Freezing embedding and slicing method suitable for plant tissues

Abstract

The invention relates to the technical field of biology, in particular to a freezing embedding and slicing method suitable for plant tissues. The method comprises the following steps: fully infiltrating a plant tissue by using a pre-cooled freezing embedding agent so as to reduce the condition that the plant tissue is separated from the freezing embedding agent during slicing, and using a cylindrical or prismatic container without a bottom surface and a top surface as a mold during embedding, pre-cooled thermally conductive carrier planes are used as the bottom and top surfaces of the mold to directly contact the frozen embedding medium to reduce ice crystals generated by the tissue during embedding and reduce the time required for freezing. According to the method disclosed by the invention, quick freezing and embedding outside a freezing microtome can be realized, and the plant tissue frozen section with a complete tissue structure and relatively good cell morphology can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to a method for cryo-embedding and sectioning of plant tissues. Background Art

[0002] Spatial transcriptome sequencing technology, also known as spatial omics technology, can correspond the gene expression information of cells with their spatial position information one by one. By superimposing samples taken at different time points, such technology can provide information in both time and space dimensions, so it is also called "spatiotemporal omics" technology. Currently, spatiotemporal omics technology has been widely applied in different types of tissues of various species (especially humans, animals, etc.). However, in the field of botany, the application of spatiotemporal omics technology is still in its infancy. In recent years, spatiotemporal omics technology has been mainly applied in the fields of plant organ development, plant cell spatial localization, etc. in plants, including species such as Arabidopsis thaliana, peanuts, orchids, etc., involving tissue types such as roots, stems, leaves, flowers, etc. However, due to the relatively rigid cell walls and large water content in plant tissues, and the significant differences between different plant species and tissue organs, it is difficult to establish a universal pretreatment scheme for plant spatiotemporal omics technology. Therefore, there are still few studies on the application of spatiotemporal omics technology in plant tissues at the present stage.

[0003] The main methods for pretreating sample tissues in spatiotemporal omics technology are cryo-embedding methods, including direct embedding method, liquid nitrogen quick-freezing method, isopentane cryo-embedding method, etc.: (1) Direct embedding method: Wrap the tissue material with the surface liquid blotted dry after washing with a cryostat embedding medium, and perform cryogenic freezing treatment in a -20°C cryostat or on dry ice; (2) Liquid nitrogen quick-freezing method: After the material is washed, first wrap it with a cryostat embedding medium in an appropriately sized embedding cassette, place it in liquid nitrogen and quickly freeze for 20 seconds, and then put the sample into a -20°C cryostat for quick-freezing for 20 to 30 minutes; (3) Isopentane cryo-embedding method: Place the isopentane container in liquid nitrogen and cool for 3 to 5 minutes until the isopentane changes from a flowing liquid to a viscous liquid state. Then use a cryostat embedding medium to wrap the material in an embedding cassette, put the embedding cassette into isopentane. When the embedding medium changes from transparent to completely opaque white, take out the embedding block and store it in an -80°C refrigerator. Among these three cryo-embedding methods, the more commonly used method is the direct embedding method. The direct embedding method uses a pre-cooled cryostat embedding medium to wrap and freeze fresh sample tissues under low-temperature conditions. After the embedding medium is completely solidified, a cryostat is used to section the tissue embedding block to obtain a frozen section of the sample tissue suitable for spatiotemporal omics technology.

[0004] However, in the existing plant tissue cryo-embedding method, without any other treatment, during the embedding process, only a layer of cryo-embedding agent is wrapped around the tissue for cryo-embedding. When directly embedding soybean seeds, the surface of soybean seeds is smooth, and it is difficult for the tissue to be fully infiltrated and contacted with the cryo-embedding agent. During the cryosectioning process, the tissue and the cryo-embedding agent are not firmly adhered, are easily separated, and cannot support the tissue, resulting in tissue wrinkling and curling, and the morphology of the tissue sheet is poor, which is not conducive to morphological observation or other experiments. In addition, the dry ice freezing speed of the tissue embedding cassette is relatively slow, and using liquid nitrogen for freezing is likely to cause over-freezing and cracking of the embedding block. If a quick-freezing stage is used for embedding in a cryostat, the space is narrow, and it is inconvenient to operate some plant samples.

[0005] Therefore, there is an urgent need in the art for a method for pre-treating plant tissues for plant tissue cryo-embedding, in order to obtain cryo-embedded blocks in which the tissue and the cryo-embedding agent are not easily separated and are well adhered during sectioning, and to obtain plant tissue sections with uniform texture, no tissue wrinkling and curling, and complete tissue morphological structure. Summary of the Invention

[0006] In view of the deficiencies of the prior art, in the first aspect, the present invention provides a method for cryo-embedding plant tissues, and the method includes the following steps:

[0007] 1) Immerse the plant tissue to be cryo-embedded in a cryo-embedding agent pre-cooled to 0°C to 4°C, and soak the plant tissue in the cryo-embedding agent for 10 minutes to 1 hour;

[0008] 2) Place a heat-conducting carrier pre-cooled to at least -70°C and having at least one plane with the plane facing up, then place an embedding mold on the upward-facing plane of the heat-conducting carrier, and add a first volume of cryo-embedding agent pre-cooled to 0°C to 4°C to the embedding mold, and the embedding mold is a container without a bottom surface and a top surface;

[0009] 3) Put the plant tissue soaked in step 1) into the embedding mold, and add a second volume of cryo-embedding agent pre-cooled to 0°C to 4°C, and the sum of the first volume and the second volume is greater than or equal to the volume of the embedding mold;

[0010] 4) Place another heat-conducting carrier pre-cooled to at least -70°C and having at least one plane in contact with the upper part of the embedding mold above the embedding mold and keep it for a period of time until all the cryo-embedding agent in the embedding mold solidifies.

[0011] In the second aspect, the present invention provides a method for making cryosections of plant tissues, and the method includes:

[0012] a) Performing the method as described in the first aspect of the present invention to obtain embedded plant tissue;

[0013] b) Placing the embedded plant tissue on a cryostat and performing cryosectioning on it.

[0014] In a third aspect, there is provided the use of a cryosection prepared by the method for preparing a cryosection of plant tissue as described in the second aspect of the present invention in spatial and temporal omics analysis, tissue cell observation, in situ hybridization, immunofluorescence localization, or histochemical reaction.

[0015] The cryoembedding method of the present invention uses a pre-cooled cryoembedding agent to fully infiltrate the plant tissue. Further, in the case of seeds, after the seeds germinate, the seed coats are peeled off, which can reduce the separation of the plant tissue from the cryoembedding agent during sectioning; when embedding, a container without a bottom surface and a top surface, such as a cylindrical or prismatic container without a bottom surface and a top surface, is used as a mold, and the plane of a pre-cooled heat conduction carrier is used as the bottom surface and the top surface of the mold to directly contact the cryoembedding agent, which can achieve the purpose of rapid freezing and solidification, reduce the generation of ice crystals in the tissue during the embedding process, and reduce the time required for freezing, providing technical support for the subsequent application and research of plant tissue in spatial and temporal transcriptomics.

[0016] In addition, the method of the present invention can achieve rapid cryoembedding outside the cryostat. Using this method to make tissue embedding blocks can significantly improve the quality of cryosections of plant tissue, significantly reduce the separation of sectioned tissue from the cryoembedding agent, and further reduce the folding and curling of tissue sections to obtain plant tissue cryosections with complete tissue structure and good cell morphology. Description of the Drawings

[0017] The present invention is described in detail through the following drawings.

[0018] Figure 1 It is a bright-field image of a cryosection of soybean embryo tissue prepared by using the method of the present invention.

[0019] Figure 2 It is a bright-field image of a cryosection of soybean embryo tissue as a control prepared without removing the seed coat.

[0020] Figure 3 It is a bright-field image of a cryosection of soybean embryo tissue as a control prepared without infiltration.

[0021] Figure 4 It is a bright-field image of a cryosection of soybean embryo tissue as a control prepared by freezing with dry ice instead of two heat conduction carriers.

[0022] Figure 5 Showing the RNA Qsep 400 detection results of soybean seeds. Detailed implementation manners

[0023] The present invention will be described in detail and specifically below in conjunction with the accompanying drawings and embodiments, so as to better understand the present invention. However, the following description is not intended to limit the scope of the present invention.

[0024] As described above, there are many disadvantages in the existing methods for cryo-embedding plant tissues, and it is not easy to produce cryosections that meet the requirements. Therefore, there is an urgent need in the art for a method for pre-treating plant tissues for cryo-embedding plant tissues, in order to obtain cryo-embedded blocks in which the tissues and the cryo-embedding agent are not easily separated during sectioning and are well adhered, and further obtain plant tissue sections with uniform texture, no wrinkling or curling of the tissues, and complete tissue morphological structure.

[0025] In a first aspect, the present invention provides a method for cryo-embedding plant tissues, the method comprising the following steps:

[0026] 1) Immerse the plant tissue to be cryo-embedded in a cryo-embedding agent pre-cooled to 0°C to 4°C, and soak the plant tissue in the cryo-embedding agent for 10 minutes to 1 hour;

[0027] 2) Place a heat-conducting carrier having at least one plane pre-cooled to at least -70°C with the plane facing up, then place an embedding mold on the upward-facing plane of the heat-conducting carrier, and add a first volume of cryo-embedding agent pre-cooled to 0°C to 4°C to the embedding mold, the embedding mold being a container without a bottom surface and a top surface;

[0028] 3) Put the plant tissue soaked in step 1) into the embedding mold, and add a second volume of cryo-embedding agent pre-cooled to 0°C to 4°C, the sum of the first volume and the second volume being greater than or equal to the volume of the embedding mold;

[0029] 4) Place another heat-conducting carrier having at least one plane pre-cooled to at least -70°C in contact with the upper part of the embedding mold with the plane and keep it for a period of time until all the cryo-embedding agent in the embedding mold solidifies.

[0030] In the method of the present invention (including the methods described below), the various steps involved are numbered with numbers. However, it should be understood that the numerical numbers here are only for the purpose of distinction and are not intended to indicate a sequential relationship between the steps, unless such a sequential relationship can be determined according to the context of the present application or the method of the present invention. For example, in the method of the present invention, steps 1) and 2) can be carried out in any order, for example, step 1) is carried out first and then step 2); or, step 2) is carried out first and then step 1); or, steps 1) and 2) can be carried out simultaneously.

[0031] Step 1): Immerse the plant tissue to be cryo-embedded in a cryo-embedding agent pre-cooled to 0°C to 4°C, and infiltrate the plant tissue in the cryo-embedding agent for 10 minutes to 1 hour.

[0032] As described in the background art, in the existing plant tissue cryo-embedding method, without any other treatment, during the embedding process, only a layer of cryo-embedding agent is wrapped around the tissue for cryo-embedding. Since the seed surface is smooth, it is difficult for the tissue to be fully infiltrated and contacted with the cryo-embedding agent. During the cryosectioning process, the tissue and the cryo-embedding agent do not adhere firmly and are easily separated. In response to this, the method of the present invention includes infiltrating the plant tissue to be cryo-embedded in the cryo-embedding agent for 10 minutes to 1 hour, such as 20 minutes, 30 minutes, 40 minutes or 50 minutes or any time period therebetween, depending on the specific situation. By this step, the separation between the tissue and the cryo-embedding agent during sectioning can be reduced, and thus the wrinkling and curling of the tissue sections can be reduced.

[0033] The plant tissue can be any plant tissue suitable for the method of the present invention. In one embodiment, the plant tissue is a seed from a leguminous plant or a fruit, stem, or leaf from a solanaceous plant or a cucurbitaceous plant. In a preferred embodiment, the leguminous plant is soybean, pea, broad bean, peanut, or lentil. In a preferred embodiment, the solanaceous plant is eggplant, tomato, potato, or wolfberry. In a preferred embodiment, the cucurbitaceous plant is cucumber, towel gourd, pumpkin, wax gourd, zucchini, or calabash.

[0034] In one embodiment, the cryo-embedding agent is OCT cryo-embedding agent.

[0035] OCT (optimal cutting temperature compound) embedding agent is a water-soluble mixture of polyethylene glycol and polyvinyl alcohol. It has been widely used in immunohistochemistry laboratories. Its use is to support the tissue during frozen sectioning to increase the continuity of the tissue, reduce wrinkles and fragmentation. Also, because the OCT mixture is water-soluble, it can dissolve in water during floating of the slides, so it will not increase background staining in subsequent staining.

[0036] In one embodiment, the pre-cooling of the cryo-embedding agent can be carried out by pre-cooling on ice or in a 4°C refrigerator. In a preferred embodiment, the pre-cooling of the cryo-embedding agent is pre-cooling on ice.

[0037] In one embodiment, the plant tissue is the seed of a leguminous plant, especially a dry seed, and the cryo-embedding method further includes step 1') before step 1): soaking the seed in water for a period of time to germinate it, and then removing the seed coat from the seed. After removing the seed coat, sufficient infiltration of the seed with the cryo-embedding agent can reduce the separation between the tissue and the cryo-embedding agent during sectioning.

[0038] Step 2): Place a heat-conducting carrier having at least one plane pre-cooled to at least -70 °C with the plane facing up, then place the embedding mold on the upward-facing plane of the heat-conducting carrier, and add a first volume of cryo-embedding agent pre-cooled to 0 °C to 4 °C to the embedding mold. The embedding mold is a container without a bottom surface and a top surface.

[0039] The so-called "heat-conducting carrier" refers to a carrier made of a material with high thermal conductivity that can effectively transfer heat. In one embodiment, the heat-conducting carrier having at least one plane is a heat-conducting metal block, including an aluminum block, an iron block, or a copper block, but not limited thereto. Any other heat-conducting carrier capable of conducting heat is within the scope of consideration of the present invention. In an exemplary embodiment, the heat-conducting carrier is an aluminum ice box.

[0040] In one embodiment, the pre-cooling of the heat-conducting metal carrier can be carried out by dry ice pre-cooling, liquid nitrogen pre-cooling, -80 °C refrigerator pre-cooling, or liquid nitrogen isopentane pre-cooling. In a preferred embodiment, the pre-cooling of the heat-conducting metal carrier is dry ice pre-cooling.

[0041] The embedding mold can be made of any suitable material. In one embodiment, the embedding mold is made of plastic, metal, or kraft paper.

[0042] It can be understood that a suitable freezing rate is desired. On the one hand, it is hoped that no excessive ice crystals are generated at such a freezing rate, and on the other hand, it is hoped that such a freezing speed can be fast enough to complete the embedding as soon as possible.

[0043] In the present invention, the freezing rate depends on the height of the embedding mold on the one hand. Therefore, in one embodiment, the embedding mold is configured to be at least 4 mm to 5 mm higher than the plant sample to be embedded. In an exemplary embodiment, the height of the embedding mold is 1 - 2 cm.

[0044] In the present invention, the freezing rate also depends on the pre-cooling temperature of the heat-conducting carrier. Therefore, in one embodiment, the heat-conducting carrier is pre-cooled to a temperature of at least -70 °C.

[0045] In addition, it can also be understood that the area of the bottom or top surface of the embedding mold should be less than or equal to the area of the plane of the heat-conducting carrier; otherwise, it is difficult to achieve effective cryo-embedding of plant tissues. Additionally, it can be understood that there are no special restrictions on the shape of the embedding mold, as long as it can accommodate plant tissues inside. In one embodiment, the embedding mold can be a cylindrical or rhombic columnar container without a bottom and a top surface.

[0046] In one embodiment, in step 2), the heat-conducting carrier is placed on the cold source with the plane facing upwards. In a preferred embodiment, the cold source is dry ice or liquid nitrogen.

[0047] It should be understood that preferably, step 3) is carried out immediately after completing step 2), at which time the first volume of the cryo-embedding agent has not completely solidified. In this way, it can be ensured that the whole plant tissue fits better with the cryo-embedding agent.

[0048] Step 3): Place the plant tissue infiltrated in step 1) into the embedding mold, and add a second volume of the cryo-embedding agent pre-cooled to 0°C to 4°C. The sum of the first volume and the second volume is greater than or equal to the volume of the embedding mold.

[0049] In this step, for the pre-cooling of the cryo-embedding agent, reference can be made to the relevant content in step 1), which will not be elaborated here.

[0050] It can be understood that before placing the plant tissue into the embedding mold, the plant tissue can be appropriately processed, such as washing it with sterile water and drying the moisture on the tissue surface.

[0051] After placing the plant tissue into the embedding mold, the position of the plant tissue in the embedding mold can be appropriately adjusted so that it is preferably located at the center of the mold.

[0052] Regarding the first volume and the second volume of the cryo-embedding agent, it should be noted that there are no special requirements for the first volume and the second volume in this article, as long as the sum of the two is greater than or equal to the volume of the embedding mold, so as to better conduct heat. In one embodiment, the first volume can be 1 / 3 to 1 / 2 of the volume of the embedding mold, and the second volume is 2 / 3 to 1 / 2 or even more of the volume of the embedding mold. As an example, the first volume can be 1 / 3 of the volume of the embedding mold, and the second volume is 2 / 3 or more of the volume of the embedding mold.

[0053] After adding the second volume of the pre-cooled cryo-embedding agent, it is necessary to ensure that no bubbles are generated in the cryo-embedding agent. In the case of bubble generation, tools such as a medicine spatula are needed to pick out the bubbles.

[0054] Step 4): Place another heat-conducting carrier with at least one plane pre-cooled to at least -70°C above the embedding mold in such a way that the plane contacts the upper part of the embedding mold and continue for a period of time until all the frozen embedding agent in the embedding mold solidifies.

[0055] In one embodiment, during the embedding process, it is necessary to timely clean the frost that may form on the surface of the heat-conducting carrier.

[0056] Different from the single-sided freezing on the quick-freezing table of a cryostat in the prior art, the method of the present invention is double-sided freezing from the top and bottom surfaces of the embedding mold. Through this freezing method, the freezing of the plant tissue to be embedded can be achieved faster with substantially no ice crystals formed.

[0057] In a second aspect, the present invention provides a method for preparing frozen sections of plant tissues, wherein the method comprises:

[0058] a) Performing the method as described in the first aspect of the present invention to obtain the embedded plant tissue;

[0059] b) Placing the embedded plant tissue on a cryostat and performing frozen sectioning on it.

[0060] It can be understood that for the implementation of step a), reference can be made to the description of the method for frozen embedding of plant tissues in the first aspect of the present invention above, and details will not be repeated here.

[0061] In addition, for step b), conventional frozen sectioning methods in the art can be used, and there are no special requirements for it. In one embodiment, the sectioning direction and section are adjusted according to the actual research purpose during sectioning.

[0062] In a third aspect, there is provided the use of the frozen sections prepared by the method for preparing frozen sections of plant tissues described in the second aspect of the present invention in spatio-temporal omics analysis, tissue cell observation, in situ hybridization, immunofluorescence localization, or histochemical reaction.

[0063] Through the frozen embedding and sectioning method of the present invention, frozen embedding blocks in which the tissue and the frozen embedding agent are not likely to separate during sectioning and have good adhesion can be obtained; frozen sections of plant tissues with complete structure and good morphology can be obtained; and tissue freezing and embedding can be carried out without relying on a cryostat.

[0064] Examples

[0065] Unless otherwise specified, the methods in the examples are all conventional methods, and the reagents used are all conventional commercially available reagents or reagents prepared according to conventional methods.

[0066] Example 1: Spatiotemporal Omics Study of Soybean Seeds

[0067] 1. Pretreatment of soybean seeds: Soak dry soybean seeds and germinate them for 96 hours for later use.

[0068] 2. Embedding and sectioning: Embed the germinated soybean seeds soaked as follows:

[0069] 1) Preparation before experiment : Pre-cool the cryoembedding agent OCT on ice. Place a 90-mm-diameter cell culture dish filled with sterile water on ice for pre-cooling, and also place a 60-mm-diameter cell culture dish filled with cryoembedding agent OCT on ice for pre-cooling. Additionally, pre-cool two 96-well aluminum ice boxes in dry ice for later use.

[0070] 2) Mold preparation : Take a 50-ml disposable sterile syringe, and use a craft knife to cut its outer sleeve into a hollow cylinder with a height of 1 - 2 cm (determined according to the tissue size) as the embedding mold for later use.

[0071] 3) Sample preparation : Use flat-headed wide-mouth forceps to put the soybean seeds germinated for 96 hours into a 90-mm-diameter cell culture dish filled with sterile water for cleaning. After cleaning, remove and wash its seed coat, then gently dry the water on the surface of the soybean seeds with lint-free paper, and then put them into a pre-cooled 60-mm-diameter cell culture dish filled with cryoembedding agent OCT for infiltration for 10 min. Note: During infiltration, ensure that the tissue is surrounded by cryoembedding agent OCT and the cryoembedding agent OCT completely covers the tissue.

[0072] 4) Sample embedding : Place a 96-well aluminum ice box face down on dry ice, put the prepared embedding mold on the flat surface of the back of the ice box, add about one-third volume of cryoembedding agent OCT to the mold, immediately put the infiltrated soybean seeds into the mold, gently adjust the position of the soybean seeds with forceps or a spatula and fill the mold with cryoembedding agent OCT. Pay attention to observing whether bubbles are generated during this process. If bubbles are generated, use a medicine spatula to pick out the bubbles in the cryoembedding agent OCT in time. Then, cover the mold with another 96-well aluminum ice box pre-cooled in dry ice and let it stand until the cryoembedding agent in the mold completely solidifies. Then, remove the ice box and the embedded block, and the required sample name, sectioning direction and other information can be marked on the surface of the embedded block or the mold, that is, the embedding is completed (the frost on the surface of the ice box needs to be cleaned in time during the embedding process). The embedded tissue block can be directly sectioned or stored in a -80 °C refrigerator for later use.

[0073] 5) Sectioning operation:Put the freshly embedded tissue blocks or the embedded tissue blocks taken out from the -80°C refrigerator into the cryostat. (In the case of taking out from the -80°C refrigerator, first equilibrate and rewarm the embedded tissue on the cryostat for at least half an hour), and then section. After the soybean cotyledons and embryos are cut simultaneously, the section collection can begin. Note that the sectioning direction and section surface can be adjusted according to the actual research purpose.

[0074] 3. Microscopic detection: Collect a section and attach it to an adhesive glass slide, and use a microscope to detect the integrity of the tissue cell morphology and structure.

[0075] Figure 1 To show the microscopic bright-field image of the frozen section of the soybean embryo tissue prepared by the method of the present invention under a 10× objective lens, the section thickness is 10 μm. It can be seen from the figure that the cotyledons and hypocotyls of the soybean embryo tissue have complete morphological structures and clear tissue textures, and can be used for subsequent RNA analysis.

[0076] Figure 2 The microscopic bright-field image of the frozen section of the soybean embryo tissue prepared by the method without removing the seed coat under a 10× objective lens, the section thickness is 10 μm. It can be seen from the figure that the soybean embryo embedded by this method is separated from the seed coat, and at the same time, some tissues of the section are wrinkled, and the effect is poor, which is not suitable for continued subsequent RNA analysis.

[0077] Figure 3 The microscopic bright-field image of the frozen section prepared by the direct embedding method without infiltration under a 10× objective lens, the section thickness is 10 μm. It can be seen from the figure that the soybean embryo tissue embedded by this method is severely separated from the OCT, and the section morphology is uneven, which is not suitable for continued subsequent RNA analysis.

[0078] Figure 4 The microscopic bright-field image of the frozen section prepared by the embedding method using dry ice instead of two heat conduction carriers under a 10× objective lens, the section thickness is 10 μm. It can be seen from the figure that the tissue of the soybean embryo embedded by this method is partially separated from the OCT, and the section morphology is partially fragmented, and it is also not suitable for continued subsequent RNA analysis.

[0079] From the comparison of the above results, it can be seen that the removal of the seed coat, the pre-infiltration of the embedding agent, and the use of two heat conduction carriers in the embedding method of the present invention can effectively obtain frozen sections of soybean embryo tissue with complete morphological structures and clear tissue textures, which is beneficial to subsequent RNA analysis.

[0080] Further collect 10 - 20 frozen sections of soybean embryo tissue, extract the total RNA of the sections using the Trizol method, and use a fully automatic nucleic acid fragment analyzer Qsep400 (or the same type of detection instrument) to detect the RNA integrity, and the results are shown in Figure 5As can be seen from this figure, the 18S and 28S rRNA bands of the soybean embryo tissue processed by the method of the present invention are clear, and the RNA integrity is relatively high.

[0081] 4. Permeabilization: After passing the RNA quality inspection, use a cryostat to section the sample, attach the sections to the BGI STOmics fluorescence chip, and use the STOmics fluorescence reagent kit for permeabilization testing. Use a fluorescence microscope to determine the results of the permeabilization test and select an appropriate permeabilization time.

[0082] 5. Library construction: After selecting an appropriate permeabilization time, use a cryostat to section the sample, attach the sections to the STOmics gene expression chip, and use the STOmics gene expression reagent kit (BGI) for library construction to obtain cDNA (concentration: 26.6 ng / μL). After fragmenting and constructing the library with the cDNA, sequence it on the DNBSEQ-T1 platform.

[0083] The reagents and consumables used in Example 1 are shown in the following table.

[0084]

Claims

1. A method for cryo-embedding plant tissues, the method comprises the following steps: 1) Immerse the plant tissues to be cryo-embedded in a cryo-embedding agent pre-cooled to 0°C to 4°C, and soak the plant tissues in the cryo-embedding agent for 10 minutes to 1 hour; 2) Place a heat-conducting carrier with at least one plane pre-cooled to at least -70°C with the plane facing upwards, then place an embedding mold on the upward-facing plane of the heat-conducting carrier, and add a first volume of cryo-embedding agent pre-cooled to 0°C to 4°C to the embedding mold. The embedding mold is a container without a bottom surface and a top surface, such as a cylindrical or prismatic container without a bottom surface and a top surface; 3) Put the plant tissues soaked in step 1) into the embedding mold, and add a second volume of cryo-embedding agent pre-cooled to 0°C to 4°C. The sum of the first volume and the second volume is greater than or equal to the volume of the embedding mold; 4) Place another heat-conducting carrier with at least one plane pre-cooled to at least -70°C in contact with the upper part of the embedding mold with the plane and keep it for a period of time until all the cryo-embedding agent in the embedding mold solidifies.

2. The method for cryo-embedding plant tissues according to claim 1, wherein the plant tissues are seeds from leguminous plants or fruits, stems, and leaves from solanaceous plants and cucurbitaceous plants; preferably, the leguminous plants are soybeans, peas, broad beans, peanuts, and lentils, the solanaceous plants are eggplants, tomatoes, potatoes, and wolfberries, and the cucurbitaceous plants are cucumbers, loofahs, pumpkins, wax gourds, zucchinis, and gourds.

3. The method for cryo-embedding plant tissues according to claim 1, wherein the plant tissues are seeds of leguminous plants, especially dry seeds, and the cryo-embedding method further comprises step 1') before step 1): soak the seeds in water for a period of time to germinate them, and then peel off the seed coats from the seeds.

4. The method for cryo-embedding plant tissues according to any one of claims 1-3, wherein the embedding mold is made of plastic, metal, or kraft paper, and the embedding mold is configured to be at least 4 mm to 5 mm higher than the plant sample to be cryo-embedded.

5. The method for cryo-embedding plant tissues according to any one of claims 1-4, wherein, the heat-conducting carrier with at least one plane is a heat-conducting metal block, such as an aluminum block, an iron block, or a copper block.

6. The method for cryo-embedding plant tissues according to any one of claims 1-5, wherein the cryo-embedding agent is an OCT cryo-embedding agent.

7. The method for cryo-embedding plant tissues according to any one of claims 1-6, wherein the pre-cooling of the cryo-embedding agent is carried out by pre-cooling on ice or in a 4°C refrigerator; the pre-cooling of the heat-conducting carrier is carried out by pre-cooling with dry ice, liquid nitrogen, in an -80°C refrigerator, or by pre-cooling with liquid nitrogen and isopentane.

8. The method for cryo-embedding plant tissues according to any one of claims 1-7, wherein in step 2), the heat-conducting carrier is placed on a cold source with the plane facing upwards. Preferably, the cold source is dry ice or liquid nitrogen.

9. A method for preparing frozen sections of plant tissues, wherein the method comprises: a) performing the method according to any one of claims 1-8, thereby obtaining embedded plant tissues; b) placing the embedded plant tissues on a cryostat and performing cryosectioning thereon.

10. Use of the frozen sections prepared by the method for preparing frozen sections of plant tissues according to claim 9 in spatio-temporal omics analysis, tissue cell observation, in situ hybridization, immunofluorescence localization, or histochemical reaction.

Images