Assessing Structural Traits in Triticum aestivum and Zea mays for C₃ and C₄ Photosynthetic Differentiation Using Free-hand and Semi-thin Sections

Our research focuses on plant structure function relationships. We aim to better understand the limitations to photosynthesis across plant functional types and how they can be improved for climate-resilient crops. New technology in software are expensive and often inaccessible, which requires a degree of trial and innovation to accomplish the same resolutions in imaging different plant functional types.

The identification of anatomical structures that differentiate C3 and C4 leaves, particularly in Triticum aestivum and Zea mays, as well as troubleshooting the preparative steps for microscopy. To begin, grow wheat and maize plant species in an environment-controlled growth chamber. Select three mature, non-senescent leaves from the wheat plant and keep them in distilled water.

Then, place the leaf specimen on a glass plate on a piece of dental wax to stabilize the sample and avoid slipping. Move the blade straight down on the leaf specimen to cut cleanly through cells and keep the cross-sections in distilled water. To mount the samples on a glass slide, place them on a water droplet and cover them with a glass coverslip for microscopic imaging.

Now, view the slide under a compound light microscope at 10X and 20X magnification. Save images as per the software guide along with the relevant scale. Free-hand sections of leaves from the C3 plant Triticum aestivum and the C4 plant Zea mays revealed that maize's bundle sheath cells are rounder and greener, indicating a higher chloroplast presence.

Begin by cutting sections from intercostal areas alongside the midrib of leaves from Triticum aestivum on a glass plate. Ensure that the length of the cut section runs along the grain of the leaf. Then, place the plant material in a syringe with one milliliter of fixation buffer.

Hold the syringe vertically, remove the air, and pump the syringe to create a vacuum, holding a finger on the tip. After performing the complete infiltration protocol as per the resin manufacturer's instructions, cover the cavities of a flat embedding mold with 100%resin and place the samples on one end of the cavity. Prepare pencil-written paper labels for each sample and place them on the other end of the cavity.

Straighten the samples and labels if they moved. Cover the mold with the embedding film and polymerize in an oven as per acrylic resin product guidelines. Then, set the polymerized block with the sample in the ultramicrotome specimen holder.

Using a rough glass knife, trim the block until the tissue becomes visible and excess resin is eliminated. Next, using a glass or diamond knife, cut semi-thin sections transversely. With a metal inoculation loop, collect the sections from the water's surface and place them on a glass slide.

Dry the slide on a hot plate to fix the sections onto the glass. Stain the fixed sections with toluidine blue for five seconds. Semi-thin cross-sections of Triticum aestivum and Zea mays showed that all leaf tissues were visible and measurable.

Zea mays exhibited Kranz anatomy with mesophyll cells around veins and chloroplast-filled bundle sheaths facilitating rapid metabolite exchange. After staining the Triticum aestivum leaf section with toluidine blue, place the slide on the microscope stage. Using a 40X objective magnification, focus on the section and adjust the brightness to ensure all cell structures are visible.

Then, use the Navigator function to set the section's location area to ensure the entire section is imaged, then click the Stitch button. After that, click Done and Run to allow the microscope to image the entire section. Opened the image analysis software and the tiles taken by the microscope.

Using the Align feature, ensure that the tiles do not overlap. After generating the image, adjust the positioning, brightness, and rotation using the Adjust tab. Then, print the scale on the image before saving it in TIFF format.

Open ImageJ software and drag the image into the window to load it. Using the Line Tool, draw a line over the scale bar, choose Analyze, and click Set Scale. Change the known distance to the scale bar's length and the unit of length to the correct unit.

To measure the prospective trait, select the Line Tool, draw a line across the area of interest, and press the M key. For area measurements, use the Polygon Selection Tool to outline the tissue of interest, then press the M key to conclude. A pop-up window displaying the measurements will appear, which can be copied to a spreadsheet for further data analysis.

Box plots illustrated significant anatomical differences between Triticum aestivum and Zea mays, particularly in interveinal distances, bundle sheath diameter, the fraction of bundle sheath cells, non-photosynthetic material, and vein frequency, reflecting expected C3 and C4 leaf anatomy distinctions.