Light microscopy (LM) observation of grain structure
Grains samples were fixed immediately in 0.1 M phosphate buffer solution (pH 7.2), containing 2.5% glutaraldehyde (v/v) and 2% paraformaldehyde (w/v). Afterwards, they were dehydrated in a graded ethanol series, transferred to acetone, and embedded in low-glutinosity Spurr’s resin (Spurr Low-Viscosity Embedding Kit, Sigma-Aldrich, St. Louis, USA). The sample blocks were polymerized at 70°C for 8 h. Semi-thin sections (2 μm) were cut at the longitudinal plane (embryo) and the transversal plane (endosperm) of the grains, using a Leica Ultrathin Microtome (EM UC7, Germany). Samples were stained with 1 % methyl violet, observed and photographed under a BX53 light microscope (Olympus, Tokyo, Japan).