Establishment of High Frequency Regeneration and Genetic Transformation System of Medicago Sativa

摘要

The establishment of alfalfa (Medicago sativa) high frequency regeneration system and genetic transformation of BADH gene mediated by Agrobacterium tumefaciens to get saline resistant transgenic alfalfa was studied. The results indicated that the embryogenic callus and embryoid induction rate of leaf of Zhongmu 1 variety was the highest among hypocotyl, leaf and cotyledon of 5 alfalfa varieties. The best medium for embryogenic callus induction was improved SH+2,4-D 4.0 mg.L~(-1)+BA 0.5 mg.L~(-1). The best one for embryoid induction and plant development medium was MSO, on which the embryoid could grow, differentiate and roots. The 5 selected genotypes that had higher embryoid induction rate and regeneration rate were used to establish asexual clone, and the leaves of the regeneration plants were used for genetic transformation. During the genetic transformation, GUS coloration analysis was used to optimize the alfalfa transformation conditions. The result was that dark culture time was 20 days including 5 day pre-culture and 3 day co-culture time, 10 mg·L~(-1) AS could improve the transformation rate obviously, Kan 50 mg.L~(-1) was suitable for screening. The leaves infected by Agrobacterium tumefaciens with BADH gene were subcultured on the selective medium 3-4 times for total 90～120 days and the Kan resistant shoot were achieved. The PCR and PCR-Southern blot analysis results showed that the BADH gene was integrated into the alfalfa genome.