The Research On Agrobacterium-mediated Transformation Of AtPCS1 Into Medicago Sativa.L

Plants have many natural properties that make them ideally suited to clean up heavy metal polluted soil in the process called phytoremediation.But most native hyperaccumulators can not be used broadly for their slow growth and little biomass. We tried to use transgenic approach for transferring the metal-accumulate related gene(AtPCS1)to the plant with advantages of high biomass and easy grown—alfalfa, to obtain transgenic plant which can accumulate heavy metals.Firstly,cotyledon and hypocotyl of nine alfalfa genotypes(Longdong,Gannong No.3,A'ergangjin,Sandeli,Gannong 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, Longdong and Gannong No.1 are the two genotypes that have the highest regeneration ability,their callus induction percentage were 85.0%and 90.0%,and regeneration percentages both 55.0%;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 Longdong and Gannong No.1,and node and cotyledon node 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 10 mg/L sucrose.And then,we transferred Agrobacterium LBA4404 with plasmid pBI121-AtPCS1,identified the positive strains by bacterial colony PCR and transferred AtPCS1 gene into alfalfa Longdong and Gannong 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,and Vacuum treatment can improve the transformation frequency.Explants transformed could sprouting directly on F medium supplemented with kanamycin and carbencillin and take root on 1/2 MS medium.The primary results of histological detection of GUS activity and AtPCS1-specific PCR to branches and leaves of some transgenic plants suggested that the AtPCS1 had been transferred into alfalfa.Further research would be necessary to testify whether the gene has transcription and translation in transgenic alfalfa,and the characteristic of transgenic alfalfa under the heavy metal(such as Cd²⁺)pressure.