Cryopreservation of zygotic embryo axes and somatic embryos of european chestnut

This work describes experiments demonstrating the feasibility of long-term conservation of Castanea sativa germplasm through cryopreservation of embryonic axes or somatic embryo clumps. Between 93% and 100% of excised embryonic axes of recalcitrant chestnut seeds survived storage in liquid nitrogen (LN) following desiccation in a laminar flow cabinet to moisture contents of 20-24% (on a fresh weight basis), and some 63% subsequently developed as whole plants. Desiccation to moisture contents less than 19% produced damage resulting in loss of organized plant development after cryostorage, allowing only root growth. When 6- 8 mg clumps of globular or heart-shaped somatic embryos were precultured for 7 days on high-sucrose medium and then desiccated to a moisture content of 25% before storage in LN, the embryogenesis resumption level after thawing was 33%. When the embryo clumps were precultured for 3 days on high-sucrose medium followed by 60 min application of PVS2 vitrification solution before cryostorage, the post-storage embryogenesis resumption level was 68%.