In vitro propagation and morphological responses of a winter-hardy fern (Phegopteris connectilis (Michx.) Watt) under varying strength of MS medium and vitamin supplementation

Abstract Background Phegopteris connectilis is a perennial fern distributed throughout the Northern Hemisphere, including Eurasia and North America. Although Phegopteris connectilis is widely distributed, local populations occurring in the Western Suwałki Lakeland region of North Eastern Poland are considered critically endangered, highlighting the need for efficient propagation methods to support conservation efforts. Because this species reproduces apogamously, the development of a reliable in vitro propagation system is important for both conservation and research. This study aimed to determine optimal Murashige and Skoog medium (MS) strength (MS, 1/2MS, 1/4MS and 1/8MS) combined with no, half, or full concentrations of vitamins for sporophyte induction. Results Gametophytes exhibited a filamentous morphology, and sporophytes were induced within 6 weeks of culture. Different strengths of MS and vitamin formulations significantly affected growth parameters, including biomass accumulation, surface area, chlorophyll and carotenoid content. During stage-I, MS supplemented with vitamins (MS + V) enhanced sporophyte induction, whereas half-strength MS with half-strength vitamins (1/2MS + 1/2V) resulted in increase in biomass and gametophyte size. All analyzed nutrients, except Na, showed very strong positive correlations with sporophyte production, and all relationships were highly significant. In stage 2, transferring gametophytes from initial treatments to MS + V, gametophytes previously (in stage-I) cultured on MS demonstrated the highest multiplication factor. Notably, culturing gametophytes on MS medium for 10 weeks followed by transfer to MS + V enhanced sporophyte formation and increase on gametophyte by 2.29-fold. Additionally, gametophytes from 1/4MS and 1/8MS suggested a possible compensatory growth response after transferring on MS + V in stage-II. Conclusions These findings demonstrate that nutrient composition strongly influences apomictic sporophyte induction and provide an efficient, reproducible protocol for sporophyte induction in P. connectilis , supporting its conservation and potential biotechnological applications.