The Primary Research On Agrobacterium Mediated Transformation Of AtPCS1 Into Alfalfa And The Establishment Of Regeneration Systems

Phytoremediation is a technology that using plants to remove heavy metals from the soil or reduce their toxicity. The use of plants to repair the heavy metal pollution of soil is a kind of green, cheap, feasible environmental restoration techniques. This experiment is proposed by means of genetic engineering ,aiming to transfer the phytochelatin synthase gene(AtPCS1) to the plant with advantages of easy grown-alfalfa,grow quickly and high biomass, so as to obtain AtPCS1 transgenic plant which can accumulate heavy metals,which is beneficial to provides the foundation materials for repairing heavy metal contaminated soil in the method of biotechnology for future.Firstly, the hypocotyl,cotyledon and leaf of three alfalfa genotypes(Long- dong,Gannong No.1 and Gannong No.3)were evaluated for their callus induction capacity and plant regeneration ability on different media. The results indicated that the cotyledon of three alfalfa genotypes has higher regeneration ability than hypocotyl and leaf. The highest regeneration ability of alfalfa is in Long-dong, its callus induction percentages is 92.0%, and the regeneration percentages is 50.0%; Furthermore, we optimized the callus induction and differentiation medium and found that the VI medium sited by this experiment was respectively the cotyledon's most suitable medium for induction and differentiation medium, in the VI medium, the cotyledons differentiation time is short, and a number of cluster buds exist; The experiments also found that regeneration buds can differentiate into seedlings on the original culture medium(the callus induction and differentiation medium)directly and take root in 1/2 MS medium, supplemented with 15.0 g/L sucrose.Secondly, the experiment is on the basis of establishing good alfalfa genetic transformation system through by leaf infection method, making use of agrobacterium-mediated to transfer AtPCS1 gene into cotyledon of alfalfa Longdong. By GUS histochemical staining and AtPCS1-specific PCR to some regeneration transgenic plants, the preliminary results indicated that the AtPCS1 had been transferred into alfalfa genome. However, the further research would be necessary to testify the transcription, the translation of gene in transgenic alfalfa, and the tolerance of transgenic alfalfa under the heavy metal pressure.