Histological Analysis of Plant Stem Nodes

For histological analyses, the first basal stem nodes were collected from the main stem from the wild-type and transgenic plants to generate paraffin sections, according to the procedure described by Han et al. [31 (link)]. WT and transgenic plant samples were fixed in formaldehyde/acetic acid/alcohol fixation buffer (FAA; 70% ethanol: formaldehyde: acetic acid = 90:5:5) for 24 h at 4 °C and then stored in 70% ethanol. The stem nodes were gradually dehydrated with 70%, 80%, 95%, and 100% ethanol for 1h in each step. Then, 0.1% safranine O in ethanol was added to pre-stain the xylem, and embedded in paraffin (Sigma-Aldrich). The plant material was cut into 8 µm ultrathin sections on a Leica RM2016 microtome, dewaxed in dimethylbenzene, mounted in neutral balsam after drying, and observed under a Leica DCF500 microscope. Micrographs of sections from 3 specimens were examined. Statistical differences were determined. Xylem thickness and phloem thickness were measured using ImageJ software (https://imagej.nih.gov/ij, accessed on 12 March 2022). Statistical differences were determined using Student’s t-test.