Preliminary Studies On Transformation Of AVP1 Gene Into Medicago Sativa

Today, soil salinity and desertification has became the cosmopolitan problem in the earth. Planting salt- and drought-tolerant plants is an crucial approach to ameliorate salinity and desertification land. Alfalfa (Medicago sativa), a perennial high quality legume, is widely planted worldwide, and it is difficult to grow under salinity and drought conditions for its lower salt- and drought-resistant. Therefore, breeding salt- and drought-tolerant alfalfa cultivar via biotechnology has the vital significance for improving soil quality, developing graziery, and reconstructing ecological environment in Northwest China.Overexpression of the AVP1 gene in the model plant Arabidopsis thaliana results in that transgenic plants are much more resistant to high concentrations of NaCl and to water deprivation than the isogenic wild-type strains because the plant tonoplast proton pumps, the V-H⁺-ATPase and the V-H⁺-PPase, establish an electrochemical H⁺-gradient across the tonoplast that drives the transport of Na⁺ against the concentration gradient from the cytoplasm into the vacuole by Na⁺/H⁺ antiporter. Undr salt stress, vacuolar compartmentalization of Na⁺ reduces the toxic effects of this cation in the cytosol and at the same time increases the osmotic pressure of the plant. The aims of this work was to transfer the AVP1 gene into alfalfa mediated by Agrobacterium tumefaciens, to optimize the transformation system, and to screen out the positive plants, which is first critical step to obtain salt- and drought-tolerant transgenic alfalfa cultivars.Chinese local alfalfa Hetian cultivar was selected as material, which has the superordinary agronomic characters. The A VP1 gene was transformed into Hetian cultivar through Agrobacterium-mediated. We have studied many factors which affect transformation efficiency and established and optimized the transformation system. The main results were as following: (1) 200mg/L Carb could inhibit the reproduction of the agrobacterium strain GV3101. No obvious effect of Carb on embryogenic callus induction was found. (2) The optimum selection pressure of kanamycin for hypocotyls callus and somatic embryo induction were 75 mg · L^-1 and 50 mg · L^-1, respectively. Lingeringly screening was prior to prophase screening. (3) The pre-culture period was 3⁴d, thebacterial concentration was OD6oo=0.5⁰.7, the negative pressure treatment was 3⁴x2min/30s, and the co-culture period was 3⁴d, all of which were essential components of the optimum transformation system. (4) It was fond that AS can't induce transformation and even high concentration AS inhibit transformation in co-culture medium. (5) Up to now, 42 kanamycin-resistant regeneration plants have been obtained.