小麦成熟胚培养方法的优化及其在小麦遗传转化中的应用

Optimization of the Tissue Culture Method Using Wheat Mature Embryos and Its Application in Wheat Transformation

为了优化小麦成熟胚培养体系,在前人报道的小麦成熟胚培养方法的基础上对小麦成熟种子处理及取胚方法进行了改进,发现胚切伤略带胚乳的取胚方法及浸泡过夜后4℃低温处理法可以提高小麦成熟胚的再生效率;从19个小麦品种(系)中筛选出5个在此培养体系下具有较高再生率的基因型,分别为农大408、农大3787、保丰104、农大3965和京花1号,其再生率分别为73.11%、68.30%、65.30%、63.40%和57.60%。进一步利用基因枪轰击法对小麦品种辽春18和保丰104成熟胚诱导的愈伤组织进行了转化,分别获得转GUS基因和转Gcn5RNAi基因的小麦T0代植株,经分子检测其转化率分别达到1.35%和1.80%。证明优化后的小麦成熟胚培养技术可以应用于小麦遗传转化研究。

Compared with wheat immature embryo,the cultured mature embryo as the explants for wheat transformation has the advantages of being independent of growing season and easy to handle.In this study,based on the reported efficient culture and regeneration system of wheat mature embryo,the preparation method of isolating embryo were modified and the genotypes with high regeneration frequency were screened.We found that the method of excising embryo with some endosperm residues after 4℃ overnight in soaking could improve the frequency of callus induction and regeneration in this wheat mature embryo culture system.Among the 19 wheat cultivars（strains） screened using the system established,the 5 genotypes with relative higher regeneration frequencies of the mature embryos were Nongda408,Nongda3787,Baofeng104,Nongda3965,and Jinghua 1,the frequencies of which were 73.11%,68.30%,65.30%,63.40% and 57.60%,respectively.In addition,the cultured mature embryos of Liaochun10 and Baofeng 104 were transformed by bombardment.The transformation rates were 1.35% for GUS gene transformation in the T0 transgenic plants of Liaochun10 and 1.80% for Gcn5RNAi gene in Baofeng 104.Our study here proved that the system of mature embryo culture could be applied to wheat genetic transformation research.