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.
Research Highlights: The Norway spruce somatic embryogenesis (SE) pipeline is suitable for multiplication of material with root rot resistance traits. Background and Objectives: Heterobasidion root rot is the economically most severe forest pathogen in Europe, reducing the benefit of planting elite forest material. In this study, the SE-propagation ability of elite Norway spruce material carrying root rot resistance traits was studied. Materials and Methods: We analyzed the presence of the root rot resistance locus PaLAR3B among 80 Finnish progeny-tested Norway spruce plus-trees used for SE-plant production as well as in 241 SE lines (genotypes) derived from them. Seven full-sib families with lines having either AA, AB, or BB genotype for PaLAR3 locus were further studied for their SE-plant propagation ability. Results: The results indicate that 43.8% of the studied elite trees carry the PaLAR3B allele (41.3% are heterozygous and 2.5% homozygous). The resistance allele was present among the SE lines as expected based on Mendelian segregation and did not interfere with somatic embryo production capacity. All embryos from PaLAR3 genotypes germinated well and emblings were viable in the end of first growing season. However, in three families, PaLAR3B homo- or heterozygotes had 23.2% to 32.1% lower viability compared to their respective hetero- or PaLAR3A homozygotes. Conclusions: There is no trade-off between root rot resistance locus PaLAR3B and somatic embryo production ability, but the allele may interfere with Norway spruce embling establishment.
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