Transformation Of Medicago.sativa By Agrobacterium Tumefaciens And Regeneration Via Somatic Embryogenesis Of Transgenic Plants With BADH Gene

Saline soil is widespread in China. The development of saline soil is of significance with the increasing population and decreasing arable land. Cultivated alfalfa ( Medicago. sativa.L ) , perennial legume crop,is wildly planted in the world. The development and application of gene engineering creates a new path for alfalfa salt-resistant breeding.In this research, ZhongMuYiHao .selected from five salt-resistant varieties, has regenerated via somatic embryogenesis by two steps. The genotype H2 was selected for transformation with its high regenerative capacity. On the basis of acquired regenerating system of somatic embryogenesis, the BADH gene was transformed through agrobaterium-mediated. We have studied many factors which affect transformation frequency and established and optimized transforming system by histological detection of GUS activity after co-cultivation. The main results were as following:1 The regeneration system of somatic embryogenesis has been established. The leaf explant has the highest regenerative capacity among explants of ZhongMuYiHao. The modified SH medium containing 4.0mg/L 2,4-D and 0.5mg/L BA induced embryogenic callus very well. MSO medium with sugar 2% and no hormone is the most effective medium for embryo induction. The supplement of 15mg/L glutathione promote the formation of embryo. The embryo converted into plant in die MSO medium. Five genotypes with high regenerative capacity were selected from 200 genotypes.2 The transformation system has been established. In the agrobacterium-mediated gene transformation, leaves of genotype H2 was transformed for its high transformation frequency. And with the histological detection of GUS activity, we optimized the transformation procedure. The wounded leaves pro-cultivated on MC for 5 days. A fresh liquid culture of A.tumefaciens was centrifuged and the pellet was resuspended in liquid MC medium to me ODgoo of 0.3 to 0.5.The wounded leaves were immersed in the bacterial suspension for 5 to 10 minutes and then to-cultivated on MC for 3 days. After washing the explants to remove excessive bacterium, callogenesis was induced on MC supplemented with 50mg/L Kan as the selective agent for transformed tissue and 400mg/L Cef to inhibit bacteria] growth. After 90 days selection eight regenerated Kan-resistant plants were abtained.