Ultracut rotary microtome

Three spikes per plant of a minimum of 10 lines of WT and the mutant were selected for phenotyping. The middle section of spikes at W8.5 (Waddington stage 8.5) stage was collected for cytological observations. Sections of 8 μm were prepared longitudinally along the spike axis by using Leica Ultracut rotary microtome. The number of rachis cells was counted by selecting all cells from one node and cell lengths were measured by selecting the similar regions on the rachis of the mutant and WT spikes. The WSEEN Grain Test System (WSeen)¹ was used to measure grain length, grain width, and thousand-grain weight.

For cytological observations, the grains of TAA10 and XX329 at 2, 4, 6, 8, 10 and 15 DAP were collected with three biological replicates. Each replicate contained the grains in eight central spikelets of one spike. Grains from different samples were fixed in 50% (v/v) ethanol, 5% (v/v) acetic acid, and 3.7% (w/v) formaldehyde over 12 h at 4 °C, followed by dehydration and embedding in paraffin. Cross sections (3 μm) from the middle part of grains were cut using a Leica Ultracut rotary microtome and stained with Periodic acid Schiff (PAS). Photographs were taken with Pannoramic MIDI (3DHISTECH, Ltd., Hungary).
Cross sections of three grains for each developmental stage (2, 4, 6, 8, 10 and 15 DAP) were used to measure cell area. On each cross section, the cell area of the pericarp was measured by selecting three regions within five-row cells from the epicarp inwards, and the cell area of the endosperm was measured using three randomly selected regions of the endosperm. In each of the measured regions, between 20 and 40 cells were measured with CaseViewer 2.0 (3DHISTECH, Ltd., Hungary).