This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.
This protocol allows seeds to be sliced intact so they don't crumble, permitting visualization of starch granules in any seed that can fit into a pipette tip. The malleable plastic cone supports the native structure of the dry tissues and organs, so this cheap and easy manifold allows many samples to be processed in a short time. To go on from the forensic microscopy advantage here, resistant starch, something that we're all looking for, accessibility of enzymes to that raw starch, amylolysis, glycemic index, and glucose spikes, these are core parameters for breeding better rice varieties to fight Type 2 diabetes around the world today.
And now it's a great pleasure to introduce Dr.Kiah Barton to you. Dr.Barton is from our lab and she will use the faculty core imaging facility to show you how this technique is deployed. Begin by de-husking the dry kernels.
Place a single kernel onto a flat rubber stopper on a work bench, and make sure that this stopper remains stationary. Use a second flat rubber stopper to abrade the kernel by twisting it against the first rubber stopper, then remove husks from the kernel, taking care not to shatter the endosperm nor grinding the seeds too aggressively between the two rubber bungs. Remove any remaining husks using fine forceps and insert an individual husked kernel into a plastic 250 microliter pipette tip.
Make sure that the embryo end of the kernel is facing towards the conical end of the pipette tip. Insert a second 250 microliter pipette tip to force the kernel into the first pipette tip and to keep the kernel immobile during sectioning, taking care not to damage the kernel or bend the second pipette tip. Lay the pipette tip assemblage flat on a workbench and hold it in place by hand.
With the other hand, use a sharp scalpel blade to slice through the center of the kernel, cutting off the end of the pipette tip. Slice downwards through the assemblage and then orient the scalpel blade vertically so the hand section's two new exposed faces are parallel. Then use the scalpel to cut one millimeter thick sections of the rice kernel.
To perform reflected light microscopy, place the transverse rice sections on a piece of heavy gauge black paper. Obtain light images of the transverse sections using a stereo microscope with mounted goosenecks for oblique illumination, using at least 10 times magnification. Wild-type Nipponbare and ssg1 sections were examined under 260 times, 920 times, and 4, 200 times magnifications.
When properly prepared, the rice sections were approximately 0.9 millimeters thick with minimal shattering of the endosperm and intact pericarp and aleurone layers. This technique allows for the preparation of sections of sufficient quality to observe the entire endosperm cell, compound starch granules, and individual sub-granules. Translucent kernel producers, such as the wild-type resistant starch hybrid line, Zhehui 7954 and cobalt generated mutant RS111, produced tightly packed polyhedral starch granules, which is the normal rice endosperm phenotype.
The SEM images of chalky kernel producers, commercial variety Ye Tang and RS4, displayed starch granules that were round and loosely packed. Wild-type Xiushiu 11 and its mutant KMD1, which expressed the Cry1Ab gene to inhibit insect predation had sections and endosperm morphotypes similar to the translucent resistant starch lines. This technique can be applied to seeds of other species.
The model monocot, Brachypodium distachyon, produces very hard seeds, but it was still possible to obtain an intact transverse section. Intact transverse sections were also produced from soft, white winter wheat. This rapid, cheap screening method for looking inside biological structures can be applied to insect legs, locus bean tree spikes, hawthorns, fish spines, or any field of forensic microscopy that can profit from this sample support setup.
After performing this protocol, sputter coating for electromicroscopy, staining with specific fluorescent dyes, and application of calcafluor to stain for a beta glucan cell wall thickness in mutant oat and barley lines are some quick ways of approaching plant breeding with rapid phenotype screening.
