Puccinellia Chinampoensis Catalase Gene Genetic Transformation Of Alfalfa

Soil salinization is one of the important non-biological stress factors affecting agricultural production and ecological environment.To increase salt-tolerance of plants has become one of important research subjects in crop genetics and breeding.As a kind of perennial legume forages known as “ King of Forages ”,alfalfa plays an essential role in protecting ecological environment,reinforcing soil,protecting slopes and preventing water loss and soil erosion.It is an important research that how to increase stress resistance and enlarge planting scale of alfalfa has been being focused.At present,the development and application of genetic engineering has explored a new pathway for alfalfa breeding for stress tolerance.A new CAT homologous genes was separated by RT-PCR in(puccinellia chinampoensis),named:PuCAT and created p3300-35s-PuCAT-NOS plant expression vector at research institute of transgenic afalfa in Jilin academy of agricultural sciences.In this study,I selected Medicago sativa L.cv.Gongnong No.5 to optimize a more efficient regeneration method of alfalfa and used PuCAT gene into alfalfa by Agrobacterium-mediated genetic transformation,To gain the new breeding of alfalfa.1.The“Gongnong 5” alfalfa tissue culture regeneration system was optimized.The sterile seedling cotyledon that had been cultivated for 7 days was selected as the explant.UM+2,4-D 2mg·L-1+ KT 0.25mg·L-1 was used as callus medium.Differentiation of the cultivated callus in different proportions of differential media was analyzed.The results showed that UM + KT 1.5mg·L-1 was the optimum induced differential medium.2.Glyphosate was selected as the screening reagent in the experiment,the optimal screening pressure concentration of which was 2.0mg·L-1 for callus.3.Observation and statistics of the experiment suggested that 2 days of pre-culturing the “Gongnong5” alfalfa sterile seedling cotyledon cultivated for 7 days before Agrobacterium tumefaciens infection could effectively increase the metabolic capacity of cells and facilitate the transformation of Agrobacterium tumefaciens.4.The best Agrobacterium tumefaciens infection concentration for the pre-cultured explant was(OD600?0.8)while the optimum infection time was 10 min.5.Co-culturing the infected explant and Agrobacterium tumefaciens for 3d resulted in the most effective Agrobacterium tumefaciens transfer to the explant.6.The resistant callus was induced by putting the alfalfa cotyledon into the UM+2,4-D 2mg·L-1 +KT 0.25mg·L-1medium supplemented with 500mg·L-1 cephalosporin after co-culture.Subsequently,it was transferred to the UM+KT 1.5mg·L-1 medium for callus differentiation induction and to MS medium for rooting and seedling.Later,the alfalfa seedling was hardened and transplanted for outdoor culture.7.The target gene PuCAT and the marker gene bar were detected in the transgenic alfalfa plant survived after hardening and transplanting by double PCR.The results indicated that the target gene PuCAT and marker gene bar had been integrated into the “Gongnong 5” alfalfa genome.30 transgenic alfalfa plants were obtained from the 56 differentiated alfalfa seedings.