Trehalose Synthase Gene Driven By Drought Responsive Promoter Genetic Transformation Of Sugarcane

Sugarcane(Saccharum officinarum L.), which is the important industrial material crop in China, is affected by drought stress at some time and some degree every year. Drought is the most serious environmental factor limiting production of sugarcane because more than 85% sugarcane growing area is upland and seasonal drought happens frequently. Moreover sugarcane is a nonsexual propagation crop of highly ployploidy and complex genetic background, use of traditional breeding techniques to drought tolerance will be a formidable task. In contrast, genetic engineering enables the specific up- or down-regulation of naturally occurring process or genetic modifications involving the ectopic expression of genes from non-plant sources, which will be an alternative pathway accessible.A plant expression vector, pCRBT, was constructed by ligating a drought responsive promoter, Prd29A, from Arabidopsis thaliana and a trehalose synthase gene from a basidiomycete, Grifola frondosea. It was introduced into the super-poison Agrobacterium tumerfaciens EHA105 through the "Triparental mating" method. The protocol of transformation has been optimized. Sugarcane's embryonic calli was transformed by Agrobacterium tumerfaciens-mediated method. The transformed shoots were screened under the selective pressure of PPT and 1526 shoots resistant to PPT were obtained. We used the method of PCR to detect the trehalose synthase gene and there were 85 PCR-positive plants in the 100 shoots resistant to PPT. The integration of foreign DNA into the genome of two bigger transgenic plants confirmed by Southern blotting analysis. Drought-resistance experiments showed: transgenic sugarcanes had more shoots and lower relative electrical conductivity, which demonstrated that transgenic sugarcanes showed higher level of resistance to drought-stress than non-transgenic ones.