The Establishment Of Regeneration Systems And Anti-drought Gene Transformation Of Alfalfa

Saline soil is a very important environmental factor influence plants growth and agricultural production. Alfalfa is the biggest for the growth area and the highest quality forage grass. Using the gene-transfer technology is the most effective method to breed anti-salt variety alfalfa in the present of forage grass breeding. The purpose of this research was to improve the salt-tolerance of alfalfa by means of transfering the anti-drought gene into it .After comparison of different regeneration systems in 2 alfalfa varieties including Gan-nong No.2 and Gan-nong No.3, an efficient alfalfa regeneration system was established based on the organogenesis,according to which, anti-drought gene was transferred into alfalfa mediated by Agrobacterium, and Kan-resistance plants were obtained. The main results are as follows:1.The establishment of regeneration systems. To different explants, hypocotyl has the best ability to induce callus. For Gan-nong No.2 , the effective media for inducing callus was on MS supplemented with 2,4-D 3.0 mg·L-1 and NAA 1.5 mg·L-1. The optical time of 2,4-D upon callus inducing is 15 days. The effective media for bud inducing was MS containing KT 0.5 mg·L-1 and NAA 0.1 mg·L-1 and sucrose 20 g·L-1 and 7% agar . Otherwise about Gan-nong No.3 , media MS supplemented with 2,4-D 2.0 mg·L-1 and NAA 1.0 mg·L-1 and 30 g·L-1sucrose and 7% agar had highy callus inducing frenquency. The effective media for bud inducing was MS containing NAA 0.3 mg·L-1 and KT 0.5 mg·L-1 and sucrose 20 g·L-1 and agar 7%. It indicated that the regeneration ability of Gan-nong No.3 stronger than Gan-nong No.2 after comparison .2.Agrobacterium mediated gene transformation of anti-drought gene. Cultivated 7 days hypocotyls of Gan-nong No.3 as explants and agrobacterium tumefaciens strain as mediator , the optimized genetic transformation system is as follows: The selective pressure was 20mg L-1and choose late was optional. 300 mg·L-1 Carb could restrained growing of agrobacterium effectively in callus inducing period and 200 mg·L-1 in buds inducing period.10 minutes infection time and OD 0.3～0.5 agrobacterial density,Preculture 3 days , 3 days coculture and 20 mg·L-1 AS concentration was suitable. In a series of replicated experiment, 23 Kanamycin resistant of Gan-nong No.3 were obtained,moreover , 21 transgenic SacB gene and 2 BADH gene. The result of PCR analysis stated that the detected Kanamycin resistant plants transgenic SacB gene were all negative.