Staining and Microscopy of Plant Ovules

The procedure for preparing the plant material was as previously described by Musiał and Kościńska-Pająk (2017 (link), 2019 (link)). Florets were isolated from capitula and placed in 80 % ethanol for 1 h. Then the plant material was softened in 1 N NaOH at 37 °C for 4 h. Samples were rinsed with distilled water three times and once with 0.1 M K₃PO₄, stained overnight at room temperature with decolorized aniline blue (DAB; 0.1 % w/v). After rinsing with 0.1 M K₃PO₄, florets were placed on a microscope slide in a drop of K₃PO₄ mixed with a drop of glycerol. Subsequently, the ovules were isolated from ovaries and squashed under a cover slip. The samples were observed under UV light using a Nikon Eclipse E400 microscope fitted with an Epi-Fl Filter Block N UV-2 A which consists of an excitation filter EX330–380, a dichroic mirror DM400 and a barrier filter BA420.