| Chrysanthemum × morifolium is one of the most famous flower in China and cut flower in the world. There are many breeding achievements every year including cut flowers and pot plants. Flower color is a important character, breeding of blue flower cultivar of Chrysanthemum is the dream of many breeders. A main reason for the lack of blue flowers is the absence of 3',5'-hydroxylated anthocyanins, which are synthesized by a key enzyme, flavonoid 3',5'-hydroxylase (F3'5'H). PCFH is a F3'5'H homologue gene cloned from Pericallis cruenta. Thus, it's significant to breed blue flower cultivar of chrysanthemum with introducing PCFH gene by genetic engineering technologies. This report describes tissue culture of 4 white flower and 6 pink flower cultivars. High regeneration frequency protocol of 4 cultivars were developed, they are 'Reagan Elite White', 'Vamp Time Pink', 'Reagan Elite Improved' and 'He Lan Bai'. It was the first time to establish genetic transformation protocol for chrysanthemum 'Reagan Elite White' by Agrobacterium and transgenic lines were got. The main research work and results were as follows:1. Stems and flower petals were isolated and sterilized in 0.1～0.15% and 0.3% NaOCl solution for 10～15min. The optimal propagation media for 'Tao hua Jian' is MS + 6-BA 0.2mg/L + NAA 0.02 mg/L, others are MS without plant growth regulator.2. The medium composition for shoot regeneration of chrysanthemum was 6-BA and NAA basing on MS culture medium. The optimal concentration proportion of 6-BA and NAA for different cultivars and the factors which would influence shoot regeneration such as gene types and different explant types were studied. The results showed that: regeneration of chrysanthemum was genotype dependent. Chrysanthemum cultivar'Reagan Elite Improved' could regenerate shoots highly through leaves and tTcls, and the shoots regenerate frequence was 87.4% and 87.2%. Adventitious shoots were regenetated from stems of 'Vamp Time Pink', tTcls of 'Reagan Elite Improved' and leaves of 'He Lan Bai', regenerate frequence was 82.2%, 76.2% and 66.6%. Optimal rooting media of these cultivars is 1/2MS + NAA 0.1 mg/L.3. Susceptibility of 'Reagan Elite White' to Kanamycin and Carbenicillin had been studied. The critical concentration for shoot regeneration of 'Reagan Elite White' was 10 mg/L Kanamycin. 10mg/L Kanamycin was the critical concentration for reducing root of 'Reagan Elite White'. The influence of Carbenicillin on shoot regeneration and the root of chrysanthemum were discussed. The results showed that over 400mg/L Carbenicillin influenced regeneration frequency and quantities of shoot regeneration.4. Transgenic protocol for 'Reagan Elite White' was developed. Leaf explants were pre-cultured for 2 days was best for transformation by Agrobacterium. The suitable concentration of Agrobacterium and the time for transforming was OD600 0.5 and 5～10 minutes. Cocultivated for 2 days at 23～25℃ after infection and delayed cultivation for 3 days were suitable for transformation. It was important to increase antibiotic from 5mg/L Kan to 10 mg/L Kan step by step. One centimeter transgenic shoot regeneration was transformed into 1/2MS with 10 mg/L Kan. The integration of PCFH gene into Chrysanthemum was identified by PCR in some transgenic lines.This study provides the basis for breeding new chrysanthemum cultivars through molecular breeding. |
