根癌农杆菌转化棉花花粉的研究  

Study on Transformation of Cotton Pollen Using Agrobacterium Tumefaciens

作　　者：李晓[1] 王学德[1] 

机构地区：[1]浙江大学农业与生物技术学院,杭州310029

出　　处：《棉花学报》2004年第6期323-327,共5页Cotton Science

基　　金：国家高技术研究发展计划(863计划;2001AA212081);国家自然科学基金(30170058);国家教育部科研基金(0114);浙江省科技攻关基金(021102089)资助

摘　　要：用根癌农杆菌感染棉花花粉获得的含有外源基因的花粉可与卵细胞自然结合,产生的转化细胞体不存在转化细胞与非转化细胞间的嵌合现象等问题,且转化方法简单,易操作。由于这种感染发生在花粉培养液中,为保证感染过程中花粉不破裂并具有生活力,以及使农杆菌有较高的感染力,必须有适宜的培养液,包括适宜的营养元素和渗透压。研究表明,在含有0.1%H3BO3、0.3%Ca(NO3)2、0.2%MgSO4·7H2O、0.1%KNO3以及45%蔗糖的培养液中培养的花粉,能够保证有95%以上的花粉不破损且有生活力,且其萌发率平均可达58.5%。转化后的花粉经瞬时GUS检测和转化后花粉破损率表明,在附加有1‰体积比的Tween 20的含有农杆菌的花粉培养液中进行抽真空处理的方法是较有效的转化方法。所获得的种子萌发后经卡那霉素筛选、GUS稳定表达检测以及Northern杂交验证,获得4棵转基因植株。The pollen-mediated transformation approach could not only circumvent the tedious tissue culture procedures like in Agrobacterium infection, but also circumvent the incertitude of the pollen tube path-way. So,its transformants combined from the transformed pollen and eggs would not be chimera.At last this approach is simple, easy to operate, and could be widely used in practice. For the transformation happened in the pollen culture medium, the solution with a suitable solute has to be selected. This study showed the medium consisting of (0.1%) H_3BO_3 、(0.3%) Ca(NO_3)_2、(0.2%) MgSO_4·7H_2O、(0.1%) KNO_3 and 45% sucrose could maintain normal turgidity of the pollens and facilitate them to be germinated. There are more than 95% pollens don't dispose in this medium and the germinating rate was up to 58.5%. A transient GUS expression assay and the exploded pollen rate proved that the transformation method with 1‰ Tween-20 in the medium using vacuum-infiltration was a better method. Finally, four transformed seedlings were confirmed by Kanamycin selection, GUS expression and Northern blot. Therefore this transformation approach is available.

关 键 词：棉花 花粉 根癌农杆菌 基因转化 真空渗透 生活力 

分 类 号：S562.35.3[农业科学—作物学]