Transferring ClCBF4 Into Chrysanthemum Grandiflorum ’ZiYan’ Mediated By Agrobaeterium Tumefaeiens

Chrysanthemum grandiflorum is the rise of a new species in recent years. It has not only characteristics such as plant low, florescence long, flower type tight, but also advantages such as strong cold, drought, and salt resistance. In order to improve the stress resistance, an increasing number of scholars transfer resistance factor into Chrysanthemum grandiflorum using molecular biological technique, so that make florescence longer and play a more important role in the process of greening. This study aims at this goal. On the basis of analyzing the gene function of C1CBF4, a species which has higher leaf differentiation rate is screened out by the plant tissue culture technique. After the good genetic transformation system established, the CICBF4 is transferred into this species mediated by Agrobacterium tumefaciens. This practice aims at obtaining the transgenetic seeding which have the good stability. The test results are as follows:1. The transgenetic and wild-type Arabidopsis are treated in drought, salt, and low temperature environment. The CICBF4 gene functional identification is conducted in terms of seed germination rate, seed roots length, seedling type, and physiological indexes. The results show that:Under higher concentration drought and salt stress, the seed germination rate and seed roots length of transgenetic Arabidopsis are higher than that of wild-type. But the two shows no obvious change under cold stress. In the test of seeding stage, the transgenetic plants are no obvious injury under the three kinds of stresses and maintain obvious vitality. The enzyme indexes match with phenotypic analysis. The enzyme activity of wild-type Arabidopsis is lower than that of transgenetic Arabidopsis while the MDA content and electrical conductivity is higher.2. Chrysanthemum grandiflorum’ziyan’and’niu 9722’are used as experimental material. The best time of explants disinfection, the most suitable leaf differentiation and plant rooting medium are explored. The high efficient regeneration systems are established of the two species, so that the best species are screened out for genetically modified. The results show that: The best time of explants disinfection for’ziyan’and’niu 9722’is 6min and 8min. The most suitable leaf differentiation medium for’ziyan’is MS+6-BA 2.0mg·L-1+NAA 1.0mg·L-1. The differentiation rate is nearly 93% and the number of shoots is nearly 8.0. The differentiation rate of’niu 9722’is lower, nearly 50%. But the stem proliferation number is 10.05 when the stem is in the MS+6-BA 2.0mg·L-1+NAA 0.5mg·L-1. The most suitable rooting medium for ’ziyan’ and ’niu 9722’ is 1/2MS added in NAA 0.2mg·L-1, 0.3mg·L-1. The plants grow well after transplanted and the survive rate reach 100%.3. Based on the establishment of high efficient regeneration system of ’ziyan’, the antibiotics sensitivity is tested. The results show that:The most suitable concentration of Km for leaf differentiation and plant rooting is 4 mg·L-1 and 6mg·L-1. The most suitable concentration of Carb for inhibiting the growth of Agrobacterium tumefaciens is 200mg·L-1. The best genetic transformation system of ’ziyan’ is established:pre-culture 2days, concentration of bacterium 0.5, infection time 8min, co-culture 2days, and delay-culture 3days. After screening the roots of resistant buds,11 resistant plants are obtained. At last,10 positive plants are obtained after PCR testing with DNA. That proves preliminary that the foreign gene CICBF4 has already transferred into Chrysanthemum grandiflorum ’ziyan’. The plants all survive after transplanted and grow well.