| In this work, the variant cell line of Astragalus cicer L. resistant to methionine was obtained by in vitro mutant selection. The conditions of isolation and culture of protoplasts from resistant Astragalus cicer L. cell line, Helianthemum Songaricum Schrenk and Zygophyllum xanthoxylum were studied. Based on them, somatic hybridization between Astragalus cicer L. and Helianthemum Songaricum Schrenk was carried out. The main achievements were as follows:1. The variant cell line of Astragalus cicer L. resistant to 100mmol/L methionine was isolated from calluses that were treated with NaN3. Plants were regenerated from the selected cell line. After growing for 6 months on a medium without selection pressure, the variant calluses were cultured for 30 days in the medium containing 100mmol/L methionine, and the relative growth rate was 11.9 fold of the control's. The content of free methionine in the variant calluses was 5.65 fold of the control's, other amino acids of the asparate family in the variant cell line increased obviously in comparison with the control's. Analyses of SDS-PAGE pattern and isozyme patterns of peroxidase and cytochrome oxidase revealed the differences between the variant cell line and the control.2. Protoplasts were isolated from the variant cell line of Astragalus cicer L.resistant to methionine. The highest yield of protoplasts (3.5×106/g F.Wt.) was obtained from 12-day-old friable calluses after subcultured on fresh medium. The enzyme combination of 1.5% Cellulase (Onozuka R-10), 1% Hemicellulase (Sigma) and 1.5% Pectinase (Serva) was more effective for protoplast isolation. Protoplasts were induced to undergo sustained division in DPD medium supplemented with 1.0 mg/L 2,4-D, 0.2mg/L 6BA, 2%(w/v)sucrose, 0.4mol/Lmannitol and 500mg/L casein hydrolyaste at a plating density of 2×105/ml. The division frequency was over 52.3%. The regenerated calluses still preserved resistance to methionine and ethionine.3. Protoplasts were isolated from the calluses of Helianthemum SongaricumSchrenk. The highest yield of protoplasts (1.9×106/g F.Wt.) was obtained from 10-day-old calluses after subculturing on fresh medium. The optimum combination of enzyme mixture was 1% cellulase, 1% hemicellulase and 1.5% pectinase. The viability of protoplasts reached to over 70%. The proper phytohormone combination was 3.0mg/L NAA and 0.2mg/L 6BA, in which the division frequency 31.8% was gotten.4. In this study, the calluses and leaves of Zygophyllum xanthoxylum were used as materials for protoplast preparation. The effetcts of some factors, such as different combination of enzymes, culture methods and phytohormones in medium, on protoplasts isolation and culture were investigated. The results demonstrated that protoplasts with high yield and quality were obtained by treating the calluses with a mixture of 2% cellulase, 1% hemicellulase and 1% pectinase for 13h. The liquid thin layer culture method was more suitable for protoplasts division. Protoplasts were induced to undergo sustained division in DPD medium added with 1.0 mg/L 2,4-D, 0.2mg/L 6BA, 0.2mg/L KT, 2%(w/v) sucrose, 0.5mol/Lmannitol and 500mg/L casein hydrolysate at a plating density of 2.0×105/ml. The division frequency was 28.4%.5. Somatic hybrid cells were obtained between methionine resistant cell line of Astragalus cicer L. and cell line of Helianthemum Songaricum Schrenk. using protoplast fusion by PEG-high pH, high Ca2+ method. The hybrid cells were selected efficiently using IOA pretreatment of protoplasts and the genetic characteristic of mutant Astragalus cicer L.. A method that can select somatic hybrids effectively was established. Three calluses of hybrid were produced.
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