Agrobacterium-mediated Genetic Transformation Of Rice Cultivar Taijing 9 With IPT And PEPC Gene

The majority of terrestrial plants assimilate atmospheric CO2 through the C3 photosynthetic pathway, and the enzyme of primary CO2 fixation in this pathway is ribulose 1,5-bisphosphate carboxylase/oxygenase(Rubisco). In addition to the C3 pathway, C4 plants use the C4 photosynthetic cycle to elevate the CO2 concentration at the site of Rubisco. One of the key enzymes in C4 pathway is phosphoenolpyruvate carboxylase (PEPC) that has higher affinity for CO2 than Rubisco. The C4 plants have up to twice photosynthetic rate than C3 plants. Consequently, it has long been expected that the transfer of C4 traits to C3 plants could improve the photosynthetic performance of C3 species.The photosynthetic rate decreases due to leaf senescence during the late growth period of rice. It is reported that the biomass can be elevated by 2% if the senescence can be delayed for one day. Cytokinin is an important phytohormone involved in delaying senescence of plant leaf, and isopentenyl transferase (IPT) gene encodes the key enzyme that catalyzes cytokinin biosynthesis. Transformation of chimeric gene which consists of leaf-specific promoter (SAG 1 and SAG 2) of the senescence expression and IPT gene can specifically increase cytokinin of transgenic rice and thus delay the leaf senescence effectively.The regenerating system of rice cultivar Tai 9 is established in this experiment. Using an Agrobacterium-mediated transformation system, we haveintroduced both the chimeric gene pSAG12-IPT and marker-free pCAMBIA1300-Ubi-PEPC-NOS into the embryogenic calli of elite japanica rice variety Taijing 9 through co-transformation method. The calli were selected on the NB media plus with hygromycin. And then 36 clones of calli resistant to hygromycin were obtained and differenciated into the plantlets. A total of 41 primary transgenic rice plants were obtained from antibiotic-resistant cells. Sixteen transgenic rice plants were determined to be positive through histochemical GUS assay and PCR assay, and 10 rice plants contained IPT gene and 6 rice plants contained PEPC gene, and 3 plants contained both IPT and PEPC gene among them. The co-transformation rate was 18.75%.The maize intact PEPC gene (8.5 kb) including all exons, introns, promoter and terminator was constructed on plant binary expression vector pCAMBIA1300 during rice genetic transformation. The second genetic transformation will be conducted on the basis of obtaining marker-free transgenic rice plant containing PEPC.Photosynthetic rates of 16 transgenic rice plants were measured under ambient conditions. The results showed that 8 of them were higher 6.26%~73.22% and 2 of them were lower 9.17% and 20.67% tha.n that of the control among all transgenic rice containing IPT gene, and that 7 of them were higher 48.76%~63.46% and 2 of them were lower 16.89% and 31.88% than that of the control among all transgenic rice containing PEPC gene in terms of photosynthetic rate. The transgenic rice plants containing both IPT and PEPC gene had a higher photosynthetic ratethan that of control and the highest one was improved by 88.65%.