Production of Naphthoquinone Pigments in Cell Suspension Cultures of Arnebia euchroma (Royle) Johnston: Influence of pH on Growth Kinetics and Acetylshikonin

In this paper, the effect of pH on in vitro cell growth and naphthoquinone pigment production in cell suspension cultures of Arnebia euchroma (Royle) Johnston was evaluated under a two-stage culture system. Friable callus derived from leaves of in vitro established shoots was maintained on MS medium supplemented with BAP (10 μM) and IBA (5 μM) and the same medium was used for raising cell suspension cultures. An acidic pH (i.e. 5.0-6.50) of the medium helped cell growth, while that having alkaline pH (7.25-9.50) favoured pigment production. Almost double cell biomass (198.67 g, FW.L) was recorded in medium at physiological pH (5.70), when compared to neutral (138.55 g FW.L) or alkaline levels (130.56 g FW.L). Acetylshikonin (I) was found to be the major shikonin derivative in these cultures. The maximum content i.e., 2.049% was recorded at alkaline pH (9.5) in comparison to 0.803% at acidic pH level (5.75) on a fresh weight basis, as revealed by HPLC analysis of the cell suspension cultures. Microscopic examination of the cell suspensions grown at different pH revealed that higher pH adversely affected growth and morphology of the cells. Importantly, the induction of pigment in the alkaline range of pH was independent of the growth phase and pH showed a regulatory role in its biosynthesis. _____________________________________________________________________________________________________________