Paraffin Sectioning of Plant Stem Bases

Paraffin sections of stem bases were prepared as previously described (Xu et al., 2017 (link); Cheng et al., 2020 (link); Jin et al., 2022 (link)), with some modifications. The bases of ‘M9T337’ stem cuttings were collected at 6, 9, and 12 days after transplanting on 1/2 MS medium with 0.5 mg L-1 IBA and 0.1 mg L-1 NAA. The bases of tobacco stem cuttings were excised at 2, 4 and 6 days after subculture on hormone-free MS medium. The samples were fixed in FAA solution (70% ethanol: formaldehyde: acetic acid, 95:5:5 [v/v/v]) for 2 days at room temperature, and store at 4 °C. Then samples were dehydrated with a graded ethanal series (50%, 70%, 85%, 95%, and 100%), infiltrated with xylene, and embedded in paraffin. Cross sections with a 10 μm in thickness were cut with a Leica RM2245 (Leica Microsystems, Wetzlar, Germany) rotary microtome, transferred onto glass slides, deparaffined with xylene, and re-hydrated through an ethanol series, and stained with toluidine blue. Slides were observed using an optical microscope DM2500 (Leica Microsystems, Wetzlar, Germany) and photos were obtained using an attached digital camera DFC420 (Leica Microsystems, Wetzlar, Germany).