NAA和2,4-D对甘草愈伤组织细胞染色体倍性及次生代谢物的影响

Effect of naphthalencacetic acid and 2,4-dicholorophenoxyacetic acid on callus cell ploidy and secondary metabolites of Glycyrrhiza uralensis

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 the system of high frequency chromosome doubling and to lay the fundation for obtaining the strain of licorice with a high active ingredient content.Methods Exogenous hormone of naphthalencacetic acid（NAA） and 2,4-dicholorophenoxyacetic acid（2,4-D） were used to induce callus of Glycyrrhiza uralensis explants from cotyledons,hypocotyls,and radicle,and the effect of NAA and 2,4-D were tested on callus cell ploidy and content of secondary metabolites.Results The result showed that there were significant effects of NAA and 2,4-D with different concentrations on cell ploidy of different explants callus.The frequency of cell with chromosome 2n 14 and 2n = 28 was significantly higher in hypocotyls callus than those in cotyledons and radicle.They were up to 42.38% and 25.19%,respectively.Meanwhile,the frequency of cell with 2n 14 and 2n = 28 in cotyledons,hypocotyls,and radicle callus all reached the highest under the concentration of 2.0 mg/L NAA,2.0 mg/L 2,4-D,and 1.0 mg/L 2,4-D,respectively.They were up to 61.50%,39.20%,63.00%,36.23%,63.50%,and 37.50%,respectively.The doubling rate of callus cell increased by the extension of cultured time.Furthermore,there were significant effects of NAA and 2,4-D on promoting the contents of glycyrrhizic acid and licorice flavanone.The contents of glycyrrhizic acid and licorice flavanone of cotyledons callus reached the highest on MS medium with 1.0 mg/L NAA and 1.0 mg/L 2,4-D,they were up to 52.13 mg/g and 8.36 mg/g,respectively.And the contents in hypocotyls callus reached the highest on MS medium with 2 mg/L NAA,they were up to 49.01 mg/g and 8.54 mg/g,respectively.Besides the contents of glycyrrhizic acid and licorice flavanone showed positive correlation with chromosome doubling frequency of callus cell.Conclusion There are significant effects of NAA and 2,4-D with different concentrations to promote chromosome doubling and the content increasing of glycyrrhizic acid and licorice flavanone in different explants callus.It suggests that perhaps the new material in licorice with higher pharmacodynamic components will be obtained by chromosome doubling.