Cryopreservation of Rye Protoplasts by Vitrification

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Abstract
A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20°C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0°C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN2); and (d) recovery of the protoplasts from LN2 and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN2 if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3±2%. In contrast, survival of ACC protoplasts following recovery from LN2 was 34 ± 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 ± 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.