Establishment And Optimization Of Agrobacterium-mediated Transformation Of Alfalfa

Salt-alkali stress is one of the most important factors in inhibiting the productivity of crops in worldwide. Many researchers are aiming toward the breeding of salt-tolerant crop cultivars to utilize saline soil. However, salt tolerance is complex physiological traits in plant. Traditional breeding method is difficulty to improve salt tolerance of plants. The problem may be dissolved by application of the genetic engineering. At present, with molecular biology and biotechnology developments, some genes responding to salt-stress have been found in many plant species and the application of these genes have obtained a large numbers of improving salt-tolerant in transgenic plants. Alfalfa (Medicago sativa. L) is the most famous herbage in worldwide. Alfalfa (Medicago sativa. L) is the most famous herbage in worldwide. It has important significance to herbage development in our country, which application of genetic engineer breeds new alfalfa cultivar and enhances salt tolerance of alfalfa. In this study, we successed in establishing and optimizing genetic transformation system of alfalfa, and transferred vacuolar Na+/H+ antiporter (Suaeda corniculata) into alfalfa genome. These results will set up the base of genetic engineering of alfalfa. The primary results of our experiment as list below:Genotype is one of important inhibitor factor for plant genetic transformation, and 7 cultivars were taken as donor material to screen the best genotype. The result indicated that GN-1 had higher regeneration frequency than other cultivars. The suspension culture system of embryogenesis callus was established in alfalfa. We optimized the two key factors of effecting embryogenesis callus regeneration, including inoculation amount of hypocotyls callus and rotating speed of suspension culture. Ultimately, the 3g/40ml and 120r/min were determined for suspension culture.Embryogenesis callus transformation system using sonic assisted Abrobacterium- mediated method (SAAT) was established and optimized. We took suspension embryogenesis callus as material, succeeded in establishing alfalfa transformation system using SAAT method. During the test, we optimized many factors effecting on genetic transformation, including the time of sonic treatment (8s), the AS concentration (100uml/L), the co-cultivate time (5 days) and kanamycin concentration (50mg/L).Total 15 resistant plants obtained in this experiment were assayed using PCR. The test result confirmed that exogenous gene had integrated into chromosome of alfalfa. In this study, we succeeded in establishing the high frequency regeneration system of alfalfa, and obtained transgenic plants via Abrobacterium-mediated method using the regeneration system. This will supply effective method for the theory and application study of genetic transformation in alfalfa.