Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips.

Abstract

Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at -196 °C by one-step vitrification. After preculturing at 5 °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 M glycerol and 0.4 M sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 M sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0 °C and, following cryopreservation, rewarmed at 40 °C and washed in 1.2 M sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μM N6-benzyladenine plus 0.5 μM gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μM indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips.