A method for dissecting the structure of mature cereal grains

Abstract

The present invention discloses a method for dissecting the structure of mature cereal grains, belonging to the technical field of cereal grain dissection. The method of the present invention includes steps such as soaking, fixing, rinsing, dehydration, embedding, slicing, spreading, staining, and observation. The quick and simple cryosection method is adopted, with simple steps, safe reagents, little damage to the grain structure, complete morphology, and high component differentiation degree. It is suitable for observing the structure of mature cereal grains, and can also provide a reference for subsequent research on the impact of cereal grain processing on nutritional quality and digestive characteristics. The present invention applies the cryosection technology to the slicing of mature cereal grains, and establishes a quick slicing method applicable to mature grains of various cereals and a simple method for structure observation. The steps of the present invention are simple, and the solvents used are safe and harmless, which can provide a reference for the determination of the nutritional quality of cereal processing and provide new ideas for further studying the role of cereal grain structure in the digestion process of nutrients.

Description

Technical Field

[0001] The present invention relates to a method for dissecting the structure of mature cereal grains, belonging to the technical field of cereal grain dissection. Background Art

[0002] Whole grains are considered to be closely related to low blood sugar response and reduced risks of obesity and type II diabetes. Recent studies have recognized that the cell walls enclosing macronutrients (starch, protein, and lipids) can not only effectively inhibit the gelatinization of starch but also hinder the hydrolysis of starch. The composition, thickness, porosity, and compactness of cell arrangement in the cell wall directly affect the transfer of water and heat during the heat treatment process and the enzyme penetration during the digestion process. Therefore, observing the structure of cereal grains at the cell biology level is of great significance for analyzing the gelatinization and digestion behaviors of endosperm starch in whole cereal grains.

[0003] To observe the structure of cereal grains, effective slicing of cereal grains is required first. Currently, the main method for studying the structure of plant seeds is the paraffin section method, which is applicable to early-stage grains with high water content and soft texture. However, mature cereal grains have a high content of endosperm starch and are hard. Using the paraffin section method is likely to cause section fragmentation and it is difficult to obtain a complete cross-section of cereal grains. At the same time, this method also has disadvantages such as time-consuming, cumbersome steps, the need to use toxic reagents such as xylene, and the high-temperature melting of paraffin is likely to damage the tissue structure of cereal grains. Cryosectioning is a method of slicing samples after reaching a certain hardness under low-temperature conditions. Compared with the traditional paraffin section method, this method does not require operations such as clearing and impregnation with wax, and can also avoid the damage to the grain structure and the gelatinization of endosperm starch caused by high-temperature melting of paraffin, and the operation is simpler, faster, and more efficient. However, due to the high water content in plant tissues and organs, large hardness after freezing, and easy section fragmentation, cryosectioning is currently mainly applied to animal tissue sections and sections of soft-textured plant organs such as roots, stems, and leaves, and there is little research on its application in plant seed sections. Patent CN103776657A discloses a method for making cryosections of coconut leaves. Liu Yu et al. also used cryosectioning to simply and quickly obtain sections of Cistanche deserticola and Cistanche tubulosa seeds. However, compared with leaves and Cistanche deserticola seeds, mature cereal grains have low water content, high starch content, large cell wall thickness, firm structure, and dense arrangement of endosperm cells, and are hard in texture. Therefore, it is difficult for fixatives and embedding agents to penetrate, and it is more difficult to obtain complete sections.

[0004] In addition, observing the structure of cereal grains requires corresponding staining of the sections to localize cells. Common staining methods include iodine staining or fluorescence staining. The iodine staining method can only stain starch and cannot well characterize other components inside the cells. Patent CN114324323 discloses a method for observing the section structure of white sorghum seeds by staining with a staining solution. The method uses iodine solution to stain the sections of sorghum seeds. Although the staining and observation method is simple, it does not distinguish different components and fails to highlight the cell wall structure. Fluorescence staining is divided into single staining and double staining. Single staining can only observe the distribution of a certain component, and double staining can achieve the observation of the distribution of multiple components and the interaction between components. However, double staining has higher requirements for the concentration of the staining agent. To avoid problems such as similar excitation light wavelengths or the inability of the staining agent probe to specifically recognize, the concentrations of two or more staining agents need to meet a certain proportional range. Otherwise, misstaining and missed staining are likely to occur, resulting in image distortion. Currently, there are few studies on using a compound fluorescent staining solution to simultaneously characterize starch and cell wall structures inside mature cereal grains. Therefore, a staining method using a compound fluorescent staining solution will be of great significance for effectively analyzing the structure of mature cereal grains. Summary of the Invention

[0005] The present invention provides a method for dissecting the structure of mature cereal grains, which not only simplifies the steps of slicing cereal grains, reduces the damage to the original structure of the grains during the embedding and slicing processes, effectively improves the quality and efficiency of slicing, but also achieves a high degree of differentiation of different components and cell wall structures of mature cereal grains through a compound fluorescence staining method. The present invention can not only be used for dissecting the structure of mature cereal grains, but also be applied to monitoring the changes in the grain structure during the cooking process of cereals, and has guiding significance for studying the loss and release of nutrients during the cereal processing process.

[0006] The first object of the present invention is to provide a pretreatment method for mature cereal grains, the method comprising soaking, fixing, rinsing, dehydrating, embedding, slicing, spreading and staining; the soaking is to make the moisture content of the cereal grains reach 20-25%; the slicing is carried out in an environment of -20°C; the staining is a compound fluorescence staining of fluorescein isothiocyanate and fluorescent brightener 28.

[0007] The present invention provides a pretreatment method for cereal grains, the method comprising the following steps:

[0008] (1) Soaking: Soak the mature cereal grains in water to make the moisture content in the grains 20-25%;

[0009] (2) Fixing and rinsing: Place the soaked grains in a fixing solution for fixing for 8-16 h, and then wash the grains with a phosphate buffer solution with a pH of 6.5-7.5.

[0010] (3) Dehydration and embedding: After the rinsed grains are soaked in ethanol for dehydration, the grains are mixed with an embedding agent for embedding;

[0011] (4) Sectioning: The embedded grains are sectioned at -15°C to -30°C;

[0012] (5) Staining: The sections are stained with a mixed solution of fluorescein isothiocyanate and fluorescent brightener.

[0013] In one embodiment of the present invention, step (1) is that the soaking process is to soak mature cereal grains in deionized water at 4°C.

[0014] In one embodiment of the present invention, in step (1), the mature cereal grains include grains such as sorghum, millet, broomcorn millet, brown rice, oats, barley, wheat, and hulless barley.

[0015] In one embodiment of the present invention, the fixing solution in step (2) is prepared by mixing 50% ethanol, paraformaldehyde, and glacial acetic acid in a volume ratio of (80 - 90):(10 - 5):(10 - 5).

[0016] In one embodiment of the present invention, the embedding agent in step (3) includes but is not limited to: OCT embedding agent, ordinary glue, or medical coupling agent.

[0017] In one embodiment of the present invention, the fixing in step (2) is to soak the soaked and softened grains in a blend of paraformaldehyde, 50% ethanol, and glacial acetic acid for 12 h for fixation.

[0018] In one embodiment of the present invention, the rinsing in step (2) is to thoroughly wash the fixed cereal grains 3 times with 0.1 moL / L phosphate buffer solution (pH 6.5 - 7.5).

[0019] In one embodiment of the present invention, the pH of the phosphate buffer solution is 7.2.

[0020] In one embodiment of the present invention, the dehydration in step (3) is to soak the rinsed cereal grains in ethanol solutions with concentrations of 30%, 50%, 70%, 90%, 100%, and 100% in sequence for dehydration, and each concentration is soaked for 10 - 30 min.

[0021] In one embodiment of the present invention, each concentration is soaked for 15 min.

[0022] In one embodiment of the present invention, the embedding in step (3) is to embed the dehydrated cereal grains in a mold using OCT embedding medium, and place the mold in a -80°C refrigerator for freezing for 2 - 8 hours. The injection of the embedding medium should be slow to avoid the generation of air bubbles.

[0023] In one embodiment of the present invention, the OCT embedding medium is a water-soluble mixture of polyvinyl alcohol and polyethylene glycol.

[0024] In one embodiment of the present invention, the sectioning in step (4) is to section the cereal grains fixed on a freezing tray using a cryostat. The section thickness is 20 μm, and the temperature of the cryostat is set to -20°C.

[0025] In one embodiment of the present invention, in step (5), before staining, there is also a flattening step. The flattening is to bring the glass slide close to the section in parallel. The section adheres to the glass slide at a certain speed and unfolds on the glass slide, and the section is naturally dried at room temperature and adheres to the glass slide.

[0026] In one embodiment of the present invention, in step (5), the mixed solution is prepared by compounding fluorescein isothiocyanate solution and fluorescent brightener 28 dye solution in a volume ratio of (1 - 10):(10 - 1).

[0027] In one embodiment of the present invention, in step (5), the mixed solution is prepared by compounding fluorescein isothiocyanate solution and fluorescent brightener 28 dye solution in a volume ratio of 1:1.

[0028] In one embodiment of the present invention, the fluorescein isothiocyanate solution is obtained by dissolving fluorescein isothiocyanate in acetone to obtain a fluorescein isothiocyanate solution with a mass-volume ratio of (0.01% - 1%); the fluorescent brightener 28 dye solution is obtained by dissolving fluorescent brightener 28 dye in deionized water to obtain a fluorescent brightener 28 dye solution with a mass-volume ratio of (0.01% - 1%).

[0029] In one embodiment of the present invention, the fluorescein isothiocyanate solution is obtained by dissolving fluorescein isothiocyanate in acetone to obtain a fluorescein isothiocyanate solution with a mass-volume ratio of 0.01%; the fluorescent brightener 28 dye solution is obtained by dissolving fluorescent brightener 28 dye in deionized water to obtain a fluorescent brightener 28 dye solution with a mass-volume ratio of 0.01%.

[0030] In one embodiment of the present invention, in step (5), the whole process of staining with the staining solution is carried out in the dark. The section is infiltrated with the staining solution gradually. After staining, it is repeatedly rinsed with deionized water until the droplets flowing down along the glass slide are colorless. Then, a cover glass is covered, and observation is carried out using a laser confocal microscope.

[0031] In one embodiment of the present invention, the observation is carried out using a laser confocal microscope. When setting the excitation light wavelength to 488 nm to observe fluorescein isothiocyanate-labeled starch, the starch appears green; when setting the excitation light wavelength to 405 nm to observe β-glucan located in the cell wall labeled with fluorescent brightener 28, the cell wall appears blue; the dual-wavelength channel is simultaneously opened to observe the starch and cell wall structures.

[0032] The present invention also provides a method for dissecting mature cereal grains, which is characterized in that the above method is applied for pretreatment and then observation is carried out.

[0033] In one embodiment of the present invention, the observation is carried out under a laser confocal microscope.

[0034] In one embodiment of the present invention, the following steps are included: (1) Soaking: Selecting plump and intact cereal grains and soaking them in deionized water at 4°C, so that the moisture content of the soaked cereal grains is 20-25%, and after soaking, taking out the grains and blotting the surface moisture of the grains with absorbent paper;

[0035] (2) Fixing: Soaking the soaked mature cereal grains in a mixed solution of paraformaldehyde, absolute ethanol and glacial acetic acid for 12 h for fixation;

[0036] (3) Rinsing: Thoroughly washing the fixed cereal grains 3 times with 0.1 moL / L phosphate buffer solution (pH 6.5-7.5);

[0037] (4) Dehydration: Soaking the fixed grains in ethanol with concentrations of 30%, 50%, 70%, 90%, 100% and 100%, 10-30 min for each stage;

[0038] (5) Embedding: Placing the dehydrated grains in a mold, slowly injecting OCT embedding agent, and freezing at -80°C for 2-8 h;

[0039] (6) Sectioning: Fixing the embedded cereal grains on a low-temperature tray, setting the temperature of the cryostat to -20°C, and the section thickness to 20 μm;

[0040] (7) Spreading: Bringing the glass slide close to the section in parallel, and the section attaching to the glass slide at a certain speed and spreading on the glass slide, and naturally drying at room temperature;

[0041] (8) Staining: Staining according to the required purpose, using the method of infiltrating the section and rinsing with deionized water;

[0042] (9) Observation: Observing using a laser confocal microscope or an inverted fluorescence microscope, and setting the dual-wavelength excitation light channel according to the selected staining solution.

[0043] The present invention also provides the application of the method in the food field, such as the influence of steaming on the grain kernel structure, the prediction of the digestive characteristics of grain kernels, and the determination of the nutritional quality of grain kernels.

[0044] Beneficial effects

[0045] (1) The present invention applies the frozen section technique to the sectioning of mature grain kernels, and establishes a rapid sectioning method applicable to mature kernels of various grains and a simple method for structure observation. By soaking, the hardness of the kernels is reduced; by using a compound fixing solution, the toughness of the kernels is enhanced and the loss of kernel structure is reduced; by gradient dehydration with gradient ethanol, the dehydration method is simplified; by embedding the kernels with OCT embedding agent, the toughness of the sections is effectively retained and it is beneficial for the dyeing agent to diffuse into the sections; by using the frozen section method, high-quality sections with complete structure and high differentiation between components are obtained; by compounding the staining solution, different components inside the kernels are successfully labeled; by setting up a dual-wavelength channel of a laser confocal microscope, the observation and effective differentiation of the kernel structure are realized.

[0046] (2) Compared with paraffin sectioning, the operation of the present invention is simple, time-consuming is short, steps are few, and steps such as clearing and impregnating with wax are not required. At the same time, no toxic solvents such as xylene are used, which is less harmful to the human body and meets the requirements of green chemistry. In addition, the effect of melting wax at a relatively high temperature on the internal components and structure of the kernels is avoided, and the deformation of starch grains caused by starch gelatinization and the swelling of cell walls are reduced.

[0047] (3) The present invention provides a new rapid analysis method for mature grain kernels, with simple steps and safe and harmless solvents used, which can provide a reference for the determination of the nutritional quality of grain processing and provide new ideas for further studying the role of grain kernel structure in the digestion process of nutrients. Description of the drawings

[0048] Figure 1 : Structure diagram of oat kernels with 21% moisture content under a laser confocal microscope.

[0049] Figure 2 : Structure diagram of hulless barley kernels with 23% moisture content under a laser confocal microscope.

[0050] Figure 3 : Structure diagram of cooked oat kernels under a laser confocal microscope.

[0051] Figure 4 : Structure diagram of cooked hulless barley kernels under a laser confocal microscope.

[0052] Figure 5 : Structure diagram of unsoaked oat kernels under a laser confocal microscope.

[0053] Figure 6: Structure diagram of oat grains with 15% moisture content under a laser confocal microscope.

[0054] Figure 7 : Structure diagram of oat grains with 25% moisture content under a laser confocal microscope.

[0055] Figure 8 : Structure diagram of oat grains with 30% moisture content under a laser confocal microscope.

[0056] Figure 9 : Structure diagram of hulless barley grains fixed for 4 hours under a laser confocal microscope.

[0057] Figure 10 : Structure diagram of hulless barley grains fixed for 8 hours under a laser confocal microscope.

[0058] Figure 11 : Structure diagram of hulless barley grains fixed for 16 hours under a laser confocal microscope.

[0059] Figure 12 : Structure diagram of hulless barley grains fixed for 20 hours under a laser confocal microscope.

[0060] Figure 13 : Structure diagram of oat grains with fluorescein isothiocyanate: fluorescent brightener 28 = 3:1 under a laser confocal microscope.

[0061] Figure 14 : Structure diagram of hulless barley grains with fluorescein isothiocyanate: fluorescent brightener 28 = 1:3 under a laser confocal microscope.

[0062] Figure 15 : Section structure diagram of oat grains under a laser confocal microscope in the control example.

[0063] Figure 16 : Section structure diagram of hulless barley grains under a laser confocal microscope in the control example. Detailed implementation mode

[0064] The present invention will be described in detail below with reference to the accompanying drawings.

[0065] The oat grains and hulless barley grains involved in the following examples were purchased from Yesanpo Food Co., Ltd.

[0066] The OCT embedding agent involved in the following examples was purchased from Haimen Rongxing Biotechnology Co., Ltd., ethanol, paraformaldehyde and glacial acetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd., fluorescein isothiocyanate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and fluorescent brightener 28 was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0067] Example 1: Pretreatment method for cereal grains

[0068] The specific implementation method is as follows:

[0069] (1) Soaking: Soak the raw oat grains in deionized water for 30 minutes at 4°C, and the water content in the grains is detected to be 21%;

[0070] Soak the raw hulless barley grains in deionized water for 1 hour at 4°C, and the water content in the grains is detected to be 23%;

[0071] (2) Fixation:

[0072] 1) Prepare the fixing solution

[0073] After mixing 50% ethanol, paraformaldehyde and glacial acetic acid in a volume ratio of 89:5:6, the fixing solution is prepared.

[0074] 2) Soak the oat grains and hulless barley grains obtained in step (1) in the fixing solution respectively for 12 hours;

[0075] (3) Rinsing: Thoroughly wash the fixed cereal grains 3 times with 0.1 moL / L phosphate buffer solution (pH 7.2);

[0076] (4) Dehydration: Soak the rinsed grains in ethanol with concentrations of 30%, 50%, 70%, 90%, and 100% in sequence for dehydration treatment, and the treatment time for each concentration level is 15 minutes;

[0077] (5) Embedding: Place the dehydrated grains in a mold, slowly inject OCT embedding medium, and freeze at -80°C for 2 hours;

[0078] (6) Sectioning: Fix the embedded cereal grains on a low-temperature tray, set the temperature of the cryostat to -20°C, and the section thickness to 20 μm;

[0079] (7) Spreading: Place the glass slide close to the section in parallel, and the section adheres to the glass slide at a certain speed and unfolds on the glass slide, and air-dries naturally at room temperature;

[0080] (8) Staining:

[0081] 1) Prepare the staining solution

[0082] Dissolve fluorescein isothiocyanate with acetone to obtain a fluorescein isothiocyanate solution with a mass-volume ratio of 0.01%;

[0083] Dissolve fluorescent brightener 28 dye with deionized water to obtain a fluorescent brightener 28 dye solution with a mass-volume ratio of 0.01%;

[0084] A staining solution was prepared by mixing fluorescein isothiocyanate solution and fluorescent brightener 28 dye solution in a volume ratio of 1:1.

[0085] 2) Using the staining solution obtained in step 1), gradually infiltrate the sections, stain for 5 min in the dark, and rinse with deionized water; during the staining with the dye solution, keep it in the dark throughout the process. Infiltrate the sections with the dye solution gradually, and after staining, rinse repeatedly with deionized water until the droplets flowing down the glass slide are colorless, then cover with a cover slip.

[0086] (9) Observation: Observe using a laser confocal microscope, and simultaneously turn on the channels with excitation light wavelengths of 488 nm and 405 nm. The results are as Figure 1 and Figure 2 shown. Figure 1 and Figure 2 are images of sections of mature oat and hulless barley grains observed under a laser confocal microscope at excitation light wavelengths of 488 nm and 405 nm, respectively. The pictures present the distribution of different components and structures under the labeling of the fluorescent staining agent.

[0087] The results show that the sections of oat and hulless barley grains prepared by the method of the present invention are intact, the cell structure is obvious, and different components can be effectively distinguished after fluorescent staining.

[0088] In the oat image, the outermost aleurone layer cells and the inner endosperm cells can be clearly observed. Starch granules are embedded in the cytoplasmic matrix, and the cytoplasmic matrix is surrounded by the cell wall structure;

[0089] while in the hulless barley image, endosperm cells with a more compact arrangement than that of oats can be observed.

[0090] Example 2: Pretreatment method for cooked cereal grains

[0091] The specific implementation method is the same as that in Example 1, except that cooked cereal grains (oat grains, hulless barley grains) are used instead of raw oat grains and hulless barley grains in Example 1. Without soaking, directly immerse them in the fixing solution prepared in step (2), and according to the method of Example 1, prepare stained sections. Observe using a laser confocal microscope, and the results are as Figure 3 and 4 shown. Figure 3 and Figure 4 are images of sections of cooked oat and hulless barley grains observed at excitation light wavelengths of 488 nm and 405 nm, respectively. The pictures present the distribution of different components and structures under the labeling of the fluorescent staining agent.

[0092] The results show that the sections of cooked oat and hulless barley grains prepared by the method of the present invention are intact, the cell structure is obvious, and different components can be effectively distinguished after fluorescent staining.

[0093] Compared with the slices of raw oats and hulless barley grains, the starch granules in the cooked cereal grains swell, the cell arrangement is no longer tight, and the cell walls are deformed.

[0094] Example 3: Pretreatment method of cereal grains

[0095] 1. The specific implementation method is the same as that of Example 1, except that the soaking step is omitted, and raw oat grains without soaking are directly immersed in the fixing solution prepared in step (2). According to the method of Example 1, stained oat grain slices are prepared. Among them, after detection, the moisture content in the raw oat grains is: 12%;

[0096] After observation with a laser confocal microscope, the results show ( Figure 5 ) that the oat grain slices are highly fragmented and it is difficult to observe the cell wall structure.

[0097] 2. The specific implementation method is the same as that of Example 1, except that the soaking time of the oat grains in step (1) is adjusted to: 20 min to make the moisture content in the oat grains: 15%; according to the method of Example 1, stained oat grain slices are prepared;

[0098] After observation with a laser confocal microscope, the results show ( Figure 6 ) that the oat grain slices are incomplete, there are multiple evenly distributed cavities inside the slices, and the cell wall structure is also not obvious.

[0099] 3. The specific implementation method is the same as that of Example 1, except that the soaking time of the oat grains in step (1) is adjusted to: 35 min to make the moisture content in the oat grains: 25%; according to the method of Example 1, stained oat grain slices are prepared;

[0100] After observation with a laser confocal microscope, the results show ( Figure 7 ) that the oat grain slices are relatively complete, there are fewer pores, and the cell walls are clear and tightly arranged.

[0101] 4. The specific implementation method is the same as that of Example 1, except that the soaking time of the oat grains in step (1) is adjusted to: 45 min to make the moisture content in the oat grains: 30%; according to the method of Example 1, stained oat grain slices are prepared;

[0102] After observation with a laser confocal microscope, the results show ( Figure 8 ) that the oat grain slices are relatively complete, but the surface is rough and not smooth, the cell walls of the aleurone layer are complete, and the cell walls of the endosperm are not obvious.

[0103] It can be seen that in the method of the present invention, the soaking time of oat grains should be: 20 to 45 minutes, so that the moisture content in the oat grains is: 20 to 25%.

[0104] 5. The specific implementation method is the same as that of Example 1, except that the fixing time of the hulless barley grains in step (2) is adjusted to: 4 hours; according to the method of Example 1, the stained hulless barley grain sections are prepared;

[0105] After observing with a laser confocal microscope, the results show ( Figure 9 ): The hulless barley grain sections are severely damaged, with many holes and gaps, the starch granules are dispersed, and the cell walls are significantly damaged.

[0106] 6. The specific implementation method is the same as that of Example 1, except that the fixing time of the hulless barley grains in step (2) is adjusted to: 8 hours; according to the method of Example 1, the stained hulless barley grain sections are prepared;

[0107] After observing with a laser confocal microscope, the results show ( Figure 10 ): The integrity of the hulless barley grain sections is high, the cell wall structure is clear, and the starch granules are tightly packed in the cytoplasmic matrix.

[0108] 7. The specific implementation method is the same as that of Example 1, except that the fixing time of the hulless barley grains in step (2) is adjusted to: 16 hours; according to the method of Example 1, the stained hulless barley grain sections are prepared;

[0109] After observing with a laser confocal microscope, the results show ( Figure 11 ): The integrity of the hulless barley grain sections is high, and the cell wall structure is clearly visible.

[0110] 8. The specific implementation method is the same as that of Example 1, except that the fixing time of the hulless barley grains in step (2) is adjusted to: 20 hours; according to the method of Example 1, the stained hulless barley grain sections are prepared;

[0111] After observing with a laser confocal microscope, the results show ( Figure 12 ): The integrity of the hulless barley grain sections is high, but the structure of some cell walls is not obvious.

[0112] It can be seen that in the method of the present invention, the fixing time of the hulless barley grains should be: 8 to 16 hours.

[0113] 9. The specific implementation method is the same as that of Example 1, except that the ratio of the staining solution for the oat sections in step (8) is adjusted to: fluorescein isothiocyanate solution: fluorescent brightener 28 dye solution = 3:1; according to the method of Example 1, the stained oat grain sections are prepared;

[0114] After observation with a laser confocal microscope, the results showed ( Figure 13 ): The blue color in the oat grain sections was not obvious, the green color was significant, and some cell wall structures that should have been blue were greenish under the microscope.

[0115] 10. The specific implementation method is the same as that in Example 1, except that the ratio of the staining solution for the oat sections in step (8) is adjusted to: fluorescein isothiocyanate solution: fluorescent brightener 28 dye solution = 1:3; according to the method in Example 1, the stained oat grain sections are prepared.

[0116] After observation with a laser confocal microscope, the results showed ( Figure 14 ): The oat grain sections were overstained with blue, the blue color of the cell walls was too significant, and the green starch color development was dim.

[0117] Control Example

[0118] The method for making sections of oat and hulless barley grains uses the traditional paraffin section method, which includes steps such as fixation, dehydration, wax infiltration, embedding, sectioning, and staining. Among them, the wax melting temperature used during wax infiltration is carried out at 60 - 70 °C (for the specific method, refer to Ogawa Y, Sugiyama J, Kuensting H, et al., 2001. Advanced Technique for Three-Dimensional Visualization of Compound Distributions in a Rice Kernel[J / OL]. Journal of Agricultural and Food Chemistry, 49(2):736–740[2022–08–30].).

[0119] Figure 15 and 16 The figures of the obtained oat and hulless barley grain sections show that: both the hulless barley and oat sections are incomplete, with holes of uneven sizes. The oats have lost most of their cell wall structures, while the hulless barley cell wall structures are loose and not compact, and the starch granules are all swollen and deformed, losing their original shapes.

[0120] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A pretreatment method for cereal grains, characterized in that, The method includes the following steps: (1) Soaking: Soak the grains in water to make the moisture content in the grains 20 - 25%; (2) Fixing and rinsing: After soaking the grains, place them in a fixing solution for 8 - 16 h, and then wash the grains with a phosphate buffer solution with a pH of 6.5 - 7.5; The fixing solution is prepared by mixing 50% ethanol, paraformaldehyde, and glacial acetic acid in a volume ratio of (80 - 90):(10 - 5):(10 - 5); (3) Dehydration and embedding: After soaking the rinsed grains in ethanol for dehydration, mix the grains with an embedding agent for embedding; The embedding agent includes, but is not limited to: OCT embedding agent, ordinary glue, or medical coupling agent; (4) Sectioning: Section the embedded grains at - 15°C to - 30°C; (5) Staining: Stain the sections with a mixed solution of fluorescein isothiocyanate and fluorescent brightener; 2. The method according to claim 1, wherein The cereal grains include sorghum, millet, broomcorn millet, brown rice, oats, barley, wheat, and highland barley; 3. The method according to claim 1, wherein In step (4), sectioning is performed by using a cryostat to section the cereal grains fixed on a freezing tray, and the temperature of the cryostat is set at - 20°C; 4. The method according to claim 1, wherein In step (5), the mixed solution is prepared by compounding a fluorescein isothiocyanate solution and a fluorescent brightener 28 dye solution in a volume ratio of (1 - 10):(10 - 1); The fluorescein isothiocyanate solution is a fluorescein isothiocyanate solution with a mass - volume ratio of (0.01% - 1%) obtained by dissolving fluorescein isothiocyanate in acetone; The fluorescent brightener 28 dye solution is a solution with a mass - volume ratio of (0.01% - 1%) obtained by dissolving fluorescent brightener 28 dye in deionized water; 5. A method for dissecting mature cereal grains, characterized in that, Apply the method described in any one of claims 1 - 4 for pretreatment and then observe; 6. The method according to claim 5, wherein The observation is carried out under a laser confocal microscope; 7. The method according to claim 6, characterized in that, Including the following steps: (1) Soaking: Soak the cereal grains in deionized water at 4°C, so that the moisture content of the soaked cereal grains is 20 - 25%, and after soaking, take out the grains and blot the surface moisture of the grains with absorbent paper; (2) Fixing: Soak the soaked mature cereal grains in a mixed solution of paraformaldehyde, 50% ethanol, and glacial acetic acid for 12 h; (3) Rinsing: Thoroughly wash the fixed cereal grains 3 times with a 0.1 moL / L phosphate buffer solution with a pH of 6.5 - 7.5; (4) Dehydration: Soak the fixed grains in ethanol with concentrations of 30%, 50%, 70%, 90%, 100%, and 100% for 10 - 30 min for each level; (5) Embedding: Place the dehydrated grains in a mold, slowly inject OCT embedding agent, and freeze at - 80°C for 2 - 8 h; (6) Sectioning: Fix the embedded cereal grains on a low - temperature tray, set the temperature of the cryostat at - 20°C, and the section thickness is 20 μm; (7) Spreading: Bring the glass slide close to the section in parallel, and the section adheres to the glass slide at a certain speed and unfolds on the glass slide, and air - dry at room temperature; (8) Staining: Stain the sections by the method of infiltrating the sections and rinsing with deionized water; (9) Observation: Observation was carried out using a laser confocal microscope, and a dual-wavelength excitation light channel was set up.

8. Use of the method according to any one of claims 1 to 7 in the pretreatment of cereal grains in the food field.

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