Agrobacterium Tumefaciens

Being one of important oil crop, soybean is suffered from many kinds of fungus. Traditional method of breeding has many limitations such as long period, low efficiency, environmental pollution, so the traditional methods are not ideal of plant resistance breeding. With the developing of genetic engineering technology, Agrobacterium-mediated transformation is emerged as the main breeding means to improve disease resistance. Gastrodia antifungal protein (GAFP) is a protein with broad anti-fungal activity, it can strongly inhibit many specific pathogens responsible for plants fungal disease, therefore, GAFP has great application value in the field of resisting fungal diseases in the plant genetic engineering.In this study, an efficient regeneration system of soybean cotyledonary node was established and optimized. The GAFP 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 of this research were as follows:1Establishment of the regeneration system of soybean cotyledonary node(1) Selection of the method for sterilization of soybean seeds:The best sterilization method was using Cl2for its low pollution rate and less damage of soybean seeds.(2) Selection of genotypes:There were large differences of regeneration frequency between soybean genotypes. Regeneration frequency ranged from7.27%to69.09%in7soybean genotypes. Xiaolidou was chosen to be the research materials in this study for the highest regeneration frequency.(3) Selection of the concentration of6-BA:The concentration of6-BA directly affected the shoot induction and differentiation from cotyledonary node. When the concentration of6-BA was Omg/L, the cotyledonary node could not induce any shoot; when the concentration of6-BA was1.67mg/L,t he regeneration frequency was the highest, up to84.6%.(4) Selection of the concentration of IBA:The frequency of root regeneration was remarkably increased by the addition of IBA at the root regeneration stage. When the concentration of IBA was1mg/L, the root regeneration frequency was highest, up to79%. 2Establishment of Agrobacterium-mediated transformation system and discussion of main factors influencing transformation rate:(1) Determination of best antibiotics concentration and antibacterial type. The suitable concentration of Hyg used in resistant shoots selection was10mg/L and Cef500mg/L. And the late selection was used to overcome the difficulty of adventitious shoot differentiation.(2) Optimization of transformation condition of soybean:The suitable proportion for the time of infection and co-culture was30min and3d, respectively.3Detection of transformants of soybean with GAFP:203regenerated plants were tested by PCR,2positive plants with GAFP gene were detected, the transformation frequency was1.96%.