本生烟组织培养及遗传转化体系的建立  

Construction of tissue culture and genetic transformation systems of Nicotiana benthamiana

作　　者：崔海涛[1] 李春霞[1] 王红艳[1] 陈红运[2] 竺晓平[1] 时呈奎[1] 李向东[1] 

机构地区：[1]山东农业大学植物保护学院,山东泰安271018 [2]国家质检总局动植物检疫实验所,北京100029

出　　处：《山东科学》2006年第1期23-27,共5页Shandong Science

基　　金：国家自然科学基金(30100117);山东省优秀中青年科学家奖励基金(2004BS06002)

摘　　要：本文研究了通过农杆菌侵染获得本生烟（Nicotiana benthamiana）转基因植株的方法。将本生烟中上部叶片消毒后切成5mm×5mm左右的小块作为外植体，在含有6-BA（3mg／L）和NAA（0．2mg／L）的MS培养基中培养2—3d，用含有表达载体的农杆菌侵染后共培养3～4d，转入含有6-BA（3mg／L）、NAA（0．2mg／L）和卡那霉素（100rag／L）的MS培养基中培养2～3周。将外植体边缘产生的愈伤组织和芽点转移到只含有卡那霉素（100mg／L）的MS培养基中继续培养，2周后分化出大量不定芽。继续培养2周，当芽长1-3cm时，将芽转移到含IBA（0．1mg／L）的MS培养基中分化生根，得到苒生植株。当再生植株高8～10cm、根长4～5cm以上时，移栽到经过灭菌的营养土中。通过实时荧光PCR扩增，从获得的植株中检测35S启动子和NOS终止子外源基因片段，判定所得的植株的确为转基因植株。最后对本生烟组织培养中应该注意的问题进行了讨论。The procedure to obtain regenerated Nicotiana benthamiana plants was explored in this paper. About 5mm × 5mm tobacco leaf discs were excised and inoculated to solid MS medium containing 6-BA （3mg/L） and NAA （0.2mg/L） for 2 - 3 days, then co-cultivated in dark for 3 - 4 days with agrobacterium strain LBA4404 harboring the plasmid pROK Ⅱ that carrying 35S promoter, NOS terminator and kanamycin resistant genes. The leaf discs were transferred to solid MS medium that contained 6-BA （3mg/L）, NAA （0.2mg/L） and kanamycin （100mg/L） as selective markers for transgenic plants. After ca. 2 - 3 weeks, resistant calli could be obtained. They were further transferred to solid MS medium that only contained kanamycin （100mg/L）. The calli were expected to become shoots. Two weeks later, the shoots that were 1 - 3cm long were transferred to MS rooting medium that contained 1BA （0.1mg/L）. Those shoots usually developed roots in 2 weeks. When the plantlets were about 8 - 10cm high and had 4 - 5cm longroots, they were transferred into pots filled with sterilized nutrient soil. Through amplification of 35S promoter and NOS terminator genes by Real-time fluorescent PCR, the regenerated N. benthamiana were confirmed to be transgenic ones. Some considerations on the key steps in regenerating N. benthamiana plants were discussed.

关 键 词：本生烟 组织培养 MS培养基 实时荧光PCR 

分 类 号：S572.035.3[农业科学—烟草工业]