Establishment Of Regeneration System Of Alfalfa And Effecting Factors Of Transformation By With SacB Gene

In the face of present serious ecology environment,to cultivate new alfalfa breed which is able to survive in drought and salina areas will contribute to the restoration of ecology environment. Also the cultivation and development of the new breed will benefit environment,society and economy. So gene engineering is an important method to improve the quantility and stress-tolerance of alfalfa. Tissue culture thechnique of five alfalfa breeds was researched and the sacB gene was transferred to alfalfa by Agrobacterium-mediated transformation in this paper. The main achievements of our rearch are following: 1 Regeneration tissiue culture system establishment: To different breeds,the regeneration ability isn't same. Mi-lu alfalfa had the strongest regeneration ability and Fu-ping alfalfa took the second place, Gan-nong No.3 is lower than Gan-nong No.2 them but is higher than Gan-nong No.2 and Gan-nong No.1. The regeneration ability of Gan-nong No.1 is lowest. To different explant, Cotyledon has the best ability to induce callus. To Mi-lu,Fu-ping,Gan-nong No.3,the effective media for inducing callus was on MS supplemented with 2,4-D 2.0 mg·L^-1 and NAA 1.0 mg·L^-1 and sucrose 30 g·L^-1, otherwise on media MS supplemented with 2,4-D 4.0 mg·L^-1 ,Gan-nong No.1 and Gan-nong No.2 had highy callus inducing frenquency. In bud inducing period, the indcing rate of cotyledon is higher than other explants. The effective media for bud inducing was MS containing KT 1.0 mg·L^-1 and NAA 0.5 mg·L^-1 and sucrose 20 g·L^-1 or inducing somatic embryo on media MS containing BA 4.0 mg·L^-1 than transfer to media MS supplement BA 0.5 mg·L^-1 to induce bud. The bud inducing rate of Mi-lu,Fu-ping,Gan-nong No.3 was about 70 percent , Gan-nong No.1 and Gan-nong No.2 was only about 30 percent. The effective root inducing meida was on 1/2 MS supplemented with IBA 1.0 mg·L-1. 2 Transformation system of sacB gene establishment: 60 mg·L-1 Kanamycin restrained the bud regeneration when it exsisted in callus inducing period, so we delayded 15 days to select resistant plant and this method improved the transformation frequency. 500 mg·L-1 Carb could restrained growing of agrobacterium effectively. According to we established transformation system: preculture 2 days, OD 0.5 agrobacterial density, 10¹5 minutes infection time and 3 days coculture. After more than 80 days selection 3 regenerated Kan-resistant plantlets were abtained.