Abstract
Cryopreservation (also called as cryobanking) of gel-embedded shoot tips on the cryo-plate dehydrated by
vitrification solution (V cryo-plate method) was examined for saving plant resources of hybrid aspen.
Cold-hardened shoot tips of in vitro grown hybrid aspen were precultured for 1 day at 25℃ on solidified 1/2
Murashige and Skoog (MS) medium containing 0.3 M sucrose. Pretreated shoot tips were placed in elliptical
wells on an aluminium cryo-plate and embedded in calcium alginate gel. Osmoprotection treatment was
performed in a loading solution containing 2.0 M glycerol and 1.0 M sucrose for 30 min at 25℃. After the
shoot tip samples on the plates were treated with the plant vitrification solution 2 for 20 min at 25℃, the
samples on the plates were directly plunged into liquid nitrogen. For rewarming, shoot tip samples on the
cryo-plates were immersed in 1.0 M sucrose solution at 25℃. Then, shoot tips in the gels were regrown at
25℃ on solidified 1/2 MS medium. The regrowth rate of cryopreserved shoot tips was almost 100%, and
normal shoot growth was observed without callus formation. This protocol appears to be a promising
technique for cryobanking of hybrid aspen genetic resources.
