Application of temporary immersion system RITA® for efficient biomass multiplication and production of artificial seeds for ex situ conservation of Linnaea borealis L

Linnaea borealis L., belonging to the Linnaeaceae family, in some European countries including Poland, is a protected species. Phytochemical studies indicate the presence of pharmacologically important secondary metabolites, which may indicate undiscovered biological activity and untapped therapeutic potential of this species. Harvesting the raw material from natural sites is impossible due to the protection status of this glacial relict in many countries. The aim of the current work was to develop an efficient protocol of Linnaea borealis L. var. borealis shoot multiplication via the development of axillary buds in an agitated culture and a temporary-immersion system as well as to establish the production of artificial seeds via alginate encapsulated nodes for short-time storage. The highest results of growth parameters were obtained for shoots agitated in MS (Murashige & Skoog) liquid medium enriched with cytokinin, auxin and gibberellin (18.3 ± 0.4 shoots per explant and 2185 ± 98% biomass increase, respectively). The growth of shoots in the RITA® bioreactor was highly efficient, especially when hormone supplementation in MS medium was used, the amount of medium in the culture vessel was 100 or 150 ml, and the immersion time of the shoots was 1 or 2 min. The survival and recovery rates of L. borealis artificial seeds, regardless of encapsulation matrix used, subsequently decreased with the increased storage duration up to 6 months (from 100 to 60% at 4 °C and from 100 to 54% at -18 ºC, respectively). This is the first report on twinflower shoot multiplication in a temporary-immersion system and artificial seeds production of this species.