Isolation And Cultivation Of Protoplasts In Populus Alba L. Var. Pyramidalis.

In the research, the induction of calli from the blades and stems of sterile seedlings and the establishment of suspension cultures using of Populns alba L. van pyramidalis were investgated. The factors influencing protoplasts isolation and purification were studied, and protoplasts culture was performed initial. The main results were described as follows:1. Establishing the aseptic seedling system, calli system, suspension cultures system of P. alba L. var. pyramidalis.It is the first to establish the aseptic seedling system. The optimal stems activate medium was 1/2MS + NAA 0.2mg/L; the optimal blades differential medium was 1/2MS + BA 1.00mg/L + TDZ 0.05 mg/L; the rooting medium was 1/2MS + IBA 0.03mg/L.There were almost no differences in the ratios of callus induction among different site of materials used in the experiments. The optimal calli induced medium was MS + 2,4-D 3.0mg/L, the inductivity ratio is 100%. The calli were vivid light yellow granules and grew fast after 2～4 subculture periods, which was ideal to establish suspension culture.It was indicated that the plant hormone levels, initial inoculation capacity and subculture periods influenced the results of P. alba L. var. pyramidalis suspension culture. The optimal medium was MS + 2,4-D 1.5mg/L + ZT 1.0mg/L + NAA 0.02mg/L + MES 600mg/L + CH 500mg/L + L-Glutamine 200 mg/L + L-Aspartic acid 150mg/L, with the starting density of 2g embryonal calli per 30ml liquid medium, the initial inoculation density of lml suspension cells per 30ml liquid medium, and a subculturing period per 8～10 days.2. Establishing the protoplasts isolation and purification technology system of the sterile seedlings blades, calli and suspension cells of P. alba L. var. pyramidalis. Based on the tenique, high-quality protoplasts were obtained.It was showed that different materials needed various components and concentrations of enzyme. The blades used 3.0% Cellulase R-10 + 0.5-1.5% Macerozyme R-10 + 0.5% Hemicellulase + 0.1-0.5% Pectolase Y-23; the calli used 0.5% Cellulase RS + 3.0% Cellulase R-10 + 1.0% Macerozyme R-10 + 0.5%-1.5% Hemicellulase + 0.5%-1.5% Pectolase Y-23; the suspension cells used 3.0% Cellulase R-10 + 1.0% Cellulase RS + 1.0% Hemicellulase + 2.0% Pectolase Y-23 + 1.0% Macerozyme R-10, plus 0.6mol/L Mannitol, 600mg/L MES, 1g/L boyine serum albumen, pH value 5.8 for 8 hours.The different materials and the subculture time distinctly influenced the yield and vitality of protoplasts. The yield of the protoplasts isolated from the 2～5 leaves of sterile seedling after subculturing for 30 days was the highest (1.57×10⁷/g · FW), and the protoplasts showed the strongest vitality (79.41). After suspension cells subcultured for 4～10 days, the yield of protoplasts was the highest (1.25 × 10⁷/g ·FW) and the vitality was 81.56%. When calli subcultured after 2～4 periods for 20 days, the yield of protoplasts was the highest (6.52×10⁶个/g · FW) and the vitality was 81.02%. The best protoplaste purification methods was the rising method of sucrose isodensity centrifugation, the yield was 2.23 × 10⁶/g · FW and the vitality was 65.55%.3. The protoplasts culture was done initially, and got some calli.It was showed that different plant hormone and concentrations distinctly influenced the protoplasts division. The best cultured method was shallow liquid culture, and the optimal cultured medium was KM_8p + 2,4-D 3.0mg/L + ZT 1.0 mg/L + 6-BA 0.5 mg/L + NAA 0.5 mg/L+ MES 600mg/L + boyine serum albumenlg/L + CH 500mg/L + L-Glutamine 200 mg/L + L-Aspartic acid 150mg/L+0.6mol/L Glucose, the culture density was 2.5×10⁵/ml. The highest division frequency and plating efficiency were 5.83% and 4.1% respectively. Moreover, many small calli were obtained according to the protocals.