Protonema suspension cultures of the medicinal moss Polytrichum juniperinum

The moss Polytrichum juniperinum was widely used by native North Americans and in traditional Chinese medicine to treat several illnesses including burns, wounds, bleeding, fever, kidney stones, and gallstones. This paper reports the efficient establishment of a protonema suspension culture of this moss and evaluation of key factors such as culture medium, trophic condition, initial pH, and inoculum size for increasing biomass production, which has scarcely been studied in this type of biological system. No significant differences were found for the maximum specific growth rate (μ_max~0.09 d⁻¹) and the total phenolic content between MS, Knop, and PPNH₄ media, although an effect on tissue differentiation was observed. Growth rate in a mixotrophic condition was (μ_max 0.27 d⁻¹) three times greater than in autotrophic and heterotrophic conditions, reaching high cell densities (~11 g DW L⁻¹). Moreover, simple sugars were secreted into the medium during the growth phase. P. juniperinum protonema cultures tolerated a wide initial medium pH range (4.5–8). The tissue growth index significantly decreased from ~7.7 to 1.9 with increased inoculum size (45–300 mg DW L⁻¹) under photoautotrophic conditions. Similar responses were obtained under mixotrophic conditions with different sucrose concentrations (15–45 g L⁻¹), but no responses to sucrose concentration and inoculum sizes were seen under heterotrophic growth conditions. Finally, this high cell density culture of P. juniperinum is suitable for further studies aimed at exploring and establishing a production platform for high-value secondary metabolites.