氮源型態及濃度對丹參毛狀根生長與丹參酮產量之影響

Effect of Nitrogen Source on Biomass and Tanshinone Production of Salvia miltiorrhiza in Hairy Root Culture

詹効松(Hsiao-Sung Chan) ； 羅子庭(Tzu-Ting Lo) ； 陳威臣(Uei-Chern Chen) ； 夏奇鈮(Chi-Ni Hsia) ； 蔡新聲(Hsin-Sheng Tsay)

《台灣農業研究》 61卷2期 (2012/06) Pp. 100-111

https://doi.org/10.6156/JTAR/2012.06102.03

摘要

利用具有快速生長特性之轉殖毛狀根做為培養材料是組織培養大量生產植物二次代謝物之新趨勢，本研究以B5鹽類配方作為丹參（Salvia miltiorrhiza）毛狀根之基礎培養基，探討B5培養基中兩種型態之氮源-硝酸鉀（KNO3）與硫酸銨[（NH4）2SO4）]及其濃度對於丹參毛狀根生質量、丹參酮含量及其產量之影響。將B5鹽類配方中之硝酸鉀濃度分別調整為0-49.5 mM，B5配方中之其餘成分及濃度維持不變，毛狀根經8週培養後，顯示以12.4-37.1 mM硝酸鉀處理具有較高之生質量；丹參酮的含量及產量則以37.1 mM硝酸鉀處理最高，為對照組B5培養基（24.7 mM硝酸鉀）的2倍。將B5鹽類配方中之硫酸銨濃度分別調整為0-2.0 mM進行毛狀根培養，B5配方中之其餘成分及濃度維持不變，結果顯示毛狀根於不含硫酸銨之培養基中可獲得最高之生質量10.25 g/L；在丹參酮的含量及產量方面，則以0.5 mM硫酸銨處理最高，分別為對照組B5培養基（1.0 mM硫酸銨）的2.8倍及4.1倍。因培養結果顯示硫酸銨對毛狀根的生長具抑制作用，因此採用二階段培養方法，亦即將丹參毛狀根先於不含硫酸銨的B5培養基中培養4週後，再以饋料方式添加硫酸銨於培養液中，使其濃度為1.0 mM及2.0 mM並繼續再培養4週，結果顯示在培養4週後以饋料添加1.0 mM硫酸銨培養方式，相較全程8週培養於初始濃度為1.0 mM硫酸銨之B5培養基之對照組，在毛狀根生質量方面可提高1.3倍，在總丹參酮產量方面則提高2.2倍。以上結果顯示，氮源型態及濃度對丹參毛狀根生長與丹參酮產量有明顯之影響，提高B5鹽類配方中之硝酸鉀濃度、降低硫酸銨濃度、或以二階段培養方式將毛狀根於不含硫酸銨之培養基中培養，生長後期利用饋料添加硫酸銨，皆能促進毛狀根生長，並增加丹參酮的累積，是現階段較佳之培養策略。

關鍵字

丹參 ； 毛狀根 ； 丹參酮 ； 氮源 ； 二階段培養

並列摘要

There is a new tendency to produce secondary metabolites of plants in tissue culture, using fast growing hairy roots. The objective of this study was to investigate effect of potassium nitrate (KNO3) and ammonium sulfate [(NH4)2SO4] in the culture medium on growth of hairy roots of Salvia miltiorrhiza Bunge and production of tanshinone. Roots of S. miltiorrhiza were grown in a modified B5 medium at 70 rpm on a shaker under darkness for eight weeks and data were collected. Results showed that growth of hairy roots varied with concentrations of KNO3 in the medium and the optimum concentration of KNO3 was 12.4 mM, which resulted in maximum production of root biomass (8.90 g dw/L). However, the highest tanshinone content was obtained in the culture containing 37.1 mM KNO3, representing a 2-fold increase in tanshinone production, compared to the control concentration of 24.7 mM KNO3 in the B5 medium. When hairy roots were cultured in the B5 basal medium containing different concentrations of (NH4)2SO4 from 0 to 2.5 mM, the maximum production of root biomass (10.25 g dw/L) was the B5 medium without (NH4)2SO4. Although growth of hairy roots was inhibited by (NH4)2SO4 at all the concentrations (0.5-2.5 mM) tested, an increase in tanshinone contents was observed in the treatment of (NH4)2SO4 at lower concentration of 0.5 mM. The tanshinone production in the treatment of 0.5 mM (NH4)2SO4 was 4.1-fold higher than the treatment of 1.0 mM (NH4)2SO4 representing the control concentration of (NH4)2SO4 in the B5 medium. Since root growth was found inhibited by (NH4)2SO4 in the B5 medium, in subsequent experiment hairy roots were grown in the B5 medium without (NH4)2SO4 for 4 weeks before adding 1.0 mM or 2.0 mM (NH4)2SO4 for another 4 weeks culturing. This two-stage model [weeks 1-4 without (NH4)2SO4 and weeks 5-8 with 1.0 mM (NH4)2SO4] increased root biomass of S. miltiorrhiza by 1.3 fold and tanshinone production by 2.2 fold, compared to the control treatment of 1.0 mM (NH4)2SO4 in the B5 medium for the entire culturing period of 8 weeks. In conclusion, biomass and tanshinone production in hairy root culture of S. miltiorrhiza were affected significantly by nitrogen sources in the B5 medium. Increasing concentration of KNO3, reducing concentration of (NH4)2SO4 in the B5 medium or using the two-stage model of application of (NH4)2SO4 is useful in enhancing biomass as well as tanshinone production in hairy root culture of Salvia miltiorrhiza.

並列關鍵字

Salvia miltiorrhiza ； Hairy root ； Tanshinone ； Nitrogen sources ； Two-stage feeding model