RESUMO
For Castanea sativa (European chestnut), a species with recalcitrant seeds that is not easily propagated vegetatively, cryopreservation is one of the most promising techniques for maintaining genetic resource diversity and for conservation of selected germplasms. Long-term conservation of selected seeds and valuable embryogenic lines can be achieved through the cryopreservation of zygotic embryonic axes and somatic embryos, respectively. This chapter describes methods for the desiccation-based cryostorage of zygotic embryonic axes, and the vitrification-based cryopreservation of somatic embryos. For zygotic embryonic axes, the highest post-thaw survival and plantlet recovery rates are obtained by desiccation in a laminar flow hood to 20-25% moisture content, followed by direct immersion in liquid nitrogen. For somatic embryos, embryogenesis resumption rates of over 60% are achieved by preculture of embryo clumps for 3 days on solid medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 60 min at 0°C, and direct immersion in liquid nitrogen. Plantlet recovery from cryostored embryogenic lines requires proliferation of the thawed embryos and subsequent maturation before germination and conversion into plantlets.
Assuntos
Fagaceae/embriologia , Sementes/crescimento & desenvolvimento , Criopreservação , Meios de Cultura , Dessecação , Germinação , VitrificaçãoRESUMO
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 %.