Dry wheat seed semi-thin slice preparation method

Abstract

The invention discloses a preparation method of a wheat dry seed semi-thin slice. The preparation method comprises the steps of pre-fixation, fixation, secondary fixation, rinsing, dehydration, resin permeation, embedding, polymerization, block trimming, slicing, slice unfolding and dyeing observation. The method has the advantages that the dry seeds are softened through pre-fixation, and the original forms of the cells of the dry seeds are not affected; due to double fixation, gas in the dry seeds is completely exhausted, the cell fixation effect is better, and slices are complete; and the replacement time of gradient ethanol and gradient resin is prolonged, so that the final resin can fully permeate intercellular spaces, and the section is complete and flat. Aiming at the characteristics that the wheat dry seeds are hard and full of starch, the method gradually solves the problems of incomplete resin infiltration, slice breakage, slice shrinkage and the like encountered by a conventional method for manufacturing the semi-thin slices of the dry seeds, the complete semi-thin slices of the wheat dry seeds with clear tissue structures are obtained, and cell morphology observation and quantitative analysis of different parts can be further carried out. Therefore, technical support is provided for research on the wheat dry seed structure.

Description

Technical Field

[0001] The invention relates to the technical field of microtissue sections, in particular to a method for preparing semi-thin sections of dry wheat seeds. Background Art

[0002] In addition to starch and protein, wheat seeds are also rich in minerals and vitamins, providing abundant nutrients. Wheat seeds absorb water, germinate, and establish seedlings, completing the transmission of genetic information, which is of great significance to wheat genetic breeding.

[0003] The study of wheat seeds involves many aspects such as grain development, seed dormancy, seed life, and seed germination. The study of seed structure is a very important research content throughout the entire seed life cycle. Wheat is mainly composed of three parts: cortex, embryo, and endosperm. The cortex is composed of epidermis, pericarp, seed coat, etc., which wraps around the outside of the entire grain to protect the embryo and endosperm. The embryo consists of four parts: plumule, hypocotyl, radicle, and scutellum. It is 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 substances such as starch and protein 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 embryonic surrounding region (ESR). The development of the endosperm determines the yield and quality of wheat (Zheng Y, Wang Z. Contrast observation and investigationof wheat endosperm transfer cells and nucellar projection transfer cells[J]. Plant Cell Reports, 2011, 30: 1281-1288.).

[0004] In the study of wheat seed structure, non-destructive testing and tissue sectioning provide very important technical means for fine structure research. Tomography technology can observe the internal structure of seeds without damaging the seeds and perform 3D modeling. High-resolution tomography can also observe the 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 measure 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 can quickly and non-destructively detect seed contents, 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 DJ. Water movement into dormant and non-dormant wheat (Triticum aestivum L.)grains[J]. Journal of experimental botany, 2009, 60(6): 1619-1631.). Nondestructive testing technology can detect moisture and oil in wheat seeds during their development and maturation; monitor changes in moisture and oil inside the seeds during storage to assess the suitability of storage conditions; and observe the water absorption process during wheat seed germination. Nondestructive testing technology can perform rapid testing without damaging the seeds, but it cannot observe the seed structure at the cellular level. In tissue section experiments, paraffin sections can achieve the preparation of sections with a thickness of 10 to tens of microns (μm). The preparation is simple, the continuous sectioning takes a short time, and the morphological structure of tissue cells can be clearly displayed. The sections can also 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.); The thickness of semi-thin sections is usually between 0.5-3 μm. The image clarity and resolution are much better than paraffin sections, and the field of view is larger than that of ultra-thin sections. Cell morphology and internal structure can be observed (Wang Wei, Zhu Peiyan. Research on the method of preparing plant semi-thin sections using Spurr resin embedding [J]. Anhui Agricultural Science Bulletin, 2014, 20(16): 16-131.); Ultra-thin sections can achieve sections with a thickness of nanometers (nm). They are mainly used in electron microscopy technology for ultrastructure observation. Their production process is more complex and professional (Li Xiaojuan, Yao Xiaomin, Lin Jinxing, et al. A whole-body staining method for ultrathin sections of plant tissues, CN201910644850.8).Since the clarity and resolution of semi-thin sections are much better than those of traditional paraffin sections, more detailed and accurate image information can be obtained when observing tissue and cell structures. Semi-thin sections also have a larger field of view, which makes it more convenient and efficient to observe complex structures or large-scale tissues. Therefore, semi-thin section technology is widely used in the biological field (Zhao Lingxiao. Slicing technology of mature and intact grains of cereals and its application in the study of corn caryopsis starch [D]. Yangzhou University [2024-07-29].).

[0005] Semi-thin sectioning technology is used to study wheat seeds. In developing seeds, the endosperm cellularization process after fertilization can be observed; the development process of nucellar protrusion cells, endosperm cells, endosperm transfer cells, and aleurone cells; the change process of cortical cells, and the accumulation process of starch, protein, and oil in cells. In germinating seeds, the changes in cell content and cell morphology after water absorption are observed, such as the decomposition of starch and oil and the programmed death of aleurone cells (Liu Datong, Yu Xurun, Zhu Dongmei, et al. Relationship between "fast filling" of Yangmai 16 and caryopsis microstructure and endogenous auxin [J]. Journal of Wheat Crops, 2017, 37(6): 11.). Since wheat dry seeds have low water content, are filled with starch and protein, and have densely arranged cells with thick cell walls and high hardness, it is difficult to penetrate the samples, and the prepared slices are very easy to break, making it difficult to maintain the integrity of the seed structure. At present, there are few studies on semi-thin sections of dry wheat seeds. A resin sectioning method for mature grain seeds invented by Wei Cunxu et al. can obtain dry wheat seed sections, but the sections have many wrinkles, which affects the observation of seed structure and contents. The resin sectioning method for mature wheat grains invented by Zhong Yingxin et al. can complete 1μm thick dry wheat seed sections in a relatively short period of time, but soaking dry seeds in water to soften them may cause deformation of internal tissues and cells, and the seed cortex in the sections is not clear enough, which is more suitable for observing protein and starch in seeds.

[0006] Semi-thin sections were prepared from dry wheat seeds to observe the differences in the internal structure of dry seeds at the cellular level and to study the changes in the internal structure of dry seeds at the cellular level during storage. This has application prospects in screening wheat materials with special structures and understanding the structural changes of wheat seeds during storage. Summary of the invention

[0007] The invention aims to provide a method for preparing dry wheat seed semi-thin slices.

[0008] In order to achieve the object of the present invention, the present invention provides a method for preparing semi-thin slices of dry wheat seeds, comprising the following steps: (1) Pre-fixation: Place dry wheat seeds completely in 4% paraformaldehyde fixative, evacuate for 20-30 min, slowly release the air, replace with 4% paraformaldehyde fixative, and fix for 10-12 h to soften the seeds. (2) Fixation: Cut the middle part of the softened seeds in (1) into a thin slice sample with a thickness of 2-3 mm, place it in a 4% paraformaldehyde fixative and evacuate for 4-6 h, then replace the 4% paraformaldehyde fixative and fix it at 4°C for 12 h; (3) Secondary fixation: Replace the fixative in (2) with FAA, evacuate for 20-30 min, then replace with FAA fixative and fix at 4°C for 12 h; (4) Rinse: Rinse the fixed samples in (3) three times with 0.1 M PB buffer to remove the fixative; (5) Dehydration: Dehydrate the sample rinsed in (4) with 20%, 40%, 60%, 70%, 80%, and 90% gradient ethanol for 50 min each time, and finally dehydrate with 100% ethanol for 3 times, 1 h each time; (6) Resin infiltration: The dehydrated samples in (5) were infiltrated with 25%, 50%, and 75% gradient LR white resin prepared in anhydrous ethanol for 4 h each time, and finally infiltrated with 100% LR white resin for 3 times, each time for 15 h; (7) Embedding: Place the sample fully soaked with the resin in (6) on the cap of a 0.5 mL centrifuge tube with the cut surface facing downward for embedding.

[0009] (8) Polymerization: Invert the centrifuge tube containing the sample in (7) and place it in a 65 °C oven for 10-12 h to perform polymerization; (9) Block trimming: Use an ultrathin slicer with the speed set to 8-10 and the slice thickness set to 2-4 μm to trim the sample in (8) to remove excess resin and expose the complete sample; (10) Slicing: Use an ultra-thin slicer with the speed set to 1 and the slice thickness set to 1-2 μm to slice the sample after trimming in (9) so that the complete slice falls into the water tank; (11) Spreading the slices: drip water on a glass slide, take out the intact slices from (10), place them in the water drops on the glass slide, bake the slices, and evaporate the water; (12) Staining observation: Use 0.1% toluidine blue solution to stain the sections in (11) for 5-10 min, rinse with running water for 1-5 min, bake the sections to dry, evaporate the water, examine under a microscope, and seal the sections for observation.

[0010] The invention adopts a new sample fixation, dehydration and resin infiltration method, retains the original structure and cell state of dry wheat seeds, and can realize complete slice preparation of hard dry seeds; uses a centrifuge tube as a container to embed samples, which is suitable for dry wheat seed samples and is easy to trim and slice after polymerization; provides Leica ultrathin slicer to efficiently complete the trimming and slicing speed and thickness parameters of continuous slicing; the invention improves the efficiency of slice spreading, and can obtain complete and flat slices; the invention overcomes the problems of slice breakage, slice spreading difficulty, and excessive slice wrinkles caused by incomplete resin penetration inside the sample when making wheat dry seed resin slices by conventional methods, and can obtain complete and clear images of wheat dry seed structure, tissue, and cells, thereby realizing cell level observation and providing technical support for wheat dry seed research.

[0011] According to a further preferred embodiment of the technical solution of the present invention, in step (1), the dry seeds are pre-fixed in a fixing solution to soften the dry seeds, thereby achieving the goal of cutting the hard dry seeds into thin slices of 2-3 mm thickness, and the fixing solution softening the dry seeds does not affect the original morphology of the internal cells.

[0012] According to a further preferred embodiment of the technical solution of the present invention, in step (2), the preparation method of 4% paraformaldehyde fixative comprises: dissolving 4 g paraformaldehyde in 90 mL 0.1 M PB at pH 7.6-7.8, adding glutaraldehyde, and mixing evenly, wherein the final concentration of glutaraldehyde in the fixative is 2.5%. The fixative of this formula has a good effect on tissue and cell fixation.

[0013] According to a further preferred embodiment of the technical solution of the present invention, in step (3), the preparation method of the FAA fixative comprises: evenly 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. In view of the fact that dry wheat seeds are difficult to fix, the FAA secondary fixation can better fix the inside of the seeds.

[0014] According to a further preferred embodiment of the technical solution of the present invention, in steps (2) and (3), vacuum is applied to allow the gas in the dry seeds to be fully discharged, and after vacuum is applied, a new fixative is replaced and the seeds are fixed at 4°C so that the fixative can fully penetrate into the cells and intercellular spaces.

[0015] In a further preferred embodiment of the technical solution of the present invention, the 20%, 40%, 60%, 70%, 80%, 90% gradient ethanol in step (5) is prepared by volume ratio of anhydrous ethanol and UP water. The 25%, 50%, 75% gradient resin in step (6) is prepared by volume ratio of pure resin and anhydrous ethanol. The gradient ethanol dehydration time and the gradient resin penetration time are appropriately increased to ensure that the inside of the dry seeds is fully penetrated and the tissue gaps and cell gaps are filled with resin, thereby solving the problems of sample breakage and incomplete slicing during slicing, and obtaining complete wheat dry seed slices.

[0016] According to 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 cover during the resin polymerization process, and the sample is located on a flat surface after embedding, which is convenient for trimming.

[0017] According to a further preferred embodiment of the technical solution of the present invention, in step (9), the ultrathin slicer is configured to automatically slice at a speed of 8-10 and a slice thickness of 2-4 μm, so that excess resin on the sample surface can be removed quickly and smoothly, and automatic trimming is more efficient and convenient.

[0018] According to a further preferred embodiment of the technical solution of the present invention, in step (10), the ultrathin slicer automatically slices at a speed of 1 and a slice thickness of 1-2 μm. Automatic slicing ensures uniform force during slicing, prevents sample breakage, and achieves continuous and complete slicing.

[0019] In a further preferred embodiment of the technical solution of the present invention, a polylysine glass slide is used in step (11). The use of a polylysine glass slide can make the water droplets present a full round shape, and the water droplets are not dispersed, which facilitates the slices to be spread on the curved surface of the water droplets, thereby solving the problem that the final slices are prone to shrinkage.

[0020] According to a further preferred embodiment of the technical solution of the present invention, in step (11), the slices are baked at 40-60°C for 5-10 min to evaporate the water, and the slices are flatly unfolded and tightly attached to the glass slide, and are not easily deformed or fallen off, thereby solving the problem of slice deformation and falling off caused by the staining and rinsing process.

[0021] According to a further preferred embodiment of the technical solution of the present invention, in step (11), a thin-tipped brush is used to pick up the slices, thereby avoiding damaging the slices and allowing the slices to be flattened, thus solving the problem of slice breakage and shrinkage.

[0022] According to 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°C to dry out moisture, so as to avoid moisture affecting microscopic observation, so that the final image is clear and high-quality, and cell morphology and contents can be observed.

[0023] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) The method of the present invention uses a fixing liquid to soften dry wheat seeds to avoid affecting the original internal structure and cell morphology thereof.

[0024] (ii) The present invention prolongs the vacuuming time, can fully expel the air inside the dry seeds, and uses two fixatives for double fixation, thereby retaining the original structure and cell state of the dry wheat seeds.

[0025] (III) The present invention prolongs the time of 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.

[0026] (iv) The present invention uses a centrifuge tube as a container for embedding, providing a plane of suitable size to support the dry wheat seed sample, which is convenient for block trimming and slicing after polymerization.

[0027] (V) The present invention provides Leica ultra-thin sectioning machine block and sectioning speed and thickness parameters, which can efficiently complete continuous sectioning.

[0028] (VI) The present invention utilizes the characteristics of poly-lysine glass slides and the tension of water to carry out film spreading, which simplifies the film spreading process, shortens the film spreading time, improves the film spreading efficiency, and can obtain complete and flat film spreads. The slices are tightly attached to the glass slide and are not easy to deform or fall off. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an image of a semi-thin section (thickness 1 μm) of dry wheat seeds using the original method (method before optimization) of Example 1 of the present invention.

[0030] Figure 2 This is an image of a semi-thin section of dry wheat seeds (1 μm thick) after the optimized method of Example 1 of the present invention.

[0031] Figure 3 Detailed images of different tissues of dry wheat seeds after the optimization method of Example 1 of the present invention (thickness 1 μm): AL: aleurone layer; NP: nucellar process; ETC: endosperm transfer cell; TE: cortex. DETAILED DESCRIPTION

[0032] The present invention provides a method for preparing semi-thin sections of dry wheat seeds, and completes microscopic observation of semi-thin sections with a thickness of 1-2 μm, so that the cortex, endosperm, nucellar protrusions, endosperm transfer cells, and aleurone cells of the dry wheat seeds can be clearly observed, thereby providing a new method for studying the structure of dry wheat seeds.

[0033] The present invention adopts the following technical solution: The method for preparing semi-thin slices of dry wheat seeds provided by the present invention mainly comprises the following steps: (1) Pre-fixation: Place dry wheat seeds completely in 4% paraformaldehyde fixative (PFA), evacuate for 20-30 minutes, slowly release the air, replace with 4% paraformaldehyde fixative (PFA), and fix at 4°C for 10-12 hours to soften the seeds.

[0034] (2) Fixation: Take out the softened dry wheat seeds and cut them into thin slices with a thickness of 2-3 mm. Place three thin slices in 2 mL of 4% paraformaldehyde fixative (PFA). Open the lid, evacuate for 4-6 h, slowly release the air, replace the 4% paraformaldehyde (PFA) fixative, and fix at 4 ℃ for 12 h.

[0035] (3) Secondary fixation: Pour out the 4% paraformaldehyde (PFA) fixative and replace it with 2 mL FAA fixative. Place on ice, cover with perforated tin foil, evacuate for 20-30 min, slowly deflate, replace with new FAA fixative, and fix at 4°C for 12 h.

[0036] (4) Rinse: Pour away the FAA fixative and wash three times with 0.1 M PB buffer, 10 min each time.

[0037] (5) Dehydration: Pour away the PB buffer and dehydrate with 20%, 40%, 60%, 70%, 80%, and 90% ethanol for 50 min each time. Finally, dehydrate with 100% ethanol three times for 1 h each time.

[0038] (6) Resin infiltration: Pour off the ethanol and infiltrate with 25%, 50%, and 75% gradient LR white resin (prepared with anhydrous ethanol) for 4 hours each time, and finally infiltrate with 100% LR white resin three times for 15 hours each time. LR white resin can be replaced by Technovet 7100 resin.

[0039] (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 surface facing down, add 500 μL of resin mixed with embedding agent to the centrifuge tube, cover the tube tightly, and fix the centrifuge tube upside down so that the cut surface of the sample is in close contact with the centrifuge tube cap.

[0040] (8) Polymerization: Invert the centrifuge tube, fix it without shaking, and place it in a 65 ℃ oven to dry for 10-12 h to carry out polymerization.

[0041] (9) Trimming: Peel open the centrifuge tube, take out the sample marker, fix the sample on a Leica ultrathin slicer (purchased from Leica, instrument model EM UC7), with the sample cut surface facing outward. Adjust the glass knife and the sample cut surface to be parallel. Set the automatic slicing speed to 8-10 and the slice thickness to 2-4 μm. Trim the embedded block and remove excess resin until a flat surface is trimmed and a complete sample can be cut out.

[0042] (10) Slicing: Stick the glass knife to the water tank, set the automatic slicing speed to 1, and slice at a thickness of 1-2 μm. When a complete and smooth sample can be cut out, fill the water tank with water and continue slicing, dropping the complete slice into the water tank.

[0043] (11) Spreading the slice: Drop water on a poly-L-lysine slide, use a fine-tipped brush to pick up the intact slice from the water tank, and gently place it on the curved surface of the water drop so that the slice is completely spread out without wrinkles. Bake the slice at 40-60 °C for 5-10 min to evaporate the water.

[0044] (12) Staining observation: Use 0.1% toluidine blue solution to stain the sections on the slide for 5-10 min, rinse with running water for 1-5 min, dry the slides at 40-60 °C, and evaporate the water. Perform a preliminary microscopic examination to ensure the integrity of the sample, seal the slides with sealing glue, and take photos using a Zeiss inverted fluorescence microscope.

[0045] The present invention adopts 4% paraformaldehyde (PFA) and FAA fixative, which are commonly used histological specimen fixatives and play a key role in cell and histological research. 4% paraformaldehyde (PFA) fixative can fix proteins in cells and tissues in a covalent cross-linking manner, forming stable chemical cross-links to maintain 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 to maintain the morphology and structure of tissues and cells, and is more widely used in plants. The applicant found that during the fixation process, the vacuum pressure is too low and the time is too short, and the gas in the dry wheat seeds cannot be completely discharged. If the vacuum time is too long, the starch granules in the dry wheat seeds will precipitate. The applicant found that the use of 4% paraformaldehyde (PFA) or FAA fixative alone causes the sample to be broken during the sectioning process due to insufficient fixation. The formaldehyde and ethanol in the FAA fixative fix for too long a time may cause excessive fixation of the tissue and changes in the morphology of the cell nucleus. Therefore, the applicant first used 4% paraformaldehyde (PFA) fixative to vacuum for 4-6 h and fix for 12 h, and then used FAA fixative to vacuum for 20-30 min and fix for 12 h, 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 complete sample slicing.

[0046] The present invention uses gradient ethanol to gradually remove the water in the sample to avoid deformation and loss of tissue. Use gradient resin to gradually replace ethanol until it is finally replaced with 100% resin. In this process, the resin penetrates into the sample, replaces the ethanol, and gradually fills the gaps in the sample. The applicant has extended the time for gradient ethanol replacement and gradient resin penetration, so that the wheat dry seeds are fully soaked in ethanol, and then replaced by resin, and the gaps in the final dry seed sample are filled with resin. The internal contents of the wheat dry seeds are relatively large. If the ethanol dehydration time and the resin penetration time are short, they cannot completely penetrate into all the gaps inside the sample, which will cause the sample to break when sliced ​​after embedding, and starch granules to precipitate. The method of the present invention ensures the integrity of the sample after embedding on the basis of maintaining the morphology of tissues and cells, and can prepare complete slices.

[0047] The present invention uses 65°C for embedding, which not only promotes the curing of the resin, but also does not cause thermal damage to the sample. The applicant found that if the embedding time is too long, the sample will be too hard, which will easily cause the glass knife to wear out too quickly and the slices to be incomplete during the slicing process; if the embedding time is too short, the sample will be soft, the slices will be discontinuous and not smooth, and it will be easy to curl and the slices will have too many wrinkles. After adjusting the embedding time to 10-12 hours, continuous slicing can be performed quickly and smoothly.

[0048] The present invention uses an ultra-thin slicer for slicing, and can achieve the target 1-2 μm thickness automatic slicing. The applicant has found that if the slice thickness is too thick or the speed is too fast during the automatic slicing process of the Leica ultra-thin slicer, the glass knife will be worn too quickly. If the slice thickness is too thin or the speed is slowed down, the slicing time will be too long and the efficiency will be reduced. When trimming, the automatic slicing speed is set to 8-10, and the slice thickness is set to 2-4 μm, and the trimming process can be completed with the highest efficiency. The applicant has found that during the formal slicing process, too fast speed causes scratches on the sample surface, affecting the observation and image quality of the sample. The automatic slicing speed is set to 1, and the slice thickness is set to 1-2 μm, which can complete higher quality slices. The parameters of automatic trimming and slicing are very necessary for efficiently obtaining high-quality slices.

[0049] The invention uses a fine-headed writing brush to scoop and spread slices, thereby avoiding damage to the slices. The slices are spread evenly by utilizing the tension of water drops on a polylysine glass slide, and then the water is dried to keep the flat slices attached to the glass slide, which is simple to operate and has better effects.

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present 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.

[0051] The LR white resin used in the following examples was purchased from Beijing Hyde Innovation Technology Co., Ltd.

[0052] Example 1 Preparation method of semi-thin slices of dry wheat seeds This embodiment provides a method for preparing semi-thin slices of dry wheat seeds. The experimental materials involved in the following embodiments are sourced from common wheat, which is preserved and provided by the Seed Development and Germination Team of the Wheat Research Institute of Sichuan Agricultural University.

[0053] The specific implementation steps are as follows: (1) Preparation of stationary phase: 0.1 M PB buffer solution was prepared by weighing 3.5814 g Na 2 HPO 4 12H 2 O, dissolve in 100 mL UP water, weigh 1.5601 g NaH 2 PO 4 ·2H 2 O, dissolved in 100 mL UP water, mixed with Na 2 HPO 4 12H 2 O solution and NaH 2 PO 4 ·2H 2 O solution until the pH is within the range of 7.6-7.8 and store at room temperature.

[0054] 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 a pH of 7.6-7.8, heat it in a 65°C water bath until dissolved, cool it to room temperature, open it in a fume hood, add 10 mL of 25% glutaraldehyde, mix well, and store it at 4°C.

[0055] The formula of FAA fixative is shown in Table 1 and stored at 4°C.

[0056] Table 1 FAA fixative formulation

[0057] (2) Pre-fixation: Place dry wheat seeds in a 2 mL centrifuge tube, fill it with 4% paraformaldehyde fixative (PFA), evacuate at -80 kPa for 20-30 min, slowly deflate, replace with 4% paraformaldehyde fixative (PFA), and fix at 4 °C for 10-12 h to soften the seeds.

[0058] (3) Fixation: Use tweezers to take out 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 three thin slices in a 2 mL centrifuge tube and add 2 mL of 4% paraformaldehyde fixative (PFA). Open the lid of the centrifuge tube, evacuate at -80 kPa for 4-6 h, slowly release the air, replace with new 4% paraformaldehyde (PFA) fixative, and fix at 4 ℃ for 12 h.

[0059] (4) Secondary fixation: Pour out the 4% paraformaldehyde (PFA) fixative in the centrifuge tube and replace it with 2 mL FAA fixative. Place the centrifuge tube on ice, open the lid of the centrifuge tube, cover the centrifuge tube with perforated tin foil, evacuate at -80 kPa for 20-30 min, slowly deflate, replace with new FAA fixative, and fix at 4 ℃ for 12 h.

[0060] (5) Rinse: Pour out the FAA fixative in the centrifuge tube, add 2 mL of 0.1 M PB buffer, cover the centrifuge tube tightly, and place it on a horizontal shaker to allow the buffer to vibrate and wash the sample. Wash three times, each time for 10 min.

[0061] (6) Dehydration: Pour out the PB buffer and add 2 mL of 20%, 40%, 60%, 70%, 80%, and 90% gradient ethanol to the centrifuge tubes, respectively. Close the tube caps and place them on a horizontal shaker to shake the ethanol solution to dehydrate the samples for 50 min each time. Then use 100% ethanol for dehydration three times, each time for 1 h.

[0062] (7) Resin penetration: Pour out the ethanol and add 2 mL of 25%, 50%, and 75% gradient LR white resin (prepared in anhydrous ethanol) into the centrifuge tubes respectively. Cover the tubes tightly and place them on a horizontal vibrator to allow the resin to fully penetrate the sample. Repeat for 4 h each time. Finally, use 100% LR white resin for penetration three times, each time for 15 h.

[0063] (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, use tweezers to place the sample on the cap of the centrifuge tube, with the cut surface facing down, add 500 μL of the resin mixed with the embedding agent to the centrifuge tube, and also add a small amount of the resin mixed with the embedding agent to the cap of the centrifuge tube, close the cap tightly, and invert the centrifuge tube so that the cut surface of the sample is in close contact with the cap of the centrifuge tube.

[0064] (8) Polymerization: Invert the centrifuge tube, fix it without shaking, and place it in a 65 ℃ oven to dry for 10-12 h to carry out polymerization.

[0065] (9) Trimming: Peel open the centrifuge tube, take out the polymerized sample and mark it, fix the sample on the sample hole of the Leica ultra-thin slicer, with the plane containing the sample facing outward. Fix the glass knife, turn on the bottom light and top light, and adjust the glass knife to be parallel to the sample section according to the reflective strip of the glass knife edge on the sample plane. Set the automatic sectioning speed to 8-10 and the section thickness to 2-4μm. Trim the embedded block and remove the excess resin until a plane is trimmed and a complete sample can be cut out.

[0066] (10) Slicing: Stick the glass knife to the water tank, set the automatic slicing speed to 1, and slice at a thickness of 1-2 μm. When a complete sample can be cut out, fill the water tank with water and continue slicing, dropping the complete slice into the water tank.

[0067] (11) Spreading the slice: Drop water on a poly-L-lysine slide, use a fine-tipped brush to pick up the intact slice from the water tank, and gently place it on the curved surface of the water drop so that the slice is completely spread out without wrinkles. Bake the slice at 40-60 °C for 5-10 min to evaporate the water.

[0068] (12) Staining observation: Use 0.1% toluidine blue solution to stain the sections on the slide for 5-10 min, rinse with running water for 1-5 min, dry the sections at 40-60 °C, and evaporate the water. Perform a preliminary microscopic examination to ensure the integrity of the sample, seal the sections with sealing glue, and use a Zeiss inverted fluorescence microscope to take pictures and save the images for further observation and analysis.

[0069] Original method (method before optimization) Semi-thin section image of wheat dry seeds (thickness 1 μm) see Figure 1 . After the optimization, the semi-thin sections (thickness 1 μm) of wheat dry seeds were prepared. Figure 2 Detailed images of different tissues of dry wheat seeds (thickness 1 μm) are shown in Figure 3 .

[0070] The specific scheme of the original method (method before optimization) is as follows: (1) Pre-fixation: Place the dry wheat seeds completely in 4% paraformaldehyde fixative for 10-12 h to soften the seeds; (2) Fixation: Cut the middle part of the softened seeds in (1) into a thin slice sample with a thickness of 2-3 mm, place it in a 4% paraformaldehyde fixative and evacuate for 20-30 min, then replace the 4% paraformaldehyde fixative and fix it at 4°C for 12 h; (3) Secondary fixation: replace the fixative in (2) with FAA, evacuate for 15 min, replace with FAA fixative and fix at 4 °C for 12 h; (4) Rinse: Rinse the fixed samples in (3) three times with 0.1 M PB buffer to remove the fixative; (5) Dehydration: Dehydrate the sample rinsed in (4) with 20%, 40%, 60%, 70%, 80%, and 90% gradient ethanol for 30 min each time, and finally dehydrate with 100% ethanol for 3 times, 40 min each time; (6) Resin infiltration: The dehydrated samples in (5) were infiltrated with 25%, 50%, and 75% gradient LR white resin prepared in anhydrous ethanol for 2 h each time, and finally infiltrated with 100% LR white resin for 3 times, each time for 12 h; (7) Embedding: Place the sample fully soaked with the resin in (6) on the cap of a 0.5 mL centrifuge tube with the cut surface facing downward for embedding.

[0071] (8) Polymerization: The capsule mold containing the sample in (7) was dried in an oven at 65 °C for 12 h to perform polymerization; (9) Block trimming: Use an ultrathin slicer with the speed set to 8-10 and the slice thickness set to 2 μm to trim the sample in (8) to remove excess resin and expose the complete sample; (10) Slicing: Use an ultra-thin slicer with the speed set to 1-2 and the slice thickness set to 1-2 μm to slice the sample after trimming in (9) so that the complete slice falls into the water tank; (11) Spreading the slices: drip water on a glass slide, take out the intact slices from (10), place them in the water drops on the glass slide, bake the slices, and evaporate the water; (12) Staining observation: Use 0.1% toluidine blue solution to stain the sections in (11) for 5 min, rinse with running water for 1 min, bake the sections to dry, evaporate the water, examine under a microscope, and seal the sections for observation.

[0072] The invention provides a method for preparing semi-thin slices of dry wheat seeds, comprising the following steps: pre-fixation, fixation, secondary fixation, rinsing, dehydration, resin infiltration, embedding, polymerization, trimming, slicing, spreading, and staining observation; the method has the advantages that the pre-fixation softens the dry seeds and does not affect the original morphology of the dry seed cells; the double fixation allows the gas in the dry seeds to be completely discharged, the cell fixation effect is better, and the slices are complete; the replacement time of gradient ethanol and gradient resin is extended to ensure that the final resin fully penetrates the cell gap, so that the slices are complete and flat; detailed parameters of the trimming and slicing processes are provided, and high-efficiency continuous slicing can be achieved; in the spreading process, the slices are fished out and placed on a glass slide using a brush, and the slices are not damaged, and the operation is simple and quick; the semi-thin slice preparation method provided by the invention aims at the characteristics that the dry wheat seeds are hard and full of starch, and gradually solves the problems of incomplete resin infiltration, broken slices, and wrinkled slices encountered in making semi-thin slices of dry seeds by conventional methods, so that complete and clear tissue structured semi-thin slices of dry wheat seeds are obtained, and cell morphology observation and quantitative analysis of different parts can be further performed, thereby providing technical support for the research on the structure of dry wheat seeds.

[0073] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing semi-thin slices of dry wheat seeds, characterized in that: The following steps are involved: (1) Pre-fixation: Place dry wheat seeds completely in 4% paraformaldehyde fixative, evacuate for 20-30 min, slowly release the air, replace with 4% paraformaldehyde fixative, and fix for 10-12 h to soften the seeds. (2) Fixation: Cut the middle part of the softened seeds in (1) into a thin slice sample with a thickness of 2-3 mm, place it in a 4% paraformaldehyde fixative and evacuate for 4-6 h, then replace the 4% paraformaldehyde fixative and fix it at 4°C for 12 h; (3) Secondary fixation: Replace the fixative in (2) with FAA, evacuate for 20-30 min, and then replace with FAA fixative and fix at 4 °C for 12 h. (4) Rinse: Rinse the fixed samples in (3) three times with 0.1 M PB buffer to remove the fixative; (5) Dehydration: Dehydrate the sample rinsed in (4) with 20%, 40%, 60%, 70%, 80%, and 90% gradient ethanol for 50 min each time, and finally dehydrate with 100% ethanol for 3 times, 1 h each time; (6) Resin infiltration: The dehydrated samples in (5) were infiltrated with 25%, 50%, and 75% gradient LR white resin prepared in anhydrous ethanol for 4 h each time, and finally infiltrated with 100% LR white resin for 3 times, each time for 15 h; (7) Embedding: Place the sample fully soaked with the resin in (6) on the cap of a 0.5 mL centrifuge tube with the cut surface facing downward for embedding; (8) Polymerization: Invert the centrifuge tube containing the sample in (7) and place it in a 65 °C oven for 10-12 h to perform polymerization; (9) Block trimming: Use an ultrathin slicer with the speed set to 8-10 and the slice thickness set to 2-4 μm to trim the sample in (8) to remove excess resin and expose the complete sample; (10) Slicing: Use an ultra-thin slicer with the speed set to 1 and the slice thickness set to 1-2 μm to slice the sample after trimming in (9) so that the complete slice falls into the water tank; (11) Spreading the slices: drip water on a glass slide, take out the intact slices from (10), place them in the water drops on the glass slide, bake the slices, and evaporate the water; (12) Staining observation: Use 0.1% toluidine blue solution to stain the sections in (11) for 5-10 min, rinse with running water for 1-5 min, bake the sections to dry, evaporate the water, examine under a microscope, and seal the sections for observation.

2. The method according to claim 1, characterized in that In step (2), the preparation method of 4% paraformaldehyde fixative includes: dissolving 4 g paraformaldehyde in 90 mL 0.1 M PB at pH 7.6-7.8, adding glutaraldehyde, and mixing evenly, wherein 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 the FAA fixative solution includes: mixing 47.5 mL of anhydrous ethanol, 10 mL of 37% formaldehyde, 5 mL of glacial acetic acid and 37.5 mL of UP water.

4. The method according to claim 1, characterized in that The preparation method of 0.1 M PB in step (4) includes: weighing 3.5814 g Na2HPO4·12H2O, dissolving it in 100 mL UP water, and obtaining a Na2HPO4·12H2O solution; weighing 1.5601 g NaH2PO4·2H2O, dissolving it in 100 mL UP water, and obtaining a 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 according to 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 according to a volume ratio.

7. The method according to claim 1, characterized in that In step (11), the slices are baked at 40-60°C for 5-10 min to evaporate the water.

8. The method according to claim 1, characterized in that: In step (11), a poly-L-lysine slide was used.

9. The method according to claim 1, characterized in that: After dyeing and rinsing in step (12), the slices are dried at 40-60°C to remove moisture.

10. The method according to any one of claims 1 to 9, characterized in that: In step (11), use a fine-tipped brush to scoop up the slices.

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