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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 secti....

Protokół

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.

Wyniki

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.......

Dyskusje

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.......

Materiały

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