| Alfalfa (Medicago sativa L.) as one of perennial herb of legume, is the world’s most important forage resource and has a very high forage and ecological value. In recent years, with the expansion of alfalfa cultivation area, limit of cold resistance of alfalfa became major factor in areas in North China. Transfer the cold resistance genes into Alfalfa to improve cold resistance became an efficient breeding method to solve the problem.In this research, AtCBF1 gene, one cold-induced transcription factor cloned from Arabidopsis, were transfered into diverse varieties of Alfalfa through mediated by Agrobacterium. the genetically engineered alfalfa with cold resistance and high forage yield were selected according to molecular detection of the transformation plants, identification of cold tolerance and field forming strains with AtCBFl gene. The main results are as follows:1. Research on affection of tissues regeneration factor include alfalfa varieties, explants and hormones ratio. Results showed that the best genotype is Hunter-River alfalfa in tissue culture. The best explant is hypocotyl. The best medium for callus induction is SH+2.0mg·L-12,4-D+0.5 mg·L-1 6-BA. The best Medium for the callus subculture is MSO+0.5mg·L-1 NAA+1.0mg·L-1 6-BA+1.0mg·L-1 AgNO3 and MS+ 0.3mg·L-1 TDZ. The best Differentiation medium is MS+0.2mg·L-1 KT.2. Arabidopsis thaliana transcription factors AtCBFl gene induced by cold were Cloned.The Agrobacterium transformation of alfalfa plant expression vector PBI121-CB Fl were constructed.3. Research on genetic transformation factors showed that, the best inhibitors of Agrobacterium is 350 mg-L^Carb, best Kan concentration is 60mg·L-1, best receptor genotype is the Hunter-River alfalfa, best explant is hypocotyl, most suitable concentration OD600 Agrobacterium bacteria is between 0.4-0.6, best time of infection is lOmin, AS optimal concentration is 10 mg · L-1.4. AtCBFl gene were transfered to alfalfa by Agrobacterium-mediated. Kan-resistant transgenic alfalfa plants were obtained. PCR Detection amplified around 650bp specific bands, indicated that AtCBFl gene is already integrated into the alfalfa genome. RT-PCR showed AtCBFl foreign gene transcriptional expression in part of transgenic plants.5. Cold resistance were evaluated by determination of relative conductivity, contents of PROLINE, soluble sugar content and MDA content in transgenic alfalfa plants. Plants with AtCBF1 gene showed higher cold resistance than control plants. Among transgenic plants, T9, T11, and T3 showed relatively higher cold resistance and T9 of the strongest cold resistance.6. Cold resistance of genetically engineered alfalfa were evaluated under artificial freezing temperature, leaves victims and restore growth showed that transgenic alfalfa plants have strong resistance to low temperature stress. Over expression of AtCBFl improved cold tolerance of alfalfa.7. The production, regeneration speed and the quality research of transgenic plants showed that, Transgenic alfalfa strains have no obviously growth delay, plant short, and severe changes compared to control plants. The only obviously change is leaves length were slightly shorter. Compare to control plant in production and regeneration speed, transgenic plants and strains show some above and some below control. Strains T11 and T9 are significantly better than control and other strains in production and the regeneration speed. Nutrition quality were also improved in transgenic strains on some nutrition components like rough protein content. Based on research results, T9 and T11 are best two transgenic alfalfa strains. |
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