四倍體丹參毛狀根系之時序培養及其丹參酮生產篩選

Screening on Tanshinone Production and Time Course Culture of Tetraploidy Hairy Roots of Salvia miltiorrhiza

夏奇鈮(Chi-Ni Hsia) ； 曹進義(Chin-Yi Tsao) ； 吳姿穎(Tzu-Ying Wu) ； 陳威臣(Uei-Chern Chen)

《台灣農業研究》 67卷1期 (2018/03) Pp. 94-105

https://doi.org/10.6156/JTAR.201803_67(1).0009

摘要

丹參(Salvia miltiorrhiza)為唇形科多年生草本植物，是中醫治療心血管疾病的重要藥材。4倍體(4N)毛狀根與其2倍體(2N)相較，具有二次代謝物含量較高之特性，是離體培養大量生產植物二次代謝物的良好工具。本研究利用秋水仙素誘導所得之4N丹參毛狀根群，探討從中篩選出高丹參酮產量毛狀根系(以下簡稱根系)的可行性。首先以固體培養基進行連續3次、每次8 wk的根系培養，並依據丹參酮產量從103個4N根系中篩選出前32名，繼續進行連續3次、每次8 wk的液體培養；同時從32個4N根系中挑選出2個根系與2N根系一起進行時序培養。固體培養的結果顯示，4N根系群的平均生質量較2N根系低，僅達2N根系的67.2%，但4N根系群的平均總丹參酮含量較2N根系為高，是2N根系的4.66倍，且4N根系群的平均丹參酮產量為2N根系的3.29倍。檢視4N根系群丹參酮含量比例的變化，以隱丹參酮含量之比例增加最多，丹參酮I含量之比例減少最多，丹參酮IIA含量之比例減少其次。在固體培養中丹參酮產量表現最佳之前10名4N根系，在液體培養篩選後僅餘2根系仍保持於前10名，顯示4N根系在固、液體培養中的表現並非完全一致，建議在固體培養篩選階段保留較多的根系，作為後續液體培養篩選之用。時序培養結果顯示，4N根系的生長速度較2N根系慢，2個4N根系的生長模式亦存有差異；2個4N根系之丹參酮含量皆於第8週達到最高，較2N根系於第12週達到最高明顯提早。綜合來看，雖然4N根系之生質量平均較2N根系為低，但因4N根系具有丹參酮含量較高，且丹參酮產量累積較快之特性，因此在丹參酮的生產效率上，4N根系仍較2N根系為高，且透過篩選機制可將高產之4N根系選拔出來，作為大規模培養之基礎。

關鍵字

丹參 ； 毛狀根 ； 多倍數 ； 丹參酮 ； 離體培養

並列摘要

Salvia miltiorrhiza Bunge, a perennial herb of Labiatae Family, has been used to cure cardiology disease in traditional Chinese medicine for long history. Polyploidy hairy roots containing higher concentration of secondary metabolites than its diploidy are considered as better explants for in vitro production of valuable secondary metabolites. The objective of this study was to establish a selection strategy on tanshinone production of tetraploidy hairy root lines (4N lines) which were induced from the diploid hairy root line (2N line) by colchicine treatment. Three consecutive cultures in solid medium were first conducted on 103 lines of 4N. Among them, 32 lines with the highest tanshinone production were selected for another 3 consecutive liquid medium culture. Meanwhile, two 4N lines and the 2N line were used to conduct the time course culture in liquid medium for 12 wk span. Screening results from solid cultures showed that the average biomass of 103 lines of 4N was only counted 67.2% of the 2N line. However, average total tanshinone contents and productivity of 4N lines were counted 4.66 times and 3.29 times higher than that of the 2N line, respectively. Composition ratios of cryptotanshinone, tanshinone I, and tanshinone IIA contents on 4N lines and 2N line were compared. It was found ratio of cryptotanshinone increasing the most in contrast decreasing ratios of tanshinone I and tanshinone IIA. The ten best lines of 4N selected from solid medium on production of tanshinones were found having only 2 lines remaining in the best ten after liquid medium selection. In order not to miss elite lines, lower selection strength was recommended in the stage of solid culture on 4N lines. Results of time course culture showed that lagging growth of 4N lines than that of 2N line. Moreover, two 4N lines had slightly different growth rates. The highest tanshinone contents were found after 8 wk of culture on 4N lines which was much earlier than that of 12 wk of culture on 2N line. In conclusion, although 4N lines have lower biomass than that of the 2N line, 4N lines have higher tanshinone contents and shorter culture period on tanshinone accumulation which would provide better efficacy on tanshinone production than that of 2N lines. It is thought that selection of elite 4N lines will provide a good foundation for in vitro production secondary metabolites.

並列關鍵字

Salvia miltiorrhiza Bunge ； Hairy root ； Polyploidy ； Tanshinone ； In vitro culture