| Precocity is a necessary character in rapeseed (Brassica napus L.). With the fast development and maturation of modern biotechnology, transformation through Agrobacterium-mediation has been widely adopted in transgenic plant studies. Transformation efficiency in rapeseed was affected by many factors. Therefore, it is very important to establish an efficient and stable genetic transformation system for rapeseed. In this study, the influencing factors of transformation system of leaf petioles were studied, and the transgenic plants were positive by PCR preliminary identification. The main results are as follows:1. Leaf-petiole regeneration system of rapeseed (B. napus L. cv. ZS758) was improved and perfection to study the effect of seedling age, concentrations and combinations of exogenous hormones on leaf petiole explant regeneration efficiency. The results showed that the shoot regeneration rate of different rapeseed varieties has obvious relationship with seedling age. With the increase in time, seedling age increased and shoot differentiation frequency decline rapidly, but the downward trend and time was not found same. The best seeding age of Zheda619was six days, and the shoot regeneration rate was the65%. The best seedling age of ZS758and ZS72was five days after sowing, shoot regeneration rates were68%and65%. The study also showed that leaf petiole explants of three rapeseed varieties were very hard to have regeneration shoots when seedling age reached at ten days, thus the leaf petiole explants for the regeneration should not be too old. Callus induction ability along with2,4-D concentration increased at lower concentrations, and the calli induced by2,4-D were found green and dense. While callus induction rate decreased when the concentration of2,4-D exceeds a certain range. The favorable callus induction medium was MS+1.0mg/L6-BA+1.0mg/L2,4-D. The rooting medium with NAA concentration simulated the growth of root regeneration, and reached at a significant level as compared to rooting medium without NAA addition. The results showed that the best rooting medium was1/2MS+0.1mg/L NAA.2. The result showed that inhibitory effect on the LBA4404and EHA105on which target gene was different when same antibiotics and concentration were used. Inhibitory effect increased with the antibiotic concentration and the results varied using different antibiotics. We found that it was difficult to suppress the growth of EHA105; while growth of LBA4404was declined. So, LBA4404as target gene by using500mg/L timentin to suppress is a better choice. The studies on Agrobacterium infection time and concentration showed the browning of leaf petiole explants and pollution rate increased with the concentration of Agrobacterium and time. Browning was less and Agrobacterium was easy to control, when Agrobacterium concentration of LBA4404was OD6oo=0.2. Thus, we chose60s as infection time when Agrobacterium concentration of LBA4404was OD600=0.2. In the Kan sensitivity test, we found that Kan had inhibition on regeneration of leaf petiole explant, and callus induction rate first decreased and then increased again with the increase in Kan concentration. The results showed that the high concentration of Kan could promote leaf petiole explant callus formation, but the callus was not normal whith color and hard structure.3. The PCR results showed that the Z3OR gene in the T-DNA was fully integrated into the genome. The field experiment of transgenic plant is now under way, and further research is required in the related aspects. |
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