农杆菌介导快速、高效获得转基因烟草纯合株系

利用植物表达载体PBI121空载体,对农杆菌介导法转化烟草技术体系进行了综合改进,探索激素配比、侵染时间、分化和生根培养阶段卡那霉素的筛选压对烟草(Nicotiana tabacum cv.Petit Havana SR1)植株再生和转化效率的影响；同时根据孟德尔显性性状杂合子自交后代遗传分离规律,在转基因株系后代扩繁时用卡那霉素筛选分离,可以在T2代较快得到纯合株系.本研究通过使用整个叶片浸染、高的卡那霉素筛选剂浓度、控制生长条件以及强化管理,极大地缩短了获得纯合株系的时间,确立了该烟草品种快速、高效获得大量转基因阳性植株及快速获得转基因纯合株系的转化体系,其转化周期可减少至5-6个月,转基因植株阳性率可达到90％,其中60％的转基因植株后代卡那霉素抗性性状分离比例接近3:1.

Rapid and efficient production of homozygous transgenic tobacco plants with Agrobacterium tumefaciens

Key factors such as composition of plant growth regulators,time period of Agrobacterium infection and concentration of selective agent kanamycin in medium were studied by inoculating leaf tissues with Agrobacterium tumefaciens strain LBA 4404 containing pBI121 plasmid to optimize tobacco genetic transformation system.Flowers from primary transformants were bagged to allow self-pollination,seed capsules harvested and dried prior to normal maturation,and T2 seedlings were plated and selected on kanamycin media.T2 homozygous transgenic lines were acquired in a short time period..An efficient complete protocol for Agrobacterium tumefaciens-mediated transformation of tobacco(Nicotiana tabacum cv.Petit Havana SR1) has been established,producing T1 flowering plants homozygous for the inserted T-DNA as verified by kanamycin resistance in T2 seedlings in 6 to 7 months from the time of cocultivation with Agrobacterium.Procedures unique and important to this protocol include: a modified "whole-leaf" transformation,high levels of kanamycin in selection media resulting in few escapes,and reinforced management of water control and light condition in greenhouse.These measures greatly shortened the time to seed set.Using this protocol,a large amount of independent tobacco lines were generated in a very short time period.Of these transgenic plants,nearly 60% segregated 3:1 for kanamycin resistance to susceptibility,indicating single transgene insertion.