Some easily seen structural features of living plant cells are destroyed or badly distorted by most of the common fixatives and embedding media used in plant histology. In stained sections of plant tissues fixed in FAA (formalin-acetic acid-alcohol mixtures) and embedded in paraffin wax, for example, mitochondria and fine transvacuolar strands of cytoplasm are usually not visible. Many structural features such as these can be preserved, however, with suitable fixatives and embedding media. Fixation in non-coagulant fixatives (e.g., osmium tetroxide, acrolein, glutaraldehyde, formaldehyde) and the use of plastics as embedding media is recommended. A method of fixation in acrolein and embedding in glycol methacrylate polymer is described in detail. In a wide range of plant specimens prepared in this way, stained sections 1-3 microns thick showed excellent preservation of tissue and cell structures. 39 references, 11 figures.

Plant microtechnique: some principles and new methods

The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected.

Influence of the Testa on Seed Dormancy, Germination, and Longevity in Arabidopsis

Differential staining of paraffin embedded plant material is accomplished without removing the paraffin. Material fixed in 3% glutaraldehyde in 0.02 M phosphate buffer is dehydrated, embedded, sectioned, and mounted on glass slides using conventional methods. The slides are placed in 0.05% toluidine blue O in distilled water for 2–30 min, rinsed in water for 1 min, and allowed to air dry. Paraffin is removed with 2 changes of xylene and the cover slip mounted with resin. Lignified tissue, suberized tissue, and some tannins are stained blue-green, while nonlignified walls are stained red-purple. An advantage of the method is the short time required for staining and mounting. Also, critical counterstaining is not required.

Simple method for differential staining of paraffin embedded plant material using toluidine blue O

An optical brightener Calcofluor White M2R New has been used to stain cell walls of higher plants. It can he used either as a vital stain for intact plants or for hand sections and plastic-embedded...

The Use of an Optical Brightener in the Study of Plant Structure

Fungal plant pathogens that attempt to penetrate and feed on living cells frequently trigger a localized plant defence response that results in fungal penetration failure. In the current study we demonstrate that breakdown products of the cell wall released by the localized application of hemicellulase elicit localized responses including, sequentially, extracellular H2O2 generation; accumulation of phenolic compounds; and cross-linking of proteins in the cell wall. In a detailed time-course study of three plant-fungus interactions that result in a high frequency of penetration failure, only one plant-fungus combination displayed a similar profile of responses to that induced by localized cell-wall degradation. The additional generation of extracellular O2- in one interaction, and the absence of phenolic compounds in the cell wall in another, demonstrate that plant responses to the penetration process may be influenced by activities of the penetrating fungus. Significantly, H2O2 generation was the only response detected in all three plant-fungal combinations at the correct time and place to account for penetration failure, and in all three combinations the enzymatic removal of H2O2 resulted in increased penetration success. Pharmacological studies suggest that in two of the three interactions, H2O2 generation required cytoskeletal involvement but was independent of transcription or translation, although inhibition of the latter processes increased fungal penetration. In at least one of these two interactions, the data suggest that H2O2 generation and new gene expression act within the same penetration-inhibiting pathway, possibly through the involvement of phenolic materials. However, enzymatic removal of H2O2 from the third interaction almost completely eliminated penetration failure, while interference with cytoplasmic processes had no effect, suggesting that H2O2 generation in this system did not require protoplast involvement and, alone, was necessary and sufficient to account for fungal penetration failure.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1046/j.0960-7412.2001.01215.x

H2O2 plays different roles in determining penetration failure in three diverse plant-fungal interactions.

1.

The polychromatic staining of plant cell walls by toluidine blue O is described and illustrated.




2.

The effects of various common fixatives and the effects of the pH of the staining solution are evaluated.




3.

Simple and rapid procedures are described for preparing stained temporary mounts of fresh material, or permanent mounts of embedded and sectioned material.




4.

The relationship between the polychromatic staining observed and the lignification of the walls is discussed.

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N. Feder

Papers

Polychromatic staining of plant cell walls by toluidine blue O