Studies On Agrobacterium-mediated Genetic Transformation Of Alfalfa (Medicago Sativa L.) With GS1, GS2 And P5CS Genes

Glutamine synthetase (GS) is a pivotal enzyme in ammonia assimilation in both plants and gram-negative microorganisms. It plays an important role in resistance to abiotic stress conditions induced by herbicide and salt.Δ1-pyrroline-5-carboxylate synthetase (P5CS) is also key enzyme to synthesize proline in the plants. The transgenic plants with P5CS can also increase salt-stress tolerance. In this study, we developed the regeneration system of different four alfalfa cultivars: XinjiangDaye, Longdong, Gannong No.1 and Gannong No.3. Especially through seed-callus-embryoid pathway, we obviously improved XinjiangDaye regeneration frequency, obtained regeneration plantlets, and chimeric was also avoided.The plant expression vector carrying two glutamine synthetases (GS1 and GS2) and P5CS gene was used for alfalfa in vitro transformation. The vector p2GS-P5CS has no potentially harmful selection marker gene. Through genetic transformation mediated by Agrobacterium tumefaciens, the foreign genes were transformed into alfalfa cultivar XinjiangDaye. The transgenic alfalfas were obtained with the help of glutamine synthetases effectual expression and NaCl selection. The main experiment results are as below.1. Development regeneration system of different four alfalfa cultivars through tissue culture.2. Optimization regeneration system of XinjiangDaye through seed-callus-embryoid pathway.3. Construction the plant expression vector p2GS-P5CS with GS1, GS2 and P5CS gene and no potentially harmful selection marker gene.4. Obtaintion XinjiangDaye transgenic plantlets through the approach of Agrobacterium tumefaciens mediated genetic transformation.5. Results of PCR, PCR-Southern and Southern blotting analysis demonstrated that foreign genes had been integrated into alfalfa genome. The transcription of foreign genes in transformants was also confirmed by RT-PCR. This offers new opportunities to transfer foreign genes by expression vector without selection marker gene. It is very important when a careful biosafety appraisal is needed.In this study, we made a good foundation for improvement of drought and salt stress tolerance in alfalfa and future gain valuable safe transgenic alfalfa products by genetic engineering.