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目的建立甘草愈伤组织细胞高频染色体加倍体系,为获得高产量药效成分的甘草品系奠定基础。方法采用组织培养法对甘草子叶、下胚轴和胚根进行愈伤组织诱导和染色体加倍;利用紫外分光光度法检测愈伤组织甘草酸、甘草总黄酮的量。结果 NAA、2,4-D不同质量浓度及配比对不同外植体愈伤组织染色体倍性变异具有显著的影响,其中下胚轴2n>14和2n=28的细胞频率明显高于子叶和胚根,平均为42.38%和25.19%;且子叶、下胚轴及胚根分别在2.0 mg/L NAA、2.0mg/L 2,4-D以及1.0 mg/L 2,4-D下,2n>14和2n=28的细胞频率最高,分别为61.50%、39.20%,63.00%、36.23%,63.50%、37.50%。此外,NAA、2,4-D对愈伤组织中甘草酸、甘草总黄酮具有明显的促进效应,在1.0 mg/L NAA+1.0 mg/L 2,4-D组合下,子叶愈伤组织的甘草酸和甘草总黄酮量均达到最高,分别为52.13 mg/g和8.36 mg/g;在2 mg/L NAA下,下胚轴愈伤组织甘草酸及甘草总黄酮量最高,分别为49.01 mg/g和8.54 mg/g。相关分析表明,愈伤组织中甘草酸、甘草总黄酮量与2n=28的细胞频率呈正相关。结论 NAA、2,4-D对愈伤组织细胞染色体加倍及甘草酸、甘草总黄酮量提高具有显著的促进作用,通过染色体加倍有可能获得高产量药效成分的甘草品系。
OBJECTIVE: To establish a doubled high frequency chromosome doubling system for Glycyrrhiza callus cells and lay the foundation for obtaining licorice line with high yield and pharmacodynamic components. Methods Tissue culture was used to induce the callus and double chromosome of licorice cotyledons, hypocotyls and radicles. The contents of glycyrrhizin and licorice flavonoids were detected by UV spectrophotometry. Results Different concentrations of NAA and 2,4-D had significant effects on chromosome ploidy variation of different explants. The frequency of hypocotyls 2n> 14 and 2n = 28 was significantly higher than that of cotyledons and The average number of radicles was 42.38% and 25.19% respectively. The cotyledons, hypocotyls and radicles were incubated with 2.0 mg / L NAA, 2.0 mg / L 2,4-D and 1.0 mg / L 2,4-D The cell frequencies of> 14 and 2n = 28 were the highest, 61.50%, 39.20%, 63.00%, 36.23%, 63.50% and 37.50% respectively. In addition, NAA, 2,4-D had a significant promotion effect on glycyrrhizin and licorice flavonoids in callus. Under the combination of 1.0 mg / L NAA + 1.0 mg / L 2,4-D, the callus Glycyrrhizic acid and licorice flavonoids reached the highest levels of 52.13 mg / g and 8.36 mg / g, respectively. Under 2 mg / L NAA, the content of total flavonoids of licorice and licorice in hypocotyls was the highest, 49.01 mg / g and 8.54 mg / g. Correlation analysis showed that there was a positive correlation between the total flavonoids of glycyrrhizin and licorice in callus and the cell frequency of 2n = 28. Conclusion NAA and 2,4-D can significantly promote chromosome doubling and glycyrrhizin and licorice flavonoids content in callus cells, and it is possible to obtain licorice lines with high yield of medicinal ingredients by doubling chromosomes.