Axenic cultures of three cryptic species of A. pinguis were established from the apical parts of gametophytes. Concentrations: 1, 2, 5, 25, 35 and 50 of commercial ACE bleach as well as 50 and 35 alcohol were tested in order to find optimal method of sterilization. The best outcomes resulted from the application of a lower concentration - 2 ACE solution and a longer time of exposure - 8 min. In order to find the optimal conditions of growth for A. pinguis, three different pH values (5.6, 6.0, 7.0) of MS 1× medium and different media were tested too. Regeneration of plants of A. pinguis were successfully achieved on two hormone-free media: special liverwort medium (Lukavsky, 1991) and MS medium (Murashige & Skoog, 1962).