| Chrysanthemum Xmorifolium Ramat. is a group of chrysanthemum varieties of plants that have a low, strong resistance, extensive management features such as tight and flowers. Due to its flowering before the"October" holiday, this new chrysanthemum has been promoted and applied in many areas. However, the limit factor for this plant used in northern of our country is that its green period become shorter when planted in the open field conditions in autumn and winter. Therefore, Cultivate and apply a strong cold varieties is particularly important. Numerous studies have shown that CBF1gene can cause cold-induced gene expression, thereby improving plant resistance to cold. The experiment aims to by the research of Chrysanthemum grandiflorum ’White Snow’ efficient regeneration system and Agrobacterium tumefaciens-mediated transformation system,which with ClCBF1gene in order to obtain new varieties of strong cold resistance, lay the foundation for the extensive use of transgenic technology to improve varieties of chrysanthemum. The main results are as follows:Chrysanthemum grandiflorum ’White Snow’ is used as test material in this experiment. To study the the effects of different ratio of hormone concentration and the degree of leaf development on leaf regeneration and the differentiation and rooting inducing stem section. Thus for Chrysanthemum grandiflorum ’White Snow’ regeneration conditions were optimized to obtain a high-speed regeneration system. The results showed that the young leaves of the Chrysanthemum grandiflorum ’White Snow’ at the top of the blade have a higher differentiation rate than the leaves in base of the plant; Differentiation of the same leaf base is higher than the middle; The best medium for inducing differentiation was MS+1.0mg·L-16-BA+0.6mg·L ’NAA; The best medium for proliferation of stems seg-ment was MS+0.3mg-L-16-BA+0.2mg·L-1NAA; The subculture medium was MS. The best medium for taking root was1/2MS+0.5mg·L-1NAA; The transferred plants grew well.On the base of establishing efficient regeneration system, we did the research of genetic transformation by Agrobacterium tumefaciens-mediated. The results showed that the optimal transformation was performed, with the optimized pre-culture time1d on best medium for inducing differentiation, the leaf discs’suitable agroinfection time10min(OD600=0.5-0.6), leaf disc delay-cultivated on the filter medium of MS+0.6mg·L-NAA+1.0mg·L-16-BA+5mg·L-’Km+350mg·L-1Carb after2d’s coculturing, inoculated to selection medium of MS+0.6mg-L’1NAA+1.0mg·L-16-BA+5mg·L-1Km+200mg·L-1Carb after3d, after differentiating adventitious buds, and then transferred the shoots which were at around1.5cm to1/2MS+0.7mg·L-1NAA+4mg·L-1Km for rooting selectin. This stable genetic transformation system effectively improve the conversion efficiency. After rooting screened, the Chrysanthemum plant’s DNA were extracted to PCR detection. The PCR results suggested that transgenie has2positive plantlets and the transformation rate is about0.67%. RNA was extracted after RT-PCR detection.lt showed that the target gene of CICBF1has been translated into genome of Chrysanthemum grandiflorum’ White Snow’. Transgenic plantlets after hardening and transplanting, all survived a month later. |
