Wheat grains were placed in a Petri dish layered with filter paper and wetted with dH2O. Grains were imbibed for 4 h and cut in half with a stainless-steel razor blade, embedded into tissue freezing medium (Leica Biosystems, Illinois, United States), mounted onto a metal holder and frozen in liquid nitrogen. Grain cross-sections were cut in cryomicrotome chamber (Leica CM3050, Nussloch, Germany) to 35 μm thick sections at −24 °C. The sections were put onto pre-cooled filter paper in pre-cooled aluminium beakers with covers. Samples were transferred to liquid nitrogen and freeze-dryed as described previously [29 (link)]. Dried sections were put on object glass, and a drop of dH2O water was added. The sample was then covered with a cover glass and observed and photographed under Axioskop 2 MOT microscope equipped with Axiocam MRc colour digital camera (Carl Zeiss Vision, Halbergmoos, Germany) using AxioVision 3.1 software. For each wheat variety, cross-sections of 7 grains were examined.
Photographs were analyzed in ImageJ software, where thickness of pericarp, testa, hyaline layer, total grain coat and aleurone layer was measured.
Photographs were analyzed in ImageJ software, where thickness of pericarp, testa, hyaline layer, total grain coat and aleurone layer was measured.