Cold hardening and slow cooling: tools for successful cryopreservation and recovery of in vitro shoot tips of silver birch

Abstract

Shoot tips of silver birch (Betulapendula Roth) derived from in vitro cultures originating from mature trees were used as explant material. Several pretreatments prior to cryopreservation were studied in order to enhance shoot tip recovery after storage in liquid nitrogen. Cold acclimation and the classical slow freezing procedure proved to be essential for successful survival, while both nonhardening and vitrification resulted in minimal survival. The optimum procedure was the following: Shoots were cold hardened (5 °C on a 8 h: 16 h (light:dark) cycle) for 3 weeks, and dissected shoot tips were then precultivated on woody plant medium with 5% dimethyl sulfoxide for 72 h. The material was transferred to cryotubes, and a mixture of 10% polyethylene glycol, 10% glucose, and 10% dimethyl sulfoxide was used as cryoprotectant. The cryotubes were slowly cooled to −38 °C before immersion in liquid nitrogen for 8 d. After fast thawing, axillary buds formed shoots without callus formation, but the growth of the new shoots was delayed from a few days to several weeks. Variability in regrowth was considerable as a result of genotype differences.