Abstract:
Cell aggregate size in both Douglas-fir and poplar suspension cultures was reduced by the addition of the chelator compounds EDTA and CDTA at concentrations under 100 ppm. Reduced cell aggregate size increased growth efficiency of suspension cultures of both species. Cell aggregates 550 j.z. or smaller in size were used in the cryogenic studies.
At room temperature, the cryoprotectants dimethyl sulfoxide (DMSO) and glycerol at concentrations above 5% were toxic to cell
survival; however when these were washed out with fresh media, the growth rate of both Douglas-fir and poplar suspension cultures was increased. The optimum cryoprotectant concentration was about 1.0%. At concentrations above 5% repeated washing with fresh media did not reduce the cellular cryoprotectant concentration and cellular injury or death may have resulted solely from the presence of cryoprotectants, even in the absence of freezing. Cryoprotectant influences observed were thought to be due to cell membrane changes allowing increased nutrient flow in the case of cell death. Cold conditioning or chilling callus at +4°C allowed higher concentrations of cryoprotectants to be added without killing cells. Suspension cultures showed that Douglas-fir entered the log phase of growth 7 days and poplar 3 days after innoculation. Cells were small and densely cytoplasmic during this time. For both species slow freezing of log-growth-phase cells at 1°C/mm was better than fast freezing; a two-step freezing protocol, cooling first to -40°C and then immersion into liquid nitrogen was successful; cooling to -40°C was more effective than cooling to higher temperatures; and fast thawing to +40°C was more effective than slow thawing in air. The cryoprotectants DMSO, glycerol and sucrose are effective alone and in combination. Most effective was a combination of all three; DMSO 5%/glycerol 1%/sucrose 4% for Douglas-fir and DMSO 5%/glycerol 5%/sucrose 4% for poplar. Douglas-fir and poplar cultures on thawing were tested for
viability-by assessing ability to reduce tetrazolium salts and ability to continue growth. Poplar cultures resumed growth after
approximately 5 weeks post-thaw culture, whereas for Douglas-fir over 80 days was required. Thawed and recultured poplar callus
differentiated into shoots and roots. Cold conditioned callus Of both Douglas-fir and poplar produced high TTC reduction values directly after thawing. Cold conditioned poplar callus frozen without cryoprotectantS or liquid responded better on thawing than when frozen with cryoprotectants in liquid media. Douglas-fir buds obtained from field trees in August or seedlings cold conditioned at +4°C for 8 weeks were frozen under various conditions, but no buds survived the freezing process. The development of techniques for successful freeze preservation requires a detailed empirical examination of; prefreezing
culture conditions, physiological state and size of cells, cryoprotectant type and concentration used, freezing rate and temperature frozen to, and thaw rate and post-thaw culture conditions.
