title: Vitrification-based cryopreservation of Grammatophyllum speciosum protocorm.
abstract: 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.
authors.affiliation: Department of Biotechnology, Mahidol University, Bangkok, Thailand.
authors.forename: K
authors.initials: K
authors.lastname: Sopalun
authors.forename: K
authors.initials: K
authors.lastname: Kanchit
authors.forename: K
authors.initials: K
authors.lastname: Ishikawa
mesh_terms: D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D018744:DNA, Plant / Q000032:analysis; D003890:Desiccation / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D029595:Orchidaceae / Q000235:genetics / Q000254:growth & development*; D011003:Ploidies; D012639:Seeds / Q000254:growth & development*; D013395:Sucrose / Q000494:pharmacology; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D018744:DNA, Plant; D013395:Sucrose; D019855:Ethylene Glycol; D005990:Glycerol; D004121:Dimethyl Sulfoxide