A method for locating plant hormones in plant tissues

Abstract

A method for locating plant hormones in plant tissues comprises the following steps: adding a fixative to the plant tissue for fixation; adding a blocking agent for blocking; combining the plant tissue to be tested with a plant hormone antibody labeled with horseradish peroxidase, then adding a phosphate buffer for elution to obtain a plant hormone antigen-horseradish peroxidase-labeled antibody complex; adding a color developer to generate a color reaction, then adding a terminator to terminate the color reaction and observing and photographing. The present invention is simple and fast to operate, and the results are intuitive and can effectively avoid the interference of nonspecific staining, visualize the distribution and content of plant hormones in plant tissues, and ensure the accuracy and stability of the observation results. In addition, the method provided by the present invention does not require tissue sectioning, and further simply and quickly achieves the in situ localization of plant hormones while ensuring the integrity of plant tissues.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for locating plant hormones in plant tissues. Background Art

[0002] Plant hormones play a crucial role in multiple aspects of plant growth and development. They are also essential signaling molecules that allow plants to sense changes in the external environment, regulate their growth, and maintain survival. The distribution and concentration of plant hormones are crucial to their physiological responses; only accumulation of hormones at their sites of action can induce physiological effects. Auxin is widely involved in various morphogenic processes during plant development. Auxin regulates cell division and promotes cell elongation; Its concentration gradient influences the morphological development of individual plants and their organs. Therefore, understanding the distribution characteristics and patterns of plant hormones within tissues and organs is crucial for understanding plant growth and development and forms the basis for exploring the mechanisms by which plant hormones function in plants.

[0003] There is still little understanding of the distribution of plant hormones in plant tissues. Based on this, how to provide a simple, accurate, fast and clear method to measure the distribution and content of endogenous plant hormones is an urgent problem that technicians in this field need to solve in their research. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for locating plant hormones in plant tissues, which can observe the distribution and content of plant hormones in plant tissues simply, accurately, quickly and clearly without the need for tissue sections.

[0005] According to one aspect of the present invention, a method for locating plant hormones in plant tissue is provided, comprising the following steps: S1. adding a fixative to the plant tissue for fixation to obtain pretreated plant tissue; the fixative comprises paraformaldehyde, piperazine-1,4-diethanesulfonic acid, magnesium sulfate, and ethylenediaminetetraacetic acid, wherein the mass percentage of paraformaldehyde in the fixative is 4%, the concentration of piperazine-1,4-diethanesulfonic acid in the fixative is 50 mmol / L, and the concentrations of the magnesium sulfate and the ethylenediaminetetraacetic acid in the fixative are 5 mmol / L; S2. Add a blocking agent to the pretreated plant tissue for blocking treatment to obtain the plant tissue to be tested; S3. Combine the plant tissue to be tested with a plant hormone antibody labeled with horseradish peroxidase, then add a phosphate buffer solution for elution treatment to obtain a plant hormone antigen-horseradish peroxidase-labeled antibody complex; S4. Add a color developer containing 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide to the plant hormone antigen-horseradish peroxidase-labeled antibody complex to cause a color reaction, then add a terminator to terminate the color reaction and observe and photograph. The method provided by the present invention first solidifies and blocks the plant tissue, and then uses a specific fixative to fix the plant tissue. The fixative can quickly penetrate into the plant tissue to exert a fixing effect, effectively preventing the outflow of endogenous hormones in the plant tissue from adversely affecting the observation results, thereby ensuring the accuracy of the positioning effect. In addition, the fixative can achieve rapid fixation and in-situ fixation of the plant hormone while retaining the original state of the plant tissue, and the operation is simple and fast. After fixation and blocking, the plant tissue to be tested is combined with a plant hormone antibody labeled with horseradish peroxidase (HRP) to form a plant hormone antigen-horseradish peroxidase-labeled antibody complex, which is then eluted and a color developer containing 3,3',5,5'-tetramethylbenzidine (TMB) is added to cause a color reaction. Under the catalysis of HRP, TMB produces a soluble blue product that adheres to the plant tissue, and the color depth of the plant tissue is positively correlated with the plant hormone content. The distribution and content of the plant hormone are determined by observing the position and color depth of the blue product. The present invention is simple and fast to operate, the results are intuitive, and the interference of nonspecific staining can be effectively avoided. The distribution and content of plant hormones in plant tissues can be visualized, while ensuring the accuracy and stability of the observation results. In addition, the method provided by the present invention does not require tissue sectioning, and further achieves the in situ localization of plant hormones simply and quickly while ensuring the integrity of plant tissues.

[0006] Preferably, after adding a fixative for fixation, a phosphate buffer is added for elution.

[0007] Preferably, the blocking agent includes bovine serum albumin, wherein the mass percentage of bovine serum albumin in the blocking agent is 2%. The bovine serum albumin (BSA) in the blocking agent can block non-specific sites and improve the accuracy of the detection result.

[0008] Preferably, after adding a blocking agent for blocking treatment, a phosphate buffer is added for elution treatment.

[0009] Preferably, in S3, the concentration of the horseradish peroxidase-labeled plant hormone antibody is 8 to 15 μg / ml. Because excessively high concentrations of HRP can cause subsequent TMB staining to appear green or red, and because adding a terminator to terminate the color development reaction can easily cause precipitation, combining plant tissue with the HRP-labeled plant hormone antibody at the above concentration can improve the localization of the plant hormone, facilitating clear and accurate observation of the distribution and content of the plant hormone.

[0010] Preferably, the method for preparing the horseradish peroxidase-labeled plant hormone antibody comprises:

[0011] S3.1 dialyzing the plant hormone antibody in carbonate buffer to obtain a pretreated plant hormone antibody; the carbonate buffer has a concentration of 0.05 mol / L and a pH of 9.6; the concentration of the pretreated plant hormone antibody is 2 mg / ml;

[0012] S3.2 Add horseradish peroxidase to sodium periodate solution and ethylene glycol and carry out oxidation reaction at 4°C in the dark for 30 minutes. When the solution turns brown, the oxidation of horseradish peroxidase is complete, i.e., oxidized horseradish peroxidase is obtained. The concentration of horseradish peroxidase is 2 mg / ml, the concentration of sodium periodate solution is 0.1 mol / L, and the volume ratio of horseradish peroxidase to sodium periodate solution is 1:1.

[0013] S3.3 The pretreated plant hormone antibody and the oxidized horseradish peroxidase are mixed evenly and reacted at room temperature for 2 hours, and then sodium borohydride is added to carry out a reduction reaction at 4°C for 30 minutes to obtain a horseradish peroxidase-labeled plant hormone antibody; the concentration of the horseradish peroxidase-labeled plant hormone antibody is 10ug / ml; the molar ratio of the pretreated plant hormone antibody to the oxidized peroxidase is 1:4, the concentration of the sodium borohydride solution is 0.5mol / L; and the volume ratio of the sodium borohydride solution to the oxidized horseradish peroxidase is 1:10. This method first removes the free amino groups contained in the plant hormone antibody by dialysis, and oxidizes the sugar group of HRP into an aldehyde group, so that the aldehyde group reacts with the amino group of the antibody to form a stable structure, thereby enhancing the specificity and affinity of the plant hormone antibody, which is conducive to a more accurate and clear subsequent color development reaction, thereby improving the accuracy and stability of the results.

[0014] Preferably, the phosphate buffer comprises sodium chloride, potassium chloride, disodium hydrogen phosphate, and dipotassium hydrogen phosphate, wherein the concentration of sodium chloride is 0.14 mmol / L, the concentration of potassium chloride is 2.7 mmol / L, the concentration of disodium hydrogen phosphate is 10 mmol / L, and the concentration of dipotassium hydrogen phosphate is 1.8 mmol / L. The phosphate buffer does not contain Tris, NH4 + The free amino groups and other components in the phosphate buffer are reasonably set to effectively wash out other interfering materials, thereby avoiding non-specific staining caused by impurities such as free amino groups in the subsequent staining process, resulting in inaccurate and unclear results. At the same time, the elution treatment with the above-mentioned phosphate buffer is beneficial to improving the permeability of plant tissues, making it easier for antibodies to enter plant tissues, thereby improving the positioning effect of plant hormones.

[0015] Preferably, in S3, the temperature for binding the plant tissue to be tested and the horseradish peroxidase-labeled plant hormone antibody is 35-40°C for 25-35 minutes. This temperature allows the HRP-labeled antibody to fully bind to the plant hormone in the plant tissue while ensuring the stability and accuracy of the results. This incubation time ensures sufficient and rapid binding of the plant hormone and the enzyme-labeled antibody while avoiding interference such as precipitation after subsequent staining due to prolonged incubation, which can cause inaccurate and unclear results.

[0016] Preferably, the temperature for combining the plant tissue to be tested with the horseradish peroxidase-labeled plant hormone antibody is 37° C. and the time is 30 minutes.

[0017] Preferably, in S4, the developer is a developer A solution and a developer B solution compounded in a volume ratio of 1:1, wherein the developer A solution includes hydrogen peroxide, ethylenediaminetetraacetic acid, citric acid and disodium hydrogen phosphate; and the developer B solution includes 3,3',5,5'-tetramethylbenzidine (TMB), hydrochloric acid and polyvinylpyrrolidone. The present invention optimizes the formula of the developer solution, uses a developer composed of specific components to detect plant hormones in the plant tissue to be tested, and rationally compoundes multiple components to form a unified developer before use, thereby making it difficult to produce precipitation during color development, reducing the interference of nonspecific staining, and adding polyvinylpyrrolidone as a stabilizer to make the color development result more stable and accurate.

[0018] Preferably, in developer solution A, the concentrations of hydrogen peroxide are 0.5 mmol / L, ethylenediaminetetraacetic acid are 1 mmol / L, citric acid are 0.5 mmol / L, and disodium hydrogen phosphate are 0.5 mmol / L; in developer solution B, the concentrations of 3,3',5,5'-tetramethylbenzidine are 1 mmol / L, hydrochloric acid are 1 mmol / L, and the mass percentage of pyrrolidone carboxylic acid is 10%. By controlling the content of the developer components, the color development reaction can be completed quickly while ensuring the accuracy and stability of the results.

[0019] Preferably, the temperature of the color development reaction is 35-40° C. and the time is 5-15 minutes. The above temperature can achieve a sufficient and complete color development reaction, thereby determining the distribution and content of plant hormones in plant tissues.

[0020] Preferably, the temperature of the color development reaction is 37° C., and the time of the color development reaction is 10 minutes.

[0021] Preferably, the volume ratio of the terminator to the developer is 1:1. The terminator comprises sulfuric acid and diethylene glycol, wherein the concentration of sulfuric acid is 0.5 mol / L and the mass percentage of diethylene glycol in the terminator is 5%. This amount of terminator effectively terminates the color development reaction, preventing excessive color development, precipitation, and other phenomena that can cause unclear and inaccurate results.

[0022] Preferably, the plant hormone is a growth hormone. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the effect of color development time (0, 5, and 10 min, respectively) on IAA staining of Arabidopsis root tips in Example 1 of the present invention;

[0024] Figure 2 This is a schematic diagram showing the effect of 5-minute developer treatment on the stained area of ​​the Arabidopsis root tip in Example 1 of the present invention;

[0025] Figure 3 This is a schematic diagram of the staining of the root tips of Arabidopsis seedlings after treatment with exogenous IAA at concentrations of 0, 5, 25, and 50 nmol / L in Example 2 of the present invention;

[0026] Figure 4 This is a grayscale image of the root tips of Arabidopsis seedlings stained after treatment with exogenous IAA in Example 2 of the present invention;

[0027] Figure 5 Schematic diagram showing the effect of color development time (0, 5, and 10 min, respectively) on IAA staining of Arabidopsis root tips in Comparative Example 1 of the present invention without adding HRP-labeled antibodies. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the drawings in the embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0029] Example 1

[0030] A method for localizing plant hormones in plant tissues, comprising the following steps:

[0031] 1. Fixation: Take a 2 cm long root of plant tissue and immerse it in a sufficient amount of fixative for 30 minutes; the fixative comprises paraformaldehyde, piperazine-1,4-diethanesulfonic acid, magnesium sulfate, and ethylenediaminetetraacetic acid. The mass percentage of paraformaldehyde in the fixative is 4%, the concentration of piperazine-1,4-diethanesulfonic acid is 50 mmol / L, the concentration of magnesium sulfate is 5 mmol / L, and the concentration of ethylenediaminetetraacetic acid is 5 mmol / L.

[0032] 2. Elution: Transfer the fixed plant tissue to a sufficient amount of phosphate buffer and rinse repeatedly 3-5 times to obtain pretreated plant tissue; wherein the phosphate buffer contains 0.14 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L disodium hydrogen phosphate, and 1.8 mmol / L dipotassium hydrogen phosphate;

[0033] 3. Blocking: Transfer the pretreated plant tissue to a sufficient amount of blocking agent and react for 30 minutes; wherein the blocking agent contains 2% bovine serum albumin by weight;

[0034] 4. Elution: The operation is the same as 2 to obtain the plant tissue to be tested;

[0035] 5. Combine the plant tissue to be tested with the HRP-labeled antibody:

[0036] 5.1 Antibody Treatment: Auxin antibody was dialyzed against 0.05 mol / L carbonate buffer (pH 9.6) to a concentration of 2 mg / ml.

[0037] 5.2 HRP Oxidation: Mix equal volumes of 2 mg / ml horseradish peroxidase (HRP) and 0.1 mol / L sodium periodate, and add ethylene glycol (the volume of ethylene glycol should be 1% of the volume of the HRP and sodium periodate mixture). Incubate at 4°C in the dark for 30 minutes. HRP oxidation is complete when the solution turns brown.

[0038] 5.3 HRP-labeled IAA antibody: Mix 2 mg / ml auxin antibody and oxidized HRP solution in a molar ratio of 1:4 and react at room temperature for 2 hours; add 0.5 mol / L sodium borohydride solution (10% of the volume of HRP solution) and react at 4°C for 30 minutes to obtain 1 mg / ml HRP-labeled IAA antibody, which is diluted to 10 μg / ml for later use. Then, immerse the eluted plant tissue in the HRP-labeled IAA antibody solution and incubate at 37°C for 30 minutes.

[0039] 6. Elution: The operation is the same as 2 to obtain the auxin antigen-horseradish peroxidase-labeled antibody complex;

[0040] 7. Color development: Immerse the above-mentioned auxin antigen-horseradish peroxidase-labeled antibody complex in a color developing agent containing 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide, and incubate at 37°C in the dark for 5 minutes. The color developing agent is prepared by mixing equal volumes of color developing agent solution A and color developing agent solution B, adjusting the pH to 4.0, and filtering through a 0.22 μm filter membrane. Color developing agent solution A contains 0.5 mol / L hydrogen peroxide, 1 mmol / L ethylenediaminetetraacetic acid, 0.5 mol / L citric acid, and 0.5 mmol / L disodium hydrogen phosphate; color developing agent solution B contains 1 mmol / L 3,3',5,5'-tetramethylbenzidine (TMB), 1 mmol / L HCl, and 10% polyvinylpyrrolidone.

[0041] 8. Stop: Mix an equal volume of stop solution into the above-mentioned developer; wherein the stop solution includes 0.5 mol / L sulfuric acid and 5% by mass of diethylene glycol.

[0042] 9. Observation: Use a bright field microscope to photograph plant tissues. The distribution of auxin (IAA) in the root tip of Arabidopsis thaliana is as follows: Figure 1 As shown ( Figure 1 From left to right, the results of taking photos of the root tips of Arabidopsis seedlings using a bright field microscope after adding the color developer for 0, 5, and 10 minutes are shown. The color-developed areas are as follows: Figure 2 As shown ( Figure 2 The root tips of Arabidopsis seedlings were photographed using a bright field optical microscope after adding a color developer for 5 minutes. The dotted box is the color-developed area of ​​the root tip. Figure 2 The right picture in the figure is an enlarged picture of the left picture). As can be seen from the figure, the method provided by the present invention successfully localized auxin in the root tip of Arabidopsis thaliana without nonspecific staining. The color after adding the color developer for 5 minutes was slightly lighter than that after adding the color developer for 10 minutes. Both color development for 5 minutes and color development for 10 minutes can clearly identify the distribution of auxin and judge the concentration of auxin by the depth of color. Moreover, this method is simple and rapid.

[0043] Three replicate experiments were conducted using the above method to localize and observe plant hormones in plant tissues. The results were the same as above, demonstrating that the distribution of auxin could be quickly and clearly identified, and the concentration of auxin could be determined by color depth. This demonstrates that the method provided by the present invention for localizing plant hormones in plant tissues is highly stable and reproducible.

[0044] Example 2

[0045] This example refers to Example 1 to locate plant hormones in plant tissues. The difference between this example and Example 1 is that the plant tissue in step 1 of Example 1 is replaced by plant tissue treated with an exogenous IAA solution (the concentrations of IAA in the exogenous IAA solution are 0, 5, 25, and 50 nmol / L, respectively). Apart from the above differences, the materials and process operations used in this example are strictly consistent with those in Example 1. The distribution of IAA in the root tip of Arabidopsis thaliana is shown in FIG. Figure 3 As shown ( Figure 3 From left to right, the plant tissues were treated with 0, 5, 25, and 50 nmol / L IAA, and the root tips of Arabidopsis seedlings were photographed using a bright field optical microscope 5 minutes after the color developer treatment. The grayscale values ​​are as follows Figure 4 As shown. Figure 3 It can be seen that the method provided by the present invention successfully localized IAA in the root tips of Arabidopsis seedlings, with clear staining and no nonspecific staining. Figure 4 As can be seen, color depth is positively correlated with auxin content. As IAA content increases, the grayscale value increases and the color becomes darker. Therefore, observing the color position and color depth of IAA can be used to determine the distribution and content of auxin, and this method is simple and rapid.

[0046] Comparative Example 1

[0047] This comparative example provides a method for locating plant hormones in plant tissues, comprising the following steps: grinding and crushing Arabidopsis root tip tissue with liquid nitrogen, weighing 0.1 g into a 1.5 mL centrifuge tube, adding 180 μL PBS, using a plant auxin (IAA) enzyme-linked immunosorbent assay kit (Meimian Industrial Co., Ltd, China), adding 10 μL of a sample to be tested and 40 μL of a sample diluent to the bottom of an enzyme labeling plate, sealing the plate with a sealing film, and incubating at 37 degrees for 30 minutes. The sealing film is carefully removed, the liquid is discarded and dried, each well is filled with washing solution and allowed to stand for 30 seconds before being discarded, 50 μL of an enzyme labeling reagent is added to each well and incubated at 37 degrees for 30 minutes, the liquid is discarded, 50 μL of a color developer A is added to each well, and 50 μL of a color developer B is added. The mixture is gently shaken and mixed, and the color is developed at 37 degrees in the dark for 10 minutes. 50 μL of a stop solution is added to each well, and the absorbance (OD value) of each well is measured in sequence at a wavelength of 450 nm. This comparative example can achieve the positioning of auxin, but requires instruments to observe the distribution and content of auxin, and cannot achieve in situ positioning of plant hormones without destroying plant tissues.

[0048] Comparative Example 2

[0049] This comparative example refers to Example 1 to locate plant hormones in plant tissues. The difference between this comparative example and Example 1 is that step 5 of Example 1 (binding the plant tissue to the HRP-labeled antibody) is omitted. Apart from the above differences, the materials and process operations used in this comparative example are strictly consistent with those in Example 1. The observation results of the plant hormone localization in this comparative example are as follows: Figure 5 As shown ( Figure 5 From left to right, images of Arabidopsis seedling root tips taken with a brightfield optical microscope at 0, 5, and 10 minutes of color development after treatment with a color developer, without the addition of an HRP-labeled antibody, are shown. The observations show that the color development time of 5 and 10 minutes, respectively, corresponds to shallow auxin color, making it difficult to clearly observe the color development and depth. This demonstrates that the method provided by the present invention, using an HRP-labeled auxin antibody to localize auxin, allows for clear observation of auxin distribution and content, thereby improving auxin localization and visualizing its distribution and content.

[0050] Comparative Example 3

[0051] This comparative example uses Example 1 to localize plant hormones in plant tissue. The difference between this comparative example and Example 1 is that the fixative in step 1 of Example 1 is replaced with a color developer consisting of 4% paraformaldehyde, DMSO, and NP-40. Aside from these differences, the materials and process used in this example strictly adhere to those of Example 1. The results of the observations of the localization of plant hormones in this comparative example are as follows: auxin displayed a shallow, turbid color, making it difficult to clearly observe the color development and depth. Comparing the localization of plant hormones in plant tissues in Example 1 and this comparative example, under the same fixation time, the fixative provided in Example 1 achieved superior fixation, preventing auxin from overflowing and resulting in rapid and clear color development. Furthermore, the fixative enabled in situ observation of auxin distribution and content without tissue sectioning. This demonstrates that the method provided by the present invention, using a specific fixative to fix plant tissue, enables in situ observation of plant hormones while maintaining plant tissue integrity and improves auxin localization.

[0052] Comparative Example 4

[0053] This comparative example, referring to Example 1, localizes plant hormones in plant tissue. This comparative example differs from Example 1 in that the fixative in step 1 of Example 1 is replaced with a color developer composed of 4% paraformaldehyde, magnesium sulfate, and ethylenediaminetetraacetic acid. Aside from these differences, the materials and process used in this example strictly adhere to those of Example 1. The results of the observations of plant hormone localization in this comparative example are as follows: auxin color development is light and turbid, and the depth of color development cannot be clearly observed. Comparing the localization of plant hormones in plant tissues in Example 1 and this comparative example, under the same fixation time, the fixative provided in Example 1 exhibits superior fixation, preventing auxin from overflowing and resulting in rapid and clear color development. Furthermore, the fixative enables in situ observation of auxin distribution and content without tissue sectioning. This demonstrates that the method provided by the present invention, using a specific fixative to fix plant tissue, enables in situ observation of plant hormones while maintaining plant tissue integrity and improves auxin localization.

[0054] Comparative Example 5

[0055] This comparative example refers to Example 1 for localization of plant hormones in plant tissues. This comparative example differs from Example 1 in that step 1 (fixation) of Example 1 is omitted. The results of the observation of plant hormone localization in this comparative example are as follows: the plant tissues displayed a pale color, making it difficult to clearly observe the distribution of IAA. This demonstrates that the fixation of plant tissues using a fixative using the method provided by the present invention can preserve the antigenicity and fine structure of plant tissues, thereby improving the localization of auxins and facilitating clear and accurate observation of auxin distribution and content.

[0056] Comparative Example 6

[0057] The comparative example refers to Example 1 to locate the plant hormone in the plant tissue. The difference between this comparative example and Example 1 is that step 3 (sealing) of Example 1 is omitted. In addition to the above differences, the materials and process operations used in this comparative example are strictly consistent with those in Example 1. The observation results of this comparative example are as follows: the plant tissue color is turbid, contains impurities, and the distribution of IAA cannot be clearly observed. This shows that the method provided by the present invention is used to add a sealing agent to the plant tissue for sealing treatment, which can avoid the decomposition and non-specific adsorption of auxin, thereby improving the color development effect and ensuring the accuracy and stability of the in situ positioning of auxin.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention.

Claims

1. A method for localizing plant hormones in plant tissues, characterized in that: The steps include: S1. Adding a fixative to the plant tissue for fixation to obtain a pretreated plant tissue; the fixative comprises paraformaldehyde, piperazine-1,4-diethanesulfonic acid, magnesium sulfate, and ethylenediaminetetraacetic acid, wherein the mass proportion of the paraformaldehyde in the fixative is 4%, the concentration of the piperazine-1,4-diethanesulfonic acid in the fixative is 50 mmol / L, and the concentrations of the magnesium sulfate and the ethylenediaminetetraacetic acid in the fixative are 5 mmol / L; S2. Adding a blocking agent to the pretreated plant tissue for blocking treatment to obtain a plant tissue to be tested; S3. The plant tissue to be tested is combined with a horseradish peroxidase-labeled plant hormone antibody, and then eluted by adding a phosphate buffer solution to obtain a plant hormone antigen - horseradish peroxidase-labeled antibody complex; S4. A color developing agent containing 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide is added to the plant hormone antigen - horseradish peroxidase-labeled antibody complex to cause a color reaction, and then a terminator is added to terminate the color reaction and observed and photographed; Wherein, the plant hormone is growth hormone.

2. The method for localizing plant hormones in plant tissues according to claim 1, wherein: The blocking agent includes bovine serum albumin, wherein the mass proportion of bovine serum albumin in the blocking agent is 2%.

3. The method for localizing plant hormones in plant tissues according to claim 1, wherein: In the S3, the concentration of the horseradish peroxidase-labeled plant hormone antibody is 8-15 μg / ml.

4. The method for localizing plant hormones in plant tissues according to claim 1, wherein: The phosphate buffer comprises sodium chloride, potassium chloride, disodium hydrogen phosphate, and dipotassium hydrogen phosphate, wherein the concentration of the sodium chloride is 0.14 mmol / L, the concentration of the potassium chloride is 2.7 mmol / L, the concentration of the disodium hydrogen phosphate is 10 mmol / L, and the concentration of the dipotassium hydrogen phosphate is 1.8 mmol / L.

5. The method for localizing plant hormones in plant tissues according to claim 1, wherein: In S3, the temperature for combining the plant tissue to be tested with the horseradish peroxidase-labeled plant hormone antibody is 35-40° C. and the time is 25-35 minutes.

6. The method for localizing plant hormones in plant tissues according to claim 1, wherein: In S4, the developer is prepared by mixing developer A solution and developer B solution in a volume ratio of 1:1, wherein developer A solution includes hydrogen peroxide, ethylenediaminetetraacetic acid, citric acid and disodium hydrogen phosphate; developer B solution includes 3,3',5,5'-tetramethylbenzidine, hydrochloric acid and polyvinylpyrrolidone.

7. The method for localizing plant hormones in plant tissues according to claim 6, wherein: In the developer A solution, the concentration of hydrogen peroxide is 0.5 mmol / L, the concentration of ethylenediaminetetraacetic acid is 1 mmol / L, the concentration of citric acid is 0.5 mmol / L, and the concentration of disodium hydrogen phosphate is 0.5 mmol / L; in the developer B solution, the concentration of 3,3',5,5'-tetramethylbenzidine is 1 mmol / L, the concentration of hydrochloric acid is 1 mmol / L, and the mass percentage of polyvinylpyrrolidone is 10%.

8. The method for localizing plant hormones in plant tissues according to claim 1, wherein: The temperature of the color development reaction is 35-40° C., and the time is 5-15 minutes.

9. The method for localizing plant hormones in plant tissues according to claim 1, wherein: The volume ratio of the terminator to the developer is 1:

1. The terminator includes sulfuric acid and diethylene glycol. The concentration of the sulfuric acid is 0.5 mol / L, and the mass percentage of the diethylene glycol in the terminator is 5%.

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