The Establishment Of Regeneration System And Research Of Genetic Transformation Of Alfalfa(Medicago Sativa L.)

Alfalfa (Medicago saliva L.) is the most important legume forages and is called "queen of the forage" because of its importance as high quality perennial forage crop cultivated on worldwide. Alfalfa has strong deep roots and root nodules, which can effectively fix nitrogen, conserve water and soil, enhance soil fertility and improve the soil. But alfalfa often grow on low salt or the soil salinization, and its sait resistance ability is weak, belongs to the medium salt resistance crops, in the saline-alkali land is hard to grow. The rapid development of modern biotechnology provide a new method to alfalfa salt resistance genetically modified breeding. Six cultivars of alfalfa named "Algonquin’,’Affinity’,’muge’,’Sandhi ’Victoria’,"Golden Empress’were applied as materials to establish alfalfa regeneration system through callus induction and shoot differentiation. Agrobacterium-mediated method was used to transfer SeNHX1gene into alfalfa to build genetic transformation system. The main results in this study are following:1. Alfalfa regeneration system establishment(1) Cotyledon, hypocotyl, and leaf of six alfalfa cultivars were applied as explants cultured on MS+2mg/L2,4-D+0.2mg/L KT for callus induction. Results demonstrated that, hypocotyl had the highest callus induction rate, then was cotyledon, and leaf showed the lowest. When hypocotyl was applied as explant,’Algonquin’ and ’Affinity’ had the highest callus induction rate, while’Sanditi’had the least.’Golden Empress’,’Victoria", and ’muge’ were in the middle.(2) Callus from cotyledon and hypocotyl were cultured on the differentiation medium Rhc, Rhd and Rhe, respectively. Results showed that, the shoot differentiation rates of callus from cotyledon were higher than that from hypocotyl, and the highest shoot differentiation rate got on the medium MS+1mg/L6-BA+0.3mg/L NAA.(3) The effect of2,4-D and the combination of2,4-D and KT on alfalfa callus induction and sheet differentiation were studied. The results showed that,2mg/L2,4-D was good for callus formation of six alfalfa cultivars. Compared with using2,4-D alone, combined KT with2,4-D can improve the callus induction rate.’Muge’ got the highest callus induction rate with the combination of0.2mg/L KT and2mg/L2,4-D, while the rest five cultivars got the highest callus induction rate with the combination of0-mg/L KT and2mg/L2,4-D. The combination of2mg/L2,4-D and0.25mg/L or0.5mg/L KT was appropriate for alfalfa callus induction, then for shoot differentiation.(4) The cytoledon of six alfalfa cultivars were cultured on callus induction medium MSH1for7days,10days,15days,20days and60days, respectively, then transferred into differentiation medium Rhd. Results showed that,’Sanditi’needed15days’ induction to get the highest shoot differentiation rate, and ’Algonquin’,’Affinity’,’Golden Empress’,’Victoria’ needed20days, while’muge’needed60days.2. Cytological observation of callusThe callus got from cytoledon of ’Affinity’ was transferred into differentiation medium Rhd, scanning electron microscope and stereomicroscope were applied to observe embryogenic callus and non-embryogenic callus. Results showed that, crumb-structure of embryogenic cell was big in cell, and was round or oval, and was easily to separate, while non embryogenic callus were hard or water invasion, almost similar fibrous long flat cells, and cells were close together, difficult to separate.3. Genetic transformation system establishment with agrobacterium-mediated method assistanted by ultrasonic wave.Cytoledon of six cultivars got from aseptic seedlings germinated7days were applied as receptor materials, and gene SeNHX1was transferred into receptor matrrials with agrobacterium-mediated method assistanted by ultrasonic wave.Through the related parameters optimized influenced on the conversion efficiency, the results showed that the best transformation effect condition were ultrasonic wave treatment10min of the cotyledons before infection, the bacteria concentration for OD6000.5, infection10min, and dark co-culture3d.Agrobactrium tumefaciens growing was controlled by300mg/L Cef effectively on the alfalfa genetic transformation.The selection pressure of antibiotics (kanamycin,Kan).In the alfalfa callus induction stage, Kan at50mg/L was effective for’Algonquin’,’Affinity’,’Golden Empress’,’muge’,’Sanditi’;while’Victoria’requied light concentration of Kan for efficient selection of the transformants,which is at40mg/L..In the alfalfa callus shoot differentiation stage,Kan at40mg/L was effective for6varieties.After more than90days selection regenerated Kan-resistant callus were abtained. The result of PCR analysis stated that the detected Kanamycin resistant callus transgenic SeNHX1gene were all positive.