Agrobacterium-mediated Transformation Of AtCBF3 And PaFT Gene Into Anthurium Andraeanum

Anthurium andreanum Lind, a species of important tropical flower, is favored by people all over the world for its long-lasting flowering period and distinguished appearance. It has entailed an increased demand for both the cut flowers and potted flowers of A. andreanum in the market since it was introduced. However, its sensitivity to temperature, which means that the plant can hardly survive in the region where holds lower temperature, limits its popularization and application, which highlights the importance of improving the plant's resistance to cold; In addition, since it takes a long time for A. andreanum to bloom in the first time, which elongates the period of breeding, raises the cost of its production and reproduction. Therefore, we intend to improve the species in this study through the method of genetic engineering that transfers the AtCBF3 and PaFT genes into A. andreanum.Based on prior researches, first, shoot regeneration system of A. andreanum for genetic transformation was optimazed by orthogonal design. Besides, CBF3 gene derived from Arabidopsis thaliana and FT gene derived from Platanus acerifolia were transferred into different explants of A andreanum. The results of PCR analysis showed that several positive plants had been obtained. The main findings were as follows:1. Optimization of shoot regeneration system of A andreanum. First, the results showed that:The best medium for the induction of callus was 1/2 modified MSa+0.25 mg/L BA+0.14 mg/L KT+0.2 mg/L IAA, the best genotype was'Champion', and the suitable explant was stem segment. The highest rate of callus induction could reach up to 96.7%. Besides, the best medium for inducing adventitious buds was modified MSa+0.01 or 0.1 mg/L TDZ, and the ability to form adventitious buds of the 'Champion* was better than the'Sweetheart Red'; The best medium for the induction of embryogenic callus was modified MS3+I.5 mg/L 2,4-D+0.5 mg/L KT+4%sucrose+2% glucose+0.25%Gel. The best genotype was'Pink champion', the suitable explant was leaf. The highest rate of embryogenic callus induction could reach up to 47.9%. The best medium for the development of embryoid was modified MSa+2%sucrose+0.25% Gel. The highest rate of the development of embryoid could reach up to 16.8%.2. The results of antibiotics sensitivity tests indicated that:The optimum selection pressure of kanamycin for shoot regeneration was 75 mg/L, which hygromycin for it was 30 mg/L; Morever, the concentration of cef used for selective culture should be 400 mg/L the first time, and it should be reduced gradually in the later culture.3. According to orthogonal experiment design and analysis, the better transformation system was that:Taking stem segments of A. andreanum as explants, precultured of it for 3d, the Agrobacterium twnefaciens solution was OD600=0.4. the time of infection was 20min, the time of co-culture was 4d at 25℃, the step up the concentration of kanamycin 0 mg/L from the first time,25 mg/L from the second time, 50 mg/L from the third time,75 mg/L from the fourth time, or hygromycin 0 mg/L from the first time,10 mg/L from the second time,20 mg/L from the third time,30 mg/L from the fourth time, until resistant buds formed. During the period of inducing adventitious buds, the suitable selection pressure of kanamycin was 30 mg/L or hygromycin was 15 mg/L; Then carrying on strong seedling culture; In the stage of rooting induction culture, the proper selection pressure of kanamycin was 15 mg/L or hygromycin was 5 mg/1 until the whole resistant plant appeared.4. According to the result of GUS staining, we found that it was effective for us to transfer genes into the plants based on this optimized system, which could be used in the following experiments.5. In this study, different genotypes and explants were used for transformation. As a result,14 km-resistant plants transformed with AtCBFS gene had been acquired, in which 'Robino','Pink champion'and'Champion'was 6,3 and 5 respectively. The results of PCR analysis showed that two positive individuals were obtained, with the transformation efficiency of 0.13%. Meanwhile,16 hyg-resistant plants of'Champion' transformed with PaFT gene were acquired. It was verified by PCR that only one plant was positive, so the transformation efficiency was 0.11%.