Cryopreservation of chestnut by vitrification of in vitro-grown shoot tips

Plants of European chestnut (Castanea sativa) have been consistently recovered from cryopreserved in vitro-grown shoot apices by using the vitrification procedure. Factors found to influence the success of cryopreservation include the source of the shoot tips (terminal buds or axillary buds), their size, the duration of exposure to the cryoprotectant solution, and the composition of the post-cryostorage recovery medium. The most efficient protocol for shoot regrowth employed 0.5-1.0 mm shoot tips isolated from 1 cm-long terminal buds that had been excised from 3-5-wk shoot cultures and cold hardened at 4°C for 2 wk. The isolated shoot tips were precultured for 2 d at 4°C on solidified Gresshoff and Doy medium (GD) supplemented with 0.2 M sucrose, and were then treated for 20 min at room temperature with a loading solution (2 M glycerol + 0.4 M sucrose) and for 120 min at 0°C with a modified PVS2 solution before rapid immersion in liquid nitrogen (LN). After 1 d in LN, rapid rewarming and unloading in 1.2 M sucrose solution for 20 min, the shoot tips were plated on recovery medium consisting of GD supplemented with 2.2 μM benzyladenine, 2.9 μM 3-indoleacetic acid, and 0.9 μM zeatin. This protocol achieved 38-54% shoot recovery rates among five chestnut clones (three of juvenile origin and two of mature origin), and in all cases plant regeneration was also obtained.