CpHSP21.4Gene From Cyclamen Persicum Enhances High Temperature Tolerance In Transgenic Dianthus Caryophyllus

Dianthus caryophyllus L., also named carnation, was one of four cut flowers in world with high ornamental value and the major contributor to the cut flower markets. Carnation originated from the Mediterranean Region, which liked cool and dry habitat, and had heat resistance, so high temperature in summer seriously affected the quality of carnation cut flower, thus the breeding of heat resistance cultivars was one of important breeding goals in the study. By Agrobacterium tumefaciens-mediated method, the CpHSP21.4gene was transferred into spray carnation cultivars, followed by resistance screening and PCR detection, and the transformed plants were achieved. At the same time the preliminary analysis on the changes of physiological and chemical indexes of transgenic plants and controls under high temperature stress was conducted. The experimental results are as follows:1. Optimization of high-frequency regeneration system of five carnation varietiesThe regeneration system of five carnation varieties, such as’Xiangxiang’,’Wana’,’Anjing’,’Huaerzi’and’Huoxing’, is optimized. The results showed, the adventitious shoots were induced from leaf explants based on MS medium supplemented with0.15mg/L TDZ and7.5g/L agar and30g/L sucrose. The type of explants and the age explants affects its differentiation frequency, the adventitious shoots from leaves which in good state were much more than that from internodes, and that from young leaves more than that from old leaves. There is significant difference of the regeneration rates between cultivars, and’Xiangxiang’owns the highest rate of regeneration, up to67%, the regeneration rate of’Huoxing’is the lowest (26.7%). In the proliferation process of axillaries buds, the best medium is MS medium supplemented with0.5mg/L BA and0.5mg/L NAA and7.5g/L agar and30g/L sucrose, on which the multiplication coefficient up to4.2. Genetic transformation of carnationTaken the young leaves as explants, the Agrobacterium tumefaciens-mediated genetic transformation system of carnation was established. Leaf explants were pre-cultured side on shoot-inducing medium for2days, then immersed in Agrobacterium suspension for8-12min. The co-cultivation was carried out on medium containing100umol/L acetosyringone (AS) for3days. After that the transformants were obtained by transferring explants to selection medium supplemented with4-5mg/L hygromycin (Hyg) and400mg/L cefotaxime (Cef). Some factors that affect the transformation frequencies on selection medium were between4.3%and17.8%, but’huoxing’were0%.3. Study on high temperature stress tolerance in CpHSP21.4gene transformed carnationPhysiological changes of high temperature stress at36℃of transformed and control carnation were measured. The experimental data showed that with the postponement of the time, the protein content in transformed carnation was higher than control plantlets, however, that both on the fourth day reached the highest value which were no difference; the soluble sugar content and the level of SOD avtivity in transformed carnation was higher than control plantlets, but there was no difference in the zero stress; the post-MDA content in transformed carnation more slowly than control plantlets.