Studies On Safe And High Efficient Transformation Techniques In Brassica Napus L.

With the rapid development of molecular biology and biotechnology, a considerable progress has been made on transformation technology during the last years. More than 10 different methods have been available to genetic transformation in plants. Nowadays the problem is no longer how to obtain transgenic plants, but to high efficiently produce fertile transgenic plants with low-copy-number and marker-free. With remarkable advantages over direct transformation methods, including easy-to-operate, low-cost, low copies and preferential integration of defined T-DNA into transcriptionally active regions of chromosomes with exclusion of vector DNAs, Agrobacterium-mediated transformation has become the most commonly used method for genetic modification in rapeseed one of the first kind of crops benefit from genetic engineering since 1986. However, Agrobacterium-mediated coculture transformation requires regeneration of transformed cells which is genotype-dependent, painstaking, labor intensive, time consuming and complicated with somaclonal variation and epigenetic changes. In order to resolve these problems, more and more in planta transformation methods have been explored which are independent in dedifferentiation and redifferentiation of receptor cells and with few tissue culture steps.Due to the long tissue-culture period and too many factors involved in Agrobacterium-mediated transformation procedures, plant expression vectors which contain a gfp gene or a uidA gene with a intron from a Arabidopsis H2A1 gene were successfully constructed to provide a convenient tool for transient expression analysis. With pBILBar-iGUS, pBINPTII-iGUS, pBILBar-GFP, high efficient transient expression systems were established for rapeseed explants such as petiole with cotyledons and hypocotyls by optimizing some factors influencing Agrobacterium-mediated transformation of rapeseed. By decreasing necrotic rate and optimizing regeneration condintions, transformation protocols were established for cotyledon nodes and hypocotyls, and transgenic plants were obtained with an average efficiency of higher than 5% using pBILBar-iGUS, pBINPTII-iGUS, pBILBar-GFP, pNapin-fae1 RNAi (500, 800, 1500), pFae1-fae1 RNAi (500, 800, 1500), etc. With a view to improve the sensitivity of Brassica plants to Agrobacterium infection and delivery of T-DNA, genome sequences of plant factors VIP1 gene and H2A1 gene, which were reported to influence Agrobacterium-mediated transformation, were cloned from Arabidopsis thaliana, and analyzed with bioinformatics tools. Plant expression vectors named pBI121-VIP1 and pBI121-H2A1 were successfully constructed in order to clarify the function of VIP1 gene and H2A1 gene and to increase the transformation efficiency of Brassica plants via Agrobacterium.In planta transformation protocols, such as pollen tube pathway, floral dip, floral spray, pollen-mediated transformation, modified from some plants such as Arabidopsis thaliana, were also studied. Among them, transgenic rapeseed plants were successfully obtained with pollen tube pathway, floral dip and pollen-mediated transformation methods, with an efficiency of 0.43% 0.31% 0.19%, respectively.To take the GMO safety into account, Cre/loxP site specific recombination systems were constructed to facilitate deletion of marker genes between two direct LoxPs after successfully transformed. Transgenic tobacco, arabidopsis and rapeseed containing Cre or loxPs have been obtained.