农杆菌介导的体外萌发花粉玉米转基因方法研究

Study on Agrobacterium-Mediated Transgenic Methods for in Vitro Germination of Pollen Maize

目前,玉米转基因方法主要采用农杆菌介导法和基因枪法,但是这两种方法都需要经过组织培养,实验条件要求较高、耗时长,且受转基因受体的基因型限制。选取杂交种苏玉19为材料,在优化玉米花粉离体萌发培养基的基础上,探索通过农杆菌侵染萌发花粉将外源基因导入玉米花粉,再将农杆菌侵染后的花粉给雌穗授粉从而获得转基因玉米。通过对培养基配方、培养温度、萌发时间、农杆菌浸染时间等技术因子的优化,建立农杆菌介导的外源基因导入离体萌发花粉的玉米转基因新方法,转化率最高可达7.27%。Southern杂交试验结果表明,外源基因整合到了玉米基因组中,该方法为外源基因导入,具有操作简单、成本低、快速培育高产新品种等优点,为玉米转基因提供技术支撑。

The main methods for maize transgenesis are the Agrobacterium-mediated method and gene gun method.However,both methods require tissue culture,which is demanding and time-consuming,and they are limited by the genotypes of transgenic recipients.In this paper,the promotional hybrid Suyu19 was randomly selected as the material and the introduction of exogenous genes into maize pollen through Agrobacterium infestation of the germinating pollen was explored.Then,the female cob was pollinated with the Agrobacterium-infested pollen to obtain transgenic maize.By optimizing the medium formulation,incubation temperature,germination time,Agrobacterium infiltration time,and other technical factors,a new method of Agrobacterium-mediated introduction of exogenous genes into isolated germinating pollen for maize transgenesis has been established.The conversion rate can reach up to 7.27%,and the results of Southern hybridization test showed that the exogenous genes were integrated into the maize genome.The method is exogenous gene introduction has the advantages of simple operation,low cost,and rapid breeding of new high-yield varieties,providing technical support for maize transgenesis.