The Culture System Establishment Of Chrysanthemum Varieties And The Researches Of Transforming F3’H Gene

Flavonoid3’-hydroxylase (F3’H) is the first critical enzyme in the anthocyanin biosynthesis pathway started from dihydrokaempferol, which can catalyze aihydrokaempferol to generate purple anthocyanin (also known as cyanidin pigment) and thus the petal and fruit color show purple. In this study, F3’H gene had been clonded from light-sensitive ’Tsuda’ turnip and light-insensitive ’Yurugi Akamaru’ turnip and then we constructed the plant expression vector through Gateway technology using non-antibiotic maker. The next, we performed genetic transformation of chrysanthemum using this vector and the plant expression vector of antibiotic maker constructed by former, in order to change the color of chrysanthemum and establish the research foundation for creating new varieties of chrysanthemum color. The major findings are as follows:(1) Constructing the plant expression vector pCAMBIA1301-FM/-F3’H through Gateway technology and then performing PCR verification and enzyme digestion for this vector; transferring the correct vector verified into Agrobacterium EHA105and then indicated that plant expression vector pCAMBIA1301-PMI-F3’H had been transferred into Agrobacterium EHA105successfully through PCR verification.(2) Obtaining the sterile regeneration system from stem explants of Chrysanthemum morifolium ’Shenyun’、’Du shiliren’ and ’Fen meigui’ by the sterilization of explants and culture in vitro; and determining the best ratio of NAA and6-BA from various varieties by double factors completely random test, establishing the leaf explant regeneration system.(3) Performing genetic transformation for the various varieties of Chrysanthemum morifolium using the Agrobacterium containing the plant expression vectors pH7WG2D-F3’H and pCAMBIA1301-PMI-F3’H. The conditions of transformation are as followed:pre culture1day, Agrobacterium infection10min under the condition of OD600value of0.5, co-cultured for2days under the condition of23-25℃in dark, delayed screening for two days, screening cluture under the condition of hygromycin concentration5mg/L and mannose concentration12g/L. Regeneration plants were tested by PCR, the results indicated that obtaining4regeneration plants containing F3’H gene of ’Tsuda’ turnip and4regeneration plants containing F3’H gene of ’Yurugi Akamaru’ turnip by hygromycin-screening; also obtaining1regeneration plants containing F3’H gene of ’Tsuda’ turnip and1regeneration plants containing F3’H gene of ’Yurugi Akamaru’ turnip by mannose-screening. And obtaining some resistance shoots after genetic transformation for Chrysanthemum morifolium’Shenyun’、’Du shiliren’ and ’Fen meigui’...