In vitro production of phenolic compounds and antioxidant activity in callus suspension cultures of Habenaria edgeworthii: A rare Himalayan medicinal orchid

An in vitro method was developed for the production of phenolic compounds from callus suspension cultures of Habenaria edgeworthii Hook. f. ex. Collett, a rare Himalayan medicinal orchid. Friable and light yellow coloured callus was induced within eight weeks when seeds were cultured on Murashige and Skoog (MS) or 1/2 MS medium supplemented with 1 mu M alpha-naphthaleneacetic acid (NAA). In order to increase the biomass, following another 2 weeks these calli were repeatedly subcultured (4 week interval) onto 1/2 MS liquid medium supplemented with 0.1 mu M 6-benzyladenine (BA). The effect of BA (0.0-3.0 mu M) and methyl jasmonate (MeJA; 10-1000 mu M) treatment on callus growth, total phenolic content and corresponding antioxidant activity was also determined following 4 weeks of culture on 1/2 MS medium (liquid). Maximum callus biomass (2.61 g fresh weight) was obtained in medium containing 1.0 mu M BA. The total phenolic content varied significantly (p < 0.05) in callus grown on BA, and ranged between 10.33 and 14.30 mg gallic acid equivalent (GAE) per g dry weight (DW) (compared to control 10.17 mg GAE per g DW). Antioxidant activity, measured by three in vitro assays, i.e., 2,2'- azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging, 1,1-dipheny1-2 picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP) assays showed maximum activity in callus grown on 3.0 mu M BA containing medium. High performance liquid chromatography (HPLC) analysis showed the presence of high gallic acid content (143.63 mg/100 g DW) when compared to wild tuber (5.5 mg/100 g DW). The result indicate that callus grown on medium supplemented with 3.0 mu M BA showed high phenolic content along with increased antioxidant activity, however, callus growth was higher with 1.0 mu M BA. The result of this investigation has commercial applications as increased biomass production will yield active compounds, much needed by the pharmaceutical and neutraceutical industries. (C) 2012 Elsevier B.V. All rights reserved.