| Chrysanthemum(C.morifolium Ramat.)is a cultivar of the genus Chrysanthemum.It has a long history of cultivation in China,and has cultivated varieties with rich colors and diverse patterns.In recent years,plant genetic engineering technology has become an important means to improve varieties and study gene function.Due to the variety of chrysanthemums and the complex genetic background,the regeneration ability of different genotypes of chrysanthemums is also different.Therefore,it is necessary to study the suitable regenerative genetic transformation system of chrysanthemum.At the same time,due to the lack of blue color of chrysanthemum,the use of genetic engineering technology to generate new varieties of blue color is of great significance for enriching chrysanthemum varieties.In this study,the leaves of 11 potted chrysanthemum cultivars were used as explants.The effects of different phytohormone ratios on chrysanthemum differentiation were compared by using 9 different differentiation media,and the optimal varieties were selected and their genetic transformation system was established.The candidate cultivars for blue transgenic chrysanthemum was selected by dihydromyricetin and the regeneration genetic transformation system was established,and the vector carrying blue gene was constructed for transformation.The main findings are as follows:1.Establishment of regeneration genetic transformation system of potted chrysanthemumThe leaves of 11 potted small chrysanthemum varieties were used as explants.The differentiation medium with 9 different plant hormone concentration ratios was used to screen the cultivars ’Weifengshenhong’ with the highest regeneration frequency.The optimal differentiation medium was:MS+6-BA 1.0 mg·L-1 NAA 0.2 mg·L-1,the regeneration frequency was 95.0%;the selection pressure of leaf differentiation for kanamycin and hygromycin were 15 mg·L-1 and 10 mg·L-1;respectively,correspondingly the selection pressures for rooting resistance of plants were 10 mg·L-1 and 8 mg·L-1;pCAMBIA1301-GUS was used to verity its regeneration efficiency of‘Weifengshenhong’,the optimum range of carbenicillin was 200-400 mg·L-1.2.Transformation of chrysanthemum with blue genesTo screen blue transgenic chrysanthemum candidate varieties,8 new pink varieties and the selected‘Weifengshenhong’ were used as the material,the petals were cultured in vitro with a solution of delphinidin precursor dihydromyricetin at a concentration of 2 mg·ml-1,’Nannongfencui’ was as a positive control,and the ’Nannongzipingpang’ was selected.The optimum differentiation medium for leaf disc ’Nannongfencui’ was:MS+6-BA 2.0 mg·L-1+NAA 0.2 mg·L-1,the regeneration frequency was 81.67%;the contents of kanamycin and hygromycin for leaf differentiation both was 15 mg·L-1,for rooting ability of plants were 12 mg·L-1 and 10 mg·L-1 respectively.The optimum differentiation medium for ’Nannongzipingpang’ cluster buds was:MS+6-BA 2.0 mg·L-1+NAA 0.8 mg·L-1,the average number was 2.56 adventitious buds;the optimal content of kanamycin and hygromycin for adventitious buds were 25 mg·L-1 and 8 mg·L-1,respectively,for the rooting resistance of plants were 12 mg·L-1 and 10 mg·L-1 respectively.The F3 ’5 ’H of Campanula,Arctotis stoechadifolia and CtA3 ’5 ’GT of the butterfly pea was cloned,for constructing of the vector pORE-R4-F3HP-Ct3’5’GT-F3HP-OhF3’5’H and pORE-R4-F3HP-Ct3’5’GT-F3HP-FLCF3’5’H respectively,the Agrobacterium-mediated leaf disc transformation was used for ’Nannongfencui’,and the multiple-shoot explants was used to transform ’Nannongzipingpang’ with Agrobacterium-mediated method.After screening by kanamycin and the identification at DNA level,a total of 11 positive transgenic lines of ’Nannongfencui’ containing the F3’5’H and CtA3’5’GT was obtained. |
