植物学报 ›› 2010, Vol. 45 ›› Issue (04): 472-478.DOI: 10.3969/j.issn.1674-3466.2010.04.011

• 技术方法 • 上一篇    下一篇

农杆菌介导南瓜遗传转化体系的建立

付洪冰1; 崔崇士1*; 赵曦2; 刘琦2   

  1. 1东北农业大学园艺学院, 哈尔滨 150030; 2黑龙江省农业科学院生物技术研究所, 哈尔滨 150086
  • 收稿日期:2009-03-09 出版日期:2010-04-30 发布日期:2010-09-26
  • 通讯作者: 崔崇士

Establishment of Cucurbita moschata Genetic Transformation System by Agrobacterium tumefaciens Transfection

Hongbing Fu1; Chongshi Cui1*; Xi Zhao2; Qi Liu2   

  1. 1Horticulture College, Northeast Agricultural University, Harbin 150030, China;
    2Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
  • Received:2009-03-09 Online:2010-04-30 Published:2010-09-26
  • Contact: Chongshi Cui

摘要: 以南瓜金辉一号(Cucurbita moschata ‘Jinhui 1’)为实验材料, 利用根癌农杆菌(Agrobacterium tumefaciens)介导转化南瓜子叶节, 研究了预培养时间、侵染时间、乙酰丁香酮(AS)浓度和共培养时间, 抗生素羧苄青霉素(Carb)、头孢霉素(Cef)以及筛选剂卡那霉素(Kan)等因素对离体不定芽的影响, 建立了南瓜最适遗传转化体系。结果表明: 外植体预培养0天,侵染时间30分钟, AS浓度为100 mg·L–1, 共培养5天可获得最高遗传转化效率; 最适除菌剂为Cef, 其最适浓度为500mg·L–1; 最适Kan筛选浓度为100 mg·L–1; 在MS培养基上培养抗性芽生根, 经PCR和Southern blot检测, 证明为转基因植株。

Abstract:

Establishing a transformation system is fundamental to transgenic study. We developed an efficient Agrobacterium tumefaciens-mediated transformation and screening system for Cucurbita moschata ‘Jinhui 1’. The leaves from tissue culture were excellent explants for transformation. To construct a genetic transformation system, factors affecting the transformation frequency included pre-culture time of explants, infection time, and concentration of acetosyringone (AS). Additionally, co-culture time and the concentration of antibiotics (Carb, Cefotaxime and Kanamycin), which inhibit the growth of Agrobacterium, were also examined in the adventitious bud. The data were analyzed by DPS software, and a suitable genetic transformation system was established. Pre-culturation for 0 days, infection for 30 min, AS at 100 mg·L–1 and co-culture for 5 days produced the highest transformation efficiency. The medium with Cefotaxime (500 mg·L–1) and Kanamycin (100 mg·L–1) was optimal for selecting transgenic plant cultivation-resistant shoots on MS for eventual root induction. PCR and Southern blot results showed the Kanamycin-resistant exogenous gene had been integrated into the genome of Cucurbita moschata.