Jsm 6360lv

The Kio sperm-like cells were fixed with 2.5% glutaraldehyde (Sigma) for 2 h, then dehydrated in a graded ethanol series and sputter-coated with gold. Samples were then studied at 20 kV (JSM-6360LV, HITACHI, Japan). The specific methods were carried out as previously described (Fu et al., 2021 (link)).

Shoot apices were collected at different growth stages. Fresh samples were fixed for 24 h in 4% paraformaldehyde (Sigma, St. Louis, USA) and 2% glutaraldehyde (Sigma) in 0.1 M PBS (pH 7.2), and subsequently washed with PBS buffer (0.1 M, pH 7.2) and dehydrated through a graded alcohol series of 70, 85, 95, and 100% of ethanol, each for 30 min. To prepare them for SEM (JSM–6360LV, Hitachi, Tokyo, Japan) observation, the samples were then washed with 100% ethanol once, post-fixed in 1% (w/v) osmium tetroxide (Alfa Aesar, Massachusetts, USA) for 2 h, dehydrated in a freeze drier (JFD–310, Hitachi, Tokyo, Japan), and sputter-coated with gold palladium in 6 different 30-s bursts (JEE–420, Hitachi, Tokyo, Japan). The samples were analysed with a scanning electron microscope (S-3000N; Hitachi, Japan).

Successive internodes (I1–3) were collected from the stems of 2-month-old WT and transgenic plants for SEM (Hitachi JSM-6360LV, Japan) and histochemical analyses, respectively. For each assay, five plants per line (transgenic lines and WT) were used as one independent experiment. Ten stem cross sections (from five plants) per line were used for measuring the stem diameters and xylem lengths. Internode cross sections were stained with phloroglucinol–HCl as previously described¹⁴ . The micrographs were acquired by a Nikon DS-U3 system with a Nikon Eclipse E100 optical microscope (Nikon, Japan). Each experiment was independently repeated three times.