CRYOPRESERVATION OF INVITRO-CULTURED MULTIPLE BUD CLUSTERS OF ASPARAGUS (ASPARAGUS-OFFICINALIS L-CV HIROSHIMAGREEN (2N=30) BY THE TECHNIQUES OF VITRIFICATION

A culture line of asparagus forming green bulbous structures consisting of numerous multiple bud clusters designated "bud clusters" was induced from a meristem culture of asparagus (Asparagus officinalis L.cv. Hiroshima-green, 2n=30). Small cubic segments (2 mm3) cut from bud clusters were cryopreserved using three different cryogenic protocols. Only vitrification produced very high levels of shoot formation after cooling to -196-degrees-C. Segments were treated with a vitrification solution (PVS2) at 25-degrees-C for 45 min or at 0-degrees-C for 120 min prior to a direct plunge into liquid nitrogen. After rapid warming, the segments were expelled into Murashige and Skoog medium containing 1.2 M sucrose for 10 min and then plated on agar shoot outgrowth medium. The average rate of shoot formation of vitrified segments producing normal shoots was near 90% without any preculture and/or cold-acclimation treatment. Revived segments resumed growth within 3 days and developed about three shoots per segment. In vitro-cultured bud clusters appear promising as material for cryopreserving asparagus germplasm.