Development Of An Efficient Genetic Transformation Procedure And Pollen-specific Expression Of Cell Cycle Genes In Brassica Napus L.

Rapeseed (Brassica napus L.) is one of the most important commercial crops in China, even in the world. Conventional breeding has made tremendous contribution to the improvement of economic trait. The increasing demand on production of different oils such as edible oil, industrial oil needs new technology in rapeseed breeding to further enhance both the productivity and adaptability of new cultivars. So it was necessary to adopt new technology to transfer the new genes into rapeseed. Genetic engineering offered new possibilities to improve the crop in various special characters that were difficult to obtain by traditional breeding procedures. Novel traits, such as rapeseed yield, quality, disease and insect tolerance, male sterility, and the other special characaters, have been introduced into the crop by genetic transformation. And genetic transformation was used to detect the function of different genes. However, it was very difficult to use the present Agrobacterium-mediated transformation in many genotypes because there existed serious genotype-dependence and long-term transformation. There was no report on plant pollen development using cell cycle genes, which could influence the growth of plant.The studies explored effect of shoots regeneration and genetic transformation from cotyledonary explants in winter type rapeseed; On the basis of this speedy and efficient genetic transformation system, cell cycle gene CycD2 was transferred into a spring type rapeseed cv Westar, and pollen morphology that was affected for expression of CycD2 gene was studied. A simple and reliable method was developed for isolating pollen nuclei from rapeseed and wheat by ultrasonic treatment, and DNA content of pollen nuclei was analyzed using flow cytometry. The main results are as follows:1 Different concentrations and combinations of hormones, different media, different concentration of sucrose as well as preconditioning conditions were ompitimized for shoot regeneration from cotyledons of semi-winter type Brassica napus L.. Shoots regeneration frequency increased significantly when cotyledons were preconditioned in MSB5 medium with 1 mg/L 2,4-D and 1 mg/L BA for 3 days and then cultured in MMO medium containing 4.5 mg/L BA. Under such a condition the regeneration period was also shortened by about one third. Cotyledons from 10 winter and semi-winter type cultivars of Brassica napus were tested to evaluate the procedure and the average regeneration frequency was 80.82% for the 10 genotypes with the highest frequency 98.89%.2 According to the speedy and high efficient shoots regeneration system, a highly effective protocol for Agrobaterium-mediated transformation of winter and semi-winter type Brassica napus was established by 3-day preconditioning the cotyledons from winter and semi-winter type Brassica napus in a medium supplementing 1 mg/L 2,4-D and 1 mg/L BA for three days before inoculation with Agrobacterium tumefaciens strain GV3101 for three days. The transformation frequencies ranging from 12.09% to 28.29% were achieved among eight winter- and semi-winter-type cultivars of Brassica napus tested. By screening the factors including different concentrations and combinations of hormones, adding MES in the cocultivation media and 4-degree treatment, which affect the browning of the hypocotyls from the winter- and semi-winter-type rapeseed, a effective protocol for Agrobaterium-mediated transformation of winter and semi-winter type Brassica napus hypocotyls was also described.3 The nuclei were isolated fom pollen by ultrasonic treatment and purified through nylon mesh. Ultrasonic treatment time and media for pollen isolated were optimized for pollen at different developmental stages in rapeseed Brassica napus and mature pollen of wheat. We analyzed the nuclear DNA levels in pollen of rapeseed at three major developmental stages, and mature pollen of wheat by flow cytometry. 2C nuclei were detected in bicellulate and tricellulate pollen of rapeseed; the possible origins of 2C nuclei were due to DNA endoreduplication and the failur...