黄淮麦区推广品种小偃22农杆菌遗传转化体系的建立

GENETIC TRANSFORMATION SYSTEM OF WHEAT Xiaoyan 22 MEDIATED BY Agrobacterium tumefaciens

小偃22小麦是目前陕西省生产上的主栽品种,在黄淮麦区和其他地区也大面积推广种植。本文通过对小偃22小麦胚性愈伤组织的来源、生理状态和干燥时间,根癌农杆菌的接种浓度和侵染方式,AS的添加浓度,胚性愈伤组织与根癌农杆菌的共培养温度和共培养时间,潮霉素的筛选浓度和筛选方式等参数进行比较研究,建立了小偃22小麦农杆菌遗传转化体系。结果显示,以来源于幼穗的胚性愈伤组织作为转化受体较为适宜,愈伤组织生理状态为从幼穗直接诱导30d后继代1次并培养15d的胚性愈伤组织,农杆菌侵染前对胚性愈伤组织干燥处理30min较为适宜;农杆菌菌液浓度为0.8OD,采用抽真空侵染5min+非真空侵染10min的浸染方式,共培养温度为22℃,共培养时间为72h,AS的适宜添加浓度为150μmol/L;筛选方式选用在抗性愈伤组织筛选阶段经75mg/L潮霉素筛选后,在分化阶段不再进行筛选或采用25mg/L低浓度潮霉素筛选。通过重复转化验证试验和分子生物学鉴定,表明该遗传转化体系具有可行性。

The Xiaoyan 22 is the current main wheat variety in Shaanxi province and has been planted large-scale in other Huanghuai wheat area also. Optimization of parameters, including embryonic callus in different resources, physiological status and drying times, Agrobacterium tumefaciens in different concentrations and infection methods, AS in different concentrations, co-culture of embryonic callus and Agrobacterium tumefaciens in different times and temperatures, hygromycin in different concentrations and selection methods were studied. And a genetic transformation system of Xiaoyan 22 mediated by Agrobacterium tumefaciens was set up. The genetic transformation system was as follows： Firstly, the embryonic callus from immature spikes and the physiological status of callus from immature spike about 30 days and sub-cultured 15 days was good and the drying time was 30min before Agrobacteriu infection. Secondly, the concentration of Agrobacteriu was 0.80D and the Agrowbacteriu infection time was 5min in the evacuation and 10min in the non-evacuation. Thirdly, the co-culture time was 72h and co-culture temperature was 22℃. Lastly, the AS concentration was 150μmol/L, and 75mg/L hygromycine concentration in callus selection stage and 0mg/L or 25mg/L in cllus regeneration stage. The results showed that the genetic transformaion system was feasible by the repeat transformation tests and molecular biology analysis.