Trehalose Synthase Gene Genetic Transformation Of Sugarcane(Saccharum Officinarum L.) Mediated By Agrobacterium Tumefaciens

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% of sugarcane growing area is upland and seasonal drought happens frequently. Moreover sugarcane is a nonsexual propagation crop of highly polyploidy and complex genetic background, use of traditional breeding techniques to improve 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, pBBBT, was constructed by inserting trehalose synthase gene from a basidiomycete, Grjfoiafrondosea, into pBBB, with a 2 copy CaM V35S as promoter and bar as selectable marker gene. Then, It was introduced into Agrobacterium tumefaciens strain EHA 105 via triparental mating. The protocol of transformation has been optimized. Sugarcane抯 embryonic calli was transformed by Agrobacterium tumefaciens-mediated method. The transformed caili and shoots were screened under the selective pressure of PPT. The transformed plants were detected by PCR and dot Southern bolting. Meanwhile, Introduction of tobacco(Nicoiiana tabacum L.) transformation and Escherichia coil expression of the genes to identif~r the ectopic expression of the gene and its function. In order to compare the expression efficiency of different types of promoters and different pathways of trehalose synthesis in transgenic sugarcane, a drought responsive promoter, prd29A, form Arabidopsis thaliana and a trehalose-6-phosphate synthase gene, OtsA, from Escherichia coil were cloned. The results were as follows: I A plant expression vector, pBBBT, was constructed by inserted the trehalose synthase gene from Gnfola frondosa into pBBB, which contained a double CaMV3SS promoter and a PPT resistance gene(bar). 2 2 Transgenic sugarcane plants were recovered from co-culture of embryonic calli with Agrobacterium turn efaciens, 56 transformants were obtained after long PPT resistance selection, 8 of them have been detected by PCR and dot Southern blotting, and the DNA of 3 plants showed positive. 3 An efficient protocol for sugarcane transformation mediated by Agrobacterium turnefaciens has been established. Some factors may be important of the success:( I )Embryonic calli were induced as competent receptor cells, including subculture on fresh medium for a few days and dried treatment before infection, (2)Supervirulent strain EHA 105 was used;(3)Several transformation conditions were combined to improve the activation of vir genes and T-DNA transfer, such as added AS, fructose and glucose,and acid infection condition and low co-culture temperature;(4)bar gene, expression to resist PPT, was used as selectable marker gene. 4 The developmental process of calluses and embryoids have been investigated. 5 Trehalose synthase gene transformation of tobacco, mediated by Agrobacterium tumefaciens, was carried out. 6 A drought responsive promoter, Prd29A, from Arabidopsis thaliana and a trehaiose-6-phosphate synthase gene, OtsA, from Escherichia co/i were cloned. 7 Some obvious morphological changes including d...