Tsunetomo Matsuzawa

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°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°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°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.