Cuticle structure

For light microscopy (LM), fresh gametophyte and sporophyte fragments were fixed in paraformaldehyde (1%) and glutaraldehyde (3%) solution in sodium phosphate buffer (Karnovsky, 1964), dehydrated in serial acetone embedded in epoxy resin (Spurr, 1969). Semithin sections were made with a Leica UC6 ultramicrotome and stained with toluidine blue (O’Brien et al., 1964). For histochemical identification of lipids, semithin sections were submitted to the Sudan IV test (Bronner, 1975). The histological slides were analyzed and photographed using an IM50 image manager coupled to a Leica DMLB microscope.

For transmission electron microscopy (TEM) analyses, small fragments (± 10 mm) of the gametophyte and sporophyte were fixed in a solution containing formaldehyde (2%), glutaraldehyde (2.5%) in sodium cacodylate buffer (0.1 M), pH 7.2 (modified from Karnovsky, 1965) for 48 hours. Samples were then post-fixed in osmium tetroxide (1%) in the same buffer, dehydrated through ethanol series and propylene oxide, and embedded in EMbed 812 resin (Electron Microscopy Sciences). Semithin sections (0.3 μm thick) were made in a Leica Ultracut R ultramicrotome with a glass knife, adhered to histological slides, and stained with toluidine blue. The region of interest was selected and ultrathin sectioned (70 nm thick), adhered to copper grids (200 mesh), and contrasted with solutions of uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). Images were obtained using a Zeiss EM900 transmission electron microscope operating at 80 kV.