Cultivation Of Protoplasts And Doubling Of Somatic Chromosome In Populus Alba L. Var.Pyramidalis

In the research, the establishment of sterile micropropagation system based on the use of stem tissue culture and callus induction of Populus alba L. var. pyramidalis were investgated. The factors influencing protoplasts isolation and purification were studied, protoplast culture and somatic chromosome doubling of tchnology were performed initially. The main results were described as follows:1.Based on the establishment of sterile tissue culture system, induce callus and culture.There were almost no differences in the ratios of callus induction among different site of materials used in the experiments., callus induction rate was 100%; the optimal callus induction medium for 1/2MS+2,4-D 3.0mg/L; 2-4 times after subculture of callus from the bright color of yellow and small particles, the growth speed faster.2.Protoplasts isolation and purification technology system of the sterile seedlings blades, calli of P. alba L. var. pyramidalis.the results show that different materials needed various components and concentrations of enzyme:The blades with 1.0% Cellulase R-10+0.5% Macerozyme R-10 +1.0% Hemicellulase+1.0% Pectolase Y-23; callus with 2.0% Cellulase R-10+1.0% Macerozyme R-10+1.0% Pectolase Y-23. In the add 600mg/LMES, 1g/L bovine serum albumin, pH value of 5.8 for 8h,and yield and viability of protoplasts were higher.The different materials and the subculture time on protoplast yield and vitality have a significant effect,which used non-sterile subculture 30d 2-4 times blades protoplast yield reached 1.5×107/g·FW, vitality reached 81.6%; by following the subculture of about 2-4 times the growth of the callus material 20d,the yield can be 8.50×106/g·FW, vitality reached 81.2%; The best protoplaste purification methods was the rising method of sucrose isodensity centrifugation,the yield of protoplasts was 2.20×106/g·FW, the vitality remained at 83.6%. 3.The protoplasts culture started initially.By testing of different basic media,different hormones,different culture methods on the influence of the protoplasts culture, found the optimal culture medium was KM8p+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+bovine serum albumin 1 g/L+CH 500mg/L+glutamine 200 mg/L+ Aspartate 150mg/L+0.6 mol/L glucose with 2.5 x 105/ml of the culture density. Observed the signs of protoplasts division,but did not get callus and plant regeneration.4. The research started the thromosome doubling of the adventitious buds of leaves, selecting 1/2MS+BA 0.25mg/L+IAA0.25mg/L+TDZ 0.01 mg/L as the best medium for induction adventitious buds.Colchicine solution was used in vitro leaves which induced adventitious buds.After 8d pre-culture, with 500mg/L of 12h colchicine solution,we observed that a number of morphological variation in regeneration plants.Then, we identified the plants of morpholotical variation by flow cytometry, polyploidy was not found.