A Transformation System For Transferring BnTR1 Gene Into Indica Rice Mediated By Agrobacterium Tumefaciens

Rice (Oryza sativa L.) have brought a huge sum of economic effectiveness to the global economy, as one of the three cereal crops and the staple food by more than half of the global people. Transgenic technology has been an useful and effective strategy to enhance rice yield and quality. To date, most researches of the transgenic rice have been concentrated on japonica rice and constructed a complete genetic transformation system, but no effective approaches were found for indica rice, which is much more difficult than japonica rice. It is very significant in production practice to solve the problem of transformation in indica rice, especially some cultivars used as parents in hybrid rice breeding, such as restorer line shuhui 527(R527), maintainer line D62B.BnTRl containing a heat response element(HSE) is a gene which could improve temperance resistance of plants and other species. It may be effective to reduce the damage of production reduction of rice caused by high temperature, through transforming BnTR1 into indica rice cultivars. In this research, we were aim at exploring appropriate transformation conditions about our rice cultivars mediated by Agrobacterium tumefaciens used four vectors, a simple vector Pzh01-TR1 in Agrobacterium tumefaciens strain LBA4404, a binary vector pSB130-35S-TR1 in EHA105, a vector without any selective marker gene pCambia-35S-TR1,and a vector pCambia-35S-TR1-EPSPs used EPSPs as the marker gene.. Several indcia rice cultivars were used as materials to transform, such as restores line shuhui 527(R527), maintainer line D62B and some others. BnTR1 could be transformed successfully and verified by molecular and physio-biochemical detection:1. Overall, mature embryo was more suitable than immature embryo as explants, for the simplicity of operation, little pollution and have not a season limitation, while immature just could be used 12-15d after flowering.2. Indica rice could be induced calli used 2,4-D 2.5mg/L, in which it has a higher efficiency 80%. Calli have good qualities as dry compact and yellowish.3. Hyg (40-50 mg/L) was enough for selecting resistance indica rice calli, and the concentration was different for different cultivar, especially,40 mg/L for R527 selection and 50 mg/L for D62B. We also have found that calli's growing ability could be enhanced after several selections. Furthermore, concentration of glyphosate was 500mg/L used for indica rice selection.4. The regenerated plants had been detected by PCR transformed by the 4 vectors. There were 18 positive plants out of the 84 regenerated plants transformed by Pzh01-TR1, 24 out of 102 by pSB130-35S-TR],16 out of 57 by pCambia-35S-TR1-EPSPs, respectively. But no positive plants had been detected in the all regenerated plants transformed by pCambia-35S-TR1 without any selective maker gene.5. The transform results of the 4 vectors in this research had no obvious difference totally, just pSB130-35S-TR1 had a little higher selectable efficiency(50.61%), and pCambia-35S-TR1-EPSPs had a little larger rate of emergence (71.25%)relatively.6. It was speculated that marker gene hyg could be inherited into T1 transgenic plants, corresponding to the Mendel segregate ratio of 3:1 as a single-gene dominance.