In vitro tuber induction and growth improvement in Dactylorhiza umbrosa by osmotic pretreatments

Abstract

ABSTRACT Harvesting Dactylorhiza umbrosa from natural habitats for salep production (powdered dried tuber) has endangered the existence of this species. Mass production of tubers is possible in vitro. Different osmotic pretreatments for inducing tuber formation in D. umbrosa were investigated. Seeds were germinated in Fast medium and protocorm derived seedlings measuring 2 cm in height with roots were subjected to 2% and 4% w/v of different osmotica including sorbitol, mannitol, and polyethylene glycol 4000 (PEG), increased concentrations of sugars [sucrose 18 g·L−1+fructose 7.5 g·L−1 (sugar 1.5x); sucrose 24 g·L−1+fructose 10 g·L−1 (sugar 2.0x)] and non-pretreated control (sugars 1x; 12 g·L−1 sucrose+5 g·L−1 fructose) for 29 days. Then, the seedlings were transferred to jars containing Fast medium (similar to the control treatment) supplemented with 1 g·L−1 activated charcoal. After 70 days, survival, growth, tuber formation, shoot proline and photosynthetic pigments, and shoot and roots soluble and reserve carbohydrates were determined. PEG and mannitol 2% pretreatments increased root number and photosynthetic pigments. The pretreatments did not affect shoot proline concentration, but induced soluble and reserve sugars accumulation in tubers. According to shoot growth improvement and obtaining the highest tuber number and root carbohydrates content, PEG 2% pretreatment was suggested for D. umbrosa tuberization.