| Rapeseed (Brassica napus L.), which is an important cash crop and a major source of edible oil both in China and around the world, is the second important source of vegetable oil to soybean and one of the world's fastest developing multi-purpose crops. At present, both the cultivated area and yield of Brassica napus in China rank top around the world, but the yield of per unit area is much lower than that of some countries, such as Germany, Denmark and other European high-yield countries. In recent decades, as the population continues to roar, the dramatic deterioration of environment, and the gradual reduction of arable land, the contradiction between population and land is becoming more and more obvious and aggravating, which makes the cultivation of rapeseed breed with super high yield the most important research task at present. However, considering the fact that traditional breeding methods is time-consuming and with heavy workload, difficult to find natural mutants and uncertainty of mutation breeding, as well as the development potential is also very limited, people have begun to seek new technologies that can overcome barriers of conventional breeding methods. With the continuous development of molecular biology and the maturity of transgenic technology, the use of genetic engineering technology to achieve the desired crop varieties has become the preferred breeding method for excellent genetic improvement workers. Cytokinin can promote directional transportation of nutrients, fruit bearing during the process of seed and fruit development and accumulation of assimilates of fruit and seeds, in addition, it can also promote the development of endosperm and enlargement of fruit body. Therefore, using genetic engineering methods to control the level of endogenous cytokinin, provides a new direction for further improving the yield of rapeseed. In this study, we used stage-specific seed development gene, ipt gene expression vector, which is a fusion combination of ipt gene (Cytokinin synthesis key enzyme gene) and Napin (rape seed storage protein) gene promoter, to transfect rapeseed, making the ipt gene have a high and specific expression during oilseed development, in order to increase the endogenous cytokinin level during seed development, which will pave the way for further cultivation of super-high yield rapeseed by improving the directional transportation efficiency of nutrients and enhancing the grouting rate. The main work and results were as follows:1. Establishment of aseptic culture systemAt first, washed the rapeseed with fresh water, then washed with 75% ethanol for 45s-lmin, then washed with sterile water 2～3 times, then soaked in 0.1% HgCl2 for 10min, after that washed with sterile water 5～6 times, soaked in sterile water for 5h after that inoculated them to germination medium, consisting of MS medium,3% sucrose and 0.7% agar, pH5.8, the seedling rate amounted 100% and the contamination rate was 0.2. Establishment of regeneration systemThe sterile cotyledonary petiole and hypocotyl of the 4～7 days old low erucic acid rape cv. ZS8 were used as explants, cultured on differential medium, consisting of MS,3mg/L 6-BA,0.02mg/L NAA,3% sucrose and 0.7% agar, pH5.8, to directly induce bud initiation, and the regeneration frequency amounted 100%. Cultured the regenerated buds on rooting medium, consisting of 1/2MS,3% sucrose,0.8% agar and 0.2mg/L NAA, pH6.1, the rooting reat also amounted 100%.3. Determination of selection pressureThe sterile cotyledonary petiole and hypocotyl of the low erucic acid rape cv. ZS8 were cultured on differential medium with different concentration of PPT, the result showed that when the PPT concentration is 15mg/L, it inhibited the callus effectively.4. Establishment of Agrobacterium-mediaated transformation systemThe cotyledonary petiole with 0.1～0.2cm stem and hypocotyl of 0.5cm were used as explants, precultured for 2 days, infected with activated A. tumefaciens LBA4404, whose OD600 was 0.3, and the infection time for cotyledonary is 6～8min, while the time for hypocotyl was 0.5～1min, then co-culured in the dark for 3 days, after that washed all the explants in liquid MS containing 500mg/LCef, then washed with fresh water 4～6 times, after that transferred them to delay medium for 6 days, and then inoculated them to differentiation medium with 15mg/L PPT. When the shoots were about 1～2cm long, cut off and transferred them to rooting medium until the seedlings were about 5-7cm long, transplanted them for further identification.5. Histochemical detection of transgenic plants and PCR identificationCut young parts of transgenic plants leaves, GUS staining was done, the results showed, out of 43 resistant seedlings, there were 18 strains were dyed blue, which was a preliminary proof of the foreign gene has been inserted into the plant genome; PCR identification results showed that 11 of them were amplified the same band, which further confirmed that the foreign gene, ipt gene has been successfully integrated into the rape genome. |
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