The Construction Of Chrysanthemum Morifolium Differentiation System And The Research Of DFR Gene Genetic Transformation

Chrysanthemum morifolium is an importantant flower variety which has wide application in landscaping. DFR (dihydroflavonol4-reductase) is the first late biosynthesis gene in anthocyanin biosynthetic pathway which catalize the synthesis of colorless Leucopelargonidin, Leucocyanidin, and Leucodelphinidin. A variety of anthocyanin was furtherly synthesized and make different colors of flowers and fruit.To study the differentiation system of Chrysanthemum morifolium’Fanxingfen’, different concentration combination of plant hormone was used in the experiment. Gateway technique was employed in the study and expression vector pCAMBIA1301-PMI-DFR was constructed using PMI as selectable marker gene and DFR from ’Tsuda’ turnip and ’Yurugi Akamaru’ turnip as target genes. pCAMBIA1301-PMI-DFR and plant expression vector using antibiotic as selectable marker were introduced into Chrysanthemum morifolium’Fanxingfen’and ’Huoyan’, the main results obtained in the study are as follows:(1) The sterile leaves of’Fanxingfen’were cultured on the18gradient differentiation culture medium to observe their ability of budding and find out the optimum NAA and6-BA concentration combination for the differentiation of’Fanxingfen’leaves. The results showed that the optimum concentration combination of NAA and6-BA is:MS+0.3mg/L NAA+1mg/L6-BA.(2) Plant expression vectors pCAMBIA1301-PMI-DFR were constructed through LR reaction and introduced into EHA105agrobacterium strain via electroporation method. Plasmids were extracted and analyzed by PCR to comfirm positive clones. Resunlts showed that pCAMBIA1301-PMI-DFR were successfully introduced into EHA105agrobacterium strain.(3) To introduce the DFR genes into the genome of chrysanthemum ’Huoyan’ and ’Fanxingfen’, agrobacterium mediated genetic transformation was conducted using leaf disc transformation method, and using mannose and hygromycin as selectable marker. Chrysanthemum’Huoyan’seedlings which grew normally on mannose and hygromycin were analyzed by PCR and Southern blot hybridization. The results of the two methods showed that we obtained1positive ’Huoyan’ plant which contains ’Tsuda’ DFR from mannose medium and hygromycin medium respectively. Genetic transformation efficiency was1.0‰. While only some buds of Chrysanthemum’Fanxingfen’were obtained after the mannose and hygromycin screening.