The Research On Agrobacterium Mediated Transformation Of AtPCS1 Into Gan-su Alfalfa

Some plants have many natural properties that make them ideally suited to clean up heavy metal polluted soil in the process called phytoremediation. It was a "green"method.We tried to use transgenic approach for transferring the phytochela-tin synthase gene (AtPCSl) to the plant with advantages of high biomass and easy grown-alfalfa, to obtain transgenic plant which can accumulate heavy metals.Firstly, the cotyledon,leaf and hypocotyl of nine alfalfa genotypes (Long-dong, Gan-nong No.3, Gan-nong No.1 and so on) were evaluated for their callus induction capacity and plant regeneration ability on different media. The results indicated that cotyledon has higher regeneration ability than hypocotyl, Long-dong and Gan-nong No.1 are the two genotypes that have the highest regeneration ability, their callus induction percentage were 95.0% and 94.0%, and regeneration percentages both 45.0%; the F and G medium seted by this experiment was respectively the cotyledon and hypocotyl's most suitable medium for callus formation and differentiation. Furthermore, we compared callus induction and plant regeneration ability of different explants of Long-dong and Gan-nong No.1, leaf and cotyledon can be directly emerge on G medium within 15 days, the calluses coming from cotyledon differentiated much earlier and had more regeneration buds than leaf and hypocotyl, plant regeneration can be accomplished on the original culture medium and take root in 1/2 MS medium supplemented with 12.5 g/L sucrose.Andthen, we transferred Agrobacterium LBA4404 and EHA105 with plasmid pBI121-AtPCSl, identified the positive strains by bacterial colony PCR and transferred AtPCS1 gene into alfalfa Long-dong and Gan-nong No.1 using different explants by leaf infection method. Kan-resistant calli assays and the histological detection of GUS activity suggested that cotyledon is the ideal explant for transgene research, the transformation frequency between these two genotypes has no significant difference. Explants transformed could sprouting directly on F medium supplemented with kanamycin and cefotaxime and take roots on 1/2 MS medium.The primary results of histological detection of GUS activity and AtPCS1-specific PCR to leaves of some transgenic alfalfa suggested that the AtPCSl had been transferred into alfalfa. Further research would be necessary to testify the transcription and the translation of gene in transgenic alfalfa, and the characteristic of transgenic alfalfa under the heavy metal pressure.