百脉根农杆菌快速高效遗传转化体系的建立  

Establishment of an efficient Agrobacterium-mediated genetic transformation system of Lotus corniculatus

作　　者：刘建利[1] 张占路[1] 吴燕民[1] 唐益雄[1] 郭蔼光[2] 陈两桂 

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]西北农林科技大学生命科学院,陕西杨凌712100 [3]深圳市农科集团,广东深圳518040

出　　处：《草业学报》2006年第3期128-131,共4页Acta Prataculturae Sinica

基　　金：深圳市科技局基金项目资助

摘　　要：以百脉根品种“里奥”作为受体材料,以GUS基因为报告基因,研究了影响农杆菌介导的百脉根遗传转化的几种因素及表面活性剂(PEG 4000)、真空处理和乙酰丁香酮对提高转化效率的影响,建立了农杆菌介导的百脉根快速高效遗传转化体系,并获得了转基因抗性苗。结果表明,以子叶(带叶柄)为外植体,在OD600为0.5的菌液中,加入150 mg/L的PEG 4000,真空度6×10-2Pa,抽真空12 min,共培养3 d,转入含卡那霉素50 mg/L和羧苄青霉素300 mg/L的分化培养基中,约20 d后,50%的外植体分化不定芽,生根成苗。PCR初步检测表明有83%的植株为GUS阳性,转化率约为42%。An efficient Agrobacterium-mediated genetic transformation system of Lotus corniculatus was established and optimized using plant transformation vector pBI121. Several factors such as Agrobacteriurn concentration, infection time, infection methods used and co-culture time, which may affect the transformation frequency were optimized. The highest transformation frequency was obtained when cotyledons with petioles were used as explants. Cotyledons with petioles of L. corniculatus were first infected with Agrobacterium suspension （OD600≈ 0.5） containing 150 mg/L PEG 4000 under 6×10^-2 Pa for 12 rain. They were then co-cultured for 3 d and after further culturing for 20 d on a differentiation medium containing Kanamycin （50 rag/L） and Carbenicillin （300 mg/L）, shoots could be obtained from 50% of the explants. After several rounds of selection, roots could grow into seedlings. The GUS gene was detected in ca. 83% of the seedlings obtained by PCR, and the transformation frequency was up to 42%,

关 键 词：百脉根 遗传转化 根癌农杆菌 GUS基因 

分 类 号：S541.6[农业科学—作物学]