A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

Summary

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

Abstract

The morphology, size and quantity of cells, starch granules and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

Introduction

Plant seeds contain storage materials such as starch and protein and provide energy and nutrition for people. The shape, size, and quantity of cell and storage materials determine the weight and quality of seed. The cells and storage materials in different regions of seed have significantly different morphologies, especially for some high-amylose cereal crops with inhibition of starch branching enzyme IIb^1,2,3. Therefore, it is very important to investigate the morphologies of cells and storage materials in different regions of seed.

Paraffin sectioning is a good method to prepare the whole-seed-sized section and can exhibit the tissue structure of seed and the accumulation of storage material in different regions of seed^4,5,6. However, the paraffin sections usually have 6-8 µm thickness with low resolution; thus, it is very difficult to clearly observe and quantitatively analyze the morphology of cell and storage materials. The resin sections usually have 1-2 µm thickness and high resolution and are very suitable to observe and analyze the morphology of cell and storage materials⁷. However, the routine resin sectioning method has difficulty in preparing the whole-seed-sized section, especially for seeds with a large volume and high starch content; thus, there is no way to observe and analyze the morphology of cells and storage materials in different regions of the seed. LR White resin is an acrylic resin and exhibits low viscosity and strong permeability, leading to its good applications in preparing the resin section of seeds, especially for cereal mature kernels with large volume and high starch content. In addition, the sample embedded in LR White resin can be stained easily with many chemical dyes to clearly exhibit the morphology of cells and storage materials under light or fluorescent microscope⁷. In our previous paper, we have reported a dry sectioning method for preparing the whole-seed-sized sections of mature cereal kernels embedded in LR White resin. The method can also prepare the whole-seed-sized section of developing, germinated and cooked cereal kernel⁸. The obtained whole-seed-sized section has many applications in micromorphology observation and analysis, especially for clearly viewing and quantitatively analyzing the morphology differences of cell and storage materials in different regions of seed^8,9.

This technique is appropriate for researchers who want to observe the microstructure of tissue and the shape and size of cells, starch granules, and protein bodies in different regions of seed using light microscope. The images of whole-seed-sized sections stained specifically for exhibiting cells, starch granules, and protein bodies can be analyzed by morphology analysis software to quantitatively measure the morphology parameters of cells, starch granules, and protein bodies in different regions of seed. In order to demonstrate the technical applicability and whole-seed-sized section applications, we have investigated the mature seeds of maize and oilseed rape and the developing, germinated, and cooked kernels of rice in this study. The protocol contains four processes. Here, we use mature maize kernel, which is the most difficult in preparing the whole-seed-sized sections due to the large volume and high starch content, as a sample to exhibit the processes step by step.

Protocol

1. Preparation of resin-embedded seed (Figure 1)

1. Fix six maize mature kernels in 10 mL of 2.5% phosphate-buffered glutaraldehyde (0.1 M, pH7.2) at 4 °C for 48 h. The researchers can choose other fixative mixtures, fixative concentrations, and fixation conditions according to their research objectives and tissue types.
2. Take out the kernels and slice them longitudinally or transversally to 2-3 mm thickness using a sharp double-sided blade, and fix them in 10 mL of 2.5% phosphate-buffered glutaraldehyde (0.1 M, pH 7.2) again for 48 h.
3. Wash the samples three times with 10 mL of 0.1 M phosphate buffer (pH 7.2) for 30 min every time.
4. Dehydrate the samples in increasing grades of ethanol aqueous solution (10 mL) from 30% to 50%, 70%, 90% once, and 100% three times for 30 min every time.
5. Infiltrate the samples in 10 mL increasing grades of LR White resin solution diluted with ethanol from 25% to 50%, 75% once, and 100% twice at 4 °C for 12 h every time.
6. Prepare the pedestals for samples before embedding. Add 0.25 mL of 100% LR White resin into a 2-mL centrifuge tube, and polymerize it at 60 °C for 48 h.
7. Successively add the pure LR White resin (0.5 mL) and the infiltrated sample into the centrifuge tube with a pedestal. Straighten the samples with the anatomical needle, and polymerize them at 60 °C in an oven for 48 h.

2. Dry sectioning for preparing whole-seed-sized section (Figure 1)

1. Take out the embedded kernels from the centrifuge tube and cut out the excess resin around the sample using a sharp blade.
2. Clamp the resin block in the sample holder of ultramicrotome (EM UC7), and trim off the superfluous resin on the surface of the sample and around the sample with a blade.
3. Polish the surface of the sample finely with a glass knife until a complete section can be formed.
4. Put a small copper hook about 2 mm above the blade edge before cutting and cut the sample into a 2 µm section. The role of the hook is to avoid the curling upward of the section.
5. Put the hook under the section to support it when the section becomes long.
6. Add 100 µL of water on an unpretreated slide, and carefully transfer the complete and unbroken section to the water with the tweezers.
7. In order to smooth the wrinkled section, heat and dry the sample on the flattening table at 50 °C overnight.
   1. If the section crumbles or tears, extend the time for each resin infiltration of the sample from 12 h to 24 h or 48 h.
   2. 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.

3. Staining and observation of the section

NOTE: In order to observe the tissue structure and morphology of cells, starch granules, and protein bodies, stain the sections with specific stains according to the purpose of the research. Here, we use the fluorescent brightener 28, iodine solution, and Coomassie brilliant blue R250 to stain the cell walls, starch granules, and protein bodies, respectively.

1. For observing the morphology of cells, stain the section with 40 mL of 0.1% (w/v) fluorescent brightener 28 aqueous solution in a 70 mL compact glass staining jar at 45 °C for 10 min, and then rinse it with running water for 5 min. Observe and photograph the section under a fluorescence microscope equipped with a CCD camera.
2. For observing the morphology of starch granules, stain the section with 40 µL of iodine solution (0.07% (w/v) I₂ and 0.14% (w/v) KI in 25% (v/v) glycerol) for 1 min, and cover the sample containing iodine solution with a coverslip. View and photograph the sample under a light microscope equipped with a CCD camera.
3. For observing the morphology of protein bodies, immerse the section with 40 mL of 10% (v/v) acetic acid in a 70 mL compact glass staining jar for 10 min at 45 °C, and then stain it in 40 mL of 1% (w/v) Coomassie brilliant blue R250 in 25% (v/v) isopropanol and 10% (v/v) acetic acid for 15 min at 45 °C. Wash the stained sections with running water for 5 min, and dry it. Observe and photograph the section under a light microscope equipped with a CCD camera.

4. Quantitative analysis of morphology parameters

1. Process and quantitatively analyze the photographed images for area, long/short axis, and roundness of cells, starch granules, and protein bodies in different regions of seed using morphology analysis software (Image-Pro Plus 6.0 software) following the procedures of Zhao et al.⁹ exactly.

Results

Simple dry sectioning method for obtaining a whole-seed-sized section
We establish a simple dry sectioning method for preparing a whole-seed-sized section of seed embedded in LR-white resin (Figure 1). The method can prepare transversal and longitudinal whole-seed-sized sections with thickness of 2 µm (Figure 2-5, Supplementary Figure 1-4). For examples, the mature seed of oilseed rape ca...

Discussion

The seeds are the most important renewable resource for food, fodder, and industrial raw material, and are rich in storage materials such as starch and protein. The morphology and quantity of cells and the content and configuration of storage materials affect the weight and quality of seeds^7,12. Though the stereology and image analysis technology can measure the size and quantity of cells in a tissue region, they are lacking in many laboratories. The paraffin and...

Materials

Name	Company	Catalog Number	Comments
Acetic acid	Sangon Biotech (Shanghai) Co., Ltd.	A501931
Compact glass staining jar (5-Place)	Sangon Biotech (Shanghai) Co., Ltd.	E678013
Coomassie brilliant blue R-250	Sangon Biotech (Shanghai) Co., Ltd.	A100472
Coverslip	Sangon Biotech (Shanghai) Co., Ltd.	F518211
Double-sided blade	Gillette Shanghai Co., Ltd.	74-S
Ethanol absolute	Sangon Biotech (Shanghai) Co., Ltd.	A500737
Flattening table	Leica	HI1220
Fluorescence microscope	Olympus	BX60
Fluorescent brightener 28	Sigma-Aldrich	910090
Glass strips	Leica	840031
Glutaraldehyde 50% solution in water	Sangon Biotech (Shanghai) Co., Ltd.	A600875
Glycerol	Sangon Biotech (Shanghai) Co., Ltd.	A600232
Iodine	Sangon Biotech (Shanghai) Co., Ltd.	A500538
Isopropanol	Sangon Biotech (Shanghai) Co., Ltd.	A507048
Light microscope	Olympus	BX53
LR White resin	Agar Scientific	AGR1281A
Oven	Shanghai Jing Hong Laboratory Instrument Co.,Ltd.	9023A
Potassium iodide	Sangon Biotech (Shanghai) Co., Ltd.	A100512
Slide	Sangon Biotech (Shanghai) Co., Ltd.	F518101
Tweezers	Sangon Biotech (Shanghai) Co., Ltd.	F519022
Sodium phosphate dibasic dodecahydrate	Sangon Biotech (Shanghai) Co., Ltd.	A607793
Sodium phosphate monobasic dihydrate	Sangon Biotech (Shanghai) Co., Ltd.	A502805
Ultramicrotome	Leica	EM UC7