Induction, Elicitation and Determination of Total Anthocyanin Secondary Metabolites from In vitro Growing Cultures of Arbutus andrachne L

Abstract

Anthocyanin pigments are important secondary metabolites that produced in many plant species. They have wide range of uses in food and pharmaceutical industries as antioxidant and food additives. Medically, they prevent cardiovascular disease and reduce cholesterol levels as well as show anticancer activity. This study aims at utilizing a rare medicinal tree, A. andrachne in the production of anthocyanin pigments via plant in vitro culture techniques. The effects of different physical and chemical factors on total anthocyanin content (TAC) in callus and cell suspension culture of A. andrachne have been investigated. Anthocyanins have been detected in the callus of A. andrachne with the presence of light. McCown Woody Plant medium (Mcc) supplemented with 0.5 mg/l NAA, 2.00 mg/l TDZ and 1.0 g/l PVP resulted in red colored callus occurred after 16 days of culture. The highest anthocyanin concentration was observed in MCc salt mixture, sucrose and a mix of starch and agar for gelling. Cell suspension growth and anthocyanin production was the highest in Gamborg’s B5 media that supplemented with 0.2mg/l Kineitin, 0.1 mg/l NAA, 0.25 g/l casein hydrolysate and 0.3% w/v sucrose. The effect of osmotic stress by using sorbitol enhanced the production of anthocyanin pigments at earlier stage than control (sucrose alone). Elicitation of anthocyanins with salicylic acid at 1.0 mg/l for 48 hours showed significant increase in TAC from 2.6 to 3.8 mg/l. This study outlined a protocol for large scale anthocyanin production via plant in vitro culture technique. Key words: Anthocyanin, Arbutus andrachne L., callus culture, cell suspension culture, elicitation