Cryopreservation of Zygotic Embryonic Axes and Somatic Embryos of European Chestnut

  • Ana M. Vieitez
  • M. Carmen San-José
  • Elena Corredoira
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 710)

Abstract

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.

Key words

Castanea sativa Chestnuts Cryostorage Cryopreservation Embryo desiccation European chestnut Plant regeneration Somatic embryogenesis Vitrification Zygotic embryos 
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Copyright information

© Humana Press 2011

Authors and Affiliations

  • Ana M. Vieitez
    • 1
  • M. Carmen San-José
    • 1
  • Elena Corredoira
    • 1
  1. 1.Instituto de Investigaciones Agrobiológicas de Galicia, CSICSantiago de CompostelaSpain

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