延胡索叶片愈伤组织诱导及其2种活性成分含量测定

Callus Induction of Corydalis yanhusuo Leaf and Content Determination of Its Two Active Ingredients

目的:以叶片为外植体诱导延胡索(Corydalis yanhusuo)形成愈伤组织,测定愈伤组织生长指标,检测生物碱类成分含量,为研究延胡索生物碱代谢调控机制和培育优良品系提供依据。方法:运用2,4-二氯苯氧乙酸(2,4-D)与6-苄基氨基嘌呤(6-BA)诱导形成愈伤组织,采用重量法测定生物量,组织化学染色法定位生物碱,高效液相色谱法测定延胡索乙素和脱氢紫堇碱含量。结果:延胡索叶片经MS+2 mg/L 2,4-D+1 mg/L 6-BA处理,愈伤组织诱导率可达75%;外观呈黄白色,表面呈颗粒状,生长迅速;继代培养的愈伤组织鲜重(2.7153±0.4398)g,增殖倍数4.43;干重(0.1552±0.0207)g,含水量94.16%。愈伤组织薄壁细胞内含能被碘化铋钾染色的橘黄色斑块,提示生物碱的存在;愈伤组织中延胡索乙素含量达(0.8600±0.3502)mg/g DW(干重),脱氢紫堇碱含量为(0.1744±0.0372)mg/g DW。结论:利用叶片作为外植体获得的延胡索愈伤组织诱导率高、生长迅速,是快速、稳定获取延胡索乙素等生物碱类活性成分的适宜材料。

Objective:The leaf of Corydalis yanhusuo was used as explants to induce callus,the growth parameters and contents of alkaloids in the callus were measured,to provide a basis for the study of alkaloids metabolic regulation and superior varieties breeding of C.yanhusuo.Methods:The combination of 2,4-dichlorophenoxyacetic acid(2,4-D)and 6-benzyladenine(6-BA)were used for callus induction.The callus biomass was measured by weight method.Localization of alkaloids was performed by histochemical staining method.The contents of tetrahydropalmatine and dehydrocorydaline were determined by high-performance liquid chromatography method.Results:The callus induction rate of C.yanhusuo reached 75%by Murashige and Skoog medium with 2 mg/L 2,4-D and 1 mg/L 6-BA treatment.The callus was yellowish-white with a granular surface and grew rapidly.The fresh weight of subcultured callus was(2.7153±0.4398)g,with the multiplication coefficient of 4.43.The dry weight was(0.1552±0.0207)g,and the water content reached 94.16%.Callus parenchyma cells contained orange patches stained with Dragendorff’s reagent,indicating the presence of alkaloids.The content of tetrahydropalmatine in the callus reached(0.8600±0.3502)mg/g DW(dry weight),meanwhile,dehydrocorydaline content reached(0.1744±0.0372)mg/g DW.Conclusion:The callus of C.yanhusuo induced by using leaves as explants showed a high induction rate and rapid growth,which was a suitable material for deriving alkaloids active ingredients such as tetrahydropalmatine quickly and stably.