Study On Factors Affecting The Agrobacterium-mediated Soybean Cotyledonary Node Transformation Of Chitinase Gene

In this research, ten varieties of soybean were choose and the cotyledonary node of these varieties were used to be the target explants of chitinase gene transformation. The factors affecting the Agrobacterium-mediated transformation and the shoot regeneration were studied. The resistant plants were obtained from the two steps selection Seven positive plant was obtained.The positive result of PCR test show that the chitinase gene was integrated into soybean抯 genome.The main results of this research:1 The establishment of regeneration systema.The high regeneration frequency genotypes were Hefeng 35, Hefeng 25, Heinong40.b.The differentiation mediums of different genotypes are:Hefeng 35, Hefeng 25: MSO + 1.5mg/i BA+ 0.2mg/I IBA Heinong 40: M SO +1 .2mg/I BA ?0.2mg/I lEAc The full cotyledonary is the best explant size for shoot regeneration. d.The root regeneration medium of shoot is : MSO + 2.0mg/I IBA2. The selection pressKm is a more effective selection press than Neo.The Km concentration of different genotype respectively is 1 00mg/I for Hefeng 25 or 125mg/I for Hefeng 35 and Heinong 40 in the period of shoot differentiation and in the root differentiation period is 10mg/I for Hefeng 25 or 15mg/i for Hefeng 35 and Heinong4O.3. The antibiotic for restrain the propagation of excess AgrobacteriumThe Cef is a good antibiotic to restrain the propagation of excess Agrobacterium after co-culture period.It has not significant influence on the regeneration of soybean.4. The establishment of Agrobacterium-mediated transformation systema.The ultrasonic treatment has no significantly influence on the regeneration of shoot when the treatment time is shorter than 1 2s.b.Among the three genotype hefeng 35 is the best tangert for Agrobacterium-mediated transformation.c.Between the two strains EHA1O5 has higer transformability than LBA4404.d.The result of orthogonal expenment shows that the group of Suc 6%, SAAT 12s,2OO~ ~ 5 ~0D600=O.5, Co-culture time 3d is the better group. The main affecting factor is the Co-culture time.5. The PCR test of resistant shoot and plantSeven positive plants were obtained. The PCR positive rate of resistant plant which had been selected by two steps is higher than that of resistant shoot which had been selected only one step.