Vitrification, a complementary cryopreservation method for Betula pendula Roth

Cryopreservation-the storage of plant germplasm in liquid nitrogen-provides a modern tool for the conservation of forest genetic resources. It is especially applicable for species in which their micropropagation can be initiated from mature tree buds, e.g., silver birch (Betula pendula Roth), thus enabling the conservation of specific genotypes: endangered elite trees and trees expressing rare, valuable or interesting characteristics. The aim of the present study was to develop a vitrification protocol applicable for the cryostorage of silver birch that avoids the use of expensive sophisticated freezers. The average recovery of vitrified axillary silver birch buds was 71% using a protocol that started with four-week cold hardening of bud-bearing in vitro donor shoots on modified medium under short day conditions. After cold hardening, the excised axillary buds were precultivated on medium containing 0.7 M sucrose for 24 h under the same conditions as during the cold hardening period. Following preculture, the buds were treated with loading solution containing 2 M glycerol and 0.4 M sucrose for 20 min at room temperature. Finally, the buds were dehydrated with PVS2 cryoprotectant for 120 min followed by direct immersion in liquid nitrogen. According to the morphology and the RAPD profiles of regenerated plants in the greenhouse, the genetic fidelity of the vitrified birch material seems to have remained unchanged.