Preparation and Imaging of Rice Spikelets

Paraffin-embedded sections of spikelet samples were prepared according to Kim et al. (2014 (link)), with minor modifications. The materials were fixed in FAA and stored at 4 °C for 24 h. The fixed spikelets were dehydrated in a gradient ethanol series, and were then incubated in 100 % ethanol overnight. Dehydrated spikelets were embedded in Paraplast Plus (Sigma). The transverse sections of each spikelet were stained with 0.5 % toluidine blue, and viewed using an SZX10 stereomicroscope (Olympus, Tokyo, Japan). For scanning electron microscopy analysis, spikelet hull and endosperm samples were processed according to Wang et al. (2015b (link)) and Li et al. (2014b (link)). Young spikelets were fixed in 4 % (w/v) paraformaldehyde and 0.25 % glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, at 4 °C overnight. Fixed spikelets were dehydrated in a graded ethanol series, and 100 % ethanol was replaced with 3-methylbutyl acetate (Toriba et al. 2010 (link)). Milled rice grains were transversely cut in the middle with a knife and were coated with gold under vacuum conditions. Samples were dried at their critical point, sputter coated with platinum, and observed with the XL-30-ESEM instrument at an accelerating voltage of 5 kV.