Staining Maize Epidermal Leaf Peels with Toluidine Blue O

Abstract

Maize is an important model organism for understanding cell patterning and development.The regular patterning of maize leaf epidermal cells has previously been characterized using the dye toluidine blue O (TBO). The polychromatic dye TBO differentially stains plant cells depending on the chemical composition of cell components and has been used to identify differences in development, cell shape and wall composition in maize. This protocol provides step-by-step instructions to fix maize leaf tissue, to peel the maize epidermis, to stain epidermal peels with TBO and finally to image using a standard light microscope. The benefit of using epidermal peels is to generate high-quality micrographs of epidermal cells for quantitative analysis. TBO staining highlights cell walls, nuclei, and differential staining of different cell types that are simple to compare, and measure.

Preparation of isolated guard cells, containing cell walls, from Vicia faba

“…Of these three enzymes, only Driselase successfully yielded iGCs, pectolyase and cellulase showed similar results (S3 Fig in S1 File ). We also experimented with enzyme digestion time, expecting too short a duration to result in a lack of cell separation and too long resulting in individual cells falling apart [ 30 ]. Initially, we tried 60 min, which yielded iGCs, but later found 30 min to also be sufficient (S4 Fig in S1 File ).…”

“…Three different enzymes were used after blending and filtering, including Driselase, pectolyase, and cellulase. Initially, we had anticipated pectolyase to be sufficient in separating cells by exploring the fact that cells are held together by pectin, as Bellinger et al had done [30], but this did not prove to be the case for Vicia faba. Of these three enzymes, only Driselase successfully yielded iGCs, pectolyase and cellulase showed similar results (S3 Fig in S1 File).…”

Heterotrimeric G-protein α subunit (RGA1) regulates tiller development, yield, cell wall, nitrogen response and biotic stress in rice

“…Toluidine blue (TBO) staining is known to examine the cuticular properties of plant 85 . Experiments were performed as described earlier 86,87 using seeds 88 . TBO solution was prepared with 10 mM sodium citrate, pH 4.4; 0.05w/v toluidine blue and 0.4 v/v tween 20.…”