苜蓿植株再生和农杆菌遗传转化条件的研究

Study on the Plant Regeneration and Agrobacterium tumefaciens-mediated Transformation Conditions of Alfalfa

[目的]建立苜蓿植株再生体系和农杆菌遗传转化体系。[方法]以"猎人河"苜蓿品种为受体材料,对影响苜蓿植株再生和遗传转化的相关因素进行了研究。[结果]最佳愈伤诱导培养基为改良SH+4.0 mg/L 2,4-D+0.5 mg/L 6-BA,最佳继代培养基为MSO+0.5mg/L NAA+1.0 mg/L 6-BA+1.0 mg/L AgNO3,而最佳分化培养基为MS,苜蓿植株再生的最佳外植体是下胚轴。苜蓿农杆菌遗传转化的选择压确定为60 mg/L卡那霉素(Kan),最适宜的抗菌素为350 mg/L羧苄青霉素(Carb),农杆菌菌液的最适OD600为0.6,最佳侵染时间为10 min。[结论]该研究为今后苜蓿的基因工程研究奠定了基础。

[Objective] The research aimed to establish the plant regeneration and Agrobacterium tumefaciens-mediated transformation system of alfalfa.[Method] Using alfalfa variety ＂Lierenhe＂ as recipient material,the related factors that affected the plant regeneration and genetic transformation of alfalfa were studied.[Result] The optimal callus induction medium was improved SH＋4.0 mg/L 2,4-D＋0.5 mg/L 6-BA,the optimal subculture medium was MSO＋0.5 mg/L NAA＋1.0 mg/L 6-BA＋1.0 mg/L AgNO3 and the optimal differentiation medium was MS.The optimal explant for the plant regeneration of alfalfa was hypocotyl.The best selection pressure for the genetic transformation of alfalfa mediated by A.tumefaciens was confirmed as 60 mg/L Kanamycin（Kan）.The optimal antibiotic was 350 mg/L carbenicillin（Carb）.The optimal OD600 of A.tumefaciens liquid was 0.6 and the best infection time was 10 min.[Conclusion]The research laid the foundation for further study on the genetic engineering of alfalfa.