The Establishment Of Regeneration Systems And Primary Analysis On Transformation Of Iron Uptake Gene In Azuki Bean

The establishment of regeneration system is the base of gene analysis and transformation.In this research,through comparison of different regeneration systems in 6 Azuki bean varieties,Bao278,P254,JN5,JN6,JN8,Jihong9.JN5 has the best ability to induce adventitious shoots.At the same time,Cultivated 5 days hypocotyls of JN5 as explants,Agrobacterium mediated gene transformation was established. The main results are as follows:1. Through comparison of different regeneration systems in 6 Azuki bean varieties, JN5 hypocotyl has the best ability to induce adventitious shoots, The rate of shoots induction is 82.5%.2. The establishment of regeneration systems. The rate of shoots induction of JN5 was effected by explants type,seedling age and hormone concentration.It stated that the best seedling age of JN5 that hypocotyl induced cluster shoots was 5 days. The effective media for bud inducing was MS containing NAA 2 mg·L-1,sucrose 30 g·L-1and 7% agar.3. Agrobacterium mediated gene transformation of iron related gene.Cultivated five days hypocotyls of JN5 as explants and agrobacterium tumefaciens strain as mediator, the optimized genetic transformation system is as follows: The selective pressure was 25 mg·L-1.300 mg·L-1Carb could restrained growing of agrobacterium effectively in callus inducing period and 200 mg·L-1in buds inducing period.Fifteen minutes infection time and OD 0.3～0.5 agrobacterial density, preculture two days, three days coculture and 100 mg·L-1AS concentration was suitable. In a series of replicated experiment, six hygtnmycin resistant JN5 plants were obtained. The result of PCR analysis stated that the two detected Hyg resistant plants transgenic AtbHLH38 gene were negative.