Corn leaf nuclei dissociation solution suitable for flow cytometry analysis and application thereof

Abstract

The application discloses a corn leaf cell nucleus dissociation solution suitable for flow cytometer analysis and application thereof, and belongs to the technical field of flow cytometer analysis. The corn leaf tissue is treated by using a compound dissociation solution for infiltration, and then is subjected to blade scratch operation, compared with traditional liquid nitrogen grinding and digestion technology, the problem of large material loss is greatly solved, sample waste is reduced, tissue fragment pollution is reduced, cell nucleus purity and quality are improved, and a reliable sample basis is provided for subsequent experiments; compared with a mechanical cutting separation method, dissociation is more sufficient, the requirement for tissue amount is greatly reduced, and the method is of great significance for precious and scarce samples, is beneficial to scientific research of a small amount of samples, and promotes the expansion of related research fields. The fluorescent probe prepared by the application does not contain heavy metal elements, reduces cell toxicity, has excellent light stability, has small size and can enter the cell nucleus, has good solubility in a complex water environment, is suitable for various experimental conditions, and provides strong support for precise and stable cell nucleus imaging.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of flow cytometry analysis, and particularly relates to a corn leaf cell nucleus dissociation solution suitable for flow cytometry analysis and application thereof. BACKGROUND

[0002] In the field of cross-research between modern biology and agricultural science, flow cytometry, as a powerful analytical tool, plays a key role in cell-level research, especially for the analysis of corn leaf cell nuclei, which can provide indispensable high-precision data and in-depth insights for plant genetics, developmental biology, and crop breeding and many other research directions. The cell nuclei of plant tissues, if directly separated from tissues or organs, such as corn leaf tissue cell nuclei, have irreplaceable value in scientific exploration. Their natural state is completely preserved, which means that the preservation of the original genomic information of the cell nuclei is almost complete, providing the most authentic and reliable sample basis for the study of genetic material. At the same time, in the actual operation process, this direct separation method is relatively simple and efficient, without the need for complex technology and equipment support, so that various types of research laboratories can more conveniently carry out related research work, greatly promoting the popularization and in-depth research of corn leaf cell nuclei.

[0003] At present, common plant cell nucleus separation methods include liquid nitrogen grinding method, mechanical method, and protoplast preparation method, etc. These methods aim to successfully separate the cell nuclei from the leaf tissue, so as to be directly applied to subsequent scientific research, such as chromatin accessibility analysis, chromatin immunoprecipitation, and other transformation experiments. Directly separated corn leaf tissue cell nuclei have significant advantages, with short off-body time, effectively avoiding changes in cell physiological state and loss of genetic information caused by long off-body time. Moreover, since it does not undergo immortalization process, its biological characteristics are highly consistent with the real state in vivo, with stable and reliable genetic characteristics, so as to provide researchers with data closest to the actual physiological function of the cell nuclei in vivo, which is of great significance for in-depth understanding of the life activity rules and genetic regulation mechanisms of corn leaf cells.

[0004] However, when using flow cytometry to analyze corn leaf cell nuclei, the key step of cell nucleus staining faces many technical difficulties. Since the cell nucleus is tightly wrapped by a special lipid bilayer membrane, the fluorescent probe must penetrate this barrier to achieve effective staining of the cell nucleus, and then meet the detection requirements of flow cytometry. In theory, the size of the fluorescent probe should be less than 8nm of nuclear pore, so as to have the possibility of entering the cell nucleus, but in fact, just meeting this size condition is far from enough, because whether the probe can successfully enter the cell nucleus is also significantly affected by many factors such as its surface properties.

[0005] In traditional nuclear staining studies, organic dyes such as 4',6-diamidinyl-2-phenylindole and Hurst dyes have been widely used for staining the nuclei of cancer cells and normal cells due to their biocompatibility. However, these organic dyes have insurmountable drawbacks: their poor water solubility leads to aggregation in aqueous environments, making it difficult to ensure staining uniformity and thus affecting the accuracy and reliability of flow cytometry. Furthermore, their insufficient photostability causes a rapid decrease in fluorescence intensity during flow cytometry analysis, especially under prolonged laser irradiation (photobleaching), resulting in unstable detection signals. This makes them unsuitable for high-precision, long-term detection and analysis of cell nuclei, severely limiting their further application in flow cytometry analysis of maize leaf cell nuclei. On the other hand, semiconductor quantum dots, such as cadmium telluride and cadmium selenide quantum dots, have also shown potential in the field of cell fluorescence imaging. However, these quantum dots pose serious safety risks. They contain heavy metals, such as cadmium, which are highly toxic and may trigger a series of adverse physiological reactions in living organisms. This can interfere with normal cellular metabolism and function and threaten the health of the entire organism, leading to strict safety limitations in bioimaging, especially in flow cytometry. Furthermore, without complex modifications, semiconductor quantum dots have low efficiency in penetrating the nuclear membrane and entering the cell nucleus, making it difficult to achieve efficient staining and accurate analysis of the cell nucleus, thus failing to meet the high-quality staining requirements of flow cytometry for nuclear samples. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention provides an efficient and simple dissociation solution and dissociation method for maize leaf cell nuclei, which can be used for flow cytometry detection of maize leaf cell nuclei.

[0007] To achieve the above objectives, the following technical solution is adopted: This invention provides an application of maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis, comprising the following steps:

[0008] a. Collect fresh corn leaves with 2 true leaves, place the leaf tissue on a pre-cooled glass plate, add 1 mL of pre-cooled dissociation solution, and make rapid incisions with a sterile scalpel blade for 2 minutes. After chopping, rinse the chopped sample with 4 mL of pre-cooled dissociation solution into a sterile 15 mL EP centrifuge tube, and dissociate the corn leaf tissue on a shaker at room temperature for 5 minutes to obtain the corn leaf tissue nucleus solution.

[0009] b. Centrifuge the maize leaf tissue cell nucleus solution at low speed in a pre-cooled centrifuge at 4°C, remove the supernatant, and obtain maize leaf tissue cell nucleus precipitate;

[0010] c. Adding 5 mL of dissociation cleaning solution to the corn leaf tissue cell nucleus precipitate for resuspension, and centrifuging at low speed in a 4℃ pre-cooled centrifuge to remove the supernatant, thereby obtaining the purified corn leaf tissue cell nucleus precipitate;

[0011] d. Adding 1 mL of dissociation cleaning solution to the purified corn leaf tissue cell nucleus precipitate for resuspension, mixing, and then using a pipette gun to suck 10 μL of the cell solution into a new EP tube, and then adding an equal volume of carbon quantum dot fluorescent probe for staining, and observing the cell nucleus integrity under a microscope;

[0012] e. Collecting the corn leaf tissue cell nucleus and performing flow cytometry analysis;

[0013] Further, the carbon quantum dot fluorescent probe is prepared by the following steps:

[0014] S1. Adding 3,3',6,6'-tetracarboxylic acid-2,2'-dipyridyl to a mixed solution of ethylene glycol and DMF, and then ultrasonically treating for 10-15 min to obtain a first solution, and then adding the first solution into a polytetrafluoroethylene hydrothermal reactor, adding 0.5 mol / L of a DMF solution of perfluoroiodohexane and an organic metal initiator, and hydrothermally reacting at 180-220℃ for 3-15 h;

[0015] S2. After the reaction is completed, the hydrothermal reactor is cooled to room temperature to obtain a second solution, which is filtered and dialyzed for purification;

[0016] S3. The dialyzed and purified solution is subjected to solvent removal under reduced pressure, and then dried to obtain the carbon quantum dot fluorescent probe.

[0017] Further, the mass ratio of 3,3',6,6'-tetracarboxylic acid-2,2'-dipyridyl, ethylene glycol and DMF in the first solution of step S1 is 0.5-2:10-20:20-50.

[0018] Further, the mass ratio of the first solution, the DMF solution of perfluoroiodohexane and the organic metal initiator in step S1 is 10-30:5-10:0.01-0.05.

[0019] Further, the organic metal initiator is selected from one of acetylferrocene, tris(acetylacetone) iron, diethyl zinc and dichlorobis cyclopentadienyl zirconium.

[0020] Further, the filtration in step S2 uses a water-based filter membrane with a pore size of 0.2 μm or 0.22 μm.

[0021] Further, the dialysis in step S2 uses a dialysis bag with a cut-off amount of 1 kD for dialysis for 12-18 h.

[0022] The present invention also provides a maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis, the dissociation solution being composed of the following components: 4 mol / L MES-KOH solution with pH=5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution, 0.5 mol / L EDTA solution, and 4 mol / L sucrose solution.

[0023] Furthermore, the volume ratio of MES-KOH solution, NaCl solution, KCl solution, MgCl2 solution, EDTA solution and sucrose solution in the dissociation solution is 5:4:20:6:10:125.

[0024] Furthermore, the dissociation cleaning solution consists of the following components: 4 mol / L MES-KOH solution with pH=5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution, and 4 mol / L sucrose solution.

[0025] Furthermore, the volume ratio of MES-KOH solution, NaCl solution, KCl solution, MgCl2 solution and sucrose solution in the dissociation cleaning solution is 5:4:20:6:125.

[0026] The beneficial effects of this invention are:

[0027] a. This invention develops a novel method for dissociating maize leaf cell nuclei. Using a compound maize leaf cell nuclei dissociation solution, the maize leaf tissue is immersed in the solution, and then dissociation is achieved by scratching with a blade. This method allows for rapid acquisition of maize leaf cell nuclei in a short time. Compared with traditional liquid nitrogen grinding and digestion techniques, this invention effectively solves the problem of excessive material loss, significantly reduces sample waste, and significantly reduces the contamination level of tissue fragments, improving the purity and quality of the obtained cell nuclei. This provides a more reliable sample basis for subsequent experimental research. Furthermore, compared with mechanical cutting and separation methods, the dissociation effect of this invention is more complete, and the required tissue volume is greatly reduced, no longer requiring more than 1g of tissue. This is of great significance for precious and scarce samples, greatly saving sample resources and enabling scientific research to be carried out smoothly even with small sample sizes, providing strong support for the further expansion of related research fields.

[0028] b. This invention uses 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine as the key starting material to prepare carbon quantum dots. During the preparation process, an organometallic initiator is introduced to trigger the decomposition of perfluoroiodohexane to generate fluorine radicals or fluoride ions, thereby doping the carbon quantum dots with fluorine. The carbon quantum dot fluorescent probe prepared by this invention has a unique structure with dual nitrogen and fluorine doping. First, it does not contain heavy metal elements, reducing cytotoxicity and minimizing interference with cellular physiological states in applications such as cell imaging, ensuring the accuracy and reliability of experimental results. Second, it exhibits excellent photostability, maintaining stable fluorescence performance even under prolonged illumination, providing a solid guarantee for long-term, high-precision nuclear imaging. Third, its small size allows it to easily penetrate the cell nucleus membrane structure, meeting the requirements for staining and imaging within the cell nucleus. Furthermore, this fluorescent probe exhibits good solubility in complex aqueous environments containing various metal ions, ensuring its stability and reliability in practical applications, adapting to various complex experimental conditions, and providing reliable support for precise and stable nuclear imaging. Attached Figure Description

[0029] Figure 1 This is a graph showing the particle size analysis results of the carbon quantum dot fluorescent probe in Example 3 of the present invention;

[0030] Figure 2 This is a graph showing the cytotoxicity analysis results of the carbon quantum dot fluorescent probe of Example 1 of the present invention;

[0031] Figure 3 This is a graph showing the cytotoxicity analysis results of the carbon quantum dot fluorescent probe of Example 2 of the present invention;

[0032] Figure 4 This is a graph showing the cytotoxicity analysis results of the carbon quantum dot fluorescent probe of Example 3 of the present invention;

[0033] Figure 5 This is a diagram showing the fluorescence staining results of cell nuclei obtained from maize leaf tissue according to the present invention.

[0034] Figure 6 This is a flow cytometry analysis result of obtaining cell nuclei from maize leaf tissue according to the present invention.

[0035] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0039] Example 1

[0040] Composition of maize leaf cell nucleus dissociation solution and dissociation washing solution suitable for flow cytometry analysis

[0041] The dissociation solution consists of the following components: 4 mol / L MES-KOH solution with pH=5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution, 0.5 mol / L EDTA solution, and 4 mol / L sucrose solution.

[0042] The volume ratio of MES-KOH solution, NaCl solution, KCl solution, MgCl2 solution, EDTA solution and sucrose solution in the dissociation solution is 5:4:20:6:10:125.

[0043] The dissociation cleaning solution consists of the following components: 4 mol / L MES-KOH solution with pH=5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution and 4 mol / L sucrose solution.

[0044] The volume ratio of MES-KOH solution, NaCl solution, KCl solution, MgCl2 solution and sucrose solution in the dissociation cleaning solution is 5:4:20:6:125.

[0045] Example 2

[0046] Composition and preparation of carbon quantum dot fluorescent probes

[0047] The carbon quantum dot fluorescent probe is prepared by the following steps:

[0048] S1. 2g of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine was added to a mixed solution of 20g ethylene glycol and 50g DMF, and sonicated for 15min to obtain the first solution. Then, the first solution was added to a polytetrafluoroethylene hydrothermal reactor, along with 10g of a 0.5mol / L perfluoroiodohexane DMF solution and 0.05g of zirconium dichloroethylene as an organometallic initiator. The reactor was hydrothermally reacted at 220℃ for 15h.

[0049] S2. After the reaction is complete and the hydrothermal reactor is cooled to room temperature, filter the mixture using a 0.22μm aqueous filter membrane, and then dialyze it for 18 hours using a dialysis bag with a 1kD cutoff for purification.

[0050] S3. Remove the solvent from the dialysis purified solution under reduced pressure and dry it to obtain the carbon quantum dot fluorescent probe.

[0051] Example 3

[0052] Composition and preparation of carbon quantum dot fluorescent probes

[0053] The carbon quantum dot fluorescent probe is prepared by the following steps:

[0054] S1. 0.5 g of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine was added to a mixed solution of 10 g ethylene glycol and 20 g DMF, and sonicated for 10 min to obtain the first solution. Then, the first solution was added to a polytetrafluoroethylene hydrothermal reactor, along with 5 g of a 0.5 mol / L perfluoroiodohexane DMF solution and 0.01 g of acetylferrocene as an organometallic initiator. The reactor was hydrothermally reacted at 180 °C for 3 h.

[0055] S2. After the reaction is complete and the hydrothermal reactor has cooled to room temperature, filter the mixture using a 0.2 μm aqueous filter membrane, and then dialyze it for 12 hours using a dialysis bag with a 1 kDa cutoff for purification.

[0056] S3. Remove the solvent from the dialysis purified solution under reduced pressure and dry it to obtain the carbon quantum dot fluorescent probe.

[0057] Example 4

[0058] Composition and preparation of carbon quantum dot fluorescent probes

[0059] The carbon quantum dot fluorescent probe is prepared by the following steps:

[0060] S1. 1 g of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine was added to a mixed solution of 15 g ethylene glycol and 30 g DMF, and sonicated for 12 min to obtain the first solution. Then, the first solution was added to a polytetrafluoroethylene hydrothermal reactor, along with 7 g of a 0.5 mol / L perfluorohexane DMF solution and 0.03 g of tri(acetylacetone)ferric as an organometallic initiator. The reactor was hydrothermally reacted at 200 °C for 8 h.

[0061] S2. After the reaction is complete and the hydrothermal reactor has cooled to room temperature, filter the mixture using a 0.2 μm aqueous filter membrane, and then dialyze it for 15 h using a dialysis bag with a 1 kDa cutoff for purification.

[0062] S3. Remove the solvent from the dialysis purified solution under reduced pressure and dry it to obtain the carbon quantum dot fluorescent probe.

[0063] Experimental Example 1

[0064] Carbon quantum dot fluorescent probe particle size analysis

[0065] The average particle size distribution of Example 4 was measured using a dynamic light scattering particle size analyzer, and the results are shown below. Figure 1 .

[0066] Depend on Figure 2 It is evident that the carbon quantum dot fluorescent probe prepared in Example 3 has a particle size of less than 8 nm, which meets the requirements for entering the cell nucleus. This result directly demonstrates the advantages of the carbon quantum dot fluorescent probe preparation scheme of this invention. By precisely controlling the proportion of raw materials and reaction conditions, this invention successfully prepared probes with suitable particle sizes. The smaller particle size allows them to penetrate the lipid bilayer membrane of the cell nucleus, overcoming the obstacle of traditional fluorescent probes being unable to enter the cell nucleus due to particle size issues. This provides the possibility for subsequent precise staining and analysis of the cell nucleus, ensuring that more accurate and detailed information about the inside of the cell nucleus can be obtained. This is of great significance for in-depth research on the structure and function of the cell nucleus. For example, when studying the distribution and state of chromatin, it can more clearly present its true situation within the cell nucleus, avoiding information loss due to the inability of the probe to enter.

[0067] Experimental Example 2

[0068] Carbon quantum dot fluorescent probe cytotoxicity analysis

[0069] The carbon quantum dot fluorescent probes from Examples 2-4 were prepared into solutions with concentrations of 0, 1000, 500, 250, 125, 75, 40, 20, 10, 5, and 2 μg / mL, and cell viability was detected. The results are shown in [Figure 1]. Figures 2-4 .

[0070] Depend on Figures 2-4Free of heavy metals, this invention exhibits low cytotoxicity. The carbon quantum dot fluorescent probe utilizes an organometallic initiator for fluorine doping during preparation, avoiding the use of heavy metals in traditional semiconductor quantum dots and significantly reducing cytotoxicity. In applications such as cell imaging, low cytotoxicity ensures that cells maintain normal physiological states and functional activities, allowing experimental results to accurately reflect the characteristics and behavior of the cell nucleus under natural conditions. During long-term cell culture observation experiments, cells do not exhibit abnormal metabolic changes, abnormal gene expression, or cell death due to probe toxicity. This provides a reliable guarantee for accurately studying the changes in the cell nucleus during cell cycle and cell differentiation processes, improving the credibility of experimental data and the accuracy of research conclusions.

[0071] Experimental Example 3

[0072] The nuclei of corn leaf tissue cells were extracted using the nucleus dissociation solution and dissociation washing solution from Example 1, and the nuclei were stained and observed using the carbon quantum dot fluorescent probe from Example 4. The specific steps included:

[0073] a. Collect fresh corn leaves with 2 true leaves, place the leaf tissue on a pre-cooled glass plate, add 1 mL of pre-cooled dissociation solution, and make rapid incisions with a sterile scalpel blade for 2 minutes. After chopping, rinse the chopped sample with 4 mL of pre-cooled dissociation solution into a sterile 15 mL EP centrifuge tube, and dissociate the corn leaf tissue on a shaker at room temperature for 5 minutes to obtain the corn leaf tissue nucleus solution.

[0074] b. Centrifuge the maize leaf tissue cell nucleus solution at low speed in a pre-cooled centrifuge at 4°C, remove the supernatant, and obtain maize leaf tissue cell nucleus precipitate;

[0075] c. Add 5 mL of dissociation washing solution to the corn leaf tissue cell nucleus precipitate for resuspending, centrifuge at low speed in a pre-cooled centrifuge at 4℃, remove the supernatant, and obtain the purified corn leaf tissue cell nucleus precipitate.

[0076] d. Resuspend the purified maize leaf tissue cell nucleus precipitate in 1 mL of dissociation washing buffer, mix well, and pipette 10 μL of the supernatant into a new EP tube. Add an equal volume of carbon quantum dot fluorescent probe for staining. Observe the cell nucleus integrity under a microscope. The results are shown in the figure. Figure 5 .

[0077] Depend on Figure 5As can be seen, the extracted cell nuclei are relatively intact, and tissue fragments are almost completely removed, meeting the needs of subsequent scientific research. The dissociation solution and unique blade scratch dissociation method used in this invention represent a novel and highly efficient cell nucleus extraction scheme compared to traditional liquid nitrogen grinding and digestion techniques. On the one hand, it effectively solves the problem of excessive material loss, greatly reducing sample waste. For precious and scarce maize sample resources, a small amount of sample is sufficient to meet experimental needs, which is a significant advantage when germplasm resources are limited or specific genetic research materials are scarce. On the other hand, it significantly reduces the degree of contamination from tissue fragments, improving the purity and quality of obtained cell nuclei, providing a more reliable sample basis for subsequent experiments. Simultaneously, compared to mechanical cutting and separation methods, the dissociation effect is more complete, the requirement for tissue volume is greatly reduced, and it no longer relies on large amounts of tissue material, making the experimental operation simpler, more flexible, and less costly. The carbon quantum dot fluorescent probe of this invention can effectively stain intact cell nuclei, further ensuring accurate morphological observation and analysis of cell nuclei. It provides clear and accurate sample images for studying the basic characteristics of maize leaf cell nuclei, such as morphology and chromosome number, and helps to carry out in-depth research in fields such as cytogenetics.

[0078] Test Example 4

[0079] Nuclei were collected from maize leaf tissue and analyzed by flow cytometry. The results are shown in [Figure number missing]. Figure 6 .

[0080] Depend on Figure 6 The fluorescence value of the maize leaf tissue cell nucleus sample was 127805, with a CV value of 3.84%, indicating diploidity, consistent with literature values. These results fully demonstrate the effectiveness and reliability of the combined scheme of the cell nucleus dissociation solution, dissociation washing solution, and carbon quantum dot fluorescent probe in flow cytometry analysis. High-quality cell nucleus dissociation and staining provide a stable and uniform fluorescence signal for flow cytometry, enabling the instrument to accurately detect and analyze various parameters of the cell nucleus, such as cell cycle distribution and DNA content. Compared with traditional cell nucleus processing and staining methods, the technical solution of this invention better meets the stringent sample quality requirements of flow cytometry, improving the accuracy and repeatability of the detection results. In plant cell biology research, this is of great value for a deeper understanding of the proliferation, differentiation, and genetic stability of maize leaf cells.

[0081] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0082] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. An application of a maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis, characterized in that: Includes the following steps: a. Collect fresh corn leaves with 2 true leaves, place the leaf tissue on a pre-cooled glass plate, add 1 mL of pre-cooled dissociation solution, and make rapid incisions with a sterile scalpel blade for 2 minutes. After chopping, rinse the chopped sample with 4 mL of pre-cooled dissociation solution into a sterile 15 mL EP centrifuge tube, and dissociate the corn leaf tissue on a shaker at room temperature for 5 minutes to obtain the corn leaf tissue nucleus solution. b. Centrifuge the maize leaf tissue cell nucleus solution at low speed in a pre-cooled centrifuge at 4°C, remove the supernatant, and obtain maize leaf tissue cell nucleus precipitate; c. Add 5 mL of dissociation washing solution to the corn leaf tissue cell nucleus precipitate for resuspending, centrifuge at low speed in a pre-cooled centrifuge at 4℃, remove the supernatant, and obtain the purified corn leaf tissue cell nucleus precipitate. d. Add 1 mL of dissociation washing solution to the purified maize leaf tissue cell nucleus precipitate, resuspend, mix well, and then use a pipette to transfer 10 μL of the cell solution to a new EP tube. Add an equal volume of carbon quantum dot fluorescent probe for staining and observe the cell nucleus integrity under a microscope. e. Collect cell nuclei from maize leaf tissues and analyze them by flow cytometry; The carbon quantum dot fluorescent probe is prepared by the following steps: S1. Add 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine to a mixed solution of ethylene glycol and DMF, and then sonicate for 10-15 min to obtain the first solution. Then add the first solution to a polytetrafluoroethylene hydrothermal reactor, add 0.5 mol / L of perfluoroiodohexane DMF solution and organometallic initiator, and hydrothermally react at 180-220℃ for 3-15 h. S2. After the reaction is completed, wait for the hydrothermal reactor to cool to room temperature to obtain the second solution, and then filter and dialyze it for purification. S3. Remove the solvent from the dialysis purified solution under reduced pressure and dry it to obtain the carbon quantum dot fluorescent probe.

2. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 1, characterized in that: In step S1, the mass ratio of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine, ethylene glycol, and DMF in the first solution is 0.5-2:10-20:20-50.

3. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 2, characterized in that: In step S1, the mass ratio of the first solution, the DMF solution of perfluoroiodohexane, and the organometallic initiator is 10-30:5-10:0.01-0.

05.

4. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 3, characterized in that: The organometallic initiator is selected from one of acetylferrocene, tri(acetylacetone)ferro, diethylzinc, and zirconium dichlorocerocene.

5. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 4, characterized in that: In step S2, the filtration uses a 0.2μm or 0.22μm aqueous filter membrane.

6. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 5, characterized in that: In step S2, dialysis is performed using a dialysis bag with a 1 kDa cutoff for 12-18 hours.

7. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 1, characterized in that: The dissociation solution consists of the following components: 4 mol / L MES-KOH solution with pH=5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution, 0.5 mol / L EDTA solution, and 4 mol / L sucrose solution.

8. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 7, characterized in that: The volume ratio of MES-KOH solution, NaCl solution, KCl solution, MgCl2 solution, EDTA solution and sucrose solution in the dissociation solution is 5:4:20:6:10:

125.

9. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 8, characterized in that: The dissociation cleaning solution consists of the following components: 4 mol / L MES-KOH solution with pH=5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution and 4 mol / L sucrose solution.

10. The application of the maize leaf cell nucleus dissociation solution suitable for flow cytometry analysis according to claim 9, characterized in that: The volume ratio of MES-KOH solution, NaCl solution, KCl solution, MgCl2 solution and sucrose solution in the dissociation cleaning solution is 5:4:20:6:125.

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