| Alfalfa is leguminous perennial shrub and the world's most important forage crop.The process of alfalfa growthusually was infected by pathogenic microorganisms.The yield of alfalfa will loss seriously when disease erupted heavily. The resistance gene was transformed into alfalfa cultivars variety, it can improve the resistance ability of alfalfa to disease in the short term.Taken the alfalfa of Gan-nong No.1 and Gan-nong No.3 as the objects to study their regeneration and disease-resistant gene transformation conditions. And mastered the conditions of regeneration.Determine the best culture medium for regeneration of two varieties of alfalfa.Lyz disease-resistant gene and GFP reporter gene were inducted into two varieties of alfalfa through the Agrobacterium-mediated. And studied conditions of gene conversion systematicly.Established the transformation system of alfalfa.The calls and plants of alfalfa were detected by the fluorescent microscope and polymerase chain reaction.The result indicated that the PBI121-Lyz-GFP dual gene has been transferred into two varieties of alfalfa and expressed successfully.The main results are as follows:1.The establishment of regeneration systems. Utilize Gan-nong No.1 and No.3 alfalfa as research material ,conducted the callus induction,differentiation and rooting. The result indicated that hypocotyl was the best explants for the regeneration of Gan-nong No.1 and No.3, the optimum medium for induction was MS+2 mg/L2,4-D+0.5mg/LKT +0.7 %agar +2.5 %sucrose and the optimum time of callus inducing is 30 days; The best medium for shoot differentiation was MS +0.3 mg / LNAA +0.7% agar +20% sucrose; The best medium for rooting was 1/2 MS +0.3 mg / LNAA +0.7 % agar +2.0 % sucrose. The best callus induction and differentiation of Gan-nong No.1 were 100 % and 28 %; The best callus induction and differentiation of Gan-nong No.3 were 100 % and 25 %.2. Gene transformation of PBI121-Lyz-GFP by Agrobacterium-mediated and the establishment of gene transformation system.Cultivated 7～10 days hypocotyls of two varieties of alfalfa were used as explants and Agrobacterium tumefaciens strain were used as mediator, conducted Lyz-GFP dual gene conversion,the optimized genetic transformation systems were as follows:The Kan screening concentration was 75mg/L and screening methods was delayed choice.Cef was better inhibitory and the optimum concentration was 300mg/L;The time of pre-culture and co-culture were same as 3 days for explant;The infection methods and time for the transformation was 10min on shaking bed, appropriate concentration of Agrobacterium OD600 = 0.4～0.5. 4 Kanamycin resistant seedlings of Gan-nong No.1 and 6 seedlings of Gan-nong No.3 were gained .Of which there are 2 Gan-nong No.1 and 4 Gan-nong No.3 were expressed of green fluorescence.Conversion rates were 50 % and 67 %.3. Fluorescence detection and PCR identification of transformed callus.The convered callus and plant were fluorescence detected and PCR amplificated.2 Gan-nong No.1 and 4 Gan-nong No.3 plants were expressed of green fluorescence though fluorescence detection. PCR identification resulted showed that the size of 750bp bands appeared in both Callus of two varieties of alfalfa. Double test results indicated that the dual gene Lyz-GFP has been successfully transferred into callus of Gan-nong No.1 and No.3 alfalfa.
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