Are you sick of eating your vegetables? Why not play with them instead! Explore your least favourite food group at its smallest unit of life, THE CELL.
Plant cells are Eukaryotic cells that, while having many similar components to other Eurkaryotic cells, have their own unique structures.
While both plant and animal cells contain many of the same components: cell membrane, nucleus, cytoplasm, mitochondria, endoplasmic reticulum, ribosomes, golgi apparatus, plant cells also contain supplementary structures. Examples of such structures are chloroplasts, vacuoles and a cell wall.
These structures perform various roles in the plant cell: chloroplasts convert solar energy into energy for the plant, vacuoles regulate turgor pressure and the cell wall provides structural support for the cell.
For a first look at a plant cell, we can look at a classically studied specimen, Elodea Canadensis (or Pondweed) to study these components.
Elodea Protocol:
- Select a thin, transparent leaf
- Place on a slide with a drop of mounting medium (60% Glycerol in PBS) and cover with a coverslip
- Place on 3D Cell Explorer and image
Fig 1. The 2D Refractive index map and their corresponding digital stain. In the first image, the chloroplasts are easily identified and stained in red. When looking at the 3D reconstruction from the side, it can be seen that the chloroplasts are distributed throughout the cell. The cell wall is stained in green. In the second image, the Nuclei are stained in purple.
You may be wondering why a plant cell needs a cell wall at all (after all we do just fine without them). By removing the cell wall, we can study what the plant cell looks like without this crucial structural component. When the cell wall is removed, the cell, encased only in a plasma membrane becomes known as a protoplast. In order to remove the cell wall without damaging the delicate protoplast within, we chose to treat it with a mix of enzymes that break down the primary cell wall by acting on pectin and cellulose and hemicellulose, the main components of the cell wall. By degrading these polysaccharides, the protoplasts are released and can be studied independently from the cell wall. The following protocol explains all the steps required for protoplast isolation.
Fig 2: A plant cell wall. By degrading the pectin, cellulose and hemicellulose with enzymes, the primary cell wall falls apart, releasing the plasma membrane encased protoplast
Protoplast Protocol:
- Slice plant tissue into thin slices (around 1mm width) using a sharp blade
- place a few strands of sliced leaf tissue into a 1.5ml falcon tube and add 1ml of enzyme solution.
- Gently agitate for 30-60 min.
- Spin 5 min at 100 x g. Remove the supernatant, making sure not to disturb the protoplast pellet.
- Add Digest/Wash Solution to 50 mL, and mix by inversion.
- Spin 5 min at 100 x g. Remove the supernatant, making sure not to disturb the protoplast pellet.
- Determine the volume of the protoplast pellet, and add one volume of Digest/Wash Solution. Gently resuspend by inversion.
- Incubate protoplasts on ice for at least 60 min prior to downstream treatments. This allows the cells time to recover from the digestion of their cell walls.
- Place in Ibidi 35mm dish and image with the 3D Cell Explorer.
Fig 3. Without the cell wall for support, the plasma membrane has formed into a sphere.
