Cryopreservation of Endangered Wild Species, Aster altaicus var. uchiyamae Kitam, Using Droplet-Vitrification...

Aster altaicus var. uchiyamae Kitam is an endemic and endangered species in urgent need of a comprehensive conservation strategy.

To develop an efficient cryopreservation protocol using in vitro shoot tips to complement traditional conservation approaches in case seeds are not available or insufficient for conservation programs.

Shoot tips of in vitro plants were cryopreserved using a droplet-vitrification method following improvement of pre-culture, osmoprotection, vitrification solution (VS), unloading and post-culture treatments. The starting protocol included step-wise pre-culture with 10% and 17.5% sucrose for 55 h and 17 h, respectively, followed by osmoprotection with C4-35% (17.5% glycerol + 17.5% sucrose) for 30 min, and cryoprotection with B5-80% (40% glycerol + 40% sucrose) for 60 min.

Shoot tips of A. altaicus were found to be moderately sensitive to the osmotic stress. Pre-culture and osmoprotection were not critical for the regeneration of cryopreserved explants when either of these treatments was applied. Osmoprotection with C4-35% on ice for 60 min followed by cryoprotection with A3-80%, a modified and diluted PVS2, on ice for 60 min resulted in the highest (65.3%) regeneration of cryopreserved shoot tips. Among alternative VSs tested, A3-80% and B5-80% were superior to PVS2 and PVS3 used under the same conditions. Step-wise recovery of shoot tips on ammonium-free medium followed by GA₃-containing medium and medium without growth regulators were critical for the normal regeneration of both VS-treated and cryopreserved shoot tips.

Cryopreservation of in vitro shoot tips using droplet-vitrification was developed as a complementary conservation approach for A. altaicus. Adjustment of the composition of regrowth media depending on recovery stage was important for the regeneration of healthy plants from cryopreserved shoot tips.