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

Abstract

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 ½ MS medium supplemented with 1μM α-naphthaleneacetic acid (NAA). In order to increase the biomass, following another 2 weeks these calli were repeatedly subcultured (4 week interval) onto ½ MS liquid medium supplemented with 0.1μM 6-benzyladenine (BA).The effect of BA (0.0–3.0μM) and methyl jasmonate (MeJA; 10–1000μM) treatment on callus growth, total phenolic content and corresponding antioxidant activity was also determined following 4 weeks of culture on ½ MS medium (liquid). Maximum callus biomass (2.61g fresh weight) was obtained in medium containing 1.0μM BA. The total phenolic content varied significantly (p<0.05) in callus grown on BA, and ranged between 10.33 and 14.30mg gallic acid equivalent (GAE) per g dry weight (DW) (compared to control 10.17mg 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-diphenyl-2 picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP) assays showed maximum activity in callus grown on 3.0μM BA containing medium. High performance liquid chromatography (HPLC) analysis showed the presence of high gallic acid content (143.63mg/100g DW) when compared to wild tuber (5.5mg/100g DW). The result indicate that callus grown on medium supplemented with 3.0μM BA showed high phenolic content along with increased antioxidant activity, however, callus growth was higher with 1.0μM 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.