Establishment Of Regeneration System In M.sativa And Transformation By Agrobacterium-mediated Of Ppk Gene

In the worldwide, the effective utilization of phosphorus is generally low, phosphorus deficiency is a serious restriction of forage production quality and the ecological, economic. the study seeks to transfer gene ppk into the alfalfa, phosphorus plant alfalfa-growing areas to reduce the soil phosphate fertilizer application, increase the effective utilization of phosphorus, while at the junction of farmland and water, the establishment of buffer zone of alfalfa to control eutrophication. Based on two varieties alfalfa to establish and optimize the regeneration system, compare the best receptor and their genetic transmission of recycling system, using Agrobacterium-mediated phosphorus alfalfa Genetic transformation of genes ppk initially received Kan resistant plants. the major findings are as following:1 Regeneration systems of Xinjiang daye alfalfathe ideal experiment sterilization methods is Rinsing the seeds under running water after an hour ,then treatment with 70% alcohol 50 seconds,then 10% sodium hypochlorite shock treatment 22 min;The most appropriate explants of Xinjiang daye alfalfa is the leaves of no vaccine after 4 weeks; the most suitable medium for callus induction is: MS + 2,4-D (2.0 mg / L) + KT (0.5 mg / L); the most suitable medium for shoot differentiation is: MS + 6-BA (0.5 mg / L) + NAA (0.05 mg / L).2 Baoding alfalfa regeneration systeminduced differentiation of the most suitable explants of Baoding alfalfa is hypocoty germination for six days ; the best medium for callus induction phase is: SH + 2,4-D (2.0mg / L) + KT (0.05mg / L); The best bud differentiation medium is: SH + KT (0.4mg / L) + NAA (0.3 mg / L).3 Ideal recipient for transformationRegeneration frequency of Xinjiangdaye alfalfais is 156.76%,better than Baoding(117.03).So Xinjiangdaye alfalfa is the ideal transformation.4 Agrobacterium mediated gene transformation of anti-drought geneUsing the Aseptic seedlings leaf that training 4 weeks as explants , agrobacterium tumefaciens strain LBA4404 as mediator,the optimized genetic transformation system is as follows: The selective pressure was 20-50mg L-1. 300 mg·L-1 Carb could restrained growing of agrobacterium effectively. 15 minutes infection time and OD 0.5 agrobacterial density,3 days coculture was suitable. In a series of replicated experiment,11 Kanamycin resistant of Xinjiangdaye were obtained, in which two showed positive detected by PCR.