摘要
目的:研究水杨酸刺激下半夏试管块茎悬浮培养及其体内生物碱含量的变化。方法:以半夏悬浮试管小块茎为材料,对其处理不同浓度的外源水杨酸刺激,分析块茎的生长情况,利用高效液相色谱法(HPLC)测定块茎中生物碱的含量。检测采用Agilent C_(18)色谱柱(4. 6 mm×250 mm,5μm),流动相乙腈-水(4∶96),柱温35℃,肌苷检测波长250 nm,鸟苷检测波长260 nm,流速1. 0 m L·min~(-1),进样量10μL。结果:外源添加不同浓度的水杨酸,处理不同的时间,半夏块茎的鲜重在第25天,水杨酸150μmol·L~(-1)时达到最大7. 483 8 g。同时积累了一定量的生物碱,鸟苷在0. 03~0. 45μg(R2=0. 999 6)线性关系良好,当水杨酸浓度为50μmol·L~(-1)时,培养10 d,半夏块茎中鸟苷的质量分数达到最大值1. 353 3 mg·g~(-1);肌苷在0. 003~0. 045μg(R2=0. 999 5)线性关系良好,在水杨酸浓度为200μmol·L~(-1)时,培养30 d,半夏块茎中肌苷的质量分数达到最大值0. 149 8 mg·g~(-1)。结论:该研究结果为半夏试管块茎的组培快繁和生物碱的调控研究提供参考,对半夏产业的发展具有十分重要的意义。
Objective: To study the suspension culture of tuber and its alkaloid content based on the stimulation of salicylic acid. Method: The tubers of Pinelliae Rhizoma in suspension tube were treated with different concentrations of exogenous salicylic acid to analyze the growth status. The content of alkaloids in tuber was detected by HPLC. Test conditions: chromatographic column for Agilent Eclipse plus C_(18) column( 4. 6 mm ×250 mm,5 μm),mobile phase of acetonitrile water( 4 ∶ 96),the column temperature was maintained at 35 ℃,detection wavelength for inosine 250 nm, guanosine 260 nm, volume flow rate 1. 0 m L·min~(-1). Result: The results showed that the exogenous salicylic acid had a certain effect on the growth of suspension tuber of Pinelliae Rhizoma. When the salicylic acid concentration was 150 μmol·L~(-1),the culture lasted for 25 days and the fresh weight reached the maximum value of 7. 483 8 g. It also accumulates a certain amount of alkaloids. The linear range of guanosine was 0. 03-0. 45 μg( R2= 0. 999 6). After 10-days cultivatation in the salicylic acid concentration of 50 μmol ·L~(-1), guanosine content of Pinelliae Rhizoma tubers reached a maximum of1. 353 3 mg·g~(-1). The linear range of inosine 0. 003-0. 045 μg( R2= 0. 999 5). When the salicylic acid concentration was 200 μmol·L~(-1),cultured for 30 days,the content of inosine in Pinelliae Rhizoma tubers reached the maximum value of 0. 149 8 mg·g~(-1). Conclusion: The results of this experiment provide a reference for the study of tissue culture and rapid propagation of Pinelliae Rhizoma tubers and regulation of alkaloids,which are of great significance for the development of Pinelliae Rhizoma industry.
引文
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