Vitrification-based cryopreservation of Grammatophyllum speciosum protocorm

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 (half strength MS) containing 0.4 M sucrose at 25 2 degree C for 2 d, followed by soaking in loading solution (2 M glycerol and 0.4 M sucrose in half strength MS liquid medium) for 20 min and then dehydrated with PVS2 solution [30 percent (w/v) glycerol, 15 percent (w/v) ethylene glycol and 15 percent (w/v) dimethyl sulfoxide in half strength MS liquid medium containing 0.4 M sucrose at pH 5.7] for 30 min. For encapsulation-dehydration, encapsulated protocorms were precultured in half strength MS liquid medium containing 0.4 M sucrose on a shaker (110 rpm) at 25 f 2 degree 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 half strength MS 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 percent), followed by encapsulation-dehydration (24 percent) and encapsulation-vitrification (14 percent). Plantlets developed from these three methods did not show any abnormal characteristics or ploidy level change when investigated by flow cytometry.