Study On The Efficient Transformation System Of Chrysanthemum And Transformation Of VtF3’5’H Gene In ’Nannong Fencui’

Chrysanthemum(Chrysanthemum morifolium),native to China,a cultivated variety of Chrysanthemum of compositae,has pretty high ornamental and economic value,which is not only one of ten famous traditional flowers of China and four most important cut flowers in the world,but also important potted and garden ground cover plants.China is the center of the origin and distribution of chrysanthemum.Chrysanthemum is abundant in germplasm resources,various in cultivars and rich in flower forms and colors,however,there is no chrysanthemum with bluish violet or blue color.Therefore,it will be of great significance to produce new chrysanthemum variety with novel flower color by genetic engineering.Despite a host variety for transformation can produce delphinidin and its varieties,a high efficient regeneration and transformation system is a pre-request for chsysanthemum blue color genetic engineering.Cut-flower chrysanthemum varieties of’Nannong Feiyu’,’QX-144’,’Nannong Fenyu’ and ’Nannong Gongfen’ which can produce high content of delphinidin via DHM feeding experiment were used as experimental material.In present study,high-frequency regeneration system and genetic transformation system of ’Nannongfenyu’ was established.And cut-flower chrysanthemum’Nannongfencui’ was transformed with VtF3’5’H gene by agrobacterium-mediated leaf disc transformation.The main results are as follows:1.’Nannong Feiyu’,’QX-144’,’Nannong Fenyu’ and ’Nannong Gongfen’ were used as plant materials,effect of different differentiation medium with the different ratio of hormones on chrysanthemum adventitious bud regeneration were tested.The results showed that the optimal differentiation medium for ’Nannong Fenyu’ is MS + 6-BA 2.0 mg·L-1 + NAA 1.0 mg·L-1 with a regeneration rate of 100.00%and propagation coefficient of 8.6;Hygromycin concentrations for inhiting leaf disc regeneration in;Nannong Fenyu’ is 10 mg·L-1;Hygromycin concentrations of rooting selection in ’Nannong Fenyu’ is 8mg·L-1.2.High efficient and transformation system of cut-flower chrysanthemum ’Nannong Fencui’ was obtained.The results show that 3 day preculture,OD600 of 0.5,10min infection,co-cultivating at 25 ℃ for 3 days and a prolonged culture for 7 day are suitable for transformation.We transformed cut-flower chrysanthemum ’Nannong Fencui’ with VtF3’5’H gene by agrobacterium-mediated leaf disc transformation,40 hygromycin resistant lines were obtained,10 out of 40 lines were verified as putative transgenic lines via PCR using hygromycin primers.RT-PCR analysis showed that VtF3’5’H has been expressed in two transgenic lines,inferring the exogenous gene were introduced into the regeneration plants genome and expressed sucessfully.