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.

Representative 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 can be sectioned transversally and longitudinally (Figure 2). For cereal crops, their mature kernels are full of starch granules, leading to that it is very difficult in preparing the whole-seed-sized section. Using the present technique, the transversal and longitudinal whole-seed-sized sections of mature maize with large volume could also be prepared (Figure 4, Supplementary Figure 1). In addition, the developing kernel (Supplementary Figure 2), germinated kernel (Supplementary Figure 3), and cooked kernel (Supplementary Figure 4) of rice can be investigated using the method.

Applications of the whole-seed-sized section
Observation of tissue structure of seed
The whole-seed-sized section can be used to observe the tissue structure of seeds. For examples, the embryo of oilseed rape consists of radicle, hypocotyl, plumule, and two cotyledons. The inner and outer cotyledons are bent in half, wrapping the hypocotyl and radicle and making the embryo spherical (Figure 2A,C). The longitudinal and transversal whole-embryo-sized sections stained with safranin clearly exhibited the radicle, hypocotyl, inner cotyledon, and outer cotyledon (Figure 2B,D). The longitudinal whole-embryo-sized section of oilseed rape is prepared more difficultly than the transversal section. Therefore, the transversal sections of embryos are widely used to investigate the micromorphology of embryos in references^5,10.

Morphology and analysis of cells in different regions of seed
The whole-seed-sized section can be used to observe and analyze the morphology of cells in different regions of seed. For example, the transversal whole-embryo-sized sections of oilseed rape were stained with fluorescent brightener 28, and the cell walls were stained specifically (Figure 3A). The micromorphology of cells in any regions of embryo could be clearly displayed at high magnification (Figure 3B,C). The radicle consists of epiderm, cortex, and vascular tissues. The epidermal cells located in the outermost layer of radicle were rectangular and radially arranged. The cortical parenchyma cells were round in shape and large in size. Some distinct spaces were observed between cortical cells. The cortical cells were arranged in layers from the inside to the outside (Figure 3B). The epidermal cells of cotyledon were square and had small volume. There were no significant differences in shape and size of epidermal cells among outer and inner surfaces of inner and outer cotyledons. Some vascular cylinders were scattered in the middle of mesophyll tissues of inner and outer cotyledons. The mesophyll parenchyma cells were significantly larger than the epidermal cells and vascular cylinder cells in the cotyledon. The mesophyll parenchyma cells showed a typical palisading arrangement in the inner region of outer cotyledon and the outer region of inner cotyledon (Figure 3C). The parenchyma cells had significantly different morphologies in different regions of embryo. In order to reveal their differences in morphology, regions 1, 2, 3, 4, and 5 were chosen in the radicle cortical tissue, inner region of inner cotyledon, outer region of inner cotyledon, inner region of outer cotyledon, and outer region of outer cotyledon, respectively (Figure 3B,C). The morphology parameters of the parenchyma cells in the above 5 regions were quantitatively analyzed using morphology analysis software (Supplementary Table 1). The area, long axis length, short axis length, and roundness of parenchyma cells showed some differences in different regions of embryos.

The cells in endosperm were full of starch and storage protein. Using the whole-seed-sized resin section, it is easy in observing and analyzing the cells in different regions of endosperm. For example, the morphology of cells in any regions of maize endosperm could be viewed clearly after the transversal whole-seed-sized sections were stained with fluorescent brightener 28. The peripheral, middle, and central endosperms in the same kernel exhibited significantly different shapes and sizes of cells (Supplementary Figure 1). In order to quantitatively analyze the morphology parameters of cells in different regions of endosperm, the images of regions were analyzed using morphology analysis software; the morphology parameters of cells are presented in Supplementary Table 2. The endosperm cells in region 1 had the smallest area among four regions, those in region 2 were larger than those in region 3, but smaller than those in region 4.

Morphology and analysis of starch granules in different regions of seed
The mature seeds from most plant resources, especially for cereal crops, contain high starch content. The granule morphology and size of starch have important effects on starch properties and play a role in the quality of seed. The resin section of seed can be stained with iodine solution to exhibit the morphology of starch granules in different regions of seed. For example, the transversal and longitudinal whole-seed-sized sections of maize were prepared successfully. The sections stained with iodine exhibited the morphology of starch (Figure 4). In order to show the morphology of starch granule in different regions of endosperm, the four regions and nine regions were chosen in the transversal and longitudinal whole-seed-sized sections, respectively (Figure 4). The starch granules in different regions showed significantly different morphology, size and quantity in endosperm cells. For transversal section, region 1 had spherical starch granules, region 2 had polygonal granules, and starch granules in both regions 3 and 4 were spherical. For longitudinal section, starch granules with polygonal shape in regions 1, 4, 5, and 8 were larger than those with spherical shape in regions 3, 7, and 9, and some compound starch granules were observed in regions 2 and 6.

The quantitative analysis of morphological parameters of starch granules in four regions of transversal section was shown in Supplementary Table 3. Starch granules in region 1 had the smallest size, those in region 2 had the largest size, and those in region 3 were larger than in region 4.

Micromorphology and analysis of protein bodies in different regions of seed
The whole-seed-sized section with high storage protein can be used to obverse and analyze the morphology of protein bodies in different regions of seed. For example, the transversal section of embryo of oilseed rape was stained with Coomassie brilliant blue R250, and the storage protein was stained blue (Figure 5). The spatial distribution of storage protein in the embryo could be clearly observed at the low magnification (Figure 5A). Storage protein is present in protein bodies. At high magnification, the protein body exhibited a heterogeneous matrix with some black granules and some unstained transparent structure (Figure 5B). The protein bodies in seed have three types: the first type consists of a homogeneous protein matrix and has no inclusions, the second type contains globular crystals, and the third type contains globular crystals and pseudocrystals¹¹. The globular crystals in the protein body are composed of phytate and other inorganic salts, which are not stained. These globular crystals are black due to that the light cannot pass through them under microscope. In addition, the spherical crystal is fragile and difficult to be penetrated by the fixative and the embedding agent. When making the section, the spherical crystals sometimes burst out, resulting in a transparent cavity inside the protein body¹¹. The protein body of oilseed rape embryo contained spheroidal crystals according to its micromorphology (Figure 5B). In order to investigate the spatial distribution of protein bodies in the embryo, five regions in the whole-embryo-sized section were chosen to represent the radicle cortical tissue, inner region of inner cotyledon, outer region of inner cotyledon, inner region of outer cotyledon, and outer region of outer cotyledon (Figure 5A,C-G). The protein bodies in all regions of embryo were spherical, ellipsoidal, and irregular in shape (Figure 5C-G).

Quantitative analysis of protein bodies in the first and second lay parenchyma cells close to the epidermis in the above chosen five regions are presented in Supplementary Table 4. The area of protein body had slight difference among the chosen five regions. The roundness of protein body was significantly lower in the outer region of outer cotyledon than in the other four regions, indicating the protein body in outer cotyledon was close to sphere. The number and area index of protein body in cell were significantly higher in the radicle parenchyma cell than in the cotyledon parenchyma cell (Supplementary Table 4).

Figure 1: Preparation of whole-seed-sized resin semithin section using dry sectioning method. Please click here to view a larger version of this figure.

Figure 2: Tissue structure of embryo in mature seed of oilseed rape variety Huashuang 5. (A) Morphology of embryo. (B) Tissue structure of longitudinal whole-embryo-sized section. (C) Morphology of transversal whole-embryo-sized section. (D) Tissue structure of transversal whole-embryo-sized section. The sections were stained with safranin. H, hypocotyl; IC, inner cotyledon; OC, outer cotyledon; R, radicle. Scale bar = 1 mm. Please click here to view a larger version of this figure.

Figure 3: Morphology of cells in embryo of oilseed rape variety Huashuang 5. (A) Transversal whole-embryo-sized section stained with fluorescent brightener 28. (B) Amplification of region B in (A), showing the cell morphology and tissue structure of radicle. (C) Amplification of region C in (A), showing the cell morphology and tissue structure of inner and outer cotyledon. Scale bar = 500 µm for (A) and 100 µm for (B,C). Please click here to view a larger version of this figure.

Figure 4: Morphology of starch granules in mature kernel of maize variety Zheng 58. The transversal (A) and longitudinal (B) whole-seed-sized sections were stained with iodine solution, and their regional amplifications exhibit the morphology of starch granules in different regions of endosperm. Scale bar = 1 mm for whole-seed-sized section and 20 µm for regional amplifications. Please click here to view a larger version of this figure.

Figure 5: Morphology of protein bodies in embryo of oilseed rape variety Huashuang 5. (A) Transversal whole-embryo-sized section stained with Coomassie brilliant blue R250. (B) Amplification of protein bodies, showing their microstructure. (C-G) Amplification of region C-G in (A), showing the morphology of protein body in radicle (C), inner region of inner cotyledon (D), outer region of inner cotyledon (E), inner region of outer cotyledon (F), outer region of inner cotyledon (G). Scale bar = 500 µm for (A), 5 µm for (B) and 50 µm for (C-G). Please click here to view a larger version of this figure.

Supplementary Figure 1: Morphology of cells in mature kernel of maize variety Zheng 58. The transversal whole-seed-sized section was stained with fluorescent brightener 28, and its regional amplifications (1-4) exhibit the morphology of endosperm cells in different regions of endosperm. Scale bar = 1 mm for whole-seed-sized section and 100 µm for regional amplifications. Please click here to download this file.

Supplementary Figure 2: Morphology of developing kernel of rice variety 9311. The transversal whole-seed-sized sections at different days after flowering (DAF) were counterstained with safranin O and iodine solution. Scale bar = 0.5 mm. Please click here to download this file.

Supplementary Figure 3: Morphology of germinated kernel of rice variety Te-qing. The longitudinal whole-seed-sized section at 8 days after imbibition was counterstained with periodic acid-Schiff's and toluidine blue O, and its regional amplifications exhibit the morphology changes of endosperm in different regions of seed. Scale bar = 20 µm. Please click here to download this file.

Supplementary Figure 4: The morphology of cooked kernel of rice variety Te-qing. The transversal whole-seed-sized section was stained with iodine solution, and its outer, middle, and inner region amplifications exhibit the morphology changes of starch granules in seed during cooking process for 0, 10, 20, and 30 min. Scale bar = 20 µm. Please click here to download this file.

Supplementary Table 1: Morphology parameters of cells in different regions of oilseed rape embryo^a Please click here to download this table.

^aThe data is means ± standard deviations (n = 3), and the values in the same column with different letters are significantly different (p < 0.05).

^bThe regions are shown in Figure 3B,C.

^cLAL: long axis length; SAL: short axis length; Roundness: (perimeter ²)/(4×π×area).

Supplementary Table 2: Morphology parameters of cells in different regions of maize endosperm^a Please click here to download this table.

^aData is means ± standard deviations (n = 3). Values in the same column with different letters are significantly different (p < 0.05).

^bThe regions are shown in transversal section of maize kernel in Supplementary Figure 1.

^cLAL: long axis length; SAL: short axis length; Roundness: (perimeter ²)/(4×π×area).

Supplementary Table 3: Morphology parameters of starch granules in different regions of maize endosperm^a Please click here to download this table.

^aData is means ± standard deviations (n = 3). Values in the same column with different letters are significantly different (p < 0.05).

^bThe regions are shown in transversal section of maize kernel in Figure 4A.

^cLAL: long axis length; SAL: short axis length; Roundness: (perimeter ²)/(4×π×area).

Supplementary Table 4: Morphology parameters of protein bodies in different regions of oilseed rape embryo^a Please click here to download this table.

^aThe data is means ± standard deviations (n = 3), and the values in the same column with different letters are significantly different (p < 0.05).

^bThe regions are shown in Figure 5.

^cRoundness: (perimeter ²)/(4×π×area); Area index is the area ratio of protein body to cell.

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 resin sections give a two-dimensional (2D) picture, leading to no way in analyzing the true size and quantity of cells. However, the cells are cut randomly at their any planes, the mean size of many cells (over 100) from at least three different sections of tissue region can reflect the 2D morphology parameters (length, width, and area) of cells, and the ratio of the chosen region area to mean cell area can reflect the quantity of cells. Therefore, it is very important for in situ viewing and analyzing the morphology of cells and storage materials in different regions of seed. The paraffin section is the most suitable for preparing the whole-seed-sized section, especially for large sized seeds⁷. However, the cells are full of storage materials with seed development, leading to that it is very difficult in obtaining the good whole-seed-sized section from late developing seeds and mature seeds. In addition, the paraffin section is too thick to exhibit the morphology clearly, and is only suitable for investigating the tissue structure of seed⁷.

The resin section is thin, and can exhibit the morphology of cells, starch granules, and protein bodies clearly⁷. However, the routine resin is not suitable for whole-seed-sized section. The technique presented here represents a fast, simple, and keen approach toward preparing transversal and longitudinal whole-seed-sized sections of mature seeds embedded in resin for viewing the morphology of cells, starch granules, and protein bodies in different regions of seed using light microscopy (Figure 2-5, Supplementary Figure 1). In addition, the technique can also prepare the section of developing, germinated, and cooked seeds to in situ investigate the morphology changes of cell, starch, and protein bodies in different regions of seed.

Another distinct advantage that this technique provides is the application of whole-seed-sized sections. In the new era of phenomics and metabolomics, it is important to quantitatively measure the morphology parameters of cells, starch granules, and protein bodies in different regions of seeds. The new technique, in conjunction with morphology analysis software, allows the researcher to quantitatively analyze the morphology parameters of cells, starch granules, and protein bodies in different regions of seed (Supplementary Table 1-4).

Though the present dry sectioning method can successfully prepare the whole-seed-sized resin section, it has some limitations and shortcomings. For the paraffin section, the paraffin can be removed easily from the section; but for the resin section, the resin cannot be removed from the section, leading to the plant sample embedded in resin. Therefore, compared with the paraffin section, the present whole-seed-sized resin section is not suitable to carry out the histochemistry and immunohistochemistry. In addition, the routine resin sectioning method can cut samples into 0.5-2 µm smooth sections due to the sample block with small volume. But the present dry sectioning method is difficult to prepare the smooth sections with thickness less than 2 µm, especially for mature seeds with large volume and high starch content.

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