In vitro Incubation with Rumen Fluid and Histological Staining of Select Cell Wall Components in Alfalfa (Medicago sativa L.) Stems.

The accumulation of cell wall components as plants grow limits forage nutrition for ruminants. In particular, lignin deposition in plant tissues impedes the breakdown of the cell wall carbohydrates cellulose and hemicellulose, thereby reducing the amount of energy available from forage. The highly digestible pectin component of the cell wall, however, is readily available for degradation. Information about lignin and pectin deposition in different stem cell types may provide mechanistic insights into the factors underlying variation in stem digestibility among alfalfa lines. In vitro incubation of stem cross sections in rumen fluid simulates the processes that stem tissues undergo in the rumen. Coupled with histological staining and microscopy, this method enables visualization of changes in tissue degradation over time and the location of lignin and pectin in the stem. Wiesner stain reacts with guaiacyl (G) aldehyde units of lignin, staining them fuchsia. Similarly, ruthenium red interacts with the carboxylic groups of pectin, staining pectin in the stem in shades ranging from pink to orange and brown. This protocol describes the digestion of alfalfa stem cross-sections after exposure to rumen microbes, followed by staining to visualize lignin and pectin patterns in the cell walls. The method can be used to identify stem tissues in which lignin and pectin are present or absent, facilitating comparisons among alfalfa lines.