Adiantum plants were kept in the dark for 16 h before the preparation of epidermal fragments to induce stomatal closing. Ten to 15 leaves were harvested and blended three times in cold water using a Waring blender for 30 s at full speed. The blender contents were filtered through a 200-μm nylon mesh, and the retained samples were washed with 5 mm MES, 50 mm KCl, and 0.1 mm CaCl₂, pH 6.5. The resultant epidermal fragments were floated in the same solution in petri dishes and kept in the dark for 1 h. The epidermal fragments were irradiated for 2 h, then 1% (w/v) fluorescein diacetate in acetone was added at 0.1 μg mL⁻¹ to determine the viability of the guard cells. Micrographs of over 50 viable stomata were obtained using a Nikon fluorescence microscope equipped with a CCD camera. The stomatal aperture surrounded by viable guard cells was defined as the width to length ratio of the stomatal pores.