Improving the methods for cryopreservation of endangered conifers

Preservation of plant genetic material is essential for any biological field. Compared to serial in vitro sub-culturing, cryopreservation often represents optimal long-term conservation technique. The crucial factors influencing successful cryopreservation include 1) proper pre-treatment, 2) cryoprotectants, 3) adequate freezing pace and 4) cold-hardening. Finding the appropriate method for cryopreservation of each species is the ultimate goal of research in this field. This work is focused on finding the optimal protocol for cryopreservation of embryogenic cell masses of coniferous species bog pine (Pinus uncinata subsp. uliginosa) and optimizing the existing one for European silver fir (Abies alba), both endangered in the Czech countryside. Cryopreservation design compares effectivity of different pre-treatments and cryoprotectants, determines the role of cold-acclimation step and evaluates resistance of selected conifer cell lines to freezing and desiccation. Post-thaw recovery of Bog pine cell line BE4 was better than cell line BR1, where no living cells were visible using FDA/PI staining. Proliferation rate was higher for European silver fir than for bog pine, especially for cell line II-2-10. Cryoprotective mixture with DMSO (PGD I) proved to be more effective than the mixture with glycerol (PGG) in cryopreservation of European silver fir. PGD I also proved to be optimal cryoprotective mixture for bog pine. In both cases, 0.4M sucrose was used as a pre-treatment and cold-hardening was included