Moss Stress Response to Salinity

Axenic in vitro cultures of the experimental mosses, P. patens, E. hungaricus and H. heimii, were established and the full development of gametophores was achieved. Details of the culture procedure can be found in Sabovljević et al. [5 ,31 ] and Ćosić et al. [50 (link)]. Having achieved a sufficient number of developed gametophores to start the experiments, cultures of the selected mosses were grown on solid BCD medium (containing 0.2 M MgSO₄ × 7H₂O, 0.18 M KH₂PO₄, 1 M KNO₃, and 0.9 mM FeSO₄ × 7H₂O) with sucrose added (0.05 M) to achieve quick growth and morphologically well-developed gametophytes [5 ,52 (link)] prior to the experimental tests. The pH of the media was adjusted to 5.8 before autoclaving at 121 °C for 30 min. Further tests were conducted on minimal BCD medium supplemented with different NaCl concentrations before autoclaving (5, 10, 50, 100, 200, 300, and 500 mM NaCl). In experiment type I, the mosses were grown on BCD medium containing NaCl for 3 days, which simulated short-term stress. Experiment type II represented long-term stress, where the mosses were grown on BCD medium containing NaCl for 21 days. The control plants were grown on BCD salt-free medium. The culture conditions were set at 18 ± 2 °C, under a long-day photoperiod (16 h:8 h light/dark). All the plants tested here were grown under the same controlled laboratory conditions including the light quality and intensity (47 μmol m⁻²s⁻¹ irradiance). After 3 and 21 days, respectively, the plant material was collected and frozen at −70 °C until further analysis. All the chemicals were supplied by Sigma Aldrich, Germany.