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

ABSTRACT

BACKGROUND: Aster altaicus var. uchiyamae Kitam is an endemic and endangered species in urgent need of a comprehensive conservation strategy. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.