Many species of Viola L. and Pulsatilla Mill. are currently threatened with extinction in various regions worldwide. Their population decline due to global climatic and environmental changes. Beauty of flowers and use of plants as a source of chemicals (plants are dug up for gardens and for obtaining secondary metabolites important for medicine with various biological activity and acting as antioxidant, antimicrobial and cytotoxic agents) are the main reasons of the destruction of their natural sites. Poor seed germination and limited vegetative propagation do not favour species spread. Active protection of these species and restoration of disappearing populations are rapidly desired. In vitro culture and cryopreservation technique serve as an excellent biotechnological tool for ex situ conservation of endangered species as well as to produce pharmaceutically important secondary metabolites. Our long-term studies focused on developing efficient protocols for micropropagation (or/and callus production) of several endangered medicinal/ornamental Viola species (V. uliginosa, V. tricolor, V. lutea ssp. calaminaria, V. lutea ssp. westfalica, V. epipsila, V. stagnina) and Pulsatilla (P. vulgaris, P. patens). The initial material were plant organs (leaves, petioles) or seedling fragments (shoot tips, hypocotyls, cotyledons). Organogenesis (adventitious shoots) and/or somatic embryogenesis were induced on solidified MS (Murashige and Skoog) medium supplemented with the combination of cytokinins and auxins or with only one plant growth regulator. Rooting shoots or seedlings converted from
somatic embryos were gradually acclimatized to ex vitro and field conditions. Genetic fidelity of regenerated shoots/plantlets with the source material was confirmed by estimation of genome size by flow cytometry and molecular markers (AFLP, ISSR). Viola stagnina was successfully micropropagated (Żabicki et al. ۲۰۱۹) and shoots were introduced to preservation in liquid nitrogen. Cyclotides (cyclic peptides produced by Viola) biosynthesis during in vitro cultivation of V. uliginosa depended on tissue and of plant growth regulators type (Slazak et al. ۲۰۱۵). The great achievement was obtaining regenerants via
organogenesis and somatic embryogenesis from seeds of the endangered Pulsatilla patens and its reintroduction into the wild. Large population of this species had disappeared in Jaworzno (Poland) ~۲۰ years ago. Over ۱۰۰ individuals were successfully transplanted into their natural site enhancing biodiversity of calamine grassland and restoring the population of this species.