Screening Of Host Varieties Of Chrysanthemum For Blue Flower Color Gene Transformation And The Establishment Of Regeneration And Transofrmation System

Chrysanthemum?Chrysanthemum morifolium?,native to China,cultivated variety genus of Chrysanthemum of compositae,has pretty high value for ornament and economy,which is one of ten famous traditional famous flowers of china and four most important cut flowers in the world.China is the center of the origin and distribution of chrysanthemum.Although chrysanthemum is abundant in germplasm resources,various in cultivars and rich in flower forms and colors,there is no chrysanthemum with bluish violet or blue.Therefore,it will be of great significance to produce new chrysanthemum variety with novel flower color by genetic engineering.In present study,27 cut-flower chrysanthemum varieties conserved in "Centre of Chinese chrysanthemum germplasm resources preservation" were used as experimental material,and their petals was cultivated in DHM.Then the delphinidin content in cultured petals were determined,out of 27 varieties,'Nannongfencui' and;Nannongfenye' are of high delphinidin content,inferring which are potential host varieties for blue color chrysanthemum genetic engineering.In addition,high-frequency regeneration system and genetic transformation system of these two varieties were established.The main results are as follows:1.On the basis of HPLC,we determined an effective way of quantitative determination for delphinidin via UPLC by comparing different determine elution conditions.The results showed that anthocyanin of chrysanthemum can be extracted by 0.1 mol.L^-1 hydrochloric acid methanol,then by adding equal volume 10%formic acid,and followed by filtering through 0.22 um filter membrane,the extraction is ready for UPLC analysis.Through the optimization of elution conditions,delphinium can be separated from other anthocynins within nine minutes.Based on optimized method,delphinium concentration within 2.5?40 mg.L^-1 has a good linear relationship and the correlation coefficient is 0.999.2.27 cut-flower chrysanthemum varieties were used as experimental material,and their petals were fed with the dihydromyricetin,a precursor of delphinidin.After that,delphinidin in petals was extracted and analyzed via UPLC,the results showed that delphinidin content in 'Nannongfencui' and 'Nannongfenye' is over 10mg.L^-1,inferring they are promising host varieties for blue color flower genetic modification.3.'Nannongfencui' and 'Nannongfenye' which are of high delphinidin concentration 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 of 'Nannongfencui' is MS +6BA1.0 mg.L^-1 + NAA 0.5mg.L^-1 with a regeneration rate of 80.65%;that for 'Nannong fenye' is MS + 6BA1.0 mg.L^-1 + NAA 0.5mg.L^-1 with a regeneration rate of 66.67%.Hygromycin concentrations of leaf disc regeneration for 'Nannongfencui'and'Nannongfenye' are 10 mg.L^-1 and 15 mg.L^-1,respectively;Hygromycin concentrations of rooting regeneration for 'Nannongfencui' and 'Nannongfenye' are 8 mg.L^-1 and 10 mg.L^-1,respectively.