Cryopreservation of an endangered pharmaceutically important orchid, Cymbidium finlaysonianum Lindl. using vitrification technique

Abstract

Cryopreservation is an in vitro conservation method which has become an important tool for long-term storage of plant genetic resources. New protocorm-like bodies (PLBs) (about 4-5 mm in diameter) of Cymbidium finlaysonianum Lindl. were isolated individually from 2-month-old proliferating PLB clusters which had been cultured in VW liquid medium (VW; Vacin and Went, 1949) supplemented with 8.84 µM 6-benzyl-aminopurine were successfully cryopreserved using a vitrification method. In this cryogenic procedure, PLBs were precultured in MS liquid medium (MS; Murashige and Skoog, 1962) supplemented with 0.5 M sucrose at 25±2°C for 2 d on an orbital shaker at 110 rpm. The PLBs were treated with loading solution (2 M glycerol plus 0.4 M sucrose) for 20 min at 25±2°C to make the precultured PLBs tolerant to plant vitrification solution 2 (PVS2). Subsequently, the selected PLBs were subjected to PVS2 (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethyl sulfoxide and 0.4 M sucrose in MS medium, pH 5.8) treatment at various exposure times (0-120 min) at 0°C and plunged into liquid nitrogen (LN) for 1 d. After storage in LN, the PLBs were rewarmed and washed by MS liquid medium containing 0.5 mL of 1.2 M sucrose for 20 min. One week after rewarming PLBs, viability was determined by TTC reduction and regrowth assessed. The results showed that the PLBs precultured with 0.5 M sucrose for 2 d, followed by dehydration with vitrification solution for 60 min had the highest post rewarming viability in terms of TTC reduction (40%) and regrowth (33.5%). No survival rate of PLBs was found without vitrification treatment. Regenerated plants showed the same morphological characteristics as the control.