Cryopreservation enables long-term conservation of critically endangered species Rubus humulifolius

Ex situ storage plays an important role in the conservation of plant biodiversity. Cryopreservation at ultra-low temperatures (−  196 °C) is the only long-term ex situ preservation method for plant species that cannot be stored in seed banks. In the present study, we developed a cryopreservation protocol for micropropagated Rubus humulifolius (Rosaceae) plants representing currently critically endangered population of the species in Finland. Abscisic acid (ABA) has been found to increase the freezing tolerance of several plant species. Thus, we studied the effect of a 10-day pretreatment with 0, 2 or 4 mg/l ABA in comparison to freshly dissected buds. We also studied how the duration of in vitro subculture affects cryopreservation result. The ABA pretreatment had divergent effect on control and cryopreserved buds: the regeneration of non-cryopreserved control buds increased from 51% to 70%, 90% or 87% while the regeneration of cryopreserved buds decreased from 52% to 35%, 6% or 9% after 0, 2 or 4 mg/l ABA pre-treatments, respectively. Buds from plants subcultured for 1 month had 63% survival, which, however, decreased to 29% or nil% after 2 or 4 months subculture. The regenerated plants were successfully transferred from in vitro to in vivo conditions in common garden. Growing in garden is needed for future restoration of the species in wild. Cryostorage and other ex situ conservation actions carried out in botanical gardens may be of increasing importance as a tool to maintain plant biodiversity in the future.