Construction Of Twin T-DNA Binary Vector Of TR1Gene And Its Transformation Into Brassica Napus

Brassica napus L. is an important oil crop with high economical value. With the development of global climate warming trend, high temperature induced maturity which cccurred during the ripening of rapeseed greatly affects the quality and yield of rapeseed. Transferring thermal resistance gene TR1into rapeseed has geat application value in the field of thermal resistance in the plant genetic engineering. In this study, rapeseed hypocotyls segments were used as explants transformated by Agrobacterium-mediated transformation. Some factors affecting transformation and shoot regeneration were studied, and the regeneration system of rapeseed hypocotyls was optimized. The TR1gene was transferred into Brassica napus with Agrobacterium-mediated method. The main results were as follows:1. Establishment of the regeneration system of rapeseed hypocotylsThe effect of two different sterilization methods were studied by using the rapeseed. The results showed that the better sterilization method was using ethanol and NaCIO because it can guarantee sterilization, and not significantly lower germination rate of rapeseed. The hypocotyls of green and etiolated rapeseeds were tested for callus induction. The results showed that when green rapeseed seedlings were used as explants, the induction rate was higher than that of etiolated ones. The more effective method is that hypocotyls were inoculateed and co-cultureed with Agrobacterium tumefacience in liquid co-culture medium for3days. We also found that delayed cultivation before selection for4days could enhance the callus introduction and shoot differentiation ratios compared with selection after co-culture. The suitable concentration of Kan and Hyg were10mg/L and2.5mg/L, respectively.2. Detection of transformants of rapeseeds with TR1gene284regenerated plants were tested by PCR analysis,2positive plants with TR1gene were detected, the positive plant frequeney of transformation was3.7%. They were detected by PCR-Southern analysis and Southern blotting, and the results showed that the exogenous gene had intergrated into the genome of rapeseeds.3. In order to delete antibiotic genes used in the transformants, the twin T-DNA binary vector pSB130-TR1was constructed, and could be transformed into rapeseeds to obtain marker-free transformants.