Studies On Genetic Transformation Of Chrysanthemum Morifolium With AP1 Gene Mediated By Agrobacterium

Chrysanthemum morifolium is one of the most famous flower in China and cut flower in the world. It blooms almost in the same period—autumn.There are few cultivars which bloom in spring or summer. The growth of flower will be accelerated by the heterologous expression of flower's meristematic characteristic gene AP1. Thus, it's significant to change flowering time of chrysanthemum with introducing AP1 by genetic engineering technologies. In the past four years, high regeneration frequency protocol of three cultivars were developed and it was the first time to establish genetic transformation protocol for chrysanthemum 'Yu Ren Mian' by Agrobacterium and 11 transgenic lines were got. Two of them were selected due to their earlier blooming. The main research work and results were as follows::1. The optimal medium composition for shoot regeneration of chrysanthemum was 6-BA and NAA which selected from four different hormone compositions 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, different physiology leaves, different explant types, timing culture in vitro and AgNO₃ were studied. The results showed that: regeneration of chrysanthemum was genotype dependent. Only three cultivars 'Zi Yan' , 'Yu Ren Mian' , 'Huang Xiu Fang' from nine genotypes could regenerate shoots highly and their shoots regenerate frequence was 93%,96%,95%;Different physiological leaves had different regeneration ability. Infancy leaves from seedling of 15～25 days had the highest shoot regeneration ability;The same type of explant had different regeneration ability according to different cultivars, while different explant of the same cultivar also had different regeneration ability. Explants from leaves of in vitro-grown chrysanthemum cultivars 'Zi Yan' , 'Huang Xiu Fang' , 'Yu Ren Mian' , 'L12M57' regenerated higher than leaves of 'Shen Ma' did.Explants from leaves, petiole and stem of 'Huang Xiu Fang' regenerated highly but leaves of other cultivars regenerated more adventitious shoots than stem or pedicle explants;Timing culture in vitroinfluenced regeneration ability and the time of keeping high regeneration ability was different according to different cultivars;AgNO3 influenced the shoot regeneration of different cultivars, but the regeneration frequency and the quantities of shoots regenerated did not improved by AgNO^,- Shoots regenerated of 'Zi Yan' was influenced by unsuitable culture condition and explants with different origin. Browning of leaves was alleviated by high concentration of NAA and Vc, charcoal did not take effect.2. The plant expression vector pBl-APl was constructed and was transfered into Agrobacterium tumefaciens EHA105> LBA4404 by freeze-thaw method.3. Susceptibility of different cultivars to different antibiotics had been studied. The critical concentration for shoot regeneration of ZiYan' and 'Yu Ren Mian' was 10 mg/L G418 and 2()mg/L kanamycin for 'Huang Xiu Fang'. 20mg/L kanamycin was the critical concentration for reducing root. The influence of Cefotaxime and Carbenicillin on shoot regeneration and the root of chrysanthemum were discussed. The results showed that over 400 mg/L Cefotaxime and Carbenicillin influenced regeneration frequency and quantities of shoot regeneration. Cefotaxime had greater hamper on shoot regeneration than Carbenicillin. They also restrained growth of chrysanthemum. The number of root increased, while the length of root and height decreased as the antibiotics concentration increased.4. Transgenic protocol for'Yu Ren Mian'was developed . That the leaves explants were pre-cultured for 2⁸ hours or not pre-cultured was best for transformation by Agrobacterium. The suitable concentration of Agrobacterium and the time for transforming was OD(,oo 0.5 and 10 minutes. Cocultivated for 2 days at 23²5°C after infection and delayed cultivation for 3 days were suitable for transformation. It was important to increase antibiotic from 5mg/L G418 to 10 mg/L G418 step by step. One centimeter transgenic shoot regeneration was transformed into 1/2MS with 20 mg/L kan. The integration of API gene into chrysanthemum was identified by PCR and Southern-blotting and single insert in genome were found in the most transgenic lines. Foreign gene did not influence the growth of transgenic lines. There was no difference on height internode^ diameter of flower colour of flower and number of flower between transgenic plants and untransgenic plants. Two of transgenic plants bloomed 15 day earlier than untransgenic plants.This study provides the basis for breeding new chrysanthemum cultivars which bloom ealier by combining conventional breeding with molecular breeding.