中国农业科学 ›› 2008, Vol. 41 ›› Issue (4): 971-977 .doi: 10.3864/j.issn.0578-1752.2008.04.005

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

农杆菌介导大豆子叶节转化系统的优化

李文霞,宁海龙,吕文河,李文滨   

  1. 东北农业大学农学院
  • 收稿日期:2007-01-05 修回日期:2007-03-16 出版日期:2008-04-10 发布日期:2008-04-10
  • 通讯作者: 李文滨

Optimization of the Agrobacterium-mediated Transformation Systems of Soybean Cotyledonary Node

Wen-Xia LI Hai-Long Ning Wen-He Lu   

  1. 东北农业大学农学院
  • Received:2007-01-05 Revised:2007-03-16 Online:2008-04-10 Published:2008-04-10
  • Contact: Wen-Xia LI

摘要: 【目的】研究影响大豆(Glycine max L.)子叶节外植体遗传转化的因素。【方法】以农杆菌转化系统为平台,用直接筛选法和延迟筛选法确定卡那霉素的筛选方法和浓度,以GUS基因的瞬时表达计算子叶节区GUS阳性率。【结果】延迟7 d进行Km选择、筛选压力为75 mg•L-1时选择效果最好;菌株培养阶段和侵染浓度分别在OD600为0.6和0.5时子叶节区GUS阳性率最高;黑农35、绥农14和合丰35为易感的大豆基因型;农杆菌菌株EHA101对大豆的转化能力好于LBA4404和AGL1;共培养培养基中添加的硫醇类化合物对T-DNA的转移有极强的影响效应。【结论】本实验方法可以优化农杆菌介导的大豆子叶节转化系统。

关键词: 大豆, 子叶节, 农杆菌, 转化系统, T-DNA

Abstract: Abstract: 【Objective】The factors influencing Agrobacterium-mediated soybean (Glycine max L.) cotyledonary node transformation were studied in this research so as to improve the transformation frequency of soybean. 【Method】Delaying selection and direct selection were conducted to ascertain secelection method and density of Km. And percentage of GUS positive in cotyledonary nodes was calculated accounding to transient expression. 【Result】 The results indicated that the best selection result was reached when the explants were cultured by delaying selection 7 days and 75 ml/l of Km; The highest percentage of GUS positive in cotyledonary nodes was obtained when strain growth phases and infection concentration are at the level of OD600=0.6 and OD600=0.5, respectively; Heinong 35, Suinong 14 and Hefeng 35 were sensitive to Agrobacterium; EHA101 had advantage in transformation to soybean over LBA4404 and AGL1; Addition of thiol compounds to co-culture medium had significant effect on T-DNA delivery. 【Conclusion】This research optimized the Agrobacterium-mediated transformation systems of soybean cotyledonary node.

Key words: soybean, cotyledonary node, Agrobacterium, transformation systems, transfer-DNA