Study On Alfalfa Transformation Of DREB1C Gene Mediated By Agrobacterium Tumefaciens

The gene transformation acceptor selected in this experiment was Medicago sativa L."Baoding", which has better ability of regeneration by our selection in many years. A transcription factor of DREB1C gene, which was cloned form Arabidopsis thaliana and related to response to abiotic stress, was introduced into the genome of alfalfa"Baoding"by Agrobacterium-mediated genetic transformation, in order to gain the new breeding of alfalfa. In this experiment, the original method of tissue culture was improved and optimized, finally, a better method for the tissue cultuer of Medicago sativa L."Baoding"was established. In order to identify transgenic plants at molecular level conveniently, the original vetor was reconstructed. The transgenetic alfalfa expressing DREB1C were gained by identification. The main result were as following:An efficient method for the regeneration of Medicago sativa L. cv. Baoding has been developed. As explants, it was shown that petiole was better than cotyledon in this study, according to the percentage of calli/explnats in this experiment. At the stage of callus induction, explants were placed on UM medium and improved SH medium, after 20 days of the callus induction, the percent of callus was measured, in order to compare on which kind of medium is more effective for the callus induction. The improved SH medium was a very effective medium for the callus induction compared to UM medium. At the stage of callus induction, 2,4-dichlorophenoxyacetic acid (2,4-D) acted mostly, kinetin (KT) could assist 2,4-D to act better. At the stage of embryo induction, 0, 0.2, 0.4 or 0.6 mg/L KT in UM media and 0 or 2 g/L casein hydrolysate in UM medium or improved SH medium were compared for embryo induction and maturation. After 20 days, the maximal percent of the somatic embryo per callus was observed on UM medium with 0.2 mg/L KT and 2 g/L casein hydrolysate. Treatment with casein hydrolysate improved the quality and maturation of the embryo. 1/2 MS medium containing 15 g/L sucrose was effective for shoots to root.In order to identify transgenic plants at molecular level conveniently, the transcription factors gene (DREB1C) from Arabidopsis thaliana and green fluorescent protein gene (GFP) from jellyfish were cloned, modified and fused by gene splicing by overlap extension PCR(SOE PCR),then fusion gene was cloned into the plant expression vector pCAMBIA1301 to get the recombinant expression vector pDR1- GFP by restriction enzyme digestion and ligation,and it was confirmed by DNA sequence analysis.A transcription factor dreb1c gene from Arabidopsis thaliana, which plays important role in plant cell under stress condition and is droved by CaMV 35S promoter, was introduced into the genome of alfalfa by Agrobacterium-mediated genetic transformation. Explants of Medicago sativa were incubated with A. tumefaciens strain GV3101 harboring a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance. After co-cultured for 3 days, explants were transferred onto callus induction medium (improved SH medium) containing 2mg/L 2,4-D,0.2mg/L KT,30 mg/L hygromycin and 300 mg/L cefotaxime. After 3 weeks, hygromycin-resistant calli were transferred onto embruogenesis induction medium (UM medium) containing 0.6mg/L KT,30 mg/L hygromycin and 300 mg/L cefotaxime. Embryo and Shoots grew up from the surface of the calli after about 4 weeks. Finally, the mature embryos and shoots were transferred onto 1/2 MS medium and finished rooting. Polymerase chain reaction (PCR) and RT-PCR revealed that the dreb1c gene was integrated into the genome of regenerated plants.