Rice Leaf Sheath Assay
We thank Dr. Hironori Koga (Ishikawa Agricultural College) for his insight and help with this method for studying the fungus-plant interaction. These tissue sections are optically clear and suitable for microscopy of the fungus-plant interaction without chemical clearing. Prasanna Kankanala obtained the micrographs on our home page of invasive hyphae inside rice sheath inner epidermal cells using this method.
Here's how to do it. The transverse section of a leaf sheath is as shown below.
Step 1: Trim off the sides of the leaf sheath. (Transverse and longitudinal views of cutting)
Step 2: Trim away the mid vein.
Step 3: Holding the section on your fingers try to cut as much outer epidermal layer as you can.
The sections are ready to mount on a slide for microscopy.
Note: Try this technique with 4 to 5 week old plants before going to younger leaf sheaths. Change blade for every 3 to 4 sections if you want a very thin section.
Leaf Sheath Inoculation on Intact Plants
Inoculate a spore suspension (105 per ml in 0.25% gelatin) under leaf sheaths of intact plants using a syringe (23 G1.5 IM needle). Inoculate plants with the mid-vein region of the leaf sheath lying horizontally inside a plastic bag to maintain high humidity. Inoculate all sheaths in the pot that will be in the same orientation during incubation. Dissect leaf sheaths as described above immediately before microscopy.