Three vitrification-based methods for the cryopreservation of Grammatophyllum speciosum protocorms were invesigated: droplet-vitrification, encapsulation-dehydration and encapsulation-vitrification. Protocorms, 0.1 cm in diameter, developed from 2-month-old germinating seeds were used. For droplet-vitrification, protocorms were precultured on filter paper soaked in half strength Murashige and Skoog medium (½MS) containing 0.4 M sucrose at 25 + 2°C for 2 d, followed by soaking in loading solution (2 M glycerol and 0.4 M sucrose in ½MS liquid medium) for 20 min and then dehydrated with PVS2 solution [30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethyl sulfoxide in ½MS liquid medium containing 0.4 M sucrose at pH 5.7] for 30 min. For encapsulation-dehydration, encapsulated protocorms were precultured in ½MS liquid medium containing 0.4 M sucrose on a shaker (110 rpm) at 25 + 2°C for 2 d, followed by soaking in the same loading solution for 20 min and then exposed to a sterile air-flow at 2.5 inches/water column from the laminar air-flow cabinet for 8 h. For encapsulation-vitrification, encapsulated protocorms were precultured in ½MS liquid medium containing 0.4 M sucrose for 1 or 2 d, followed by soaking in the same loading solution for 20 min and then dehydrated with PVS2 solution for 60 min. For all three methods, preculturing with 0.4 M sucrose for 2 d resulted in a significant induction of dehydration and freezing tolerance. The cryopreservation results showed highest protocorm regrowth after droplet-vitrification (38%), followed by encapsulation-dehydration (24%) and encapsulation-vitrification (14%). Plantlets developed from these three methods did not show any abnormal characteristics or ploidy level change when investigated by flow cytometry.
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Document Type: Research Article
July 1, 2010
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CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation
The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.