Articles
Cryopreservation of an endangered pharmaceutically important orchid, Cymbidium finlaysonianum Lindl. using vitrification technique
Article number
1234_16
Pages
125 – 132
Language
English
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.
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.
Authors
S. Rittirat, S. Klaocheed, J. Suppapan, P. Chaithada, S. Kalawong, K. Thammasiri
Keywords
Cymbidium finlaysonianum, plant vitrification solution 2, cryopreservation, TTC, protocorm-like bodies (PLBs), 6-benzyl-aminopurine
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