Enhancing the Cryopreservation System of Larch Embryogenic Culture by Optimizing Pre-Culture, Osmoprotectants, and Rapid Thawing

Abstract

Cryopreservation is considered the safe and efficient strategy for the long-term conservation of embryogenic cultures. The objective of this study was to cryopreserve the embryogenic tissues of hybrid larch to overcome the result raised by rapid growth rates of conifer embryogenic cultures necessitating frequent sub-culturing. We systematically evaluated several parameters, including the pre-culture method (liquid or solid), osmoprotectant type (DMSO, sucrose, or PEG6000), duration of cryoprotection (1–3 h), and thawing temperature (4 °C, 25 °C, or 40 °C). After one month of cryopreservation, we assessed the regeneration efficiency and maturation ability of both cryo-preserved and non-cryopreserved tissues. Our optimized protocol involves pre-culturing embryonic tissue on the solid medium with 0.4 M sorbitol for 48 h, followed by treatment with 10% DMSO, 0.4 M sucrose, and 15% PEG6000 for 1 h on ice, and immersion in liquid nitrogen with rapid thawing at 40 °C. Notably, the use of solid media during pre-culturing was crucial to enhancing the success rate of cryopreservation. Using protocol optimization, we achieved high embryogenic tissue survival rates of over 80% without affecting the ability of somatic embryogenesis. This work provides a comprehensive set of steps for routine cryopreservation of embryogenic tissues for long-term conservation in hybrid larch, along with sample protocols for cryopreservation of larch. The results demonstrate that vitrification is a reliable method for preserving embryogenic tissues of hybrid larch with broader implications for the cryopreservation of other plant species. Further optimization and standardization of protocols across different species would ensure the preservation of genetic diversity and facilitate future research in plant biotechnology that benefits human health, food security, and environmental sustainability.