培养条件对三叶青愈伤组织生长及总黄酮含量的影响

Influence of Culture Conditions on the Growth of Callus and Content of Total Flavonoids in Tetrastigma Hemsleyanum

研究培养条件对三叶青愈伤组织生长及黄酮积累的影响,建立用愈伤组织生产三叶青黄酮的方法。以三叶青的叶诱导产生的愈伤组织为材料,分别采用单因素实验、正交实验的方法比较了不同光照周期、不同有机添加物、不同前体、3种基础培养基、不同6-BA与NAA浓度的培养条件下愈伤组织生长的情况及黄酮含量。用愈伤组织生产三叶青黄酮可采用两步培养法来进行:先在促进愈伤组织生长的最佳处理组合条件:12 h/d间断性光照、MS+3.0 mg/L6-BA+2.0 mg/L NAA+2 g/L蛋白胨下培养30 d,再转入最利于黄酮积累的处理组合:连续光照、B5+4.0 mg/L 6-BA+2.0 mg/L NAA+40 mg/L苯丙氨酸下培养20 d。采用该方法培养的愈伤组织中总黄酮最大浓度可以达到28.4±3.9 mg/g DW。采用愈伤组织培养方式生产三叶青黄酮,不仅可以大大地缩短生产周期,而且培养物中的总黄酮含量也明显高于原植株叶片,接近于根茎中的黄酮含量,具有较大的可行性。

To set up the optimal culture conditions for the callus growth and the accumulation of total flavonoids,the influence of light,medium,plant growth regulators and organic additives on the growth of callus and content of total flavonoids in Tetrastigma hemsleyanum was analyzed.The callus growth and the accumulation of total flavonoids under different culture conditions（photoperiods,organic additives,basic meida,concentrations of 6-BA and NAA） were studied by single factor experiment and orthogonal test.For callus growth the optimum medium was 12 h/d photoperiods,MS＋3mg/L 6-BA＋2 mg/L NAA＋2 g/L peptone;For accumulation of total flavonoids the optimum medium was 24 h/d photoperiods,B5＋4 mg/L 6-BA＋2 mg/L NAA＋ 40 mg/L Phenylalanine.Callus of Tetrastigma hemsleyanum could be cultured by a Two-step Method： callus was cultured in the former medium for 30 days and then in the latter for 20 days.By using this method,total flavonoids in cultured callus of maximum concentration could reach 28.4±3.9 mg/g DW.Tetrastigma hemsleyanum grew significantly faster in callus culture.The total flavonoids content in callus was significantly higher than that of raw plant leaves,close to the rhizomes.