紫花苜蓿毛状根转化体系的建立及应用

Establishment and Application of Hairy Root Transformation System of Alfalfa

以紫花苜蓿品种WL-525HQ为材料,用发根农杆菌介导毛状根转化,分析毛状根分化率及转化率,筛选最优的转化方案,以快速获得紫花苜蓿转基因材料。结果表明:当发根农杆菌侵染苜蓿叶片的菌液浓度OD_(600)达0.5时,毛状根分化率可达72%;筛选培养基中添加5~8mg/L潮霉素时,毛状根的分化率及转化率都相对较高,分别为30.7%~33.2%和59.7%~67.9%,转化方案可靠;在以毛状根为外植体诱导全株时,采用羧苄青霉素能够有效抑制细菌,5mg/L潮霉素对愈伤组织的分化效果最好。优化了以毛状根为外植体探究紫花苜蓿全株的转化方案,并应用于耐铝基因MsLEA2研究,为紫花苜蓿功能基因研究提供了一种快速有效的手段。

In order to obtain alfalfa transgenic materials quickly,alfalfa variety WL-525HQ was used as material,Agrobacterium rhizogenes was used to mediate hairy root transformation,the differentiation rate and transformation rate were analyzed,and the optimal transformation scheme was selected.The results showed that when the OD_(600) concentration of LBA9402 was 0.5,the differentiation rate of hairy roots was the highest(72%);When 5~8mg/L Hygromycin was added to the screening medium,the differentiation rate and transformation rate of hairy roots were relatively high,which were 30.7%~33.2%and 59.7%~67.9%respectively,indicating that the transformation scheme was reliable;When the whole plant was induced with hairy roots as explants,it was found that carbbenicillin could effectively inhibit miscellaneous bacteria,and 5mg/L hygromycin had the best effect on callus differentiation.This study optimized the transformation scheme of alfalfa whole plant with hairy root as explant,and applied it to the study of aluminum tolerance gene MsLEA2,which provide a fast and effective means for the study on functional genes of alfalfa.