The morphology science and the quality of cells, starch granules, and the protein bodies in kernel determine the weight and the quality of seed. In this study, we present as a simple population of a whole kernel embedded in LR White resin and establish a simple dry suctioning method of whole kernels. We choose LR White resin as embedded medium for two major reasons.
First, it is low viscosity and strong permeability, leading to its good applications in preparing this mighty sections of seeds, especially for cereal mature kernels with a large volume and high starch content. Second, the sample embedded in LR White resin can be stained easily with many chemical dyes to clearly exhibit the morphology of samples under light with fluorescent microscope. The prepared sections can be specifically stained to exhibit the cells, starch granules, and protein bodies clearly in emergence of whole kernel, and the morphology parameters can also be quantitatively analyzed.
The method contains studies. Here, we use mature maize kernels with larger volume and high-starch content as a sample to exhibit the processes step by step. To begin, clips kernels into a simple bottle.
Add about 10 millimeters of 2.5%phosphate-buffered glutaraldehyde. one more pH 7.2 and place it at 4 degrees centigrade for 48 hours to fix them both. Then take out these kernels and slice them longitudinally or transversally, with two to three millimeters thickness.
You need a sharp double-sided blade. And fix them for another 48 hours. Next, wash your samples three times with about 10 millimeters, so 0.1 M phosphate buffer, pH 7.2 for 30 minutes every time.
To hydrate the samples step by step, soak them in about 10 millimeters of 30%ethanol aqueous solution for 30 minutes first, and successively soak them in 10 millimeters, so 50%70%90%once, and 100%three times. To infiltrate a sample step by step, successively retrieve them with 10 millimeters of LR White resin solution diluted with ethanol of 25%50%75%once, and 100%twice at four degrees centigrade for 12 hours every time. Before embedding, we need to prepare the pedestals for samples.
Add 250 microliters of pure LR White resin into two millimeters centrifuge tube and polymerize it at six degrees centigrade for 48 hours. Next, successively add 500 microliters of pure LR White resin and infiltrate the sample into the centrifuge tube with a pedestal. After straightening the samples with anatomical needle, put the resin at six degrees centigrade in an oven for 48 hours.
Take out those embedded kernels from the centrifuge tube and cut out excess resin around a sample using a sharp blade. Place the resin block in the sample holder of microtone and trim off the superfluous resin on the surface of the sample and around the sample with the blade. Further polish the surface of the sample finally with a glass knife until a complete section can be formed.
Now it's time to prepare this amazing section with two microns thickness. Before slicing, put a small copper spoke about two millimeters above blade's edge to prevent a section from curling. As the section elongates, put a spoke under the section to support it.
Next, add the 100 microliters of water on the unprotruded slide and carefully transfer the complete and unbroken section to the water with the treaters. Heat and dry them at 50 degrees centigrade to smooth the wrinkled section. Finally, two important tips need attention.
First, if the section crumbles or tears, extend the time for each resin infiltration of the sample. Second, if the section has some lines paralleled to the knife, clamp the sample block tightly. If the section has some lines vertical to the knife, please use a new knife.
To observe the morphology of cells, starch granules, and protein bodies, we can stain the sections with fluorescent brightener 28, iodine solution, and Coomassie brilliant blue R250 respectively. For other specific stains, base it on the proposal research. Here we use iodine solution to stain starch granules as an example.
Add 40 microliters of iodine solution to immerse the section. And keep it in the jar for one minute. And then cover the sample with a cover sleeve.
And blot off the excess agent solution around it. Observe and photograph the sample immediately under a lighted microscope equipped with a CCTV camera. We can process and quantitatively analyze the photographed images for area, long or short access, and roundness of cells, starch granules, and protein bodies in different regions for kernel using morphology analysis software.
We establish a simple dry sectioning method for retaining whole kernel sized sections in LR White resin. The method can prepare transverse and longitudinal sections of the whole kernel with of two microns. or examples, the mature whole seed of oilseed rape can be sectioned transversely and longitudinally.
The sections of a mature whole kernel of maize with larger volume can also be prepared successfully. In addition, the developing kernel, cooked kernel, and the germinated kernel can also be investigated using the method. The prepared sections of whole kernels have the below applications.
First, the sections of whole kernel can be used to observe the tissue structure of seeds. For examples, the sections of whole embryo of oilseed rape stained with safranin can clearly exhibit its a radical, hyper casual, inner and outer The sections of whole kernel can be used to observe and analyze the morphology of cells. For example, the transversal sections of whole embryo of oilseed rape are stained with fluorescent brightener 28, and the cell oils are stained specifically.
The micro-morphology of cells in any regions of whole embryo can be displayed at high magnification. The morphology parameters of parenchymal cells, can be quantitatively analyzed using morphology analysis software and show some differences in diverse regions of embryo. The transversal sections of whole kernel of maize stained with fluorescent brightener 28 can also exhibit as a morphology of cells, and the morphology parameters of cells are different in diverse regions of endosperm.
sections of whole kernel can be stained with iodine solution to exhibit its morphology of starch granules, for example, the transversal and the longitudinal sections of maize kernels stained with iodine can exhibit its morphology of starch granules clearly. The starch granules in diverse regions shows significantly different morphology, size, and quantity in endosperm cells. So quantitative analysis of morphological parameters of starch granules shows that they are significantly different in diverse regions for endosperm.
The sections can be used to observe and analyze the morphology of protein bodies. For example, the starch protein is stained blue using Coomassie brilliant blue R250. The spatial distribution of starch protein in the whole embryo of oilseed rape can be observed in its a low magnification, and the micro structure can be exhibited clearly at high magnification.
In addition, the numbers error index and morphology parameters of protein bodies in chosen regions can also be quantitatively analyzed. This technique provides the first and simple preparation of resin sections of whole kernels from developing and mature seeds. The sectional applications in tissue structure of whole kernel and then investigating the morphology of cells, starch granules, and the protein bodies in different regions of a whole kernel.
The acquired images can be quantitatively analyzed to show the morphology parameters of cells, starch granules, and protein bodies and further compare them in different regions of whole kernel.
