【摘要】 将苜蓿无菌苗下胚轴切割后，在附加２ｍｇ／Ｌ２，４Ｄ的ＭＳ培养基上诱导愈伤组织。愈伤组织在附加０５ｍｇ／Ｌ２，４Ｄ的ＳＨ培养基中悬浮培养。悬浮培养物在用于转化之前，用０４５ｍｏｌ／Ｌ甘露醇处理１ｈ，然后用０１６ｍｏｌ／ＬＣａＣｌ２·２Ｈ２Ｏ洗涤两次。预处理后的悬浮培养物用ＳＨ培养基悬浮（１０ｍｌ／ｇ悬浮培养物），再加０２ｍｌ农杆菌悬浮液，于２５±２℃共培养２ｄ。共培养的悬浮培养物洗涤后在附加０５ｍｇ／Ｌ羧苄青霉素的无激素培养基上选择培养。悬浮培养天数、悬浮培养基激素组成和选择培养基种类明显影响转化频率。纸电泳分析表明７０％的转化体可以合成农杆碱和甘露碱。染色体观察显示转化组织细胞存在严重的数目和结构变异更多还原

【Abstract】 Seeds of alfalfa were germinated on MS medium without phytohormones. The hypocotyls of seedlings were excised and cultured on MS medium with 2mg/L 2,4 dichlorophenoxyacetic acid to induce callus. Calluses were suspended and cultured in SH medium supplemented with 0.5mg/L 2,4 dichlorophenoxyacetic acid. Before transformation, alfalfa suspension cultures were treated with 0.45mol/L mannitol for 1h, and washed with 0.16mol/L CaCl 2.2H 2O for two times. After pretreatment, the pellets were resuspended in SH medium without phytohormone (10ml/g suspension cultures), and 0.2ml of Agrobacterium rhizogenes suspension was added. Mixture of alfalfa cells and Agrobacteria was co cultured at 25±2℃ for two days. Transformed calluses and hairy roots were obtained by hormone free selection. Several factors, such as culturing time of suspension cultures, phytohormone constitution of suspension medium and basal selection medium, affected the transformation frequency apparently. Paper electrophoresis revealed that over 70% transformants could systhesize agropine and mannopine. A comparative cytological observation revealed number and structural alterations of chromosomes in the resulting materials.更多还原