Agrobacterium-mediated Transformation Of Transcriptional Activator Cbf3 Gene Into Anthuriun Andreanum

Anthurium andreanum is a valuable ornamental plant of Araceae monocotyledons. Anthurium andreanum is the second tropic flower, which is attractive for people beacause of its beautiful flowers and leaves. It survives at wet and warm habitat of an optimal temperature between 20℃to 28℃, but can not grow at low-temperature area such as the north of China. Hence, researchers wish to increase the plant's freezing and chilling tolerance through transgenetic engeneering. So we attempted to introduce CBF3 gene, which encode transcriptional activators that induce expression of a battery of COR (cold regulated) genes, into Anthurium andreanum so as to improve its tolerance to low temperture. Firstly, in this study, the plant regeneration system was optimized, and protocol for Agrobacterium-mediated transformation of leaves and roots was established, based on which we transferred the Arabidopsis CBF3 into Anthurium andreanum. The result of PCR analysis showed that transgentic calli were obtained. The main results are as follows:1. Agrobaeterium-mediated transformation system of leaves was established and optimized. Firist of all, we significantly improved the frequency of callus inducation from leaves, which provided a good foundation for genetic transformation. It was found that A. andreanum was sensitive to NH₄NO₃, and low ammonium/nitrate ratio was benifical for growth and shoot production. The carbon source also affected callus induction. Sucrose was suitable for callus induction in A. andreanum. The best medium for callus inducation from leaves was MS (with 0 mg/L NH₄NO₃) containing 3% sucrose, on which the induction frequency was over 90%. Secondly, We discussed the influence of preculture time, incubation time and co-culture time on genetic transformation. They were in direct proportionality on transformation frequency in a certain range, while too high or too low were both harmful to the transformation. The optimized transformation conditions of leaves were precultured for 4 d, incubated for 20 min and co-cultured for 3 d with Agrobacterium tumefaciens, with 30 mg/L kanamycin as selection pressure. After that the concerntration of kanamycin was increased to 75 mg/L. Kanamycin-resistant calli from leaves were easier to produce shoots than that from roots. PCR analysis of GUS gene was carried out for the 4 kanamycin-resistant plantlets and 2 transgenetic plantlets were obtained, finally.2. The Agrobaeterium-mediated transformation system of roots was established and optimized. The same as leaves, firstly we improved the efficiency of callus inducation from roots. The results showed that roots of A. andreanum's are sensitive to NH4NO3, and a low ammonium/nitrate ratio was benifical for callus induction. The carbon source also affected callus induction. Sucrose was suitable for callus induction from roots of A. andreanum. The best medium for callus inducation form leaves was MS (with 0 mg/L NH₄NO₃) containing 3% sucrose, on which the induction frequency was over 90%. Secondly, we discussed the influence of preculture time, incubation time and co-culture time on genetic transformation. They were in direct proportionality on transformation efficiency in a certain range, while too high or too low were both harmful to the transformation. Meanwhile the optimized conditions for transformation of roots were precultured for 5 d, incubated for 15 min and co-cultured for 5 d, with 40 mg/L kanamycin as selection pressure. The result of PCR analysis of GUS gene for the 5 kanamycin-resistant calli showed that 3 transgenetic calli were obtained at last.3. In this experiment, CBF3 gene was inserted into plasmid pMV which contains 35S promoter. And the constructed plasmid pMV-cbf3 was acquired. The CBF3 gene was transformed into A. andreanum by Agrobacterium EHA105. We have tried to introduce CBF3 gene into leaves, roots and calli. And kanamycin-resistant calli were got from roots and calli explants. The result of PCR analysis of CBF3 gene for the 5 kanamycin-resistant calli showed that 5 positive results were obtained.