Cryopreservation of european chestnut germplasm

The objective of this study was to develop a cryopreservation method for zygotic embryonic axes, somatic embryos and apices excised from shoot cultures of chestnut. Between 93 and 100% of embryonic axes survived liquid nitrogen (LN) storage following desiccation to moisture contents of 24-20% (on a fresh weight basis), and 63% subsequently developed as whole plants. The ability of desiccation- and vitrification- based procedures enables embryogenic cultures to withstand cryopreservation. A 68% recovery rate was achieved by 3-day pre-culture on high-sucrose medium followed by 60 min application of PVS2 vitrification solution prior to cryogenic storage. A 40-60% recovery rate was obtained when cryostored shoot apices 0.5-1.0 mm long, exposed to PVS2 solution for 120 min at 0°C, were cultured on Gresshoff and Doy (1972) medium supplemented with growth regulators. Cold hardening (1-2 weeks) of the stock material improved shoot regrowth.