Optimization of cryopreservation methods for the preservation of seeds of the species Parrotia persica

Background and Objective
 Cryopreservation is an effective method for the long-term preservation of plant genetic resources. Its main advantages include the preservation of genetic diversity, elimination of viruses, minimal storage requirements without the need for continuous subculturing, maintenance of phenotypic and genotypic stability, and facilitation of international germplasm exchange. Cryoprotective chemicals in cryopreservation increase plasma membrane stability, lower the freezing point, and increase cytosol viscosity while protecting cells from damage during cooling. Protecting the biodiversity of the Hyrcanian forests, as a living fossil, is of special importance. This study aimed to evaluate the possibility of preserving the seeds of the Persian ironwood (Parrotia persica) under cryopreservation conditions, which is classified as a near-threatened species by the International Union for Conservation of Nature (IUCN).
Materials and Methods
 The optimization of each cryopreservation protocol depends on the plant's inherent stress tolerance and its capacity to withstand the pretreatment procedures. For cryopreserving Parrotia persica seeds, dehydration was first carried out using various pretreatments to prevent free water crystallization and plasma membrane damage during cryogenic temperature exposure. These involved treatments with 30% glycerol, vitrification solution, and seed moisture reduction through desiccation before being immersed in liquid nitrogen, along with a non-pretreated control, were used to enhance desiccation tolerance and cryogenic processes. Each treatment comprised three replicates of 25 seeds. Pretreated and control seeds were retrieved from liquid nitrogen after 24 hours and immediately thawed at 42°C. To break dormancy, cryopreserved seeds and non-cryopreserved controls were sown in moist sand and stratified at 4°C. Germination started after five months. After assessment of viability and germination percentage, data were analyzed using one-way ANOVA based on a completely randomized design, with mean comparison using Duncan’s multiple range test (p≤0.05).
Results
 There were significant differences in germination percentage and shoot length among the cryopreservation treatments for the P. persica seeds (P<0.05). Result of the mean comparison based on Duncan's method indicated that the vitrification treatment had the highest germination percentage (71%) compared to other treatments, suggesting that this method was much more effective in preserving seed viability and germination ability. The vitrification method enhances the extracellular solution concentration, thereby reducing intracellular water, which stabilizes the cell membrane during dehydration and freezing, preventing damage to cellular structures and improving seed viability and germination. The 30% glycerol treatment exhibited the lowest germination percentage with value (24.6%), indicating greater damage to the seeds with this method. There were no significant differences among the treatments for other seed germination traits for cryopreservation. In the P. persica  species, the desiccation dehydration treatment using a desiccator reduced seed moisture to 1.79%, resulting in a germination percentage even lower than that of the cryopreserved control with value (3.8%). This treatment caused approximately a 2% reduction in seed moisture, which led to damage to the seed embryo.
Conclusion
The results of this study showed that the highest germination percentage belonged to seeds that treated by vitrification method and in this regard, the seeds of the P. persica species collected from various regions of Golestan province were subjected to optimal vitrification treatment and stored in the liquid nitrogen tank at the Biotechnology Research division of Forests and Rangelands Research Institute, Tehran, Iran. This technology aims to provide a means of conserving this species under critical conditions.