Cryopreservation of rye protoplasts by vitrification

26th

ABSTRACTS,

REFERENCES

1. Boutron, P. Cryobiology 25, 569 (1988). 2. La&se, A. Results presented at the Conference. 114. The Essential

Role of Vitrification in Cryopresof Parasitic Helminths. ERIC R.

ervation JAMES, MARTHA

AMY HODGSON-SMITH, BRAD SMITH, JACKSON-GEGAN , DAVID MCLEAN,

JAMES T. RAWLS, R. DOBINSON, MINTER,

AND

JOHN IRENE PETER

III,

RENFRO POPIEL,

ANTHONY DONALD

J. HAM

(Department

L. of

Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425). Classical cryopreservation protocols incorporating low concentrations of cryoprotectants combined with slow cooling have been used by several workers to successfully cryopreserve certain species of helminths, principally the infective third stage larvae (L3) of nematodes of the gastrointestinal tract. A significant group of organisms, however, has proved completely refractory to cryopreservation using this approach. Exhaustive studies with Schistosoma mansoni (trematoda) schistosomula (SS), Trichinella spiralis (nematoda) muscle stage larvae (MSL), and Sterinernema feltiae (nematoda) infective third stage juveniles (53) failed to yield viable organisms following cryopreservation by “classical” methodology. These are all relatively large (150-1000 X 30-50 pm) multicellular (>2000 cells) organisms normally inhabiting the tissues of their hosts-unlike the GI tract L3 nematodes which are all free-living preparasitic stages on grass or soil. For successful cryopreservation, S. mansoni SS require a two-step incubation in ethanediol (lo%, v/v, followed by 35%, v/v) followed by cooling at 5000°C min-‘; T. spiralis MSL also require a two-step cryoprotectant incubation (20% ethanediol followed by 33% ethanediol + 33% methanol in saline) and cooling at 5000°C min - ’ ; S. feltiae 53 require partial dehydration (to 97% RH), incubation in a high cryoprotectant concentration (70% methanol), and fast cooling at >2OO”C min-‘. Other studies (see abstract 66, James and Renfro, this volume) indicate that these combinations of cryoprotectant concentrations and cooling rates will produce vitrification. For these three organisms cooling rate is critical with no increase in survival at faster rates and a precipitate drop to zero survival at slower rates. 115. Cryopreservation tion.

ROBERT

of Rye Protoplasts LANGIS

AND

PETER

by VitrijkaL.

STE-

(Department of Agronomy, Cornell University, Ithaca, New York 14853). PONKUS

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 proce-

ANNUAL

575

MEETING

dure involves (1) equilibration (loading) of the protoplasts with an intermediate concentration of ethylene glycol (EG) at 2O”C, (2) dehydration of the protoplasts in a concentrated vitrification solution containing EG, sorbitol, and bovine serum albumin (BSA) at O”C, (3) placing the protoplasts into straws and quenching in liquid nitrogen (LN3, and (4) recovery of the protoplasts from LN, and removal (unloading) of the vitrification sblution. BSA was required in the solutions to minimize the decrease in survival resulting from manipulation (centrifugation, washing, and transfer to straws) of the protoplasts. Generally, losses were ~5% when BSA (0.2% minimum) was present in the medium, but reached 40% when BSA was absent. Following recovery from LN,, survival was ca. 50% for both NA and ACC protoplasts, but the optimum conditions for loading and unloading the CPAs were different. For NA protoplasts, the highest survival following recovery from LN, occurred if the protoplasts were first loaded with 1.75 M EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a 2 osm sorbitol solution. If they were unloaded in an isotonic solution, survival was 40% of the ACC protoplasts survived following unloading in an isotonic sorbitol solution. For dehydration in the concentrated vitrification solution, exposures at 0°C for less than 1 min were required for the highest survival of both NA and ACC protoplasts, and survival declined rapidly with longer exposure times. Several different vitrification solutions were used: EG + BSA; sorbitol + BSA; or two mixtures containing EG + sorbitol + BSA, either 40 + 15 + 6 wt% or 24 + 30 + 6 wt%, respectively. When compared at the minimum concentration required for vitrification and the prevention of devitrification, the sorbitol solution was the least toxic during the dehydration step, the EG solution was the most toxic, and the mixture was intermediate. However, following recovery from LN,, survival was lowest in the sorbitol solution, intermediate in the EG solution, and highest in the 24:30:6 mixture of EG + sorbitol + BSA. (Supported by a gift from RJR-Nabisco, Inc.) 116. Cryopreservation Embryos. D. V.

of Drosophila

P. L. STEPONKUS, LYNCH, R. E. PIN,

melanogaster

S. P. MYERS, T.-T. LIN, R. J.