Study On Embryogenic Suspension Culture And Protoplast Fusion Of Poplar

In this paper, calli were induced from the blades and stems of sterile seedlings of the poplars. Suspension cultures were established. The factors influencing protoplasts isolation were studied. The poplar protoplast fusion was also studied, by use of the polyethylene glycol (PEG) method. The main results described as follows:1 There were almost no differences in the ratios of callus induction among different poplar of materials used in the experiments. With the concentration of l~3mg/L 2,4-D and after five subculture periods, the calli were vivid light yellow granules and grew fast, which was ideal to establish suspension culture.2 It was showed that there were no significant differences in the time needed to establish suspension culture among different Populus species. Suspension in the liquid MS medium combined with 2,4-D 1.0~2.0mg L -1, KT 0.2mg L-1, with the starting density of 4g fresh suspension cell per 40mL liquid medium, and a subculturing period per 5~8d, were the best for establishment and maintaince of suspension culture.3 Under the condition of suspension culture, cell cluster were formed first, and then one or several embryoids were induced from embryogenic cultures. High concentration of BA can induce more embryoids than low concentration, and low concentration of BA combined with NAA promoted the growth of embryoids.4 Four enzymes:1.0% Cellulase RS, 0.5% Pectolyase Y23, 0.5% Hemicellulase, andl.0% Macerozyme R-10 combined with 0.6mol/L Mannitol, 1470mg/L CaCl2 2H2O, and 95mg/L KH2PO4,pH value 5.8, were applied to isolate protoplasts. It was indicated that components and concentrations of enzyme distinctly influenced the results of protoplasts isolation.5 It was showed that different materials and different subculture time distinctly influenced the yield and vitality of protoplasts: after suspension cells subcultured for 3 days, the yield of the protoplasts was the highest. And the yield of the protoplasts isolated from the leaves of sterile seedlings after subculturing for 25 days was the highest and the protoplasts showed the strongest vitality. Higher yield and stronger vitality of the protoplasts isolated from suspension cells than from the leaves of sterile seedlings were observed. The results also showed that the isolation time affected the yield and vitality of the protoplasts. With 8 hours isolation, the protoplasts yield of suspension cells was almost the highest. Some protoplasts began to decomposed and the vitality began to decrease after 10 hours isolation. No significant influences of osmatic pressure to the protoplasts isolation were observed.6 The results of the fusion of the selected protoplasts induced by Polyethylene glycol (PEG) showed that a relatively high fusion probability could be obtained when the protoplasts were induced by the concentration of 35% PEG and the protoplasts were not prone to decomposed.