Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

Grasses like maize have leaf primordia rolled within the shoot, making them difficult to study. We address this problem with improved protocols for preparing maize leaf transfer sections and hormones for fluorescence and confocal imaging. The first protocol uses a wire striper to cut the older leaves, allowing the measurement of the primordium prior to sectioning.

The second protocol uses clear double-sided nanotape to unroll whole leaf primordium. These protocols improve the accuracy of transverse sectioning and enable unrolling of the leaf primordia, which have been very difficult to achieve due to the rolled conical morphology. These methods will be useful for visualizing and quantifying leaf anatomical and developmental traits in maize and other grasses.

Based on these methods, we plan to develop lifestyle imaging strategies to address questions in grass leaf development. Begin by cutting the desired aged maize seedling at the mesocotyl tissue using a small knife or pair of scissors. Clean the seedling with a paintbrush.

Then hold the wire stripper with its jaws facing the chute. Position the chute to the selected hole and squeeze the stripper handles together. Slide the stripper away from the chute to trim the upper portion of the leaves.

Use a ruler to measure the length from the primordium's tip to the chute's base. To begin the leaf primordium imaging, hold the chute steady on a smooth surface under a stereo microscope at around 0.8x magnification. Using a razor blade or scalpel, make an initial cut slightly above the target region to discard the distal portion of the chute.

Then obtain around 0.25 to 0.8 millimeter thin sections at the desired points along the length of the primordium. Once done, mount the leaf section on a clean glass slide. Apply a few drops of water directly to the section using a pipette and place a cover slip on top.

Apply a few more drops through the edges of the cover slip if needed. Place the slide on the epifluorescence microscope's stage and adjust the settings to visualize the fluorophore. Using this method, transverse section sampling was standardized by measuring the primordia prior to sectioning.

A trend was discovered showing that the vanishing tassel two had a wider primordium and more veins than normal, indicating that the defect in the mutant began early in the leaf development. This method also enabled the systematic examination of the expression patterns of hormone response fluorescent protein reporters in the leaf primordia. Begin the preparation of the glass slide by cutting a rectangular piece of clear double-sided nanotape and sticking it to the center of a clean glass slide without removing the protective plastic film on the top side of the tape.

Next, dissect the chute by removing the upper portion of the older leaves with a wire stripper. Then hold the chute by the mesocotyl and carefully remove the surrounding leaves with a dental probe to extract the primordia of interest. For mounting the primordium, remove the protective film from the tape.

Lay the exposed primordium on the tape. Cut the primordium at the base using a razor blade, discarding the basal stem and the hypocotyl. For smoother unrolling, lubricate the inner or adaxial leaf surface by dipping the needle tip into 100%glycerol.

Unroll the primordium using a dental probe with a bent needle. Position the needle tip parallel to the surface and unfurl the outer basal margin by gently pressing it against the tape. With the outer margin adhering to the tape, align the needle tip parallel to the long axis of the leaf.

Gently slide the needle sideways to unroll and flatten the leaf onto the tape. Apply a drop of water to the unrolled primordium. Immediately place a cover slip on the water drop and the primordium.

Gently press down the edges of the cover slip so that they adhere to the tape. Once done, place the slide on the epifluorescence microscope stage to visualize the fluorophore. This method was followed to visualize and analyze the fluorescent protein expression in maize leaf primordia.

Complimentary to the transverse section analysis, the whole leaf mount analysis revealed tissue and stage specific patterns of hormone response during the information. Whole mount analysis method enabled qualitative and quantitative analyses of fluorescent protein expression in the maize leaf primordia, indicating the effectiveness of this method in examining the maize leaf primordia, which are difficult to image due to their rolled morphology.