基因枪法介导大白菜小孢子转基因技术研究初报

Transformation of Chinese Cabbage Microspores by Particle Bombardment

围绕基因枪微粒的制备、大白菜小孢子对基因枪轰击的耐受能力以及小孢子的胚胎发生能力开展研究,初步建立了以小孢子为外植体的遗传转化体系。高胚胎发生的大白菜材料‘CC-4’的小孢子在基因枪7.58×10~6 Pa爆破压力,9 cm的轰击距离下,利用直径0.6μm,亚精胺包裹的金粒子微弹以每次500μg的金粒子量连续轰击5次,小孢子活力不受影响,培养1 d后GUS基因瞬时表达检测,平均每皿可获得20个以上转化小孢子;小孢子培养15 d后成功获得胚状体,GUS基因瞬时表达检测,平均每皿最多获得4个遗传转化的胚状体,40个花蕾可获得20个以上转化胚状体。初步探讨基因枪法介导大白菜小孢子结果表明:大白菜小孢子数量庞大易收集,可耐受基因枪的多次轰击,并能继续保持胚胎发生的能力,作为新尝试的遗传转化受体,有可能是解决白菜类作物再生困难,提高转基因效率的有效手段。

Chinese cabbage transient GUS gene expression was achieved when microspores were bombarded with gold particles coated with plasmid DNA. Helium rupture pressures of 7.58 ×10^6 Pa and the particle size of 0.6 μm did not significantly affect the viability of microspores of Chinese cabbage. Under optimized bombardment conditions for Chinese cabbage microspores（7.58 ×10^6 Pa rupture pressure,stopper-to-target-cell distance of 9 cm,0.6 μm particle size,500 μg gold particles per bombardment,and five shots）,more than 20 GUS-positive microspores per petri dish were observed after 1 day of culture. The highest number of stably transformed embryos was four per dish in our test results. Collection of microspores of 40 buds,more than 20 transformed embryos were observed. The results of this study suggest that particle bombardment of microspores could be an alternative method for genetic transformation of Chinese cabbage,and may help advance the breeding research and the gene functional analysis of Chinese cabbage.