Microscopic Analysis of Primary Root Tissue

Primary root tissue close to the seed (the first 0.5 cm) was harvested for light microscopy and scanning electron microscopy (SEM) studies. For light microscopy, tissues were fixed in 96% ethyl alcohol and stored at 4° C. Tissues were then placed in a histocassette and gradually dehydrated in ethyl alcohol. Subsequently, they were placed in a 1:1 solution of ethyl alcohol and 100% xylol (Spintissue Processor STP120 and Histo Star Embedding Centre, Thermo Scientific). The dehydrated samples were embedded in Histoplast (cat. no. 22900700, Fisherbrand), and 5 μm-thick cross-sections were cut with a microtome (Microm HM 340E, Thermo Scientific) and incubated in a drying oven (DX402, Yamato) at 60° C for 30 min, then placed in xylene for 2 min to remove paraffin. Tissue samples were then rehydrated in 1× PBS and visualized for endogenous fluorescence by epifluorescence microscopy (DIM6000, Leica, Germany). For SEM, the samples were treated as previously described by Olivares-Garcia et al. (2020) (link).