Wheat root paraffin section and preparation method thereof

Abstract

The invention belongs to the technical field of preparation of tissue sections, and relates to a paraffin section of wheat roots and a preparation method thereof, the preparation method comprises the steps of fixing, dehydrating, transparentizing, waxing, embedding, slicing, spreading, baking, dewaxing to water, dyeing and sealing in sequence, a pre-dyeing step is carried out between the fixing step and the dehydrating step, and the pre-dyeing step is carried out between the pre-dyeing step and the dehydrating step. In the pre-dyeing step, a sarranine solution is adopted to treat the fixed wheat roots, and the treatment time is 12-20 h; treatment is carried out under the conditions of positive and negative pressure alternate circulation and temperature increase in the steps of dehydration, transparentizing and waxing; in the dyeing step, a 5% sodium sulfite solution is added for treatment, the treatment time is 0.5-1 h, and a 0.5% hydrochloric acid ethanol solution is added for treatment for 5-10 s. According to the method, the tissue structure in the obtained slice is clear and complete in combination with the optimized process, the dyeing contrast is distinct, the tissue structure of the wheat slice in a survival state is restored to a greater extent, and accurate obtaining of wheat research experiment results is facilitated.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tissue section preparation, and particularly relates to a paraffin section of wheat root and a preparation method thereof. BACKGROUND

[0002] Wheat is one of the most important food crops in the world, accounting for about one-third of the total world food production. It occupies a very key position in the human diet structure and is the main raw material for making bread, noodles, steamed buns and many other staple foods, providing basic energy and nutrients for billions of people around the world. As a global key food crop, the importance of wheat research and improvement is self-evident. From a global perspective, in-depth exploration of the characteristics of wheat and improvement of its yield and quality can effectively meet the growing demand for food by the population, greatly alleviate the pressure on food supply, and thus effectively guarantee global food security. In the field of agricultural production, research on wheat can help optimize planting techniques, cultivate excellent varieties, improve the utilization efficiency of agricultural resources, reduce the adverse effects of diseases, pests and natural disasters on wheat yield, and thus promote the stable development and benefit improvement of the entire agricultural production system.

[0003] Wheat root paraffin section preparation is of great significance in wheat research and is a commonly used experimental technique for wheat root research and observation. In terms of structural observation, it can clearly show the structure of the epidermis, cortex and vascular column of wheat root, such as the arrangement of cortex parenchyma cells and the situation of aerenchyma, the morphological distribution of xylem and phloem cells, which has a profound impact on its absorption and transport function. In terms of growth and development research, it can compare sections at different growth stages to perceive changes in cell number, elongation and tissue proportion, helping to explore the adaptive mechanisms of roots under different soil and fertilization conditions. In pathological research, it is indispensable to analyze wheat root rot and other root diseases, determine the infection site of pathogenic bacteria and the resulting cell lesions, and thus provide support for disease control.

[0004] However, the conventional plant tissue paraffin sectioning technique can prepare paraffin sections of most plant tissues, but for plant tissues with high water content such as wheat roots, especially young roots, the paraffin sections prepared using conventional plant sectioning techniques have problems such as long sectioning period, tissue loss, deformation and fragmentation of tissue structure, and poor staining effect. It is still relatively difficult to prepare high-quality paraffin sections of wheat roots. SUMMARY

[0005] The purpose of the present application is to provide a paraffin section of wheat root and a preparation method thereof, which solves the above-mentioned defects of the conventional plant tissue paraffin sectioning technique in preparing paraffin sections of wheat roots.

[0006] The technical scheme adopted by the present application is as follows: A method for preparing a paraffin section of a wheat root, comprising sequentially performing the steps of fixing, dehydrating, transparentizing, waxing, embedding, sectioning, spreading, baking, de-waxing to water, staining and mounting, wherein a pre-staining step is performed between the fixing step and the dehydrating step, the pre-staining step refers to treating the fixed wheat root with a safranin solution, the treatment time is 12-20 hours, the dehydrating, transparentizing and waxing steps are performed under the conditions of positive and negative pressure and increased temperature, the section after de-waxing is treated with a sodium sulfite solution for 0.5-1 hour during the staining and mounting step, and the section after safranin staining is treated with a 0.5% hydrochloric acid ethanol solution for 5-10 seconds.

[0007] The preparation of a paraffin section of a wheat root using conventional plant tissue paraffin sectioning techniques results in problems such as tissue loss, deformation and fragmentation of the tissue structure, and poor staining effect, mainly because the wheat root, especially the young wheat root, is small in size, fragile in texture and easy to break, and is mostly milky white, so that the tissue is not only easy to lose during the sectioning process, but also is severely damaged during the operation, and the prepared section is deformed and fragmented in structure. Moreover, the young root has a very high water content, the cells are swollen, and the intercellular space is relatively small. At the same time, although the primary wall of the young cell is mainly composed of cellulose and pectin, its structure is relatively dense, and in the conventional preparation method, the paraffin is difficult to completely penetrate into the tissue. When sectioning and staining are performed, the dye molecules are also difficult to uniformly and deeply penetrate into all cells. This can result in uneven staining, deep staining in some areas, light or incomplete staining in some areas.

[0008] The present application uses a safranin solution to pre-stain the wheat root for a long time before dehydration, and the safranin solution prepared with a low-concentration ethanol not only allows the wheat root to be slowly and stably colored, but also makes a transition for the next step of gradient alcohol dehydration. The wheat tissue pre-stained with the safranin solution is still deep red after the dehydration, transparentization and waxing processes, and is extremely easy to observe, which forms a sharp contrast with the paraffin environment, solves the problem of easy tissue loss, and makes it easier to control the operation of the wheat root tissue in the subsequent steps, greatly reducing the operation damage. In actual repeated experiments, it is found that the pre-staining before dehydration, and the subsequent dehydration, transparentization, waxing, sectioning, de-waxing to water processes, the pre-staining color is completely faded by organic reagents, especially when the pre-staining time is relatively short, the pre-staining color is basically faded after the dehydration step is completed. Therefore, the present application optimizes the pre-staining, uses 50% ethanol as a solvent to prepare the pre-staining safranin solution, and lengthens the pre-staining time, so that the sample is still deep red after dehydration, which is extremely easy to observe, forms a sharp contrast with the paraffin environment, solves the problem of easy tissue loss, makes it easier to control the operation of the wheat root tissue in the subsequent steps, greatly reduces the operation damage, preserves the integrity and beauty of the wheat root section, restores the tissue structure of the wheat section to a greater extent in the survival state, and is beneficial to the accuracy of the experimental results.

[0009] As preferred, the concentration of the safranin solution is 0.1-0.3 %.

[0010] As preferred, the safranin solution is prepared with 50 % ethanol as solvent, which provides an ethanol environment before the next step of gradient ethanol dehydration, reduces the damage of dehydration to root tissues, ensures the integrity of root tissues, and also improves the dehydration efficiency.

[0011] As preferred, the dehydration and transparency steps are performed under the conditions of pressure (about -35-35 Kpa alternating circulation) and temperature (37-45 ℃).

[0012] The -35-35 Kpa alternating circulation mentioned above means that the instrument is pressurized to about 35 Kpa and negative pressure to about -35 Kpa, and then the pressure is continuously changed from 0 to about 35 Kpa, from 35 Kpa to 0, from 0 to about -35 Kpa, and from -35 Kpa to 0.

[0013] As preferred, the wax immersion step is performed under the conditions of pressure (about -35-35 Kpa alternating circulation) and temperature (60-65 ℃).

[0014] As preferred, after the completion of the fixation step, the fixed wheat roots are washed with water for about 2 h.

[0015] As preferred, in the staining step, 5 % sodium sulfite solution is used for treatment, which makes the safranin color more stable and not easily covered by the subsequent fast green staining.

[0016] As preferred, in the staining step, 0.5 % hydrochloric acid ethanol is used for treatment, which can remove excess safranin dye and make the fast green dye color more quickly, so that the tissue staining background is clean and the contrast is clear.

[0017] As preferred, the preparation method specifically comprises the following steps: (1) Sampling: taking fresh wheat rootlets; (2) Fixing: placing the wheat rootlets in FAA fixing solution for fixation; (3) Washing: washing the fixed wheat rootlets with tap water for about 2 h; (4) Pre-staining: pre-staining the washed wheat rootlets with 0.1 % safranin solution (prepared with 50 % ethanol as solvent) at room temperature overnight (12-20 h).

[0018] (5) Dehydration: the pre-stained sample is subjected to dehydration treatment under the conditions of pressure (about -35 ~ 35 Kpa alternating cycle) and temperature (37 ~ 45℃), in turn through 60%, 70%, 80%, 90%, 95%(I), 95%(II) gradient ethanol and anhydrous ethanol(I), anhydrous ethanol(II); (6) Transparency: the dehydrated sample is subjected to transparency treatment under the conditions of pressure (about -35 ~ 35 Kpa alternating cycle) and temperature (37 ~ 45℃), in turn through xylene(I), xylene(II); (7) Wax immersion: the transparent sample is subjected to wax immersion treatment under the conditions of pressure (about -35 ~ 35 Kpa alternating cycle) and temperature (60 ~ 65℃), in turn immersed in paraffin(I), paraffin(II), paraffin(III); (8) Embedding, slicing, spreading, baking, dewaxing to water, staining and sealing, in the staining and sealing step, 5% sodium sulfite solution is used for deacidification treatment and 0.5% hydrochloric acid ethanol is used for differentiation treatment.

[0019] The application also provides a paraffin section of corn kernels, which is obtained by the above preparation method.

[0020] By implementing the above technical solution, compared with the prior art, the application has the following beneficial effects: 1. In the present application, the wheat root is pre-stained with safranin solution between the fixing and dehydration steps. The pre-staining with low concentration for a long time makes the safranin solution have bright and stable color, which is not easy to fade after gradient dehydration. This not only forms a sharp contrast with the paraffin environment, but also makes the wheat root easy to observe, and has a positive effect on the integrity of the wheat root tissue, greatly reducing the operation damage.

[0021] 2. On the basis of the pre-staining step, the application further adopts the method of increasing pressure and negative pressure cycle and increasing temperature in the dehydration, transparency and wax immersion steps, and removes the previous transition reagent mixed by equal volume. Due to the conditions of pressure and temperature, the replacement of the reagent in the tissue is more stable and fast, which not only shortens the sample processing time, but also ensures the dehydration effect, thereby improving the section efficiency.

[0022] 3. In the staining and sealing step of the present application, 5% sodium sulfite solution is used for deacidification treatment, which makes the safranin color more stable and not easy to be covered by the subsequent fast green staining. The differentiation treatment with 0.5% ethanol can not only remove the excess safranin dye, but also make the fast green dye color more quickly, so as to make the tissue staining background clean and the contrast clear. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Wheat root tissue before pre-staining; Figure 2 a wheat rootlet paraffin section prepared by the preparation method of the present application Figure 3 a wheat rootlet paraffin section prepared by the preparation method of the present application Figure 4 a wheat rootlet paraffin section prepared by the conventional preparation method; Figure 5 a wheat rootlet paraffin section prepared without increasing the pressure and temperature conditions; Figure 6 and Figure 7 a wheat rootlet paraffin section prepared without pre-staining; Figure 8 and Figure 9 a wheat rootlet paraffin section prepared without using 5% sodium sulfite solution and 0.5% hydrochloric acid ethanol solution treatment; Figure 10 a corn kernel paraffin section prepared by the preparation method of the present application. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be described clearly and completely by means of examples. Obviously, the described examples are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0025] In the following examples, the raw materials used are conventional materials in the art unless otherwise specified. The reagent preparation method is as follows: 1. FAA fixing solution is prepared by the following reagents in the volume ratio: 40% formaldehyde solution: glacial acetic acid: 50% ethanol = 5:5:90.

[0026] 2. 1% safranin staining solution (95% ethanol preparation): 1 g of safranin is dissolved in 100 ml of 95% ethanol, filtered and used.

[0027] 3. 0.1% safranin pre-staining solution (50% ethanol preparation): 1 g of safranin is dissolved in 1000 ml of 50% ethanol and used.

[0028] 4. 0.5% fast green staining solution (95% ethanol preparation): 0.5 g of fast green is dissolved in 100 ml of 95% ethanol, filtered and used.

[0029] 5. 5% sodium sulfite solution (ultra-pure water preparation): 5 g of sodium sulfite is dissolved in 100 ml of ultra-pure water and used.

[0030] 6. 0.5% hydrochloric acid in ethanol (75% ethanol): add 0.5 ml of hydrochloric acid to 100 ml of 75% ethanol and mix well. Example

[0031] This example provides a method for preparing a paraffin section, using a young wheat root as a material, comprising the following steps: (1) Sampling: take fresh wheat roots, about 0.5 cm in length.

[0032] (2) Fixing: place the wheat roots in FAA fixing solution and fix for more than 24 hours.

[0033] (3) Washing: place the fixed sample in an embedding box and wash with tap water for about 2 hours.

[0034] (4) Pre-staining: pre-stain with 0.1% safranin solution overnight (16 hours).

[0035] (5) Washing: wash the pre-stained sample with 50% ethanol for about 10 minutes.

[0036] (6) Dehydrating: under the conditions of pressure (alternating circulation of about -35 Kpa~35 Kpa, as in step (6)) and temperature 37℃, sequentially dehydrate with 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 95% ethanol I, and 95% ethanol II for 30 minutes each, and then dehydrate with anhydrous ethanol I for 30 minutes and anhydrous ethanol II for 1 hour.

[0037] (7) Clearing: under the conditions of pressure (alternating circulation of about -35 Kpa~35 Kpa, as in step (6)) and temperature 37℃, clear with xylene I and xylene II for 30 minutes each.

[0038] (8) Waxing: under the conditions of pressure (alternating circulation of about -35 Kpa~35 Kpa, as in step (6)) and temperature (65℃), immerse in paraffin I, paraffin II, and paraffin III for 1 hour each.

[0039] (9) Embedding: condense a layer of wax at the bottom of the embedding mold, gently lift the wheat roots with tweezers, and place them in the embedding mold for embedding; then cover the embedding box, condense the appropriate amount of paraffin, and remove the mold.

[0040] (10) Sectioning: paraffin sectioning with a thickness of 6-20 μm.

[0041] (11) Spreading: Use tweezers to pick up the wax strip and spread it into the spreader for 40 seconds. The spreading temperature is 42 ℃. Then use an adhesive glass slide to pick up the wax strip.

[0042] (12) Baking the slides: After the slides have been removed, place them in a drying oven at 65°C for more than 2 hours.

[0043] (13) Dewaxing to water: pass through xylene I for 8 min, xylene II for 8 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 90% ethanol for 3 min, 80% ethanol for 3 min, 70% ethanol for 3 min, and rinse with tap water for 3 min.

[0044] (14) Staining and mounting: 5% sodium sulfite solution for 30 min, 1% safranin staining solution for 30 min, rinse slightly with water, 0.5% hydrochloric acid ethanol for 5 s, rinse with water, 95% ethanol for a few seconds, 0.5% fast green staining solution for a few seconds to a few minutes, 95% ethanol for a few seconds, anhydrous ethanol for a few seconds, anhydrous ethanol for a few seconds, xylene clearing for 5 min, and mounting.

[0045] The above scheme optimizes the paraffin section preparation method for wheat seedlings in several ways. First, the wheat roots are pre-stained with a 0.1% safranin solution. After pre-staining, the tissue is red, making it very easy to observe and creating a sharp contrast with the paraffin environment. Figure 1 The image shows tissue before pre-staining; the sample is translucent and milky white. During subsequent paraffin embedding, it is difficult to accurately control the manipulation of the tissue, and it is extremely easy to lose it. For example... Figure 2 The pre-stained tissue exhibits vibrant color and sharp contrast, resolving the issue of tissue loss and allowing for more accurate control of wheat root tissue manipulation in subsequent processing steps, reducing operational damage. Secondly, this invention employs negative pressure, pressurized circulation, and increased temperature, eliminating the need for equal-volume mixing of transition reagents. The enhanced pressure and temperature conditions result in more stable and rapid reagent displacement within the tissue, shortening sample processing time while ensuring effective dehydration and improving slide preparation efficiency. Finally, the optimized staining process utilizes a 5% sodium sulfite solution for deacidification, resulting in more stable safranin staining, and a 0.5% hydrochloric acid-ethanol solution for color separation. This removes excess safranin stain and allows for faster fast green staining, leading to a clean and highly contrasting tissue staining background. This optimized method simplifies experimental steps, shortens the experimental cycle, saves costs, protects the environment, and improves slide quality and efficiency.

[0046] The obtained slices are as follows Figure 3As shown, the prepared sections after the optimization method, whether it is the epidermis and cortex, or the xylem, phloem, pericycle and endodermis, the cell structure of each layer is clear and complete, and the overall staining contrast is also relatively bright. Such high-quality sections are more conducive to accurate data results in experimental research.

[0047] Comparative Example 1 Conventional plant tissue paraffin section preparation method.

[0048] (1) Sampling: Take fresh wheat roots, about 0.5 cm in length.

[0049] (2) Fixation: Place the wheat roots in FAA fixing solution for 24 h or more.

[0050] (5) Washing: Wash the sample with tap water for about 2 h.

[0051] (6) Dehydration: Dehydrate successively in 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 95% ethanol I, 95% ethanol II for 1.5 h each, and in anhydrous ethanol I and anhydrous ethanol II for 2 h.

[0052] (7) Transparency: Mix anhydrous ethanol and xylene (1:1), xylene I, and xylene II, and make them transparent for 1 h each.

[0053] (8) Wax immersion: Immersing in the mixture of xylene and paraffin overnight at room temperature, and immersing in paraffin I and paraffin II for 2 h at 55°C, respectively.

[0054] (9) Embedding: First, condense a layer of wax at the bottom of the embedding mold, and gently hold the wheat roots with tweezers and place them in the embedding mold for embedding; then cover the embedding box and take the appropriate amount of paraffin to condense and demold.

[0055] (8) Sectioning: Paraffin sectioning with a thickness of 6-20 μm.

[0056] (9) Spreading: Hold the wax ribbon with tweezers and spread it in the spreading machine for 40 s, with a spreading temperature of 39-42°C, and then use an adhesive glass slide to lift the wax ribbon.

[0057] (10) Baking: Place the glass slide with the lifted section in a forced air drying oven at 65°C for 2 h or more.

[0058] (11) De-waxing to water: De-waxing successively in xylene I for 8 min, xylene II for 8 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 90% ethanol for 3 min, 80% ethanol for 3 min, 70% ethanol for 3 min, and tap water for 3 min.

[0059] (12) Staining and sealing: 1% safranin staining solution for 1 h, slightly washed with water, 70%, 80%, 90%, 95% gradient ethanol for several seconds, 0.5% fast green staining solution for several seconds to several minutes, 95% ethanol for several seconds, anhydrous ethanol for several seconds, anhydrous ethanol for several seconds, xylene transparency for 5 min, and sealing.

[0060] As shown in FIG. 2, the cross section of the wheat root was deformed, the cell wall outline was not clear, the epidermis and cortex cells lost their original shape, and were squeezed together in disorder. The xylem and phloem were also deformed, and there was obvious damage during the operation. The overall staining saturation was insufficient, and the contrast was not obvious, which affected the judgment of the experimental results. Figure 4

[0061] Comparative Example 2 This comparative example provides a method for preparing a paraffin section, using a wheat seedling root as a material. The difference from Example 1 is that, in the dehydration, transparency, and wax immersion steps, different pressure cycles and 37°C conditions are not performed. The obtained section is shown in FIG. 2. Figure 5 As shown in FIG. 2, the cross section of the wheat root was deformed, the cell wall outline was not clear, the epidermis and cortex cells lost their original shape, and were squeezed together in disorder. The xylem and phloem were also deformed, and there was obvious damage during the operation. The overall staining saturation was insufficient, and the contrast was not obvious, which affected the judgment of the experimental results.

[0062] Comparative Example 3 The difference from Example 1 is that no pre-staining is performed. The un-stained wheat is milky white, and it is not easy to control the operation of the wheat root, which leads to sample loss and easy operation damage. The finally prepared wheat section is not complete or is deformed by the forceps, as shown in FIG. 3. Figure 6 Figure 7 After pre-staining, the sample is deep red, which is in sharp contrast with the environment, and the operation is easy to control, which can basically avoid the above results.

[0063] Comparative Example 4 The difference from Example 1 is that 5% sodium sulfite solution and 0.5% hydrochloric acid ethanol solution are not used for treatment. Safranin staining solution is an alkaline dye, and fast green is an acidic dye. The two reagents are combined for use, which is not easy to control, and is easy to be dyed into a reddish or greenish result. For example, if the fast green staining solution is dyed for too long, it will be greenish (as shown in FIG. 4), and if it is dyed for too short, it will be reddish (as shown in FIG. 5). Figure 8 Figure 9 Example

[0064] This example provides a method for preparing a paraffin section, using a corn kernel as a material, which includes the following steps: (1) Sampling: Fresh corn kernels are taken.

[0065] (2) Fixing: The corn kernels are placed in FAA fixing solution for more than 24 h.

[0066] (3) Washing: The fixed sample is cut in half from the middle of the corn kernel with a blade, and is placed in an embedding box for more than 2 h of tap water washing. ​​​​

[0067] (4) Pre-staining: 0.1% safranin solution overnight pre-staining (20 h).

[0068] (5) Washing: The pre-stained sample is immersed in 50% ethanol for about 10 min.

[0069] (6) Dehydration: Under the conditions of pressure (about -35 Kpa~35 Kpa alternating circulation, as in step (6)) and temperature (40°C), the sample is sequentially dehydrated in 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 95% ethanol I, 95% ethanol II for 30 min each, and then dehydrated in anhydrous ethanol I for 30 min and anhydrous ethanol II for 1 h.

[0070] (7) Transparency: Under the conditions of pressure (about -35 Kpa~35 Kpa alternating circulation, as in step (6)) and temperature (40°C), the sample is sequentially dehydrated in xylene I and xylene II for 30 min each.

[0071] (8) Wax immersion: Under the conditions of pressure (about -35 Kpa~35 Kpa alternating circulation) and temperature (65°C), the sample is immersed in paraffin I, paraffin II, and paraffin III for 1 h each.

[0072] (9) Embedding: A layer of wax is condensed on the bottom of the embedding mold, and the wheat root is gently lifted with tweezers and placed in the embedding mold for embedding. Then the embedding box is covered, and an appropriate amount of paraffin is condensed and removed.

[0073] (10) Slicing: The paraffin section is cut to a thickness of 6-20 μm.

[0074] (11) Spreading: The wax tape is taken out with tweezers and spread in the spreading machine for 40 s, with a spreading temperature of 42°C. Then the wax tape is lifted with an adhesive glass slide.

[0075] (12) Baking: The glass slide with the lifted wax tape is placed in a forced air drying oven at 65°C for 2 h or more.

[0076] (13) De-waxing to water: The sample is sequentially dehydrated in xylene I for 8 min, xylene II for 8 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 90% ethanol for 3 min, 80% ethanol for 3 min, 70% ethanol for 3 min, and tap water for 3 min.

[0077] (14) Staining and mounting: 5% sodium sulfite solution for 30 min, 1% safranin staining solution for 30 min, slight water washing, 0.5% hydrochloric acid ethanol for 5 s, water washing, 95% ethanol for several seconds, 0.5% fast green staining solution for several seconds to several minutes, 95% ethanol for several seconds, anhydrous ethanol for several seconds, anhydrous ethanol for several seconds, xylene transparency for 5 min, mounting, see Figure 10 .

[0078] The general description of the invention involved in the present application and the description of the specific embodiments should not be understood as a limitation on the technical solutions of the invention. Based on the disclosure of the present application, the skilled in the art can add, reduce or combine the disclosed technical features in the general description or / and the specific embodiments (including the examples) without violating the elements of the invention involved, to form other technical solutions within the protection scope of the present application.

Claims

1. A method for preparing paraffin sections of wheat roots, comprising the sequential steps of fixation, dehydration, clearing, wax impregnation, embedding, sectioning, spreading, baking, dewaxing to water, staining, and mounting, characterized in that, A pre-staining step is performed between the fixation and dehydration steps. The pre-staining step involves treating the fixed wheat roots with safranin solution for 12–20 h. The dehydration, clearing, and waxing steps are performed under positive and negative pressure cycling and increased temperature conditions. In the staining step, the dewaxed sections are treated with sodium sulfite solution for 0.5–1 h and then treated with 0.5% hydrochloric acid ethanol solution for 5–10 s.

2. The method for preparing paraffin sections of wheat roots according to claim 1, characterized in that, The concentration of the pre-stained safranin solution is 0.1% to 0.3%.

3. A method for preparing paraffin sections of wheat roots according to claim 1 or 2, characterized in that, The safranin solution was prepared using 50% ethanol as a solvent.

4. The method for preparing paraffin sections of wheat roots according to claim 1, characterized in that, The dehydration, clearing, and waxing steps are carried out under positive and negative pressure cycles and increased temperature conditions.

5. The method for preparing paraffin sections of wheat roots according to claim 5, characterized in that, The finger pressure in the dehydration and transparentization steps is approximately -50 to 50 kPa, and the temperature is 37 to 45 °C.

6. The method for preparing paraffin sections of wheat roots according to claim 5, characterized in that, The wax impregnation step is carried out under a pressure of approximately -50 to 50 kPa and a temperature of 60 to 65 °C.

7. The method for preparing paraffin sections of wheat roots according to claim 1, characterized in that, In the staining and mounting step, a 5% sodium sulfite solution is used for treatment.

8. The method for preparing paraffin sections of wheat roots according to claim 1, characterized in that, In the staining and mounting step, differentiation is performed using 0.5% hydrochloric acid ethanol.

9. A paraffin section of a wheat root, characterized in that, The method for preparing paraffin sections of wheat roots as described in claim 1 was used.

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