| Rape is an allotetraploid plant, and can be divided into different varieties. In this experiment we seleceted "zhong shuang11" as our experiment material,"zhong shuang11" belongs to half winter Brassica napus. It is a large-scale promotion of double-low rapeseed variety, as well as through traditional breeding methods of selection of the most suitable for mechanical harvesting oilseed rape varieties. Besides, it is one of the important candidate species of transgenic modified. The transformation efficiency of oilseed rape is different under the influence of varieties and genotypes. According to the report, there is no speial transformation methods for "zhong shuang11". In this study, we had a research on a genetic transformation methods for "zhong shuang11". Research results are as follows:At first, plasmid pZP212was used to be as a template to amplify the2x35s Enhancer fragment. Then, the fragment was connected into vector pBluescript Ⅱ SK (+),the new plamid was named pWBH003. And then,another2x35s Enhancer fragment was connected into pWBH003, we get a new vector named pWBH004. pWBH004is a vector withfour forward repeat35s Enhancer fragments. At last, pWBH004was subcloned into vector pSB2GW7, we get the vector pWBH005. pWBH005is used to transform Aarabidopsis thaliana and Brassica napus "zhong shuang11". All vectors and vectors used in the middle of the experiment are correct through sequence validation.Secondly, transforming Agrobacterium GV3101with pWBH005aimed to infect Colombia wild-type Arabidopsis, then selected transgenic Arabidopsis with Basta resistance. Through this expeiment, we proof the binary-vector pWBH005can be used for transformation of plants,and it provided a groundwork for "zhong shuang11" in rape transformation. Besides, we get growing weaker, early flowering mutant type contrast with wild type.Thirdly, transforming Agrobacterium GV3101with pWBH005aimed to infect Brassica napus "zhong shuang11". At the beginning of this experiment, we test the best screening concentration of resistance genes (basta resistance) of "zhong shuang11", and went on to study the process of regeneration of transgenic plants. Experimental procedures are as follows:(1) get aseptic seedlings on1/2MS medium;(2) get hypocotyls of aseptic preparation of5d age, cut into small5-8mm, on media training for two days;(3) disseminated by Agrobacterium culture:Agrobacterium-dip30min, in training of darkness under the conditions of a total2D;(4) Screening differentiation medium for germination induced by a month;(5) Rooting medium for a month on rooting culture;(6) Acclimation of candidate transgenic plantletsThe basic medium of all of the medium used above are1/2MS medium. It was needed to add the appropriate concentration of plant hormone, screening agents, antibiotics. Sucrose content in the culture medium for15g/L or30g/L. PCR identification displayed that we got To generation of genetically modified plants. In To and T1generation plants we detected positive plants through PCR.In this experiment, due to "zhong shuang11" for winter rape, it needs vernalization of flowering induction, and in natural growing season it cannot flowered and fruited normaliy. This results in serious effects on transgenic experiments. We detected the flowering coditions of "zhong shuang11" many times to induce it flowering off-season. Then we discussed other issues such as low Luring shoots efficiecy and the detection of transgene method we Encountered in this experiment. |
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