The Transformation Of LYZ-GFP Dual Genes By Agrobacterium Mediatedand Its Expression Analysis In 3 Varieties Of Alfafa

Alfalfa(Medicago sativa L.) is the most widely planted forage legumes in China, especially in the north, northwest and northeast of the main land, which are the main producing areas of alfalfa. But disease often threatens alfalfa production. Serious disease may cause large-scale reduction of production, greatly limits the usage and nutritional potential of forage in animal husbandry. Therefore, this study used Gannong No.1 variegate alfalfa(G1), gannong No.3 alfalfa(G3), and Hetian alfalfa(HT) as materials, optimized the regeneration system of three varieties of alfalfa; LYZ-GFP dual gene was transferred into three varieties of alfalfa mediated by Agrobacterium, and the influence factors among transformation process were analyzes, optimized genetic transformation system was established; micro-detection of GFP and PCR detection were conducted, transgenic plants were successfully obtained with Gannon No.1 and Hetian alfalfa, laid a research foundation for the eventual formation of new varieties of transgenic alfalfa. The contents are listed as follows:1. Study on regeneration of 3 varieties of Alfalfa and genetic transformation,optimization of alfalfa regeneration system and genetic transformation system. Hypocotyls of three alfalfa cultivars was used as explants, disinfected method on seed growth, callus induction, and differentiation was studied, as well as factors affecting genetic transformation. The results showed that: the optimal disinfected method of G1, G3 and HT is 20% sodium hypochlorite treatment of 25 min, the germination rates were the highest, which were 93%, 84% and 97.5% respectively. The optimum regeneration system of three varieties of alfalfa was established. the callus induction rate of G1, G3, HT were 98.5%, 90% and 99.5% on MS medium added with 2mg/L 2,4-D and 0.5mg/L KT; when 0.5mg / L NAA was added in MS medium, the differentiation rate of G1 and G3 reaches a maximum, which were 74% and 47%, and when the NAA concentration was 0.05 mg / L, the differentiation rate of HT was 62% which was the highest.2. Explored the optimum concentration of growth regulator for the micropropagation of three alfalfa varieties and optimized the massive propagation conditions for transgenic plantlets. In this research, Gannong No.3(G3), Hetian(HT), Longdong(LD) alfalfa was taking as the experimental materials. Effect of different kinds growth regulator on the growth of alfalfa micro-cuttage tube plantlet was studied, different concentration of indolebutyric-such as acid(IBA), naphthalene acetic acid(NAA), ABT(ABT) was tested. The results showed that: tube plantlet of Gannong no.3 alfalfa had the highest average root number(8), maximum root length(17.5 cm), which rooting rate under 0.2 mg/L NAA treatment was 100%. Moreover, plant height and leaf number of G3 was also significantly higher than that of the control(CK) and other treatment(P<0.05). The root growth situation of Hetian alfalfa tube plantlet under 0.1 mg/L NAA treatment is obviously superior to other treatment. The rooting rate was 100%, with higher average root number(7) and maximum root length(29.6 cm). Besides, plant height and leaf number of HT was also significantly higher than that of the control(CK) and other treatment(P<0.05). Longdong alfalfa tube plantlets showed superior root growth situation under 0.2 mg/L NAA treatment to other treatment, and rooting rate was 100%, with high root number(7) and maximum root length(10.2 cm). Plant height and leaf number was also significantly higher than that of the control(CK) and other treatment(P<0.05). Therefore, under the concentration gradients of setting, the optimal growth regulator for Longdong and Gannong No.3 alfalfa on micro-cuttage tube plantlet growth is 0.2 mg/L NAA, and suitable growth regulator for Hetian alfalfa is 0.1 mg/L NAA.3. The transgenic plants containing the target gene were obtained by PCR and fluorescent micro-detection, and the positive evidence was obtained. Fluorescent micro-detection and PCR testing was conducted with transgenic plant,plants containing the dual Lyz-GFP gene was obtained. Tracking on the early stage of the transformed callus showed that callus of G1, G3 and HT can be found with green fluorescent protein. The fluorescent detection of transformed leaves showed that G1 and HT regeneration plant leaves have green fluorescence expression. PCR amplification Of GFP and LYZ genes with G1 and HT alfalfa transformed plant showed that six strains of G1 fluorescence positive plant amplified 750 bp GFP gene. Four of them amplified 500 bp Lyz gene, one strains of HT amplified Lyz gene.