This article discusses the process of lignification in plants, focusing on the deposition of lignin in cell walls to provide structural support. It details the visualization of lignin using Wiesner staining and phloroglucinol staining techniques.
In plants, lignin - a complex biopolymer - is typically deposited in the cell walls of the tissues to provide support.
Lignification is predominantly found in the secondary cell wall thickenings of water-conducting xylem cells, such as vessels and fibers, and the extra-xylary cells, such as interfascicular fibers.
Structurally, lignin is composed of crosslinked monolignol precursors. These aromatic alcohols polymerize, forming a highly branched structure. This feature enhances the plant’s strength and rigidity.
To visualize lignin deposition using Wiesner staining, take agarose-embedded dicot stem cross-sections derived from the plant’s middle or lower portion. Add them to a microcentrifuge tube containing a suitable buffer.
Add the prepared stain solution, which contains the acidified phloroglucinol and ethanol, to the stem sections. The alcohols present in the lignin react with the phloroglucinol and ethanol to yield a pink pigment. This results in the development of pink coloration corresponding to the lignin deposition in the cell walls of specialized cells.
Now, transfer the stained section onto a microscopic slide. Gently place a coverslip over the section, avoiding bubbles, to help better visualize it. Visualize the sections under a light microscope.
The pink coloration in the specialized xylem cells and the interfascicular fibers in the stem cross-sections confirm the presence of lignin in these tissues.
For Phloroglucinol staining, transfer the stem sections to a microcentrifuge tube. Add 1 milliliter of the phloroglucinol solution to the tube, and cap it immediately because the hydrochloric acid in the phloroglucinol stain is highly corrosive. Gently move the tube to ensure that all the sections are stained. Gently pipette the sections into a cut pipette tip, and onto a microscope slide. Cover the sections with a coverslip. Observe the sections under bright-field lighting. The solution dries up in 5 to 10 minutes causing a deterioration of the specimens, therefore, the imaging has to be completed within that period of time.
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