Storage of Cell Suspensions and Protoplasts of Glycine max (L.) Mem, Brassica napus (L.), Datura innoxia (Mill.), and Daucus carota (L.) by Freezing

Abstract

Summary The effect of freezing on survival was studied in fast growing cell suspensions of Glycine max (L.) Merr., Datura innoxia (Mill.), Brassica napus (L.) and Daucus carota (L.). The cells were pretreated by partial plasmolysis in culture medium containing 1.0 M sorbitol. The pretreated cells were subjected to a decrease in temperature at a rate of 1 dgC/min from 0° to -35°C. The frozen cells were stored at either -80°C or -196°C. Thawing of the cells was performed in the presence of 1.0 M sorbitol. After storage in liquid nitrogen for up to 6 months at least 19 % of the colony forming capacity was retained, as compared to non-frozen controls. After cryopreservation the four cell lines retained their growth characteristics and chromosome number. An auxotrophic mutant of soybean preserved in liquid nitrogen displayed its phenotype after thawing. Cryopreserved cells of Daucus carota were regenerated to normal plants at a high frequency. Protoplasts of G. max were frozen in medium containing 0.7 M sorbitol under otherwise identical conditions. After storage in liquid nitrogen clones developed from individual protoplasts with an efficiency of 10-3 as compared to non-frozen controls.