Journal of Taiwan Agricultural Research

Author:Hsiao-Sung Chan, Tzu-Ting Lo, Uei-Chern Chen, Chi-Ni Hsia*, and Hsin-Sheng Tsay

Abstract:

    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 modifed B5 me-dium 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 concen-tration 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, repre-senting 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 treat-ment 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 cul-turing.  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 signifcantly by nitrogen sources in the B5 medium.  Increasing concentration of KNO₃, re-ducing 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.

Key words:Salvia miltiorrhiza, Hairy root, Tanshinone, Nitrogen sources, Two-stage feeding model

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