Plant tissue embedding method, method for preparing plant space-time transcriptome sample and application of plant space-time transcriptome sample

Abstract

The invention provides an embedding method of a plant tissue, so that a space-time transcriptome slice sample of the plant tissue which is complete in slice, clear in cell structure and complete in cell tissue is obtained, and high-quality space-time transcriptome data can be obtained through space-time transcriptome sequencing. The method provided by the invention can be used for spatio-temporal transcriptome slice samples of various plants which are prone to slice rupture, can be well applied to a spatio-temporal transcriptome sequencing technology of plant tissues, has good stability, widens the application range of the spatio-temporal transcriptome technology, and has good application prospects. And an important research means is provided for multiple fields such as tissue cell functions, microenvironment interaction and development process pedigree tracking.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a method for embedding plant tissues, a method for preparing plant spatio-temporal transcriptome samples, and the application of the plant spatio-temporal transcriptome samples prepared by this method in plant spatio-temporal transcriptome sequencing. Background Art

[0002] As the Method of the Year 2020 by Nature Methods, spatio-temporal transcriptomics is a technology dedicated to quantifying transcriptional information corresponding to spatial positions. In recent years, a large number of scientific journals have successively reported major discoveries and breakthroughs of this technology in different fields. This technology will bring detailed spatial information based on traditional transcriptome sequencing, helping researchers identify the location of transcription within tissues, with a resolution accurate to the cellular level or even higher, which will promote researchers' understanding and interpretation of individual cells and cell populations within the entire tissue.

[0003] The combination of the number of countless cells with different functional types and developmental (time) and regional (space) differences constitutes the main component of transcriptional heterogeneity in biological tissues. Spatial heterogeneity is a key feature of organ function, and the positional information of cells is very important for the study of cell fate regulation mechanisms and cell lineage development processes. Therefore, in order to better understand cells, it is necessary to record their transcriptional heterogeneity and spatial coordinates simultaneously. Currently, spatio-temporal transcriptomics technology loads probes with spatial position information onto a biochip at the nanoscale resolution through sequencing. These biochips capture target nucleic acids or proteins in biological tissues, and then convert the captured information into a nucleotide sequence with a corresponding relationship through biochemical reactions and recover it. Finally, the captured information and spatial coordinates are mapped one by one through next-generation sequencing.

[0004] The main challenge hindering current spatial transcriptomics technology is that the imaging results of tissue sections are prone to section breakage problems. The reasons for section breakage are as follows: The current sample preparation methods for spatial transcriptomics mainly involve cryo-embedding, that is, fresh samples are directly cryo-embedded with an embedding agent under low-temperature conditions without any pretreatment. Direct embedding of fresh samples is relatively recommended to avoid degradation during other operations. However, the widely used cryo-embedding is not friendly to most plant samples because plant samples have structures such as vacuoles and generally have a higher water content than animal tissues. During cryo-embedding, ice crystals are easily generated, causing section breakage and making it impossible to obtain good sectioning results. Moreover, tissues with internal breakage are prone to low cell integrity problems during the experiment of spatial transcriptomics sectioning. Therefore, in the plant field, due to the characteristics of plant tissues, it is difficult to obtain high-quality sections and transcriptome libraries simultaneously, which has certain limitations. For some plant tissues, the problems of poor section quality and low cell integrity caused by section breakage are particularly prominent and difficult to solve, directly affecting the results of spatial transcriptomics.

[0005] Currently, there are sample preparation methods for pre-treating samples before embedding to reduce section breakage. Common pre-treatments include dehydration treatments, such as low-concentration sucrose and glycerol gradient dehydration treatments, which can reduce section breakage to a certain extent, but their effects are limited. In addition, paraffin embedding is also commonly used in the sample preparation of spatial transcriptomics technology, that is, paraffin sections are obtained by conventional paraffin embedding methods for spatial transcriptomics experiments. The advantage of paraffin sections is that the sections are complete and the cell structure is clear. However, the adhesion of paraffin sections on the spatial sequencing chip is extremely poor, and they are very prone to displacement. Moreover, since conventional paraffin embedding usually uses FAA (formaldehyde-acetic acid-alcohol) or PFA (4% paraformaldehyde) fixatives for long-term fixation, tissues fixed for a long time often have the situation where RNA is fixed and difficult to release. In addition, the transparency and wax infiltration steps in paraffin embedding usually need to be carried out at high temperatures, which will cause serious degradation of RNA, resulting in low RNA abundance and ultimately unable to obtain good-quality data; and the paraffin embedding protocol is cumbersome and time-consuming. Moreover, using other methods to process samples also has the risk of fixation, making it difficult to release RNA, which is not conducive to spatial transcriptomics sequencing.

[0006] Therefore, in order to solve the problems of easy section breakage and incomplete cell structure in direct freezing in the existing sample preparation of spatial transcriptomics, as well as the problems of difficult release of RNA from spatial samples, poor library quality, poor adhesion on the spatial chip, easy tissue displacement, and complex and time-consuming paraffin embedding in paraffin embedding, it is necessary to develop new sample preparation or treatment methods to enable the research on difficult samples with easy section breakage problems to be carried out, thereby broadening the application scope of spatial transcriptomics technology. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and deficiencies existing in the above-mentioned prior art, and aims to provide an embedding method for plant tissues and a corresponding method for making plant spatio-temporal transcriptome samples. Through multiple experiments, the inventors of the present invention found that when plant tissues are fixed briefly with Carnoy's fixative and subjected to gradient infiltration and embedding with polyester wax, plant spatio-temporal transcriptome samples with complete sections, clear cell structures, and intact cell tissues can be obtained, thus completing the present invention.

[0008] Therefore, in the first aspect, the present invention provides an embedding method for plant tissues, and the method includes:

[0009] (1) Fixing the plant tissue with Carnoy's fixative for 15 minutes to 25 minutes, preferably 15 minutes;

[0010] (2) Dehydrating the fixed plant tissue with a dehydrating agent;

[0011] (3) Subjecting the dehydrated plant tissue to gradient infiltration with polyester wax for 0.5 hour to 1 hour, preferably 1 hour, for each gradient;

[0012] (4) Embedding the plant tissue subjected to gradient infiltration with polyester wax.

[0013] In the second aspect, the present invention provides a method for making a plant spatio-temporal transcriptome sample, and the method includes:

[0014] i. Executing the method of the first aspect of the present invention to obtain the embedded plant tissue;

[0015] ii. Sectioning, mounting, dewaxing, fixing with methanol, and permeabilizing the embedded plant tissue.

[0016] In the third aspect, the present invention provides the application of the plant spatio-temporal transcriptome sample made by the method of the second aspect of the present invention in plant spatio-temporal transcriptome sequencing.

[0017] The present invention has one or more of the following beneficial effects:

[0018] The present invention provides a method for embedding plant tissues based on a polyester wax embedding agent to increase the number of intact cells. The method includes briefly fixing plant tissues with Carnoy's fixative and performing gradient infiltration and embedding of the fixed plant tissues with a polyester wax embedding agent, followed by sectioning, mounting, dewaxing, methanol fixation, and permeabilization, thereby obtaining a plant spatiotemporal transcriptome sample. The sections obtained by the method of the present invention are complete, and the section quality has been significantly improved compared with direct cryoembedding. In addition, the method of the present invention has good application for spatiotemporal transcriptome samples with the problem of easy section breakage, and has good effects in the production of various plant samples, can be well applied to plant spatiotemporal transcriptome experiments, and has stability.

[0019] Based on the brief fixation with Carnoy's fixative and the use of a polyester wax embedding agent, the present invention is simple, fast to operate, and can help experimenters obtain high-quality plant spatiotemporal transcriptome data. Compared with the common cryoembedding and paraffin embedding in the prior art, the advantages of the present invention are as follows:

[0020] (1) The steps of the embedding method of the present invention are more simplified, easier to operate, and take less time than those of the prior art methods. For example, the brief fixation with Carnoy's fixative does not cause nucleic acid crosslinking, and thus no subsequent de-crosslinking step is required. For another example, the polyester wax embedding agent used has a low melting point and is miscible with most organic solvents (including alcohols, ethers, esters, ketones, and hydrocarbons). Therefore, after dehydration, the plant tissues can be directly infiltrated with polyester wax as a support agent into the plant tissues without the need for a clearing agent transition.

[0021] (2) Compared with the sections obtained by conventional cryoembedding, the sections of the plant spatiotemporal transcriptome samples prepared based on the polyester wax embedding agent obtained by the present invention are complete and do not affect RNA release, and high-quality spatiotemporal transcriptome data can be obtained. Under the same tissue area, the number of intact cells is higher than that of paraffin sections, and thus a higher number of captured genes can be obtained.

[0022] (3) Compared with xylene used as a dewaxing agent in a conventional paraffin embedding protocol, methanol is used as the dewaxing agent in the present invention. Since wax can hinder the penetration of the permeabilization reagent into the tissue, thereby inhibiting the contact between proteinase K and the protein in the tissue, affecting tissue permeabilization and nucleic acid release. To eliminate the adverse effects of wax on DNA extraction and PCR amplification, the tissue needs to be thoroughly dewaxed. Xylene used as a dewaxing agent in a conventional paraffin embedding protocol is likely to cause changes such as tissue shrinkage, hardening and embrittlement during tissue processing if used improperly, which has a certain impact on the quality of tissue sections. However, methanol used in the present invention can better maintain the integrity of tissue morphology and avoid situations such as tissue morphological deformation. Brief Description of the Drawings

[0023] Figure 1It is a flow chart of a spatial-temporal transcriptome experiment involving the embedding method of the present invention.

[0024] Figure 2 It is a flow chart of a spatial-temporal transcriptome experiment involving the conventional paraffin embedding method.

[0025] Figure 3 It is a standard flow chart of a spatial-temporal transcriptome experiment involving the conventional cryo-embedding method.

[0026] Figure 4 It is an overall comparison diagram of sections obtained from cryo-embedded corn stems (control group) and sections obtained from polyester wax-embedded corn stems (test group). Among them, A and B are the bright-field images and FB (Fluorescent Brightener) images of the control group respectively, and C and D are the bright-field images and FB images of the test group respectively.

[0027] Figure 5 It is an enlarged comparison diagram of sections obtained from cryo-embedded corn stems (control group) and sections obtained from polyester wax-embedded corn stems (test group). Among them, A and B are the bright-field images and FB images of the control group respectively, and C and D are the bright-field images and FB images of the test group respectively.

[0028] Figure 6 It is a partial comparison diagram of sections obtained from cryo-embedded soybean leaves (control group) and sections obtained from polyester wax-embedded soybean leaves (test group). Among them, A and B are the bright-field images and FB images of the control group respectively, and C and D are the bright-field images and FB images of the test group respectively.

[0029] Figure 7 It is an enlarged comparison diagram of sections obtained from cryo-embedded soybean leaves (control group) and sections obtained from polyester wax-embedded soybean leaves (test group). Among them, A and B are the bright-field images and FB images of the control group respectively, and C and D are the bright-field images and FB images of the test group respectively.

[0030] Figure 8 It is an overall comparison diagram of sections obtained from cryo-embedded Arabidopsis stems (control group) and sections obtained from polyester wax-embedded Arabidopsis stems (test group). Among them, A and B are the bright-field images and FB images of the control group respectively, and C and D are the bright-field images and FB images of the test group respectively.

[0031] Figure 9 It is an enlarged comparison diagram of sections obtained from cryo-embedded Arabidopsis stems (control group) and sections obtained from polyester wax-embedded Arabidopsis stems (test group). Among them, A and B are the bright-field images and FB images of the control group respectively, and C and D are the bright-field images and FB images of the test group respectively. Detailed implementation manners

[0032] As described above, the sections obtained by direct freezing embedding are prone to breakage, with complex operations, long time consumption, and poor section quality. For paraffin embedding, it is difficult to release RNA from the sections, the library quality is poor, the adhesion to the spatial-temporal chip is poor, tissue displacement is likely to occur, and the paraffin embedding operation is complex and time-consuming. In view of the problems existing in the existing embedding techniques, the inventors of the present invention conducted repeated studies and unexpectedly found that when using Carnoy's fixative for short-term fixation and polyester wax for infiltration and embedding, a plant spatial-temporal transcriptome sample with complete sections, clear cell structures, and intact cell tissues can be obtained, thus completing the present invention.

[0033] Therefore, in a first aspect, the present invention provides a method for embedding plant tissues, the method comprising:

[0034] (1) Fixing the plant tissues with Carnoy's fixative for 15 to 25 minutes, preferably 15 minutes;

[0035] (2) Dehydrating the fixed plant tissues with a dehydrating agent;

[0036] (3) Infiltrating the dehydrated plant tissues with polyester wax in each gradient for 0.5 to 1 hour, preferably 1 hour;

[0037] (4) Embedding the plant tissues that have been infiltrated in gradients with polyester wax.

[0038] The following will describe in detail each step included in the method of the first aspect of the present invention.

[0039] Step (1): Fixation of plant tissues

[0040] The method of the present invention is particularly applicable to plant tissues, or rather, the method of the present invention is mainly proposed for plant tissues. The plant tissues used for making tissue sections are preferably fresh and intact to help reflect the true mRNA expression. Any tissue of any plant can be used in the method of the present invention. By way of example, the plant tissues may include, but are not limited to, tissues such as seeds, flowers, fruits, leaves, stems, root tissues of plants, and the like.

[0041] In the present invention, Carnoy's fixative is used to fix plant tissues. Carnoy's fixative, also known as Carnoy's Fluid / Carnoy's solution, is a fixative invented by the Belgian cytologist Jean Baptiste Carnoy in 1886. Carnoy's fixative is one of the earlier fixatives applied in histology and is a non-aqueous phase fixative prepared from alcohols (such as methanol, ethanol) and acetic acid (sometimes chloroform, i.e., trichloromethane, can also be added). This fixative needs to be prepared and used immediately because esterification reaction will occur after long-term storage, generating methyl acetate and affecting the fixing effect. Carnoy's fixative is mainly applicable to the fixation of general plant tissues and cells. It can fix cytoplasm and cell nuclei, especially suitable for the fixation of chromosomes, etc., and has a good effect on showing DNA and RNA.

[0042] Carnoy's fixative plays an important role in maintaining cell structure. Its formula is not unique, and different experimental purposes and tissue types may require different formulas and concentrations, which can be appropriately adjusted according to different material samples. There are usually two preparation methods for Carnoy's fixative. One is to mix absolute ethanol and acetic acid in different volume ratios such as 1:1, 2:1, 3:1, 4:1, and 3:2. The other is to mix absolute ethanol, chloroform, and acetic acid in a volume ratio of 6:3:1. The inventor of the present invention found that when using the Carnoy's fixative with the second formula, the tissue is prone to shrinkage and deformation during embedding, and is prone to rupture during sectioning, and the sections are dry, resulting in failure to section smoothly. It is speculated that this may be due to the inclusion of chloroform. In addition, when using the Carnoy's fixative with the first formula, except for the Carnoy's fixative prepared by mixing absolute ethanol and acetic acid in a volume ratio of 3:1, the Carnoy's fixatives prepared by mixing absolute ethanol and acetic acid in other volume ratios all have different degrees of problems and are not suitable for preparing plant spatial transcriptome samples.

[0043] Therefore, in one embodiment, Carnoy's fixative contains absolute ethanol and acetic acid in a volume ratio of 3:1.

[0044] In the prior art, when using Carnoy's fixative to fix plant tissues, it usually needs to be fixed overnight. In comparison, the method of the present invention only requires a short-term fixation of 15 minutes to 25 minutes (such as 15 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 25 minutes), especially 15 minutes, to obtain good sectioning effects, that is, it can maintain good tissue integrity and a high number of gene captures. At the same time, the method of the present invention can also greatly shorten the sample preparation time.

[0045] Thus, in one embodiment, the plant tissue is fixed for 15 to 25 minutes. In a preferred embodiment, the plant tissue is fixed for 15 minutes.

[0046] It should be noted that in this specification, when referring to a specific numerical value or numerical range, it should be understood that the end values of the numerical value or numerical range are modified by the term "about", and the term "about" can be defined as a range consisting of ±10% of the numerical value or end value.

[0047] In addition, to facilitate the penetration of the fixative into the plant tissue, the fixation is preferably carried out under vacuum conditions. Thus, in one embodiment, the fixation is carried out under vacuum conditions; preferably, the vacuum conditions are from -0.6 MPa to -0.8 Mpa.

[0048] It can be understood that the specific fixation time can be adjusted by ±1 hour according to the characteristics of the sample tissue. For example, for relatively tender tissue samples, the fixation time can be appropriately shortened, while for some relatively old tissue samples such as lignified and fibrotic tissues, the fixation time can be appropriately extended.

[0049] By using Carnoy's fixative to fix the plant tissue for a short time, nucleic acid cross-linking will not occur as in the case of using paraformaldehyde, so there is no need for a subsequent cross-linking removal step, which further simplifies the subsequent processing steps. For example, there is no need to perform the operation of cross-linking removal in the subsequent steps; at the same time, Carnoy's fixative can quickly penetrate cells, fix them and maintain the integrity of the chromosome structure, and can also enhance the basophilia of chromosomes, achieving excellent staining effects. In addition, compared with the prior art that usually uses Carnoy's fixative for overnight fixation, the method of the present invention only uses Carnoy's fixative for short-term fixation for 15 to 25 minutes, so the operation time is greatly shortened.

[0050] Step (2): Dehydration of plant tissues

[0051] The fixed plant tissue cannot be directly penetrated by wax, but the water in the plant tissue needs to be removed first. Commonly used dehydrating agents include ethanol, acetone, n-butanol, tert-butanol, etc., and a suitable dehydrating agent can be selected according to different sample characteristics. For example, for some tissue samples rich in cells, mild dehydrating agents such as n-butanol and tert-butanol should be used to avoid over-dehydration of the tissue; while for some tissue samples with relatively few cells, stronger dehydrating agents such as a mixed dehydrating agent of ethanol and acetone should be used to avoid insufficient dehydration of the tissue, resulting in the phenomenon that the tissue in the embedded wax block has different hardness from the wax block, or although a wax ribbon can be cut, the tissue in the wax ribbon is not in pieces.

[0052] In one embodiment, the method of the present invention can use ethanol such as absolute ethanol, acetone, n-butanol, tert-butanol, but is not limited thereto. Any suitable dehydrating agent can be used in the present invention.

[0053] In addition, dehydration can be carried out using dehydrating agent solutions with gradually increasing concentration gradients, or only a single concentration of dehydrating agent can be used. In the present invention, it is preferred to use absolute ethanol as the dehydrating agent, and only 100% absolute ethanol is used for dehydration because its dehydration effect is moderate and it can be miscible with the polyester wax used later, and thus there is no need to use a clearing agent for transition.

[0054] In the case of simply using 100% absolute ethanol for dehydration, this dehydration process can also be carried out multiple times. Therefore, in one embodiment, the dehydration includes: first, at least two dehydrations are carried out at 0°C to 4°C for 10 minutes to 50 minutes (such as 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes or 50 minutes) each time, preferably 50 minutes, and then at 30°C to 42°C (such as 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, 40°C, 42°C), preferably 42°C, at least one dehydration is carried out for 0 minutes to 50 minutes (such as 0 minutes, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes or 50 minutes) each time, preferably 50 minutes.

[0055] First of all, for the above-mentioned "first, at least two dehydrations are carried out at 0°C to 4°C for 10 minutes to 50 minutes, preferably 50 minutes each time", it should be understood that this method step is the dehydration carried out after replacing the Carnoy's fixative with the dehydrating agent after the fixation is completed. This dehydration can be carried out at least twice, such as twice, three times, four times, etc., and can be carried out, for example, on ice. And it can also be understood that the above-mentioned "then at 30°C to 42°C, preferably 42°C, at least one dehydration is carried out for 0 minutes to 50 minutes, preferably 50 minutes each time" is carried out under elevated temperature conditions, and its purpose is to better connect with the subsequent gradient infiltration with wax step. This step can be carried out, for example, in an oven at 42°C.

[0056] In addition, similar to the fixation step, in order to promote the penetration of the dehydrating agent into the plant tissue, it is preferred that the dehydration step is carried out under vacuum conditions. Therefore, in one embodiment, the dehydration is carried out under vacuum conditions; preferably, the vacuum conditions are -0.6 MPa to -0.8 MPa.

[0057] It can be understood that the specific dehydration time can be adjusted by ±1 hour according to the characteristics of the sample tissue. For example, for relatively tender tissue samples, the dehydration time can be appropriately shortened, while for some relatively old tissue samples such as lignified and fibrotic ones, the dehydration time can be appropriately extended.

[0058] Step (3) Gradient infiltration of plant tissues with wax:

[0059] After dehydrating the plant tissue, gradient infiltration with wax is then carried out. In this article, the so-called "gradient infiltration with wax" refers to soaking the tissue with wax solutions having gradually increasing concentration gradients, so as to gradually expel the dehydrating agent from the tissue and allow the wax to infiltrate into the tissue as a supporting agent, thereby hardening the tissue and wrapping the tissue.

[0060] In the present invention, polyester wax is used. Polyester wax is a polywax having similar sectioning properties to conventional paraffin wax.

[0061] As described above, the sections of cryoembedding are prone to breakage, complex in operation, time-consuming, and poor in section quality. To address this problem, the present inventors propose to carry out gradient infiltration with wax and embedding using polyester wax. On the one hand, the polyester wax embedding agent has a relatively low melting point and is soluble in organic solvents such as ethanol. Therefore, during the operation, the tissue can be infiltrated with wax and embedded without using a clearing agent to clear the tissue, thereby reducing the operation steps and time; and when sectioning, continuous ribbons can be formed, and it is not easy to break. The sections are more complete than the conventional cryoembedding sections, so the section quality is improved. On the other hand, polyester wax embedding does not require a crosslinking dissociation operation. Compared with conventional paraffin wax embedding sections, the operation is simple, time-consuming is short, and the library quality is good.

[0062] In one embodiment, the polyester wax can be prepared from PEG400 distearate and 1-hexadecanol at a weight ratio of 9:1. However, it can be understood that any other suitable polyester wax can also be used in the present invention.

[0063] In one embodiment, a dehydrating agent-polyester wax solution with the polyester wax concentration gradually increasing from greater than 0% to 100% is used for gradient infiltration with wax at 30°C to 45°C (such as 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, 40°C, 42°C, 45°C), preferably 45°C, for 0.5 hour to 1 hour (such as 30 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 1 hour) each time, preferably 1 hour. The polyester wax concentration can increase in a geometric ratio or a non-geometric ratio, depending on the specific situation. As an example, the polyester wax concentration can increase according to a concentration gradient composed of two or more concentrations among 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100%. In a preferred embodiment, three dehydrating agent-polyester wax solutions with polyester wax concentrations of 50%, 75% and 100% are used for gradient infiltration with wax. It should be noted that the so-called "polyester wax concentration" here refers to the proportion of polyester wax relative to the total volume of polyester wax and dehydrating agent. For example, for a "50% polyester wax concentration", it means that the dehydrating agent-polyester wax solution contains 50% polyester wax by volume.

[0064] During the gradient wax dipping process, additional incubation can better help remove the dehydrating agent. Therefore, preferably, when using 100% polyester wax for wax dipping, this step can be repeated at least once. In addition, when filtering the 50% dehydrating agent-50% polyester wax solution, boiling is likely to occur, so a layer of sealing film can be sealed at the mouth of the plastic tube and a few holes can be pierced on it to prevent the sample from splashing. Again, when filtering 100% polyester wax, the polyester wax is easy to solidify, and the oven temperature can be appropriately adjusted according to the room temperature.

[0065] It is understood that the specific wax immersion time can be adjusted by ±1 hour according to the characteristics of the sample tissue. For example, for relatively young tissue samples, the wax immersion time can be appropriately shortened, while for some relatively old tissue samples such as lignified and fibrotic tissues, the wax immersion time can be appropriately extended.

[0066] It should be noted that appropriate fixation time, dehydration time and wax immersion time are crucial to the present invention. The hardness of the slices will directly affect the quality of the slices. For example, if the slices are too soft, the support force is insufficient, and the slices cut out cannot be sliced; if the slices are too hard, the slices are easy to break, affecting the tissue integrity, and then affecting the data quality of subsequent RNA capture. A good slice should be a slice with no tissue detachment from the wax block, moderate hardness, and high tissue integrity.

[0067] In the present invention, the inventors tested the shortest fixation time of Carnoy's fixative and the best time for dehydration and wax immersion, and found that good results were obtained when the immersion treatment in Carnoy's fixative reached 15 minutes and the dehydration and wax immersion time was 6.5 hours.

[0068] In addition, similar to the above two steps, in order to promote the penetration of polyester wax into plant tissue, the gradient wax impregnation step is preferably performed under vacuum conditions. Therefore, in one embodiment, the gradient wax impregnation is performed under vacuum conditions; preferably, the vacuum conditions are -0.6 MPa to -0.8 MPa.

[0069] In a preferred embodiment, the method comprises:

[0070] (1') fixing the plant tissue using Carnoy's fixative under a vacuum condition of -0.6 MPa to -0.8 MPa for 15 to 30 minutes, preferably 15 minutes;

[0071] (2') using anhydrous ethanol as a dehydrating agent to dehydrate the fixed plant tissue under a vacuum condition of -0.6 MPa to -0.8 MPa, comprising:

[0072] a) replacing Carnoy's fixative with anhydrous ethanol and dehydrating the plant tissue at 0° C. to 4° C. for 10 to 50 minutes, preferably 50 minutes;

[0073] b) Replace the absolute ethanol with another absolute ethanol and dehydrate the plant tissue at 0 °C to 4 °C for 10 minutes to 50 minutes, preferably 50 minutes;

[0074] c) Replace the said another absolute ethanol with absolute ethanol preheated to 30 °C to 42 °C (e.g.,

[0075] 30 °C, 32 °C, 34 °C, 35 °C, 36 °C, 38 °C, 40 °C, 42 °C), preferably 42 °C, and dehydrate the plant tissue at 30 °C to 42 °C, preferably 42 °C for 0 minute to 50 minutes, preferably 50 minutes;

[0076] (3') Gradient impregnate the dehydrated plant tissue with polyester wax under a vacuum condition of -0.6 MPa to -0.8 MPa, including:

[0077] a') Replace the absolute ethanol with a 50% absolute ethanol - 50% polyester wax solution and impregnate at 30 °C to

[0078] 45 °C, preferably 45 °C for 0.5 hour to 1 hour, preferably 1 hour;

[0079] b') Replace the 50% absolute ethanol - 50% polyester wax solution with a 25% absolute ethanol - 75% polyester wax solution and impregnate at 30 °C to 45 °C, preferably 45 °C for 0.5 hour to 1 hour, preferably 1 hour;

[0080] c') Replace the 25% absolute ethanol - 75% polyester wax solution with 100% polyester wax solution and impregnate at

[0081] 30 °C to 45 °C, preferably 45 °C for 30 minutes to 2 hours;

[0082] d') Optionally, repeat step c') at least once;

[0083] (4') Embed the plant tissue subjected to gradient impregnation with polyester wax.

[0084] In a second aspect, the present invention provides a method for making a plant spatiotemporal transcriptome sample, the method including:

[0085] i. Perform the method of the first aspect of the present invention to obtain the embedded plant tissue;

[0086] ii. Section, mount, dewax, fix with methanol and permeabilize the embedded plant tissue.

[0087] In one embodiment, the dewaxing is dewaxing with methanol. For example, it can be carried out in the following manner: after the baking of the chip is completed, transfer the chip to methanol for dewaxing treatment until there is no visible solidified wax liquid on the chip after the methanol volatilizes.

[0088] The plant spatio-temporal transcriptome sample prepared by the method of the present invention has an increased number of intact cells.

[0089] When directly freezing, embedding, and sectioning tissues, tissue sections are prone to breakage, RNA is difficult to release, and the operation is complex, time-consuming, and prone to serious problems with poor section quality. Compared with existing methods, the present invention uses Carnoy's fixative for fixation and polyester wax for infiltration and embedding, and can obtain complete sections, with the section quality being significantly improved compared to direct freezing embedding.

[0090] In the third aspect of the present invention, there is provided the use of a plant spatio-temporal transcriptome sample prepared by the method of the second aspect of the present invention in plant spatio-temporal transcriptome sequencing.

[0091] In addition to the above steps, when using the plant spatio-temporal transcriptome sample for plant spatio-temporal transcriptome sequencing, many additional steps are also involved.

[0092] Figure 1 The spatio-temporal transcriptome experimental flow chart of the method of the present invention is shown. It can be seen from the figure that after embedding the tissue block, the subsequent steps further include sectioning the tissue, chip processing, tissue patching, tissue dewaxing, methanol fixation, staining and imaging, tissue permeabilization, reverse transcription, tissue removal, cDNA release, magnetic bead recovery, library construction, Make DNB, on-machine sequencing, and data analysis, so as to obtain the spatio-temporal distribution data of mRNA in the tissue.

[0093] Figure 2 The spatio-temporal transcriptome experimental flow chart of the conventional paraffin embedding method is shown. It can be seen from the figure that its Figure 1 process is basically the same, and the difference is that an additional "demethylation" step is added. As described above, this is due to the use of a fixative such as paraformaldehyde.

[0094] Figure 3 The spatio-temporal transcriptome experimental process of the conventional freezing embedding method is shown. It can be seen from the figure that its Figure 1 process is basically the same, and the difference is that it does not involve the tissue dewaxing step because no wax embedding is performed.

[0095] It should be understood that Figure 1 - 3 the method steps shown in

[0096] Examples

[0097] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0098] Materials and methods:

[0099] Preparation of polyester wax : The polyester wax is made of PEG 400 distearate and 1-hexadecanol with a volume ratio of 9:1. Specifically, the polyester wax is prepared through the following steps: melt 90 mL of PEG 400 distearate at 65 °C, and add 10 mL of 1-hexadecanol under stirring, and continuously stir for 6 hours to obtain the polyester wax.

[0100] Preparation of Carnoy's fixative : Mix absolute ethanol and acetic acid evenly according to a volume ratio of 3:1.

[0101] Related reagents : ssDNA Quantification Assay Kit QubitTM ssDNA Reagent (Invitrogen), PEG400 distearate (Sigma Aldrich, Cat.No.305413), 1-hexadecanol (Sigma, Cat.No.258741), ethanol, acetic acid, DEPC water, RNaseZap (Invitrogen), Imaging Reagent Image Reagent (MGI), STOmics Gene Expression Kit-S1, STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI), embedding cassette bottom mold (Shitai), OCT (Sakura), liquid nitrogen, isoparaffin (Sigma-Aldrich), 100% methanol (Sigma-Aldrich).

[0102] Experimental equipment : Microtome (Leica), compressed air tank (CINE EDC GEAR), PCR instrument (ProFlex PCRSystem), microscope (Mitoc PA53F36), Seq2000 sequencer (MGI), vacuum drying oven (ThermoScientific), constant temperature graphite plate (LICHEN).

[0103] Preparation of plant spatio - temporal transcriptome samples : Use embedding agents polyester wax (A), conventional paraffin (B), and OTC (C) to prepare plant spatio-temporal transcriptome samples respectively. The specific steps are as follows:

[0104] (1) Preliminary preparation: Prepare materials such as pre-cooled Carnoy's fixative, OCT, polyester wax, paraffin, constant-temperature graphite plate, absolute ethanol, acetic acid, 75% alcohol, methanol, embedding cassettes, forceps, etc.

[0105] (2) Material trimming : Take samples and trim the materials to fit the size of the embedding cassettes for subsequent operations.

[0106] (3) Sample fixation :

[0107] A. Fixation for the polyester wax-embedded sample protocol: Immerse the plant materials in Carnoy's fixative (absolute ethanol: acetic acid = 3:1, v / v) for 15 min. During the immersion process, apply vacuum to promote penetration. Observe whether the sample sinks to the bottom of the container (keep on ice). If it does not sink to the bottom, repeat this step. After determining that the sample has sunk to the bottom, replace the fixative with a new one and let it stand on ice for 1 h. The vacuum condition is -0.6 MPa to -0.8 Mpa for 15 min.

[0108] B. Fixation for the conventional paraffin-embedded sample protocol: Immerse the plant materials in Carnoy's fixative (absolute ethanol: acetic acid = 3:1, v / v) for 20 min. During the immersion process, apply vacuum to promote penetration. Observe whether the sample sinks to the bottom of the container (keep on ice). If it does not sink to the bottom, repeat this step. After determining that the sample has sunk to the bottom, replace the fixative with a new one and let it stand on ice for 1 h. The vacuum condition is 0 to 0.01 Mpa for 5 - 10 min.

[0109] C. Fixation for the conventional cryo-embedded sample protocol: Immerse the materials in Carnoy's fixative (absolute ethanol: acetic acid = 3:1, v / v) for 10 min to 30 min. During the immersion process, apply vacuum to promote penetration and change the fixative once in the middle. The vacuum condition is 0 to 0.08 Mpa for 5 - 10 min.

[0110] (4) Dehydration and gradient infiltration with wax : After aspirating and discarding the fixative in the fixed sample using a pipette or dropper, process it according to the following procedure:

[0111] A. Dehydration and gradient infiltration with polyester wax for the polyester wax-embedded sample protocol: (about 6.5 h):

[0112] ① Replace with absolute ethanol and perform vacuum filtration on ice for 50 min;

[0113] ② Replace with absolute ethanol and perform vacuum filtration on ice for 50 min;

[0114] ③ Replace with absolute ethanol preheated to 42 °C 10 min in advance and perform vacuum filtration in a 42 °C oven for 50 min;

[0115] ④ Replace with a 50% absolute ethanol - 50% polyester wax solution and perform vacuum filtration in a 45 °C oven for 1 h;

[0116] ⑤ Replace with a 25% absolute ethanol - 75% polyester wax solution and perform vacuum filtration in an oven at 45°C for 1 hour;

[0117] ⑥ Replace with a 100% polyester wax solution and perform vacuum filtration in an oven at 45°C for 1 hour;

[0118] ⑦ Replace with a 100% polyester wax solution and perform vacuum filtration in an oven at 45°C for 1 hour;

[0119] B. Dehydration, clearing, and infiltration of samples using the conventional paraffin embedding protocol (approx. 5h):

[0120] ① Wash the acetic acid residue from the fixative on the sample with 100% ethanol, transfer the sample to a special embedding cassette for paraffin sections, fasten the cassette, and label the sample name and treatment conditions.

[0121] ② Preheat the paraffin embedding machine in advance and place the embedding cassette in the heated insulation chamber of the machine (60°C - 65°C) in advance.

[0122] ③ Space the paraffin embedding cassettes apart, insert them into the cassette rack, and place the rack in a pre - opened clearing and infiltration machine for clearing and infiltration treatment. The specific process is as follows:

[0123] 100% ethanol for 40 min × 3;

[0124] Histo - Clear II (National Diagnostics) for 30 min × 3;

[0125] Paraffin for 30 min × 3

[0126] (5) Sample embedding :

[0127] A. Embedding of samples using the polyester wax embedding protocol:

[0128] ① Pour the vacuum - filtered sample and liquid together into a suitable petri dish, place it on a 45°C constant - temperature graphite plate (to prevent the wax liquid from solidifying) and wait for embedding.

[0129] ② Pipette an appropriate amount of 100% polyester wax into a suitable - sized embedding cassette, carefully pick up the sample with forceps, adjust the arrangement direction and placement method of the sample according to the required section plane during sectioning, and make the sample evenly arranged in the embedding cassette.

[0130] ③ Transfer the embedding cassette with the adjusted sample position to ice for cooling and solidification, and after solidification, place it in a sealed bag and store it in a 4°C refrigerator.

[0131] B. Conventional paraffin embedding:

[0132] ① Take an embedding cassette or culture dish of appropriate size, adjust the flow rate of the paraffin liquid, and collect the paraffin liquid with the embedding cassette or culture dish.

[0133] ② Use long forceps to take out the paraffin embedding cassette from the transparent wax impregnator and place it on a heating table (60°C - 65°C). Gently and quickly transfer the sample to the small embedding cassette on the heating table with heat forceps kept at 60°C - 65°C. When adjusting the sample position, pay attention to frequently change the preheated forceps to prevent the paraffin from solidifying when exposed to air. Arrange the samples as quickly as possible according to the section plane required for sectioning.

[0134] ③ The embedding container with the sample position adjusted can be transferred to a cooling table for cooling and solidification. After solidification, cover it with a lid or place it in a container with a lid, and store it in a 4°C refrigerator.

[0135] C. Conventional cryoembedding:

[0136] ① Mark information such as the material, time, and operator on the embedding cassette, then add a small amount of OCT embedding medium (about 1 / 3 height) to the embedding cassette and pre-cool it on ice.

[0137] ② After taking out the material, quickly dry it with dust-free paper, and then transfer it to the embedding cassette containing OCT embedding medium, making the material stick to the bottom of the embedding cassette as much as possible. Keep the operation on ice during this process.

[0138] ③ After adjusting the material position, a layer of OCT can be added to completely cover the material. During embedding, it is required that the tissue be as flat as possible, and try to remove all air bubbles.

[0139] ④ Place the embedding cassette in a vacuum filtration device, and perform vacuum filtration for 5 minutes to remove air bubbles in the OCT and make the combination of OCT and the material closer; the vacuum condition is 0 - 0.08 Mpa for 5 - 10 minutes.

[0140] ⑤ Carefully remove the air bubbles in the embedding cassette, and adjust the material position again to make it fit the bottom of the embedding cassette.

[0141] ⑥ Transfer the embedding cassette to a device containing liquid nitrogen and isopentane. After standing for 30 seconds, transfer the embedding cassette to dry ice for temporary storage. The embedded material can be stored in an -80°C refrigerator.

[0142] Screening of Carnoy's fixative

[0143] Using maize stem (Zm_B73) as the experimental material, different formulations of Carnoy's fixative were used. According to the above method steps for the polyester wax-embedded sample protocol, polyester wax embedding medium was used to prepare the spatial transcriptome samples, and the prepared embedded materials were sectioned. The specific formulations of different Carnoy's fixatives are as follows:

[0144] Test group 1: Absolute ethanol, chloroform, and acetic acid were uniformly mixed in a volume ratio of 6:3:1;

[0145] Test group 2: Absolute ethanol and acetic acid were uniformly mixed in a volume ratio of 3:1.

[0146] In addition, further tests were also conducted on the Carnoy's fixative in test group 2 which only contained absolute ethanol and acetic acid. The volume ratios of absolute ethanol to acetic acid in test groups 2.1 - 2.5 were 1:1, 2:1, 3:1, 4:1, and 3:2 respectively.

[0147] Screening of the optimal fixation time, dehydration and infiltration time for polyester wax embedding:

[0148] Using corn stems (Zm_B73) as experimental materials, polyester wax embedding agents were used to prepare plant spatiotemporal transcriptome samples. The method steps were the same as those described in the previous text for the polyester wax embedding sample protocol. The difference was that different fixation times, dehydration times, and infiltration times were further tested, specifically as follows:

[0149] (1) Setting of fixation time conditions: Test groups 1 - 5 were set to 5 min, 10 min, 15 min, 20 min, and 25 min respectively.

[0150] (2) Setting of dehydration and infiltration time conditions: Test groups 6 - 10 were set as shown in Table 1 below.

[0151] Table 1. Setting of dehydration and infiltration time conditions

[0152]

[0153] Stability of polyester wax embedding treatment

[0154] Using corn stems (Zm_B73) as experimental materials, according to the above method steps, polyester wax embedding agents were used to make spatiotemporal transcriptome samples, and a control group was made using the standard process of conventional cryoembedding. Three different experimenters were arranged to conduct the experiment, and the spatiotemporal transcriptome data obtained by the three experimenters using this protocol were compared. The experiment was carried out according to the subsequent standard process of the spatiotemporal transcriptome experiment and photographed, and chip preparation and on - machine sequencing were carried out according to the above - mentioned MGIDNB make and sequencing reagent preparation operation standard process, and then the off - machine data analysis was carried out using the BGI spatiomics visualization system.

[0155] Experimental results:

[0156] 1. Effects of polyester wax embedding and cryoembedding on tissue structure

[0157] Using experimental materials such as maize stems (Zm_B73), soybean leaves (Zhonghuang 13), and Arabidopsis stems, according to the above method steps, OCT embedding medium (as the control group) and polyester wax (as the test group) were used to prepare spatial transcriptome samples respectively, and the effects of these two embedding methods on the tissue structure of the samples were observed by taking pictures.

[0158] The photos of maize stems, soybean leaves, and Arabidopsis stems are shown respectively in Figure 4 - 5 , Figure 6 - 7 and Figure 8 - 9 . It can be seen from these figures that the images of the control group have poor clarity, single cells cannot be distinguished, and the integrity of the whole tissue is not available; while the clarity of the cells in the images of the test group is greatly improved, not only can single cells be distinguished, but also the integrity of the whole tissue is available. Especially for dense cells, the test group can clearly distinguish the cell boundaries in the dense area. Therefore, at the cellular level, the polyester wax embedding method is superior to the cryo-embedding method.

[0159] 2. Comparison of library construction concentrations of three embedding methods

[0160] Using maize stems (Zm_73) as experimental materials, according to the above method steps, OCT embedding medium (control group 1), conventional paraffin wax (control group 2), and polyester wax (test group) were used to prepare spatial transcriptome samples respectively. The control group and the test group were subjected to subsequent experiments and photographed according to the standard spatial transcriptome experiment, and chip preparation and on-machine sequencing were carried out according to the standard operation procedures of MGIDNB make and sequencing reagent preparation, and the off-machine data analysis was carried out using the BGI spatial omics visualization system.

[0161] The library construction concentration information is shown in Table 2 below:

[0162] Table 2 Library construction concentration information of control group 1, control group 2, and test group

[0163]

[0164] As can be seen from Table 2, the library concentration of control group 2 is lower than the minimum library construction requirement and cannot successfully construct a library.

[0165] The spatial omics analysis results of control group 1 and the test group are shown in Table 3 below. In terms of the average gene capture amount (mainly referring to the Bin200 Median Gene Type value in the table), the test group of the polyester wax scheme increased by 408.3% compared with control group 1, which indicates that the polyester wax embedding scheme is the best and can obtain higher data capture.

[0166] Table 3 Spatial omics analysis results

[0167]

[0168] Note: The unit (M) represents the sequencing data volume. M is often used to describe the number of reads. For example, 1M is 10 6 reads. 3. Results of spatial and temporal transcriptome analysis of polyester wax embedding and conventional cryo-embedding

[0169] Using experimental materials of maize stems (Zm_B73), soybean leaves (Zhonghuang 13), and Arabidopsis stems (Arabidopsis), following the above method steps, OCT embedding medium (as the control group) and polyester wax (as the test group) were used to prepare spatial and temporal transcriptome samples respectively. Follow the standard spatial and temporal transcriptome experimental procedures for subsequent experiments and take pictures. Prepare the chip and perform on-machine sequencing according to the standard operating procedures of MGIDNB make and sequencing reagent preparation, and then use the BGI spatial omics visualization system for off-machine data analysis.

[0170] The results of spatial omics analysis are shown in Table 4 below. Mainly referring to Bin200 Median Gene Type, among them, compared with their respective control groups, the average gene capture of the maize stem test group after using the polyester wax embedding scheme increased by 52.10%, the average gene capture of the soybean leaf test group increased by 227.45%, and the average gene capture of the Arabidopsis stem test group increased by 204.22%. Thus, it can be seen that the average gene capture of the polyester wax embedding scheme increased by 52.10% to 227.45% compared with the control group using the conventional cryo-embedding scheme. Based on this, it can be considered that the polyester wax embedding scheme adopted in the present invention has a significant effect, not only improving the cell integrity of the sections, but also obtaining higher data capture.

[0171] Table 4 Results of spatial omics analysis

[0172]

[0173]

[0174] Note: The unit (M) represents the sequencing data volume. M is often used to describe the number of reads. For example, 1M is 10 6 reads.

[0175] Based on the above results, it can be seen that the samples embedded with polyester wax by gradient infiltration and embedding have the best performance in terms of tissue integrity and the number of gene captures, that is, the number of intact cells with the same tissue area is higher, and the number of gene captures is also higher. In contrast, other schemes show great disadvantages in terms of the number of gene captures and are also slightly inferior in terms of tissue integrity.

[0176] 4. Screening results of Carnoy's fixative

[0177] The results showed that the tissue of the material samples under the conditions of Test Group 1 was prone to shrinkage and deformation during embedding, and was prone to cracking during sectioning, and the sections were dry, resulting in difficulties in smooth sectioning; the material samples under the conditions of Test Group 2 could better maintain the tissue structure of the plant material during embedding, and the sections were not easy to crack, being relatively complete and enabling smooth sectioning. In this regard, we analyzed that the possible reason was that chloroform in the conditions of Test Group 1 had strong dissolving ability, which might cause deformation and dissolution of plant tissues, and chloroform was volatile during the sectioning process, easily evaporating and causing tissue water loss, making the sections become dry. In addition, some plant materials might have high resistance to chloroform, resulting in poor tissue fixation effect and even morphological and structural changes of the tissue. Moreover, chloroform is a toxic volatile solvent, and for plant materials, especially those containing volatile components, it might cause solvent volatilization during tissue embedding and degreasing, resulting in incomplete tissue dehydration. In addition, chloroform might also cause unstable texture of the wax blocks, and cracking was easy to occur during sectioning. And chloroform and acetic acid might interact with some staining agents, affecting the staining effect.

[0178] Therefore, we initially selected the preparation method of mixing absolute ethanol and acetic acid with a volume ratio of 3:1 as the preparation scheme of Carnoy's fixative, and further optimized and tested the mixing ratio of absolute ethanol and acetic acid. The results are shown in Table 5.

[0179] Table 5 Screening results of the ratio of Carnoy's fixative (absolute ethanol: acetic acid)

[0180]

[0181] We found that an appropriate ratio of absolute ethanol and acetic acid could help maintain the morphological structure of the tissue and the connection between cells, avoiding cell swelling and deformation. If there was too much absolute ethanol, it would lead to excessive dehydration of the material, the tissue was prone to shrinkage, and the sections were easy to crack; if there was too little absolute ethanol, it would lead to incomplete dehydration of the material, affecting the impregnation of wax and the quality of the embedded block; if there was too much acetic acid, it might dissolve the fat and other components in the tissue, resulting in tissue softening, being not easy to place and adjust the position during embedding, and affecting the preparation of the embedded block. Finally, it was determined that in the preparation scheme of Carnoy's fixative, the more appropriate ratio of absolute ethanol to acetic acid was 3:1 by volume.

[0182] 5. Screening results of the best fixation time and dehydration and wax infiltration time

[0183] (1) The results of different fixation times are shown in Table 6 below:

[0184] Table 6 Test analysis results of the fixation time

[0185]

[0186] Based on the above results, the relatively appropriate fixation time was finally determined to be 15 - 25 min, and the best effect was achieved when the fixation time was 15 min. Meanwhile, during the testing process of the present invention, it was found that when the fixation time with the fixative was 20 min, there was also an obvious effect on tissue fixation, but the effect was the best when the fixation time was 15 min, that is, it could maintain good tissue integrity and a high number of gene captures.

[0187] (2) The test results of dehydration and infiltration with paraffin are shown in Table 7 below:

[0188] Table 7 Test analysis results of dehydration and infiltration with paraffin

[0189]

[0190] Based on the above results, the relatively appropriate dehydration and infiltration time with paraffin was finally determined to be 6.5 h.

[0191] Therefore, based on the above results, it can be determined that the optimal fixation time is 15 min, and the optimal dehydration and infiltration time with paraffin is 6.5 h. Under these conditions, the obtained sections have a high section integrity and less tissue dewaxing phenomenon, which can avoid the problems of easy breakage of directly frozen - embedded sections and easy tissue dewaxing of conventional paraffin - embedded sections.

[0192] 6. Stability of polyester wax embedding treatment

[0193] The spatio - temporal omics analysis results related to polyester wax embedding are shown in Table 8 below. In terms of the average gene capture amount, the average gene capture in the test group data obtained by the three experimenters is better than that in the control group: the average gene capture of the polyester wax embedding scheme is increased by 47.60% - 105.80% compared with the control group. The above data prove that the three experimenters can obtain stable effects by applying the same experimental scheme.

[0194] Table 8 Spatio - temporal omics analysis results

[0195]

[0196] Note: The unit (M) is the sequencing data volume: M is often used to describe the number of reads. For example, 1M is 10 6 reads.

[0197] Therefore, based on the above results, it can be determined that the polyester wax embedding scheme of the present invention can obtain stable effects that are superior to the conventional frozen embedding scheme.

[0198] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for embedding plant tissues, the method comprising: (1) Fix the plant tissue with Carnoy's fixative for 15 to 25 minutes, preferably 15 minutes; (2) Dehydrate the fixed plant tissue with a dehydrating agent; (3) Perform gradient infiltration of the dehydrated plant tissue with polyester wax for 0.5 to 1 hour, preferably 1 hour, for each gradient; (4) Embed the plant tissue subjected to gradient infiltration with polyester wax.

2. The method according to claim 1, characterized in that, The Carnoy's fixative contains ethanol and acetic acid, preferably containing absolute ethanol and acetic acid with a volume ratio of 3:

1.

3. The method according to claim 1 or 2, characterized in that, The dehydration includes: first dehydrating at 0°C to 4°C for at least two times, each time for 10 to 50 minutes, preferably 50 minutes, and then dehydrating at 30°C to 42°C, preferably 42°C, for at least one time, each time for 0 to 50 minutes, preferably 50 minutes.

4. The method according to any one of claims 1-3, characterized in that, Perform gradient infiltration of the dehydrated plant tissue with a dehydrating agent-polyester wax solution with the concentration of polyester wax gradually increasing from more than 0% to 100% at 30°C to 45°C, preferably 45°C, for 0.5 to 1 hour, preferably 1 hour, for each gradient; preferably, use three kinds of dehydrating agent-polyester wax solutions with the polyester wax concentration of 50%, 75% and 100% to perform gradient infiltration; preferably, the polyester wax is prepared from PEG400 distearate and 1-hexadecanol with a weight ratio of 9:

1.

5. The method according to any one of claims 1-4, wherein, The fixation, dehydration and / or gradient infiltration are carried out under vacuum conditions; preferably, the vacuum conditions are -0.6 MPa to -0.8 MPa.

6. The method according to any one of claims 1-5, wherein, The dehydrating agent is selected from ethanol such as absolute ethanol, acetone, n-butanol, tert-butanol; preferably, the dehydrating agent is absolute ethanol.

7. The method according to any one of claims 1-6, wherein, The plant tissue is the seed, flower, fruit, leaf, stem or root tissue of a plant.

8. The method according to any one of claims 1-7, wherein, The method includes: (1') Fix the plant tissue with Carnoy's fixative for 15 to 25 minutes under the vacuum conditions of -0.6 MPa to -0.8 MPa, preferably 15 minutes; (2') Dehydrate the fixed plant tissue with absolute ethanol as the dehydrating agent under the vacuum conditions of -0.6 MPa to -0.8 MPa, including: a) Replace Carnoy's fixative with absolute ethanol and dehydrate the plant tissue at 0°C to 4°C for 10 to 50 minutes, preferably 50 minutes; b) Replace absolute ethanol with another absolute ethanol and dehydrate the plant tissue at 0°C to 4°C for 10 to 50 minutes, preferably 50 minutes; c) Replace the other absolute ethanol with absolute ethanol preheated to 30°C to 42°C, preferably 42°C, and dehydrate the plant tissue at 30°C to 42°C, preferably 42°C, for 0 to 50 minutes, preferably 50 minutes; (3') Perform gradient infiltration of the dehydrated plant tissue with polyester wax under the vacuum conditions of -0.6 MPa to -0.8 MPa, including: a') Replace absolute ethanol with a 50% absolute ethanol - 50% polyester wax solution and infiltrate the wax at 30°C to 45°C, preferably 45°C, for 0.5 to 1 hour, preferably 1 hour; b') Replace the 50% absolute ethanol - 50% polyester wax solution with a 25% absolute ethanol - 75% polyester wax solution and infiltrate the wax at 30°C to 45°C, preferably 45°C, for 0.5 to 1 hour, preferably 1 hour; c') Replace the 25% absolute ethanol - 75% polyester wax solution with a 100% polyester wax solution and impregnate the wax at 30°C to 45°C, preferably 45°C, for 30 minutes to 2 hours; d') Optionally, repeat step c') at least once; (4') Embed the gradient-impregnated plant tissue with polyester wax.

9. A method for making a plant spatio-temporal transcriptome sample, the method comprising: i. Perform the method according to any one of claims 1-8, thereby obtaining the embedded plant tissue; ii. Section, attach, dewax, fix with methanol, and permeabilize the embedded plant tissue.

10. The method according to claim 9, characterized in that, The dewaxing is dewaxing with methanol.

11. Use of a plant spatio-temporal transcriptome sample prepared by the method according to claim 9 or 10 in plant spatio-temporal transcriptome sequencing.

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