Optimization of the D-Cryoplate Technique for Cryopreservation of <i>Cannabis sativa</i> Axillary Buds

Abstract Long-term conservation of elite Cannabis sativa genotypes is essential to maintain genetic fidelity in clonal propagation systems. This study aimed to evaluate and optimize the D-cryoplate technique for cryopreservation of C. sativa axillary buds, with particular emphasis on the role of explant dehydration before cryostorage. Axillary buds from micropropagated plants of two cultivars were encapsulated in calcium alginate on aluminum cryoplates, osmotically pretreated, dehydrated for 0–90 minutes, and immersed in liquid nitrogen. After cryostorage, the explants were rapidly rewarmed, recovered in vitro, and subsequently acclimatized under greenhouse conditions. Shoot regeneration was strongly dependent on dehydration time, with the highest regeneration rates (63–70%) observed after 60–70 minutes of dehydration. These optimal times corresponded to capsule water content of 21–28%. Short dehydration periods resulted in low regeneration due to freezing injury, while excessive dehydration significantly reduced explant viability due to desiccation stress. Shoots regenerated from cryopreserved axillary buds showed typical morphological development with growth parameters comparable to those of the noncryopreserved control. These results demonstrate that precise control of explant water content is critical for successful cryopreservation, and the D-cryoplate technique is an efficient and chemically safe strategy for the long-term preservation of elite C. sativa clonal germplasm.