不同基因型荞麦遗传转化体系初探

Genetic Transformation System of Different Genotypes of Buckwheat: A Preliminary Study

本研究旨在利用遗传转化基础,打破荞麦杂交困难、种质创新难的瓶颈,提升荞麦研究水平。本试验以甜荞‘PI647061’和苦荞‘ZNQ152’为材料,设置正交实验对下胚轴进行组织培养,并通过农杆菌介导法侵染愈伤组织,用组织化学染色观察GUS基因的瞬时表达。试验结果表明:MS+2.0 mg/L 2,4-D+1.0 mg/L 6-BA可使下胚轴出愈率达90%以上。10种不同基因型荞麦下胚轴在MS+0.5 mg/L NAA+1.0 mg/L 6-BA中分化率最高,再生率最高达52.6%。以携带遗传转化载体pRI201-AN-GUS的农杆菌LBA4404侵染愈伤组织,经选择培养后获得再生幼苗,进而在叶片中成功观察到GUS基因表达的蓝色斑点,转化阳性率为1.23%。本研究为探索关于荞麦优良基因的转化及建立有效的遗传再生体系提供了理论和实践基础。

The aim is to utilize the basis of genetic transformation to break the bottleneck of buckwheat hybridization and germplasm innovation, and improve the research level of buckwheat. In this experiment,common buckwheat‘PI647061’and tartary buckwheat‘ZNQ152’were used as materials, and the hypocotyls were cultured by orthogonal experiment. The callus was infected by agrobacterium-mediated transformation,and the temporary expression of GUS gene was observed by histochemical staining. The results showed that MS+ 2.0 mg/L 2,4-D + 1.0 mg/L 6-BA could make the calli ratio of hypocotyls reach more than 90%. In MS +0.5 mg/L NAA + 1.0 mg/L 6-BA, hypocotyls of ten species of buckwheat had the highest differentiation rates,and the highest regeneration rate was 52.6%. The calli were infected by agrobacterium tumefaciens LBA4404,which carried the genetic transformation vector pRI201-AN-GUS. And the regenerated seedlings were obtained after selective culture, and then the blue spots of GUS gene expression were successfully observed in the leaves. The positive rate of transformation was 1.23%. This study provides a theoretical and practical basis for the exploration of the transformation of buckwheat excellent genes and the establishment of an effective genetic regeneration system.