Studies On Alfaifa Salt-tolerant Multi-gene Genetic Transformation

Alfalfa (Medicago sativa L.) distributed widely in the world is the perennial legume and known as the king of grass. It has important significance which nutrient-rich, adaptable, improving soil nitrogen fixation and other advantages, but it’s yield varieties and resistance need to be further improved. With the increased pastures salinization and severely degraded vegetation, still particularly needs to cultivate new varieties of stress resistance alfalfa, in order to meet the increasing forage needs of grassland animal husbandry. Alfalfa is a perennial cross-pollinated plants. The rate of selfe burliness is low. To modify varieties is very inconvenient by conventional breeding. With the rapid development of molecular biology, genetic engineering of plants has become an important way that modified alfalfa traits in modern breeding. The purpose of this study is to transfer GY1, GY2, GY3, GY4gene into alfalfa varieties Golden Queen by Agrobacterium-mediated, trying to improve its salt tolerance. Identified transgenic alfalfa by molecular biology method. The main results of this study are as follows:(1) A good receptor system of alfalfa cotyledon node has been established. By summarizing the analysis, the following conclusions:alfalfa cotyledons section for the receptor material, MS medium without any hormones for bud induction medium. The rooting medium is1/2MS+1mg/LIBA+10g/LSucrose+8g/LAgar. Hygromycin screening pressure is20mg/L in cotyledon node regeneration. Cefotaxime within700mg/L as a bacteriostatic agent regeneration has little effect on alfalfa cotyledon node.(2) Optimization genetic transformation system which alfalfa cotyledons section as receptor. By summarizing the analysis, we concluded that:the9-day-old cotyledon node is best receptor material. In Agrobacterium concention OD600=0.6,disseminated time for20min, co-culture time for3d. The inhibitory concentration of Cefotaxime was500mg/L.(3) Preliminary identification of resistant regenerated plants.103resistant regenerated plants was acquired in the test.we have done PCR amplification based on screening gene Hyg and purpose gene GY1, GY2,GY3and GY4, tested shows18plant is positive. The positive rate was17.48%.; Then we chosed four of them and used southern hybridization identification.We acquired three positive lines.The copy number was1or2.