Studies On Genetic Transformation Of IPT Gene Into Grain-Straw-Dual-Use-Rice (GSDUR)

During late growth period of rice, premature leaf senescence decreases photosynthetic rate, which lowers the assimilation capability gradually and prevents rice yield from further increase. Cytokinin is an important phytohormone involved in delaying senescence of plant leaf, and IPT gene encodes the key enzyme that catalyzes cytokinin biosynthesis. Transformation of chimeric gene which consists of promoter specific to leaf senescence expression and DPT gene can specifically increase cytokinin of transgenic rice and delay leaf senescence effectively.Grain-Straw-Dual-Use-Rice (GSDUR) is a type of rice cultivar with both grain and straw useful. GSDUR was used as receptor for genetic transformation in this research. High-frequency regenerating system and genetic transformation system were established, based on which chimeric gene PSAG12-IPT was transformed and relative molecular tests were conducted. The main results of this research are as follows:1.Establishing GSDUR high-frequency regenerating systemThe effects of explant, media, ingredient of different hormones and generation number of callus subculture on GSDUR callus induction and regeneration were studied and a high-frequency regenerating system for GSDUR was established. The best explant for induction was juvenile embryo 12-15 days after pollination. The best callus-induction and subculture media was NB. The best hormone ingredients of differentiation media was 0.05mg.L"1NAA+2.00mg.L"16-BA, and differentiation rate was highest with two generations of callus subculture. The callus induction rate of the system reached 98.5% and differentiation rate 66.1%.2. Establishing effective transformation system mediated by AgrobacteriumThe effects of Agrobacterium infection solution concentration, callus precultivation days, cocultivation days and the acetosyringone concentration on GUS transient expression rate were investigated and four important parameters of GSDUR high-efficiency transformation were established. (1) The most suitable infection solution concentration was approximately OD=1.0~1.5. (2) Cocultivation time should not exceed 3 days. (3) 4 days of callus precultivation increased transformation rate. (4) The suitable concentration of the added acetosyringone in cocultivation was 100 μM.3.Transforming IPX gene to GSDUR and transgenic plants was obtainedOn the basis of the above mentioned genetic transformation system, chimeric gene PSAG12-IPT was transformed to the embryonic callus tissue of GSDUR through Agrobacterium-mediated method. After resistance screening by hygromix, the resistant calli differentiated to seedlings, and 46 resistant callus clones and 126 transgenic plants were obtained. PCR testing and GUS staining analysis showed that 80 of them were positive, the transformation frequency was 5.9 %.