Abstract: In this study, the leaf of Medicago sativa ‘Xinjiang Daye’was used as transformation receptor. BADH (betaine aldehyde dehydrogenase) gene derived from Haloxylon was transformed into the alfalfa by Agrobacterium tumefaciens. To establish the ideal transformation system, the effect of kanamycin on callus differentiation was studied. The four main factors affecting transformation efficiency were optimized through orthogonal experimental design. Results showed that the upper limit concentration of kanamycin affecting callus differentiation was 50 mg·L^-1. The treatment Ⅳ was the best combination and the ratio of resistant bud differentiation was 65.5%. Bacterium concentration had the greatest effect on the ratio of transformation among the four factors, followed by cephalosporin concentration, infection time, and the preculture-time of explants had minimal impact. The BADH gene was preliminarily proved to integrate into the genome of alfalfa by PCR and RT-PCR identification. The transformation frequency was 16.0%。

Key words: Alfalfa (Medicago sativa), Transformation, Agrobacterium tumefaciens, BADH gene, Transgenic plant

摘要： 以新疆大叶苜蓿（Medicago sativa ‘Xinjiang Daye’）叶片作为转化受体，利用农杆菌介导法将从梭梭（Haloxylon ammodendron）中获得的甜菜碱醛脱氢酶基因（BADH）转化紫花苜蓿，研究选择剂卡那霉素对愈伤组织分化的影响，并采用正交试验设计优化了影响紫花苜蓿转化的4个主要因子，建立了农杆菌介导的紫花苜蓿高效转化体系，获得了转基因植株。结果表明：卡那霉素在愈伤组织分化培养时的上限浓度以50 mg·L^-1为宜；转化过程中当浸染时间为5 min，菌液浓度OD₆₀₀为0.2，外植体预培养6 d，共培养后清洗外植体的头孢雷素（Cef）浓度为800 mg·L^-1是最佳转化方案，抗性芽分化率达到65.5%；4个因子中菌液浓度对转化率影响最大，清洗外植体的Cef浓度和浸染时间影响次之，外植体预培养时间的影响最小。经PCR和RT-PCR检测，初步证明了BADH基因已经整合到紫花苜蓿中，转化率为16.0%。

关键词: 紫花苜蓿, 转化, 农杆菌, BADH基因, 转基因植株