Plant paraffin embedding method

Abstract

The invention relates to the technical field of plants, in particular to a paraffin embedding method for reducing plant RNA degradation. In the plant paraffin embedding method provided by the invention, a stationary liquid for fixing plant tissues is prepared from absolute ethyl alcohol and acetic acid in a volume ratio of (2-5): (1-4); buffer I used for dehydration is absolute ethyl alcohol and n-butyl alcohol with the volume ratio of (1-3): (1-2); buffer II used for waxing is n-butyl alcohol and low-temperature paraffin wax with the volume ratio of (1-3): (1-2), and the melting point of the paraffin wax is 20-50 DEG C. According to the method, low-temperature paraffin with the melting point of 20-50 DEG C is adopted, and a reagent for paraffin embedding and a paraffin embedding process are adjusted, so that the plant section with relatively high RNA integrity and relatively high tissue structure integrity is obtained. The paraffin embedding method provided by the invention is suitable for different types of molecular biology experiments of space transcriptome, immunohistochemistry, immunofluorescence and the like.

Description

Technical Field

[0001] The invention relates to the technical field of plants, in particular to a plant paraffin embedding method. Background Art

[0002] In botanical research, the species, morphology, structure, and function of plants are all important data for research. Ordinary optical microscopes are most commonly used to observe plant morphology and structure at the micrometer level. However, there are many plant species, and the thickness of different plant bodies or tissue parts varies, making it difficult to transmit light and not conducive to microscopic observation. Therefore, microscopy has become an important means in botanical research. Conventional microscopy techniques include frozen sectioning techniques and paraffin sectioning techniques. The frozen sectioning technique is to clean the impurities on the surface of fresh plant tissues, and then use an embedding agent (optimal cutting temperature compound, OCT) of a water-soluble mixture of polyethylene glycol and polyvinyl alcohol to fill the gap between the tissue and the embedding box to support the tissue morphology for cryo-embedding.

[0003] The frozen section technique is simple, rapid and can better preserve the integrity of tissue RNA and various antigen activities, which is more conducive to the development of subsequent molecular biology experiments. However, the cell size, water content and tissue hardness of different tissue parts of plant samples are different. The tissue contains more water during the embedding process, and the cell structure of tissues with a high degree of lignification is easily broken during the sectioning process, affecting the integrity of the tissue structure.

[0004] Paraffin embedding technology mainly makes the tissue cell structure clear through the steps of fixation, dehydration, transparency, and wax penetration, better supports the tissue structure, has a more complete tissue morphology structure and thin slices, can be cut into continuous wax strips, and can observe the continuous changes of cells and tissue structures. However, the conventional paraffin embedding technology has a complicated preparation process, a long preparation process, and high temperatures, which leads to serious degradation of tissue RNA, affecting the subsequent molecular biology experiments. Summary of the invention

[0005] The purpose of the present invention is to provide a paraffin embedding method which can effectively reduce plant RNA degradation and simultaneously achieve a better sectioning effect.

[0006] The melting point of common paraffin is 50°C to 70°C. In order to reduce the degradation of plant RNA, the present invention uses low-temperature paraffin to prepare plant paraffin embedded blocks and obtain plant paraffin sections. The melting point of the low-temperature paraffin is 20°C to 50°C, and preferably, the melting point of the low-temperature paraffin of the present invention is 20°C to 42°C.

[0007] When selecting low-temperature paraffin to prepare plant paraffin embedding blocks, the present invention finds that conventional paraffin embedding methods are not suitable for low-temperature paraffin. If conventional fixative is used and then low-temperature paraffin is used for embedding, plant tissue RNA will still be degraded, and plant sections with high RNA integrity and high tissue structure integrity cannot be obtained.

[0008] When conventional paraffin is further used to prepare plant paraffin embedding blocks, the present invention finds that the conventional paraffin embedding process uses Carnoy's fixative to fix tissues at room temperature and uses 50-70°C paraffin embedding to cause serious degradation of plant RNA. If the normal temperature Carnoy's fixative is used for fixation and then low temperature paraffin is used for embedding, plant tissue RNA will still be degraded, and high integrity new RNA cannot be obtained. However, the use of this technical solution to fix, dehydrate, and make transparent under low temperature conditions and use low temperature paraffin for paraffin embedding of plant tissues can effectively prevent RNA degradation.

[0009] Specifically, the technical solution provided by the present invention is as follows.

[0010] In a first aspect, the present invention provides a method for embedding plant paraffin. In the method, a fixing liquid used for fixing plant tissues is anhydrous ethanol and acetic acid in a volume ratio of (2-5): (1-4); Buffer I used for dehydrating plant tissues is anhydrous ethanol and n-butanol in a volume ratio of (1-3): (1-2); Buffer II used for waxing plant tissues is n-butanol and low-temperature paraffin in a volume ratio of (1-3): (1-2), and the melting point of the paraffin is 20-50°C.

[0011] The plant paraffin embedding method provided by the present invention comprises: plant tissue fixation, dehydration, transparency, wax dipping, wax penetration and embedding; During the wax dipping or wax penetration, vacuum treatment is performed for 30 minutes to 2 hours.

[0012] In the plant paraffin embedding method provided by the present invention, the pressure of the vacuum treatment is 1-40 KPa and the temperature is 25-55°C.

[0013] In the plant paraffin embedding method provided by the present invention, the temperature of the plant tissue is less than or equal to 55°C.

[0014] In the plant paraffin embedding method provided by the present invention, the tissue transparent liquid is configured with one or more of n-butanol, xylene and methanol.

[0015] As a specific embodiment of the present invention, the plant paraffin embedding method provided by the present invention comprises: (1) Plant tissues are fixed in fixative at 0-4°C for 12-20 hours; (2) Place the plant tissue fixed in step (1) in pre-cooled Buffer I and incubate on ice for 30 min to 2 h; (3) Place the plant tissue treated in step (2) into pre-cooled tissue clearing solution and incubate on ice for 30 min to 2 h; (4) Place the plant tissue treated in step (3) into the inverted mixed Buffer II and evacuate the mixture for 30 min to 2 h; (5) placing the plant tissue treated in step (4) into a low-temperature paraffin solution and evacuating the solution for 30 min to 2 h to obtain a plant tissue paraffin-embedded block; (6) Adding the plant tissue paraffin-embedded block to an embedding box, placing it on ice to solidify and freeze, and freezing the solidified plant tissue paraffin-embedded block.

[0016] In the plant paraffin embedding method provided by the present invention, in each step, the temperature of the plant tissue is less than or equal to 55°C.

[0017] The fixation and transparency stages of the present invention are both carried out under low temperature conditions and low temperature paraffin is used to reduce the effect of temperature on RNA during the embedding process. At the same time, a vacuum concentrator is used to promote the entry of paraffin into tissue cells, which reduces the paraffin embedding process, shortens the paraffin embedding time, and reduces the effect of excessive operation processes and reagents on RNA. Experiments have shown that the embedding method can effectively reduce RNA degradation while completely preserving the morphological structure of the tissue.

[0018] In the plant paraffin embedding method provided by the present invention, Buffer II is obtained by dissolving at 30-50°C.

[0019] In the plant paraffin embedding method provided by the present invention, during the vacuuming in step (5), the low-temperature paraffin solution is replaced every 40 minutes to 1 hour.

[0020] In a second aspect, the present invention provides application of the above-mentioned plant paraffin embedding method in reducing RNA degradation in plant tissues.

[0021] In a third aspect, the present invention provides a plant paraffin section, which is prepared by using the plant paraffin embedding block obtained by the above-mentioned plant paraffin embedding method.

[0022] The beneficial effects of the present invention are: After embedding the plant tissues, RNA extraction and determination showed that the RIN values ​​of the plant tissues embedded in ordinary paraffin were all less than 5, and most RNA fragments were degraded. However, the RIN values ​​of the plant tissues embedded using this method were all above 5, and the integrity of the sections was better than that of the frozen sections.

[0023] In addition, the plant slice method with higher RNA integrity and higher tissue structure integrity obtained by the present invention is applicable to different types of molecular biology experiments including but not limited to spatial transcriptome, immunohistochemistry, immunofluorescence, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a slice diagram of a corn root plant provided in Example 2 of the present invention.

[0026] Figure 2 This is the RNA quality diagram of the corn root plant slice provided in Example 2 of the present invention.

[0027] Figure 3 This is a slice diagram of a potato stem plant provided in Example 3 of the present invention.

[0028] Figure 4 This is the RNA quality map of potato stem plant slices provided in Example 3 of the present invention.

[0029] Figure 5 This is a tobacco root plant slice diagram provided by Example 4 of the present invention.

[0030] Figure 6 This is the RNA quality map of tobacco root plant slices provided in Example 4 of the present invention.

[0031] Figure 7 This is an RNA quality map of plant slices obtained by treating rapeseed stem tissue with the fixative 1 provided in Comparative Example 1 of the present invention.

[0032] Figure 8 This is an RNA quality map of plant slices obtained by treating rapeseed stem tissue with the fixative 2 provided in Comparative Example 1 of the present invention.

[0033] Fig. 9 This is the RNA quality map of plant slices obtained by treating rice roots with paraffin at room temperature provided in Comparative Example 2 of the present invention.

[0034] Fig.10 This is the RNA quality map of plant slices obtained by treating rice roots with low-temperature paraffin provided in Comparative Example 2 of the present invention.

[0035] Fig.11 This is a RNA quality diagram of plant slices obtained by treating Impatiens balsamina stems using the method of Example 1 provided in Comparative Example 3 of the present invention.

[0036] Fig.12 This is a map of RNA quality of plant slices obtained by treating Impatiens balsamina stems using the method of Example 5 provided in Comparative Example 3 of the present invention.

[0037] Fig.13 This is a diagram of tobacco root tissue sections processed by the method of Example 5 provided in Experimental Example 1 of the present invention.

[0038] Fig.14 This is a diagram of RNA quality of tobacco root tissue sections processed by the method of Example 5 provided in Experimental Example 1 of the present invention.

[0039] Fig.15 This is a diagram of potato stem tissue sections processed by the method of Example 5 provided in Experimental Example 2 of the present invention.

[0040] Fig.16 This is a diagram of RNA quality of potato stem tissue sections processed by the method of Example 5 provided in Experimental Example 2 of the present invention.

[0041] Fig.17 This is a diagram of soybean seed tissue sections processed by the method of Example 5 provided in Experimental Example 3 of the present invention.

[0042] Fig.18 This is a diagram of RNA quality of soybean seed tissue sections processed by the method of Example 5 provided in Experimental Example 3 of the present invention.

[0043] Fig.19 This is a diagram of corn stem tissue sections processed using the method of Example 5 provided in Experimental Example 4 of the present invention.

[0044] Fig. 20 This is a diagram of RNA quality of corn stem tissue sections processed by the method of Example 5 provided in Experimental Example 4 of the present invention.

[0045] Fig.21 This is a diagram of rapeseed stem tissue sections processed using the method of Example 5 provided in Experimental Example 5 of the present invention.

[0046] Fig. 22 This is a diagram of RNA quality of rapeseed stem tissue sections processed by the method of Example 5 provided in Experimental Example 5 of the present invention. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Example 1 Conventional paraffin embedding method 1. Take a fresh tissue sample and thoroughly clean all impurities on the tissue surface with ultrapure water.

[0049] 2. Fixation: Use FAA fixative: anhydrous ethanol: acetic acid: formaldehyde (3:1:2), immerse the tissue in the fixative, and fix it at room temperature for more than 24 hours.

[0050] 3. Dehydration: After tissue fixation, rinse the tissue surface with ultrapure water to remove excess fixative, and then use five different gradients of alcohol: 70%, 85%, 95%, 100%, and 100% to dehydrate the tissue. Perform each gradient for 1-2 hours at room temperature.

[0051] 4. Transparency: After the tissue is dehydrated, it is transferred into a transparent liquid for transparency. The transparent liquid is xylene: anhydrous ethanol (1:1), and the process is carried out at room temperature for 5-12 hours.

[0052] 5. Wax dipping: Pour the material together with xylene into a small wax cup, and then gently pour in the dissolved paraffin. The paraffin solidifies on the upper layer of xylene. At this time, the small wax cup can be placed in a 35-37℃ incubator to slowly melt the paraffin until it is saturated, which takes about 1-2 days; ③ Move the wax cup into a 56-60℃ incubator, wait for the paraffin to melt, pour out the paraffin containing xylene, and then replace the melted pure wax once every 1 hour or so, and replace the pure wax 2-3 times in total. The melting point of the paraffin used is 54-60℃.

[0053] 6. Embedding: Add paraffin into the embedding box and place the tissue in the direction of the section until the paraffin solidifies.

[0054] Example 2 The stationary phase used in this embodiment is anhydrous ethanol: acetic acid = 3:1; The buffer I used was anhydrous ethanol:methanol = 1:1; The tissue clearing fluid used was methanol; The buffer II used was methanol: paraffin = 1:1; The paraffin used is paraffin with a melting point of 42-60°C.

[0055] The paraffin embedding method used in this embodiment comprises the following steps: 1. Take fresh plant tissue samples and thoroughly clean all impurities on the tissue surface with ultrapure water, and trim the tissue to a size less than 6.8mm*6.8mm and a thickness less than 1cm.

[0056] 2. Fixation: Wipe the surface of the plant tissue dry with dust-free paper and place it in pre-cooled tissue fixative. Invert and mix well. After the tissue is completely immersed, use a vacuum concentrator to evacuate for 30 minutes and fix it at room temperature overnight (15 hours).

[0057] 3. Dehydration: Pipette 4 mL of Buffer I and place on ice for pre-cooling. Take out the fixed tissue, absorb the excess liquid on the surface of the tissue with dust-free paper, place it in the pre-cooled Buffer I, and incubate on ice for 30 minutes.

[0058] 4. Transparency: Pipette 4 mL of tissue transparent liquid and place it on ice for pre-cooling. Take out the incubated tissue, absorb the excess liquid on the surface of the tissue with dust-free paper, place it in the pre-cooled tissue transparent liquid, and incubate on ice for 1 hour.

[0059] 5. Wax Immersion Ⅰ: After mixing Buffer Ⅱ by inversion, take 1.2 mL and place it in a 1.5 mL centrifuge tube. Take out the incubated tissue, absorb the excess liquid on the surface of the tissue with dust-free paper, and then place the tissue in Buffer Ⅱ. Open the centrifuge tube and place it in a vacuum concentrator for 30 minutes. The vacuum pressure is 1 KPa and the temperature is 45 ° C. After the vacuum is completed, incubate at 38 ° C for 1 hour.

[0060] 6. Wax immersion II: Mix the dissolved paraffin by inversion, take out the tissue after centrifugation, place the tissue in a 1.5mL centrifuge tube containing 1.2mL paraffin, and then put it into a vacuum concentrator for evacuation for 1h. The vacuum pressure used is 1KPa and the temperature is 45℃.

[0061] 7. Embedding: After vacuuming, take out the tissue, add paraffin into the embedding box, and place the tissue according to the slice direction. After adjustment, place it on ice to solidify and freeze. Wrap the solidified embedding block with tin foil and carefully store it at -80℃ to avoid severe bumps to obtain the plant paraffin embedding block.

[0062] Corn root plant slices were prepared by the method provided in this example, and the RIN value of plant RNA was 2.7 (as shown in Figure 2). Figure 1 and Figure 2 as shown).

[0063] Example 3 The difference between the reagents used in this example and those in Example 2 is: The buffer I used was anhydrous ethanol: xylene = 1:1; The tissue clearing fluid used was xylene; The buffer II used was xylene: paraffin = 1:1; The paraffin used is paraffin with a melting point of 42-60°C.

[0064] The difference between the paraffin embedding method used in this embodiment and that in embodiment 2 is that: Step 2 is to wipe the moisture off the surface of the tissue with dust-free paper and place it in the pre-cooled tissue fixative, invert and mix, and use a vacuum concentrator to evacuate the tissue for 20 minutes after the vacuum is completed. After the vacuum is completed, place the tissue at 4°C for overnight fixation (15 hours).

[0065] Step 5: After mixing Buffer II by inversion, take 1.2 ml and place it in a 1.5 ml centrifuge tube. Take out the incubated tissue, absorb the excess liquid on the surface of the tissue with dust-free paper, and then place the tissue in Buffer II and incubate at 38°C for 1 hour.

[0066] Step 6 is to mix the dissolved paraffin by inversion, take out the tissue after centrifugation, place the tissue in a 1.5 ml centrifuge tube containing 1.2 ml paraffin, and then put it into a vacuum concentrator for vacuuming for 1 hour. The pressure used for vacuuming is 1 KPa, the temperature is 45°C, and the paraffin is replaced every 30 minutes.

[0067] Step 8 is to wrap the solidified embedded block with tin foil and carefully store it at -20°C.

[0068] The potato stem plant slices were prepared by the method provided in this example, and the RIN value of the plant RNA was 4.9 ( Figure 3 and Figure 4 as shown).

[0069] Example 4 The difference between the reagents used in this example and those in Example 2 is: The buffer I used was anhydrous ethanol: n-butanol = 1:1; The tissue clearing fluid used was n-butanol; The buffer II used was n-butanol: low temperature paraffin = 1:1; The paraffin used is low-temperature paraffin with a melting point of 37~42℃.

[0070] The difference between the paraffin embedding method used in this embodiment and that in embodiment 2 is that: Step 2 is to wipe the moisture off the surface of the tissue with dust-free paper and place it in the pre-cooled tissue fixative, invert and mix, and after the tissue is completely immersed, place the tissue at 4°C for overnight fixation (15 hours).

[0071] Step 5 is to mix Buffer Ⅱ by inversion, take 1.2 ml and place it in a 1.5 ml centrifuge tube, take out the incubated tissue, absorb the excess liquid on the surface of the tissue with dust-free paper, and then place the tissue in Buffer Ⅱ, open the centrifuge tube and place it in a vacuum concentrator for 30 minutes. The vacuum pressure used is 1Kpa and the temperature is 45°C.

[0072] Step 6 is to mix the dissolved paraffin by inversion, take out the centrifuged tissue, place the tissue in a 1.5 mL centrifuge tube containing 1.2 mL paraffin, and then put it into a vacuum concentrator for vacuuming for 30 minutes. The vacuuming pressure is 1 KPa and the temperature is 45°C. After the vacuuming is completed, incubate at 38°C for 1.5 hours.

[0073] Step 8 is to wrap the solidified embedded block with tin foil and carefully store it at -20°C.

[0074] The tobacco root plant slices were prepared by the method provided in this example, and the RIN value of the plant RNA was 6.4 (as shown in Figure 2). Figure 5 and Figure 6 as shown).

[0075] Example 5 The difference between the reagents used in this example and those in Example 2 is: Buffer I used: anhydrous ethanol: n-butanol = 1:1; Histoclear fluid used: n-butanol; Buffer II used: n-butanol: low-temperature paraffin = 1:1; The paraffin used is low-temperature paraffin with a melting point of 20~40℃.

[0076] The difference between the paraffin embedding method used in this embodiment and that in embodiment 2 is that: Step 2 is to wipe the moisture off the surface of the tissue with dust-free paper and then place it in the pre-cooled tissue fixative, invert and mix well, so that the tissue is completely immersed and placed flat at 4°C for overnight fixation. The fixation time is 15 hours.

[0077] The incubation time on ice in step 3 is 1 h, and the incubation time on ice in step 4 is 1.5 h.

[0078] Step 5: After mixing Buffer II by inversion, take 1.2 mL and place it in a 1.5 mL centrifuge tube. Take out the incubated tissue, absorb the excess liquid on the surface of the tissue with dust-free paper, and then place the tissue in Buffer II. Open the centrifuge tube and place it in a vacuum concentrator for evacuation for 1.5 hours. The vacuum pressure is 1 KPa and the temperature is 42°C.

[0079] Step 6 is to mix the dissolved paraffin by inversion, take out the tissue after centrifugation, place the tissue in a 1.5 ml centrifuge tube containing 1.2 ml paraffin, and then put it into a vacuum concentrator for vacuuming for 1.5 hours, with a vacuum pressure of 1 KPa and a temperature of 40°C, and replace with new paraffin every 40 minutes.

[0080] After the solidification and freezing in step 7 is completed, the solidified embedded blocks are carefully placed at -20°C. After freezing for 2 hours, the embedded blocks are wrapped in tin foil and carefully stored at -80°C to avoid severe bumps.

[0081] Comparative Example 1 Different fixatives The rape stem tissue was fixed overnight with the two fixatives, and then embedded according to the embedding method of Example 5. After embedding, the tissue was sliced ​​and RNA was extracted and tested for quality. The results are as follows:

[0082] The results showed that RNA in tissues fixed with anhydrous ethanol: acetic acid: formaldehyde (3:1:2) showed degradation, with a RIN value of <5, while tissues fixed with anhydrous ethanol and acetic acid (3:1) showed no serious degradation, with a RIN value of >5. The quality detection diagram of plant RNA in plant slices obtained with different fixatives in this comparative example is shown in Figure 7 and Figure 8 , Figure 7 The quality of plant RNA obtained from plant sections using fixative 1 is Figure 8 The quality of plant RNA from plant sections obtained with fixative 2.

[0083] The experimental results show that fixative solutions containing formaldehyde and different concentration ratios cannot fix tissue RNA well, and formaldehyde can easily cause cross-linking between RNA and protein, leading to RNA degradation.

[0084] Comparative Example 2 Different paraffin waxes The rice roots were embedded using low-temperature paraffin with a melting point of 20-40°C and conventional paraffin with a melting point of 55-65°C according to the embedding method of Example 5. After embedding, the tissue was sliced ​​and RNA was extracted and tested for quality. The results were as follows:

[0085] The results showed that when conventional paraffin was used for embedding, even with the embedding reagent and process optimized by the present invention, RNA of tissue embedded in conventional paraffin was degraded, and the RIN value was less than 5, while tissue embedded in low-temperature paraffin did not show serious degradation, and the RIN value was greater than 5. The experimental results show that low-temperature paraffin can effectively prevent RNA degradation caused by excessive temperature during long-term wax immersion of tissue.

[0086] The quality detection diagram of plant RNA in plant sections obtained from different paraffin waxes in this comparative example is shown in Fig. 9 and Fig.10 , Fig. 9 The quality of plant RNA obtained from plant sections after routine paraffin embedding. Fig.10 The quality of plant RNA obtained from plant sections after low-temperature paraffin embedding.

[0087] Comparative Example 3 The Impatiens balsamina stems were taken and embedded according to the conventional paraffin embedding process of Example 5 and Example 1, respectively. After embedding, the tissues were sliced ​​and RNA was extracted and tested for quality. The results are as follows.

[0088] The results showed that RNA of tissues embedded in conventional paraffin embedding process was degraded, with RIN value less than 5, while tissues embedded in low-temperature paraffin did not show serious degradation, with RIN value greater than 5.

[0089] The quality detection diagram of plant RNA in plant slices obtained with different fixatives in this comparative example is shown in Fig.11 and Fig.12 , Fig.11 The quality of plant RNA obtained from plant sections using conventional paraffin processing. Fig.12 This is the plant RNA quality of the plant slices obtained in Example 5.

[0090] Experimental Example 1 Tobacco root tissue slices Take a tobacco root tissue sample and embed it according to the paraffin embedding process provided in Example 5 of the present invention. After embedding, perform tissue sectioning, toluidine blue staining and RNA quality inspection. The sectioning results are shown in Fig.13 , RNA quality inspection chart see Fig.14 , the RNA quality test results are as follows.

[0091] The experimental results showed that the tissue structure of the plant slices was intact, the RNA fragments had good integrity, and the RIN value was >5.

[0092] Experimental Example 2 Potato stem tissue sections Take a potato stem tissue sample and embed it according to the paraffin embedding process provided in Example 5 of the present invention. After embedding, perform tissue sectioning, toluidine blue staining and RNA quality inspection. The sectioning results are shown in Fig.15 , RNA quality inspection chart see Fig.16 , the RNA quality test results are as follows.

[0093] The experimental results showed that the tissue structure of the plant slices was intact, the RNA fragments had good integrity, and the RIN value was >5.

[0094] Experimental Example 3 Soybean Seed Tissue Section Take soybean seed tissue samples, embed them according to the paraffin embedding process provided in Example 5 of the present invention, and then perform tissue sectioning, toluidine blue staining and RNA quality inspection. The sectioning results are shown in Fig.17 , RNA quality inspection chart see Fig.18 , the RNA quality test results are as follows.

[0095] The experimental results showed that the tissue structure of the plant slices was intact, the RNA fragments had good integrity, and the RIN value was >5.

[0096] Experimental Example 4 Corn stem tissue sections Take corn stem tissue samples, embed them according to the paraffin embedding process provided in Example 5 of the present invention, and then perform tissue sectioning, toluidine blue staining and RNA quality inspection. The sectioning results are shown in Fig.19 , RNA quality inspection chart see Fig. 20 , the RNA quality test results are as follows.

[0097] The experimental results showed that the tissue structure of the plant slices was intact, the RNA fragments had good integrity, and the RIN value was >5.

[0098] Experimental Example 5 Rapeseed Stem Tissue Section Take rape stem tissue samples, embed them according to the paraffin embedding process provided in Example 5 of the present invention, and then perform tissue sectioning, toluidine blue staining and RNA quality inspection. The sectioning results are shown in Fig.21 , RNA quality inspection chart see Fig. 22 , the RNA quality test results are as follows.

[0099] The experimental results showed that the tissue structure of the plant slices was intact, the RNA fragments had good integrity, and the RIN value was >5.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for embedding plants in paraffin, characterized in that: In the plant paraffin embedding method, The fixative used for plant tissue fixation is anhydrous ethanol and acetic acid in a volume ratio of (2~5):(1~4); Buffer I used for dehydration of plant tissues is anhydrous ethanol and n-butanol in a volume ratio of (1-3): (1-2); The Buffer II used for wax impregnation of plant tissues is composed of n-butanol and low-temperature paraffin in a volume ratio of (1-3): (1-2), and the melting point of the paraffin is 20-50°C.

2. The plant paraffin embedding method according to claim 1, characterized in that: The plant paraffin embedding method comprises: plant tissue fixation, dehydration, transparency, wax dipping, wax penetration and embedding; During the wax dipping or wax penetration, vacuum treatment is performed for 30 minutes to 2 hours.

3. The plant paraffin embedding method according to claim 2, characterized in that: The pressure of the vacuum treatment is 1-40Kpa, and the temperature is 25-55°C.

4. The plant paraffin embedding method according to any one of claims 1 to 3, characterized in that: Configuration of tissue clearing fluid: one or more of n-butanol, xylene, and methanol.

5. The plant paraffin embedding method according to any one of claims 1 to 4, characterized in that: include: (1) Plant tissues are fixed in fixative at 0-4°C for 12-20 hours; (2) Place the plant tissue fixed in step (1) in pre-cooled Buffer I and incubate on ice for 30 min to 2 h; (3) Place the plant tissue treated in step (2) into pre-cooled tissue clearing solution and incubate on ice for 30 min to 2 h; (4) Place the plant tissue treated in step (3) into the inverted mixed Buffer II and evacuate the mixture for 30 min to 2 h; (5) placing the plant tissue treated in step (4) into a low-temperature paraffin solution and evacuating the solution for 30 min to 2 h to obtain a plant tissue paraffin-embedded block; (6) Adding the plant tissue paraffin-embedded block to an embedding box, placing it on ice to solidify and freeze, and freezing the solidified plant tissue paraffin-embedded block.

6. The plant paraffin embedding method according to claim 5, characterized in that: Dissolve at 30~50℃ to obtain BufferⅡ.

7. The plant paraffin embedding method according to claim 5, characterized in that: During the vacuuming in step (5), the low-temperature paraffin solution should be replaced every 40 minutes to 1 hour.

8. Use of the plant paraffin embedding method according to any one of claims 1 to 7 in reducing RNA degradation in plant tissues.

9. A plant paraffin section, characterized in that: The plant paraffin sections are prepared by using the plant paraffin embedding block obtained by the plant paraffin embedding method described in any one of items 1 to 7.

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