Method for preparing semi-thin sections of dry seeds of wheat

Abstract

The application discloses a preparation method of a wheat dry seed semi-thin section, which comprises pre-fixing, fixing, secondary fixing, rinsing, dehydrating, resin permeating, embedding, polymerizing, repairing, sectioning, spreading, dyeing and observing.The method has the advantages that the pre-fixing softens the dry seed without affecting the original morphology of the dry seed cells; the double fixing makes the gas in the dry seed be completely discharged, the cell fixing effect is better, and the section is complete; and the gradient ethanol and gradient resin replacement time is prolonged, so that the final resin can fully penetrate the cell gap, so that the section is complete and flat.The method gradually solves the problems of incomplete resin penetration, section breakage and section shrinkage in the conventional method for preparing the dry seed semi-thin section according to the characteristics of the hard and starch-filled wheat dry seed, and the complete and clear wheat dry seed semi-thin section is obtained, so that the cell morphology observation and quantitative analysis of different parts can be further carried out, thereby providing technical support for the structure research of the wheat dry seed.

Description

Preparation method of semi-thin slices of dried wheat seeds Technical Field

[0001] This invention relates to the field of microtissue sectioning technology, and more specifically, to a method for preparing semi-thin sections of dried wheat seeds. Background Technology

[0002] In addition to starch and protein, wheat seeds are rich in minerals and vitamins, providing abundant nutrients. The germination of wheat seeds through water absorption to establish seedlings and the transmission of genetic information is of great significance for wheat genetics and breeding.

[0003] Research on wheat seeds involves multiple aspects, including grain development, seed dormancy, seed lifespan, and seed germination. Seed structure research is a crucial aspect throughout the entire seed life cycle. Wheat mainly consists of three parts: the cortex, the embryo, and the endosperm. The cortex, composed of the epidermis, pericarp, and seed coat, covers the entire grain and protects the embryo and endosperm. The embryo, consisting of the plumule, hypocotyl, radicle, and scutellum, forms the basis for establishing a new plant after seed germination (Chateigner-Boutin AL, Alvarado C, Devaux MF, et al. The endosperm cavity of wheat grains contains a highly hydrated gel of arabinoxylan[J]. Plant Science, 2021, 306: 110845.). Wheat endosperm cells are divided into two types: one is the endosperm cells, which account for more than 90% of the endosperm tissue. Their main physiological function is to store starch and protein and other substances to provide nutrition for embryo development. The other is the endosperm surface cells, which account for 5%-10% of the endosperm tissue. As the grain develops, they differentiate into the aleurone layer (AL), endosperm transfer cells (ETC), and the embryo surrounding region (ESR). The development status of the endosperm determines the yield and quality of wheat (Zheng Y, Wang Z. Contrast observation and investigation of wheat endosperm transfer cells and nucellar projection transfer cells[J]. Plant Cell Reports, 2011, 30: 1281-1288.).

[0004] In wheat seed structure research, non-destructive testing and tissue sectioning provide crucial technical means for fine structure studies. Tomography can observe the internal structure of seeds without damaging them and perform 3D modeling. High-resolution tomography can also observe stomata on the seeds, which is very important for understanding the changes in tissue structure during wheat seed development (Legland D, Le TDQ, Alvarado C, et al. New Growth-Related Features of Wheat Grain Pericarp Revealed by Synchrotron-Based X-ray Micro-Tomography and 3D Reconstruction[J]. Plants, 2023, 12(5): 1038.). X-rays can be used to determine the distribution and accumulation of metal elements in wheat seeds (jiboye B., Cakmak I., Paterson D., et al. X-Ray fluorescence microscopy of zinc localization in wheat grains biofortified through foliar zinc applications at different growth stages under field conditions[J]. Plant Soil. 2015, 392, 357-370.).Nuclear magnetic resonance (NMR) can rapidly and non-destructively detect the contents of seeds, such as water content, oil content, and water-oil distribution (Abenavoli MR, Cacco G, Sorgona A, et al. The inhibitory effects of coumarin on the germination of durum wheat (Triticum turgidum ssp. durum, cv. Simeto) seeds[J]. Journal of chemical ecology, 2006, 32: 489-506; Rathjen JR, Strounina EV, Mares D J. Water movement into dormant and non-dormant wheat (Triticum aestivum L.)grains[J]. Journal of experimental botany, 2009, 60(6): 1619-1631.). Non-destructive testing (NDT) techniques can detect moisture and oil content during wheat seed development and maturation; monitor changes in internal moisture and oil content during wheat seed storage to assess the suitability of storage conditions; and observe water absorption during wheat seed germination. While NDT allows for rapid testing without damaging the seed, it cannot provide cellular-level observation of seed structure. In tissue sectioning experiments, paraffin sections can achieve a thickness of 10 to tens of micrometers (μm). Preparation is simple, continuous sections are quick to prepare, and the morphology and structure of tissue cells can be clearly displayed. Furthermore, the sections can be preserved for a long time (Lu Jinhong, Lu Yanlin, Zhao Gan, et al. Application of a new staining method in plant paraffin sections [J]. Guangdong Chemical Industry, 2021, 48(19): 28-31.). Semi-thin sections are typically 0.5-3 μm thick, with image clarity and resolution far superior to paraffin sections. The field of view is also wider than that of ultrathin sections, allowing observation of cell morphology and internal cell structures (Wang Wei, Zhu Peiyan. Study on preparation of plant semi-thin sections using Spurr resin embedding [J]. Anhui Agricultural Science Bulletin, 2014, 20(16): 16-131.). Ultrathin sections can achieve nanometer (nm) level thickness and are mainly used in electron microscopy for observing ultrastructures. Their preparation process is more complex and requires higher expertise (Li Xiaojuan, Yao Xiaomin, et al.). Lin Jinxing, et al. A whole-body staining method for plant tissues in ultrathin sections (CN201910644850.8).Because the clarity and resolution of semi-thin sections are far superior to those of traditional paraffin sections, more detailed and accurate image information can be obtained when observing tissue and cell structures. In addition, the field of view of semi-thin sections is larger, which makes it more convenient and efficient to observe complex structures or large-scale tissues. Therefore, semi-thin sectioning technology is widely used in the biological field (Zhao Lingxiao. Slicing technology of mature and complete cereal grains and its application in the study of corn caryopsis starch [D]. Yangzhou University [2024-07-29].).

[0005] The semi-thin section technique was used to study wheat seeds. In developing seeds, the endosperm cellification process after fertilization, the development process of nucellar process cells, endosperm cells, endosperm transfer cells, and aleurone layer cells, the changes in cortical cells, and the accumulation of starch, protein, and lipids in cells could be observed. In germinating seeds, changes in cell contents and cell morphology after water absorption could be observed, such as starch and lipid decomposition and programmed cell death in aleurone layer cells (Liu Datong, Yu Xurun, Zhu Dongmei, et al. Relationship between Yangmai 16 "fast grain filling" and caryopsis microstructure and endogenous auxin [J]. Journal of Triticeae Crops, 2017, 37(6): 11.). Due to the low water content of dry wheat seeds, the fact that they are filled with starch and protein, and that the cells are tightly packed with thick and hard cell walls, samples are difficult to saturate, making it easy to break the sections and maintain the integrity of the seed structure. Currently, there is limited research on semi-thin sections of dried wheat seeds. A resin sectioning method for mature cereal seeds invented by Wei Cunxu et al. can produce dried wheat seed sections, but the sections have many wrinkles, affecting the observation of seed structure and contents. A resin sectioning method for mature wheat grains invented by Zhong Yingxin et al. can produce 1μm thick dried wheat seed sections in a relatively short period, but soaking the dried seeds in water to soften them may cause deformation of internal tissues and cells, resulting in insufficient clarity of the seed coat in the sections. This method is more suitable for observing proteins and starches within the seeds.

[0006] Preparing semi-thin sections from dried wheat seeds and observing the differences in internal structure at the cellular level, as well as studying the changes in internal structure at the cellular level during dried seed storage, has promising applications for screening wheat materials with special structures and understanding structural changes in wheat seeds during storage. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing semi-thin slices of dried wheat seeds.

[0008] To achieve the objective of this invention, a method for preparing semi-thin slices of dried wheat seeds is provided, comprising the following steps:

[0009] (1) Pre-fixation: Place the dry wheat seeds completely in 4% paraformaldehyde fixative, vacuum for 20-30 min, slowly release the gas, replace the 4% paraformaldehyde fixative, fix for 10-12 h to soften the seeds.

[0010] (2) Fixation: Cut a thin slice of 2-3 mm thickness from the middle of the softened seed in (1), place it in 4% paraformaldehyde fixative and vacuum for 4-6 h, then replace with 4% paraformaldehyde fixative and fix at 4 ℃ for 12 h.

[0011] (3) Secondary fixation: Replace the fixative in (2) with FAA, evacuate for 20-30 min, replace with FAA fixative and fix at 4℃ for 12 h;

[0012] (4) Rinsing: Rinse the fixed sample from (3) three times with 0.1 M PB buffer to remove the fixative;

[0013] (5) Dehydration: The sample rinsed in (4) was dehydrated with 20%, 40%, 60%, 70%, 80% and 90% graded ethanol for 50 min each time, and finally dehydrated with 100% ethanol 3 times for 1 h each time.

[0014] (6) Resin permeation: The dehydrated sample in (5) was permeated with 25%, 50% and 75% gradient LR white resin prepared with anhydrous ethanol for 4 hours each time. Finally, it was permeated with 100% LR white resin 3 times for 15 hours each time.

[0015] (7) Embedding: Place the sample fully impregnated with resin in (6) on the cap of a 0.5 mL centrifuge tube with the cut side facing down for embedding.

[0016] (8) Polymerization: The centrifuge tubes containing the sample from (7) were inverted and dried in a 65 ℃ oven for 10-12 h to carry out polymerization;

[0017] (9) Trimming: Use an ultramicrotome with a speed of 8-10 and a slice thickness of 2-4 μm to trim the sample from (8) to remove excess resin and expose the complete sample.

[0018] (10) Slicing: Using an ultramicrotome with a speed of 1 and a slice thickness of 1-2 μm, slice the sample after trimming in (9) and let the complete slice fall into the water tank;

[0019] (11) Spreading the slide: Drop water on the slide, take out the complete slice from (10), place it in the water drop on the slide, bake the slice, and evaporate the water.

[0020] (12) Staining observation: After staining the sections in (11) with 0.1% toluidine blue solution for 5-10 min, rinse with running water for 1-5 min, bake the sections to dry, evaporate the moisture, examine under a microscope, and mount for observation.

[0021] This invention employs novel sample fixation, dehydration, and resin permeation methods to preserve the original structure and cellular state of dry wheat seeds, enabling complete section preparation from hard, dry seeds. Centrifuge tubes are used as containers for sample embedding, suitable for dry wheat seed samples, facilitating trimming and slicing after polymerization. The invention provides efficient speed and thickness parameters for trimming and slicing continuous sections using a Leica microtome. It improves slide spreading efficiency, yielding complete and flat slides. This invention overcomes the problems of incomplete resin permeation leading to section breakage, difficult slide spreading, and excessive section wrinkling associated with conventional methods for preparing resin sections of dry wheat seeds. It provides complete and clear images of the structure, tissue, and cells of dry wheat seeds, enabling cellular-level observation and providing technical support for dry wheat seed research.

[0022] In a further preferred embodiment of the present invention, in step (1), the seeds are pre-fixed in a fixative solution to soften them, thereby enabling the hard dried seeds to be cut into thin slices of 2-3 mm thickness. The fixative solution softens the dried seeds without affecting the original morphology of the internal cells.

[0023] In a further preferred embodiment of the present invention, step (2) involves preparing the 4% paraformaldehyde fixative by dissolving 4 g of paraformaldehyde in 90 mL of 0.1 M PB at pH 7.6-7.8, adding glutaraldehyde, mixing thoroughly, and achieving a final concentration of glutaraldehyde of 2.5% in the fixative. This fixative formulation has a good effect on tissue and cell fixation.

[0024] In a further preferred embodiment of the present invention, step (3) involves preparing the FAA fixative by mixing 47.5 mL of anhydrous ethanol with 10 mL of 37% formaldehyde, 5 mL of glacial acetic acid, and 37.5 mL of UP water until homogeneous. Addressing the difficulty in fixing dry wheat seeds, secondary FAA fixation better fixes the interior of the seeds.

[0025] In a further preferred embodiment of the technical solution of the present invention, in steps (2) and (3), vacuuming is performed to allow the gas in the dry seeds to be fully discharged. After vacuuming, a new fixative is replaced and the seeds are fixed at 4 ℃, so that the fixative can fully penetrate into the cells and intercellular spaces.

[0026] In a further preferred embodiment of the technical solution of the present invention, the 20%, 40%, 60%, 70%, 80%, and 90% gradient ethanol in step (5) is prepared by mixing anhydrous ethanol and UP water in a volume ratio. The 25%, 50%, and 75% gradient resin in step (6) is prepared by mixing pure resin and anhydrous ethanol in a volume ratio. By appropriately increasing the dehydration time of the gradient ethanol and the penetration time of the gradient resin, it is possible to ensure that the inside of the dry seeds is fully penetrated and that the interstitial spaces and intercellular spaces are filled with resin, thereby solving the problems of sample breakage and incomplete sections during sectioning, and obtaining complete wheat dry seed sections.

[0027] In a further preferred embodiment of the technical solution of the present invention, in step (8), the centrifuge tube is inverted so that the sample is polymerized on the plane of the tube cap during the resin polymerization process. After embedding, the sample is located on a flat surface, which is convenient for trimming.

[0028] In a further preferred embodiment of the technical solution of the present invention, in step (9), the ultrathin slicer is automatically set to a slice speed of 8-10 and a slice thickness of 2-4 μm to quickly and smoothly remove excess resin from the sample surface, making automatic trimming more efficient and convenient.

[0029] In a further preferred embodiment of the technical solution of the present invention, in step (10), the automatic slicing speed of the ultrathin slicer is set to 1 and the slice thickness is 1-2 μm. Automatic slicing ensures uniform stress during the slicing process, prevents sample breakage, and achieves continuous and complete slicing.

[0030] In a further preferred embodiment of the present invention, a poly-L-lysine slide is used in step (11). Using a poly-L-lysine slide can make the water droplets appear as full circles, preventing them from spreading and facilitating the unfolding of the slices on the curved surface of the water droplets, thus solving the problem of the final slices easily shrinking.

[0031] In a further preferred embodiment of the technical solution of the present invention, in step (11), the slides are baked at 40-60 ℃ for 5-10 min to evaporate the moisture, and the slides are spread out flat and tightly attached to the glass slide, making them less prone to deformation and detachment. This solves the problem of slide deformation and detachment caused by the staining and rinsing process.

[0032] In a further preferred embodiment of the technical solution of the present invention, a fine-tipped brush is used in step (11) to pick up the slice, which avoids damaging the slice and can flatten the slice, thus solving the problems of slice breakage and shrinkage.

[0033] In a further preferred embodiment of the technical solution of the present invention, after staining and rinsing in step (12), the slides are baked at 40-60 ℃ to dry the moisture, so as to avoid the moisture affecting the microscopic observation, so that the final image is clear and of high quality, and the cell morphology and contents can be observed.

[0034] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0035] (i) The method of the present invention uses a fixative to soften dry wheat seeds, thus avoiding affecting their original internal structure and cell morphology.

[0036] (ii) The present invention extends the vacuuming time, which can fully remove the air inside the dry seeds. It uses two fixatives for double fixation, which preserves the original structure and cell state of the wheat dry seeds.

[0037] (iii) The present invention extends the time for ethanol dehydration and resin impregnation, so that the resin can fully penetrate into the cells and gaps of the dry wheat seeds, thereby achieving complete slice preparation.

[0038] (iv) The present invention uses centrifuge tubes as containers for embedding, providing a plane of suitable size to support the dry wheat seed sample, which is convenient for trimming and slicing after polymerization.

[0039] (v) This invention provides speed and thickness parameters for trimming and slicing in the Leica ultrathin slicer, which can efficiently complete continuous slicing.

[0040] (vi) The present invention utilizes the properties of polylysine slides and the surface tension of water to spread slides, which simplifies the slide spreading process, shortens the slide spreading time, improves the slide spreading efficiency, and can obtain complete and flat slides. The slices are tightly attached to the slides and are not easily deformed or detached. Attached Figure Description

[0041] Figure 1 is a semi-thin slice image (1 μm thick) of dry wheat seeds obtained by the original method (method before optimization) in Embodiment 1 of the present invention.

[0042] Figure 2 is a semi-thin slice image (1 μm thick) of wheat dry seeds obtained by the optimized method of Embodiment 1 of the present invention.

[0043] Figure 3 shows detailed images (1 μm thickness) of different tissue parts of wheat dry seeds obtained by the optimized method of Example 1 of the present invention. AL: aleurone layer; NP: nucellar process; ETC: endosperm transfer cells; TE: cortex. Detailed Implementation

[0044] This invention provides a method for preparing semi-thin sections of dried wheat seeds, which can be observed under a microscope with a thickness of 1-2 μm. This allows for clear observation of the cortex, endosperm, nucellar protrusions, endosperm transfer cells, and aleurone layer cells of dried wheat seeds, providing a new method for the study of the structure of dried wheat seeds.

[0045] The present invention adopts the following technical solution:

[0046] The method for preparing semi-thin slices of dried wheat seeds provided by this invention mainly includes the following steps:

[0047] (1) Pre-fixation: Place the dry wheat seeds completely in 4% paraformaldehyde fixative (PFA), vacuum for 20-30 minutes, slowly release the gas, replace the 4% paraformaldehyde fixative (PFA), fix at 4 ℃ for 10-12 h to soften the seeds.

[0048] (2) Fixation: Take out the softened dry wheat seeds and cut them into thin slices with a thickness of 2-3 mm. Place 3 thin slices in 2 mL of 4% paraformaldehyde fixative (PFA), open the cap, evacuate for 4-6 h, slowly release the gas, replace the 4% paraformaldehyde (PFA) fixative, and fix at 4 ℃ for 12 h.

[0049] (3) Secondary fixation: Discard the 4% paraformaldehyde (PFA) fixative, replace it with 2 mL FAA fixative, place it on ice, cover it with perforated aluminum foil, evacuate for 20-30 min, slowly release the gas, replace it with new FAA fixative, and fix at 4 ℃ for 12 h.

[0050] (4) Rinsing: Discard the FAA fixative and wash three times with 0.1 M PB buffer for 10 min each time.

[0051] (5) Dehydration: Discard the PB buffer and use 20%, 40%, 60%, 70%, 80% and 90% graded ethanol for dehydration, 50 min each time, and finally use 100% ethanol for dehydration three times, 1 h each time.

[0052] (6) Resin infiltration: Discard the ethanol and impregnate with 25%, 50%, and 75% gradient LR white resin (prepared with anhydrous ethanol) for 4 hours each time. Finally, impregnate three times with 100% LR white resin for 15 hours each time. LR white resin can be replaced with Technovet 7100 resin.

[0053] (7) Embedding: Take 20 mL of 100% LR white resin, add 0.39 g of embedding agent, and mix thoroughly by inverting. Label the centrifuge tube, place the sample on the cap of a 0.5 mL centrifuge tube (or other embedding mold), with the cut side facing down, add 500 μL of resin mixed with embedding agent to the centrifuge tube, tighten the cap, and fix the centrifuge tube upside down so that the cut side of the sample is in close contact with the cap of the centrifuge tube.

[0054] (8) Polymerization: Invert the centrifuge tube, fix it without shaking, and dry it in a 65 ℃ oven for 10-12 h to carry out polymerization.

[0055] (9) Trimming: Peel open the centrifuge tube, remove the sample label, fix the sample on a Leica microtome (purchased from Leica, instrument model EM UC7), with the sample cut surface facing outwards, adjust the glass blade to be parallel to the sample cut surface, set the automatic slicing speed to 8-10, and the slice thickness to 2-4 μm. Trim the embedding block, remove excess resin, until a flat surface is achieved and a complete sample can be cut out.

[0056] (10) Slicing: Stick the glass blade to the water tank, set the automatic slicing speed to 1, and the slicing thickness to 1-2 μm. When a complete and smooth sample can be cut, fill the water tank with water and continue slicing. Drop the complete slice into the water tank.

[0057] (11) Spreading the slide: Drop water onto a polylysine slide, use a fine-tipped brush to scoop out the complete slice from the water bath, and gently place it on the curved surface of the water droplet so that the slice is fully spread out without wrinkles. Bake the slice at 40-60 ℃ for 5-10 min to evaporate the moisture.

[0058] (12) Staining observation: After staining the slides with 0.1% toluidine blue solution for 5-10 min, rinse with running water for 1-5 min, bake at 40-60 ℃ to dry, and evaporate the moisture. Perform preliminary microscopic examination to ensure the integrity of the sample, mount with mounting adhesive, and take pictures for observation using a Zeiss inverted fluorescence microscope.

[0059] This invention employs 4% paraformaldehyde (PFA) and FAA fixatives, commonly used histological specimen fixatives that play a crucial role in cell and histological studies. 4% PFA fixative can covalently cross-link proteins in cells and tissues, forming stable chemical cross-links and maintaining the morphology and structure of tissues and cells. FAA fixative fixes proteins in tissues through the covalent cross-linking effect of formaldehyde, while glacial acetic acid and ethanol help maintain the morphology and structure of tissues and cells, making it more widely used in plants. The applicant discovered that during fixation, excessively low vacuum pressure or insufficient time cannot completely remove gases from dried wheat seeds, while excessively long vacuum time leads to the precipitation of starch granules from the dried wheat seeds. The applicant also found that using 4% PFA or FAA fixatives alone results in sample breakage during sectioning due to insufficient fixation. Excessive fixation time with formaldehyde and ethanol in FAA fixatives may lead to over-fixation of tissues and changes in cell nuclear morphology. Therefore, the applicant first used 4% paraformaldehyde (PFA) fixative to vacuum for 4-6 hours and fix for 12 hours, and then used FAA fixative to vacuum for 20-30 minutes and fix for 12 hours, so that the air in the sample was extracted and the fixative was immersed, thereby achieving full fixation of the dry wheat seeds and completing the complete sample section.

[0060] This invention uses gradient ethanol to gradually remove moisture from the sample, avoiding tissue deformation and loss. Gradient resin is used to gradually replace ethanol until it is 100% resin. During this process, the resin penetrates the sample, replacing the ethanol and gradually filling the gaps. The applicant has extended the gradient ethanol replacement and gradient resin impregnation times, ensuring that the dried wheat seeds are fully impregnated with ethanol and subsequently replaced by resin, ultimately filling the gaps in the dried seed sample with resin. Since dried wheat seeds contain a large amount of internal contents, if the ethanol dehydration time and resin impregnation time are short, they cannot completely penetrate all the gaps inside the sample, leading to sample breakage and starch precipitation during slicing after embedding. The method of this invention maintains the morphology of the tissue and cells while ensuring the integrity of the embedded sample, enabling the preparation of complete sections.

[0061] This invention uses 65°C for embedding, which promotes resin curing without causing thermal damage to the sample. The applicant found that excessive embedding time leads to excessive sample hardness, causing rapid wear of the glass cutter and incomplete sections during slicing; insufficient embedding time results in softer samples, discontinuous and uneven slicing, and excessive curling and wrinkling. Adjusting the embedding time to 10-12 hours enables rapid and smooth continuous slicing.

[0062] This invention utilizes an ultramicrotome for slicing, enabling automated slicing to a target thickness of 1-2 μm. The applicant discovered that during automated slicing with a Leica ultramicrotome, excessively thick slices and excessively high speeds lead to rapid wear of the glass blade, while excessively thin slices and slower speeds result in excessively long slicing times and reduced efficiency. During trimming, setting the automated slicing speed to 8-10 and the slice thickness to 2-4 μm maximizes the efficiency of the trimming process. The applicant also found that excessively high speeds during the actual slicing process cause scratches on the sample surface, affecting sample observation and image quality. Setting the automated slicing speed to 1 and the slice thickness to 1-2 μm produces higher-quality slices. The parameters for automated trimming and slicing are essential for efficiently obtaining high-quality slices.

[0063] This invention uses a fine-tipped brush to pick up and spread the slides, avoiding damage. The surface tension of water droplets on a poly-L-lysine slide is used to spread the slides evenly, and then the moisture is dried, leaving the slides flat and adhered to the slide. This method is simple and yields better results.

[0064] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0065] The LR white resin used in the following examples was purchased from Beijing Haide Innovation Technology Co., Ltd.

[0066] Example 1: Method for preparing semi-thin slices of dried wheat seeds

[0067] This embodiment provides a method for preparing semi-thin slices of dried wheat seeds. The experimental materials involved in the following embodiments are common wheat, which were preserved and provided by the Seed Development and Germination Team of the Wheat Research Institute of Sichuan Agricultural University.

[0068] The specific implementation steps are as follows:

[0069] (1) Preparation of fixative: The method for preparing 0.1 M PB buffer is as follows: weigh 3.5814 g Na2HPO4·12H2O and dissolve it in 100 mL UP water, weigh 1.5601 g NaH2PO4·2H2O and dissolve it in 100 mL UP water, mix the Na2HPO4·12H2O solution and NaH2PO4·2H2O solution until the pH is within the range of 7.6-7.8, and store at room temperature.

[0070] The preparation method of 4% paraformaldehyde (PFA) fixative is as follows: Weigh 4 g of paraformaldehyde and dissolve it in 90 mL of 0.1 M PB with pH=7.6-7.8. Heat in a water bath at 65 ℃ until dissolved, cool to room temperature, open in a fume hood, add 10 mL of 25% glutaraldehyde, mix well, and store at 4 ℃.

[0071] The FAA fixative formulation is shown in Table 1. Store at 4 ℃.

[0072] Table 1. Formulation of FAA Fixative

[0073]

[0074] (2) Pre-fixation: Place dry wheat seeds into a 2 mL centrifuge tube, fill it with 4% paraformaldehyde fixative (PFA), vacuum at -80 kPa for 20-30 min, slowly release the gas, replace the 4% paraformaldehyde fixative (PFA), fix at 4 ℃ for 10-12 h to soften the seeds.

[0075] (3) Fixation: Use tweezers to remove the softened dry wheat seeds, cut them horizontally along the middle of the seeds, and cut them into thin slices with a thickness of 2-3 mm. Place 3 thin slices in a 2 mL centrifuge tube, add 2 mL of 4% paraformaldehyde fixative (PFA), open the centrifuge tube, vacuum at -80 kPa for 4-6 h, slowly release the gas, replace with new 4% paraformaldehyde (PFA) fixative, and fix at 4 ℃ for 12 h.

[0076] (4) Secondary fixation: Discard the 4% paraformaldehyde (PFA) fixative in the centrifuge tube and replace it with 2 mL of FAA fixative. Place the centrifuge tube on ice, open the cap, cover the centrifuge tube with perforated aluminum foil, evacuate at -80 kPa for 20-30 min, slowly release the gas, replace with new FAA fixative, and fix at 4 ℃ for 12 h.

[0077] (5) Rinsing: Discard the FAA fixative in the centrifuge tube, add 2 mL of 0.1 M PB buffer, tighten the cap of the centrifuge tube, place it on a horizontal shaker and shake the sample with the buffer solution to wash it 3 times, 10 min each time.

[0078] (6) Dehydration: Discard the PB buffer, add 2 mL of 20%, 40%, 60%, 70%, 80% and 90% graded ethanol to the centrifuge tubes respectively, tighten the caps of the centrifuge tubes, place them on a horizontal shaker and shake the ethanol solution to dehydrate the samples for 50 min each time, and then use 100% ethanol to dehydrate three times, 1 h each time.

[0079] (7) Resin permeation: Discard the ethanol, add 2 mL of 25%, 50%, and 75% gradient LR white resin (prepared with anhydrous ethanol) to the centrifuge tubes respectively, tighten the caps of the centrifuge tubes, place them on a horizontal vibrator to allow the resin to shake and fully permeate the samples, 4 h each time, and finally permeate three times with 100% LR white resin, 15 h each time.

[0080] (7) Embedding: Take 20 mL of 100% LR white resin, add 0.39 g of embedding agent, and mix thoroughly by inverting. Label a 0.5 mL centrifuge tube, place the sample on the centrifuge tube cap with the cut side down using tweezers, add 500 μL of the mixed embedding agent resin to the centrifuge tube, and also add a small amount of the mixed embedding agent resin to the centrifuge tube cap, tighten the cap, and invert the centrifuge tube so that the sample cut side is in close contact with the centrifuge tube cap.

[0081] (8) Polymerization: Invert the centrifuge tube, fix it without shaking, and dry it in a 65 ℃ oven for 10-12 h to carry out polymerization.

[0082] (9) Trimming: Peel open the centrifuge tube, take out the polymerized sample and mark it. Fix the sample in the sample well of the Leica microtome with the sample-containing surface facing outwards. Fix the glass blade, turn on the bottom and top lights, and adjust the glass blade to be parallel to the sample cutting surface according to the reflective strip on the sample plane. Set the automatic slicing speed to 8-10 and the slice thickness to 2-4 μm. Trim the embedding block, remove excess resin, until a flat surface is achieved and a complete sample can be cut.

[0083] (10) Slicing: Stick the glass blade to the water tank, set the automatic slicing speed to 1, and the slicing thickness to 1-2 μm. When a complete sample can be cut out, fill the water tank with water and continue slicing. Drop the complete slice into the water tank.

[0084] (11) Spreading the slide: Drop water onto a polylysine slide, use a fine-tipped brush to scoop out the complete slice from the water bath, and gently place it on the curved surface of the water droplet so that the slice is fully spread out without wrinkles. Bake the slice at 40-60 ℃ for 5-10 min to evaporate the moisture.

[0085] (12) Staining observation: After staining the slides with 0.1% toluidine blue solution for 5-10 min, rinse with running water for 1-5 min, bake at 40-60 ℃ to dry, and evaporate the moisture. Perform preliminary microscopic examination to ensure the integrity of the sample, mount with mounting adhesive, and take pictures using a Zeiss inverted fluorescence microscope to save the images for further observation and analysis.

[0086] The image of a semi-thin section (1 μm thick) of dried wheat seed obtained using the original method (before optimization) is shown in Figure 1. The image of a semi-thin section (1 μm thick) of dried wheat seed obtained using the optimized method is shown in Figure 2. Detailed images of different tissue parts of dried wheat seed (1 μm thick) are shown in Figure 3.

[0087] The original method (the method before optimization) is as follows:

[0088] (1) Pre-fixation: Place the dry wheat seeds completely in 4% paraformaldehyde fixative solution and fix for 10-12 h to soften the seeds;

[0089] (2) Fixation: Cut a thin slice of 2-3 mm thickness from the middle of the softened seed in (1), place it in 4% paraformaldehyde fixative and vacuum for 20-30 min, then replace with 4% paraformaldehyde fixative and fix at 4 ℃ for 12 h.

[0090] (3) Secondary fixation: Replace the fixative in (2) with FAA, vacuum for 15 min, replace with FAA fixative and fix at 4 ℃ for 12 h;

[0091] (4) Rinsing: Rinse the fixed sample from (3) three times with 0.1 M PB buffer to remove the fixative;

[0092] (5) Dehydration: The sample rinsed in (4) was dehydrated with 20%, 40%, 60%, 70%, 80% and 90% graded ethanol for 30 min each time, and finally dehydrated with 100% ethanol 3 times for 40 min each time.

[0093] (6) Resin permeation: The dehydrated sample in (5) was permeated with 25%, 50% and 75% gradient LR white resin prepared with anhydrous ethanol for 2 hours each time, and finally permeated with 100% LR white resin 3 times for 12 hours each time.

[0094] (7) Embedding: Place the sample fully impregnated with resin in (6) on the cap of a 0.5 mL centrifuge tube with the cut side facing down for embedding.

[0095] (8) Polymerization: The capsule mold containing the sample from (7) was dried in an oven at 65 °C for 12 h and then polymerized;

[0096] (9) Trimming: Use an ultramicrotome with a speed of 8-10 and a slice thickness of 2 μm to trim the sample from (8) to remove excess resin and expose the complete sample.

[0097] (10) Slicing: Using an ultramicrotome with a speed of 1-2 and a slice thickness of 1-2 μm, cut the sample slices after trimming in (9) and let the complete slices fall into the water tank;

[0098] (11) Spreading the slide: Drop water on the slide, take out the complete slice from (10), place it in the water drop on the slide, bake the slice, and evaporate the water.

[0099] (12) Staining observation: After staining the sections in (11) with 0.1% toluidine blue solution for 5 min, rinse with running water for 1 min, bake the sections to dry, evaporate the water, examine under a microscope, and mount for observation.

[0100] This invention provides a method for preparing semi-thin sections of dried wheat seeds, comprising the following steps: pre-fixation, fixation, secondary fixation, rinsing, dehydration, resin infiltration, embedding, polymerization, trimming, sectioning, spreading, and staining observation. The advantages are: pre-fixation softens the dried seeds without affecting the original morphology of the seed cells; double fixation ensures complete gas expulsion from the dried seeds, resulting in better cell fixation and intact sections; extending the gradient ethanol and gradient resin replacement time ensures that the final resin fully penetrates the intercellular spaces, resulting in intact and flat sections; detailed parameters for trimming and sectioning processes are provided, enabling efficient continuous sectioning; the spreading process uses a brush to scoop the sections and place them on a glass slide without damaging them, making the operation simple and quick. This invention addresses the characteristics of dried wheat seeds being hard and full of starch, gradually solving problems encountered in conventional methods for preparing semi-thin sections, such as incomplete resin infiltration, section breakage, and section shrinkage. It yields complete semi-thin sections of dried wheat seeds with clear tissue structures, enabling further observation and quantitative analysis of cell morphology in different parts, thus providing technical support for the study of the structure of dried wheat seeds.

[0101] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing semi-thin slices of dried wheat seeds, characterized in that, Includes the following steps: (1) Pre-fixation: Place the dry wheat seeds completely in 4% paraformaldehyde fixative, vacuum for 20-30 minutes, slowly release the gas, replace the 4% paraformaldehyde fixative, and fix for 10-12 hours to soften the seeds; (2) Fixation: Cut a 2-3 mm thick slice from the middle of the softened seeds in (1), place it in 4% paraformaldehyde fixative, vacuum for 4-6 hours, replace the 4% paraformaldehyde fixative, and fix at 4℃ for 12 hours; (3) Secondary fixation: Replace the fixative in (2) with FAA, vacuum for 20-30 minutes, replace the FAA fixative, and fix at 4℃ for 12 hours; (4) Rinsing: Use 0.1M... Rinse the fixed sample in step (3) three times with PB buffer to remove the fixative; (5) Dehydration: Dehydrate the sample rinsed in step (4) with a gradient of 20%, 40%, 60%, 70%, 80%, and 90% ethanol for 50 minutes each time, and finally dehydrate it three times with 100% ethanol for 1 hour each time; (6) Resin permeation: Permeate the dehydrated sample in step (5) with a gradient of 25%, 50%, and 75% LR white resin prepared with anhydrous ethanol for 4 hours each time, and finally rinse it with 100% LR white resin. (7) Implantation: Place the resin-impregnated sample from step (6) on the cap of a 0.5 mL centrifuge tube with the cut side facing down for embedding; (8) Polymerization: Invert the centrifuge tube containing the embedded sample from step (7) and dry it in a 65℃ oven for 10-12 hours for polymerization; (9) Trimming: Trim the sample from step (8) using an ultramicrotome with a speed of 8-10 and a slice thickness of 2-4 μm to remove excess resin and expose the complete sample; (10) Slicing: Use an ultramicrotome with a speed of 1 and a slice thickness of 1-2 μm to trim the sample from step (6). 9) After trimming the sample slices, let the complete slices fall into the water tank; (11) Spread the slides: drip water on the slide, take out the complete slices from (10), place them in the water droplets on the slide, bake the slides, and evaporate the water; (12) Staining and observation: use 0.1% toluidine blue solution to stain the slices from (11) for 5-10 min, rinse with running water for 1-5 min, bake the slides to dry, evaporate the water, examine under a microscope, and mount the slides for observation; in step (11), bake the slides at 40-60℃ for 5-10 min and evaporate the water; in step (12), after staining and rinsing, bake the slides at 40-60℃ to dry the water.

2. The method according to claim 1, characterized in that, In step (2), the preparation method of 4% paraformaldehyde fixative includes: dissolving 4g of paraformaldehyde in 90mL of 0.1M PB with pH 7.6-7.8, adding glutaraldehyde, mixing evenly, and the final concentration of glutaraldehyde in the fixative is 2.5%.

3. The method according to claim 1, characterized in that, In step (3), the preparation method of FAA fixative includes: mixing 47.5 mL of anhydrous ethanol with 10 mL of 37% formaldehyde, 5 mL of glacial acetic acid and 37.5 mL of UP water until homogeneous.

4. The method according to claim 1, characterized in that, The preparation method of 0.1M PB in step (4) includes: weighing 3.5814g Na2HPO4·12H2O and dissolving it in 100mL UP water to obtain Na2HPO4·12H2O solution; weighing 1.5601g NaH2PO4·2H2O and dissolving it in 100mL UP water to obtain NaH2PO4·2H2O solution; mixing the Na2HPO4·12H2O solution and the NaH2PO4·2H2O solution until the pH is in the range of 7.6-7.

8.

5. The method according to claim 1, characterized in that, The 20%, 40%, 60%, 70%, 80%, and 90% gradient ethanol in step (5) is prepared by mixing anhydrous ethanol and UP water in a volume ratio.

6. The method according to claim 1, characterized in that, The 25%, 50%, and 75% gradient resins in step (6) are prepared by mixing pure resin and anhydrous ethanol in a volume ratio.

7. The method according to claim 1, characterized in that, In step (11), a polylysine slide is used.

8. The method according to any one of claims 1-7, characterized in that, In step (11), use a fine-tipped brush to pick up the slice.

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