CRYOPRESERVATION OF ASPARAGUS (ASPARAGUS-OFFICINALIS L) EMBRYOGENIC SUSPENSION CELLS AND SUBSEQUENT PLANT-REGENERATION BY VITRIFICATION

Embryogenic cells of asparagus (Asparagus officinalis L.) were successfully cryopreserved by vitrification and subsequently regenerated plants. The cells were cryoprotected with a mixture of 2 M glycerol and 0.4 M sucrose at 25-degrees-C for 10 min and then transferred to 1.8-ml plastic cryotubes. The cryoprotected cells were dehydrated with a new vitrification solution (designated PVS3) at 0-degrees-C for 20 min prior to a plunge into LN2. PVS3 contains 50% (w/v) glycerol and 50%. (w/v) sucrose in water. The vitrified cells in LN2 were rapidly warmed in a water bath at 40-degrees-C and then expelled into LS medium supplemented with 1.2 M sucrose. The cell survival evaluated by fluoresceine diacetate and phenosafranin amounted to 80-90% of untreated, unfrozen controls. Revived cells resumed growth within 3 days after plating and regenerated plantlets via embryogenesis. Plant regeneration efficiency was nearly the same as that of the untreated, unfrozen control. Even an 80% concentration of PVS3 diluted with water produced 80% survival.