The Study On Transformation And Expression Of Lysozyme Gene Labeled With Fluorescent In Different Varieties Of Alfalfa

Alfalfa is one of the most widely distributed perennial leguminous grass in the world.Diseases seriously threaten the Alfalfa production. The happening of the disease reduce theproduction performance of Alfalfa, and damage the health of livestock, lower the quality ofgrass, cause a large number of deaths of Alfalfa when it serious. Based on this situation,agrobacterium mediated approach was used to transfer the broad-spectrum disease resistancegenes that carry fluorescent marks---the isozyme gene in to alfalfa, so as to enhance thedisease resistance of alfalfa by express the dual genes Lyz-GFP.The study on plant regeneration in vitro of3cultivars of Medicago sativa （GannongNo.1, Gannong No.3, Xinjiang da ye）, which are widely cultivated in the northwest region,was conducted. Successfully established plant regeneration system of3varieties of Alfalfa.The hypocotyls of varieties of Alfalfa were chosen as receptors for transformation of dualgenes Lyz-GFP by agrobacterium mediated, and the transgenic plants were obtained. Theinfluence factors which influenced plant regeneration and genes tranformation were alsodiscussed, and optimum conditions were defined in this paper. Furthermore, fluorescencedetection and PCR amplification test were conducted and obtained the existing proof of thetarget genes in transformational Alfalfa.The contents are listed as follows:1. Study on regeneration of3varieties of Alfalfa, high-frequency regeneration system isestablished.The different donor tissues of alfalfa were used as explants, and the callus induction,differentiation and root culture were studied. The results showed that the frequency of callusinduction and somatic embryogenesis from hypocotyls of Gannong No.1was97.2%and70.9%respectively. Xinjiang da ye take second place. Gannong No.3has lower frequency ofcallus induction and somatic embryogenesis. So the former2cultivars are optimum for plantregeneration in vitro. Hypocotyl was the optimum donor tissues for rapid and higherfrequency induction of callus. It was better to induct callus from Hypocotyl on solidified MSmedium supplemented with2.0mg/L2,4-D or2.0mg/L2,4-D and0.5mg/L KT, the highestrate of callus induction could be97.2%, and to develop somatic embryogenesis on solidifiedMS medium supplemented with2mg/L6-BA and0.5mg/L NAA, its highest frequency couldbe70.9%. It took20minutes to finish the callus induction and initiate development ofsomatic under hormone treatment mentioned above. The plants could root in1/2MS medium supplemented with1.0mg/L IBA,1%sucrose and0.7%agar. It would be good forsomatic embryogenesis on Solidified MS medium supplemented with25～30g/L sucrose.2. The transfer conditions were studied. The genetic transformation system andregeneration system of explant inoculated with Agrobacterium tumefaciens was optimized,and the transgenic Alfalfa plant were obtained.The results indicated that Kanamycin was suitable to select transformed tissue, and75～80mg/L was appropriate screening concentration for Gannong No.1and Xinjiang daye Alfalfawhile50～60mg/L was appropriate for Gannong No.3Alfalfa. Cefotaxime was appropriate asantibiotics, its action concentration was250mg/L for Gan-nong No.3and Xinjiang Daye but300mg/L for Gannong No.1. The optimum conditions for gene transformation in alfalfa wasdefined, that were Agrobacterium tumefaciens whose concentration was OD₆₀₀0.3⁰.5wasneeded to inoculate alfalfa explants for10minutes, and it was better to shake for10min inrate100r/min. The hypocotyls inoculated with Agrobacterium tumefaciens of Gan-nongNo.1and Xinjiang Daye presented stronger ability of regeneration, which were used asreceptors for genes transformation. MS supplemented with2.0mg/L2,4-D+0.5mg/L KT and2.0mg/L6-BA+0.5mg/L NAA were suitable for callus induction and redifferentiation ininoculated alfalfa respectively. The induction frequency of callus from inoculated hypocotylsof the3varieties of alfalfa achieved83.3%,87.7and81%respectively, and theirdifferentiation rate achieved43.7%,41.1%and21.4%respectivelyon in media mentionedabove.3. Follow the system of the transformation and regeneration,53resistant plants ofGannong No.1Alfalfa,5of Gannong No.3and46of Xinjiang Daye Alfalfa were obtained.The detection with fluorescent tracing taken in different development stages of transformantshowed that positive results were appeared in3plants of Gannong No.1Alfalfa and4ofXinjiang Daye Alfalfa, the gene transformation frequence was5.7%and8.7%respectively inGan-nong No.1and Xinjiang Daye. PCR amplification test were conducted on these7plants,Xinjiang Daye alfalfa transgenic plant was obtained.