| To study the related factors influencing transformation of Brassica napus by agrobacterium-mediated floral-dip method, two double - low rapeseed materials of Brassica napus, zhongshuang No. 4 and Westar were used as the recipients, Agrobacterium tumefaciens strain C58 carrying plasmid Pky171 and GUS-CCT1 (CRY1 C end) and agrobacterium tumefaciens strain GV3301 carrying plasmid pCambial302 and 35S-GFP were used as the vector system. Resistant screening with kanamycin (Km) in transformation T1 generation was carried out and the receptor genes were further tested in the transformaed recipient plants. The main results are as follow:1. The young racemes of recipient rape materials were roughly classified into five development periods, the main inflorescence, the 1~st to 2~nd , the 3~rd to 4~th and the 5~th to 6~th branches, and transformed respectively by floral-dip method. The results of variance in transformation frequency showed no significant difference between the main raceme and the 1~st plus 2~nd branches, and between the 3~rd to 4~th branches and the 5~th to 6~th branches. The differences between the main plus the 1~st and 2~nd branches and the 3rd and 4th branches plus the 5~th and 6~th branches, however, were significant. It was shown that the development periods of rape inflorescence can be roughly grouped into two. The main inflorescence and the 1st and 2nd branches were the best period (the best branches inflorescence) for floral dipping transformation in Brassica napus.2. The surfactant Silwet-77 played an infiltrating assistant role in floral-dip transformation. The experiment results with different Silwet-77 concentrations and different dipping times showed that Silwet-77 poisoned the inflorescence to some extent. The results ofvariance analyses showed that remarkable differences among the Silwet-77 concentrations 0.02%, 0.05%, 0.1%, 0.2%, 0.5% and the control (0%). Moreover, transformation frequency in 0.05% concentration of silwet-77 was up to 1. 09%, and was obviously higher than that in other concentrations and the transformation were stable.3. The results of different dipping time showed that the efficiency of transformation in treatment of 3 consecutive dipping within 7days was higher than that in 1, 2, 4 dippings within 7d. The multiple dipping treatments every another day in a week in different Silwet-77 concentrations showed that transformation efficiency of 3 consecutive dippings every another day in a week was the highest to 2.03%, and was obviously higher than 1 and 2 times, 0.65% and 1. 18%. Therefore, it was demonstrated thatthere-time dipping treatment more appropriate in improving transformation efficiency. But because of poison effect of Silwet-77, the times of treatment should not be more than 4 times. 4. The variance analysis of the transformation efficiency in different sucrose concentrations demonstrated that the difference was significant, and SSR comparisons of the means showed that 20% (g/v) sugar concentration produced significantly higher transformation efficiency than 5%, 1% and 0%. However, if the osmotic balance of pollens in sucrose was considered, only the 15% (g/v) of sucrose concentration was the best treatment concentration.5. To study the effect of cytokinin on floral-dip transformation efficiency, 6BA and KT were used in contrast experiment with five concentrations. The result showed the transformation efficiency of KT treatment was better than that of 6BA. And 0. 01 (mg/v) of 6BA and KT both were useful in improving floral-dip transformation efficiency.6. Observations of passages of agrobacterium into the recipient plants with gus and gfp reporter gene indicated that three ways were possiblefor agrobacterium to penetrate into plants: (1) through epidermis and stomata, ( 2) through nectary, and( 3) through stigma. Only was it an effective transformation way to enter recipient plant through the stigma. So it was suggested that it was the most effective way for agrobacterium to enter the recipient plants by floral-dip transformation in Brassica napus. |
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