Optimization Of Transgenic System And Establishment Of Transient Transformation System In Chrysanthemum

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the "Chinese top ten famous flowers" which is originated in China. It has occupied about 30% of the global cut flower market as the top of "four most famous cut flowers all around the world". (Chrysanthemum nankingense) is one of the diploid wild species in chrysanthemum, with the most simple genetic background involved in the origin of cultivated chrysanthemum. Due to the large chromosome number of cultivated chrysanthemum, researchers have met with many difficulties on the analysis of molecular genetics and molecular biology in it. While Ch. nankingense can be an excellent model plant to solve this problem for it’s simple genetic background. Transgenic technology has become an important means of improving crop varieties and gene function study. Genetic engineering in chrysanthemum has a rather late start, many factors can affect the efficiency result of transgenic, for example:the kind of vectors or promotor. On chrysanthemum, successful transgenic or stable exogenous gene expression in transgenic plants are rarely reported. On the study of genetic engineering in chrysanthemum, there is an urgent need to overcome difficulties such as the low efficiency of genetic transformation and low expression level of the target gene not as we expected. Research on the influence of transformation methods and promoter for chrysanthemum transgenic can contribute to finding way out of these difficulties. The main results are summarized as follows:1. Expression analysis of GUS in 35S:GUS/2×35S:GUS transgenic chrysanthemum.In this study, the sequence of 35S promoter and 2×35S promoter were cloned from pCAMBIA1301 vector by using technology of primer design and PCR. The 35S promoter and 2×35S promoter were inserted in pORE R1 Vector and pORE R2 Vector, and obtained the follow four recombinant vectors:pORE Rl-35S:GUS, pORE R2-35S:GUS, pORE R1-2 ×35S:GUS and pORE R2-2×35S:GUS. The four constructed genetic transformation vectors were introduced into the cut chrysanthemum ’Jinba’ by the agrobacterium-mediated genetic transformation system. We have transformed 4090 pieces of leaf-disc, and received 22 overexpressed transgenic lines. The target gene has been successfully inserted into the chrysanthemum genome of the 22 transgenic lines detected by PCR. Real-time quantitative PCR (qRT-PCR) analysis was used to detect the function of promotor, the results indicated that 2×35S promotor exhibited obvious advantage for exogenous gene expression than 35S promotor in the same vector, GUS expression level in the former transgenic lines were about twice of the latter. Also we can see darker blue in the former transgenic lines by histochemical evaluation of GUS expression.2. The establishment of the agrobacterium-mediated transient transformation system in chrysanthemum leaf.By using agro-infiltration method and β-glucuronidase (GUS) as a marker gene, the expression vector pORE R1-2×35S:GUS was transformed into the leaf of cut chrysanthemum ’Jinba’,’Youxiang’ and Ch. nankingense, this study discussed the effects of plant accessions, leaf positions on transient gene expression in chrysanthemum leaf. The results indicated that ’Youxiang’ leaf displayed the highest transient transformation rate of GUS (76.67%), it’s expression level was also the best; transformation rate of GUS in Ch. nankingense was the lowest (60.0%), expression level of it was worse than ’Youxiang’, and some leaves were withered to death after injection; the fourth leaf in the morphological upper part of shoot was the optimal choose for the agro-infiltration method in transient transformation. In order to facilitate the operation of other genes, the GUS gene was recombined into Gateway vectors pMDC 43 (2×35S) to further verify the effectiveness of transient expression in the above materials. The establishment of this transient transformation system can provide an effective method for the analysis of gene function in chrysanthemum leaves, and it laid a sound foundation in chrysanthemum genetic engineering for further access of highly efficient and stable transgenic chrysanthemums.In summary, the efficient expression of 2×35S in chrysanthemum ’Jinba’ was confirmed in this research; and a agrobacterium-mediated transient transformation system which can be applied to chrysanthemums and Ch. nankingense was established.