Method for separating plant tissue cell nucleus
By using liquid nitrogen freeze-grinding and stage-by-stage regulation of surfactants, combined with density gradient separation, the problem of difficult plant cell nucleus extraction was solved, and efficient and pure cell nucleus separation was achieved, which is suitable for a variety of plant cells.
Patent Information
- Application Number
- CN202511016173.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-10-17
AI Technical Summary
Existing technologies make it difficult to efficiently separate high-quality cell nuclei from plant tissues, especially due to differences in plant cell structure and the presence of cell walls, which make cell nucleus extraction difficult and contain high impurities.
Liquid nitrogen cryo-grinding combined with surfactant-free separation solution A was used to protect the cell nuclear structure. Subsequently, separation solution B containing a non-ionic surfactant was used to selectively dissolve the cell membrane. Then, separation solution C without surfactant was used to stabilize the nuclear membrane structure. A density gradient system was constructed using separation solutions D and E for centrifugation to separate the cell nucleus from other organelles.
It achieves high-purity and high-integrity cell nucleus separation, effectively removes impurities such as chloroplasts, especially chloroplasts in leaf cells, and is suitable for the extraction of various plant cells.
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Figure CN120796439A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of molecular biology technology, and particularly relates to a method for separating plant tissue cell nuclei. BACKGROUND
[0002] The nucleus is a highly specialized, complex and diverse organelle in cells. It contains most of the genetic information of the cell and controls most of the functions of the cell. With the development of single-cell sequencing technology, its application direction has gradually expanded to the field of agronomy, and the sample types have gradually diversified to various plant samples. As a key technology for epigenetic experiments, the ATAC-seq or CUT&tag technology encounters sample preparation problems when applied to plant samples. When plant cell nuclei are extracted, how to obtain high-quality cell nuclei becomes a major difficulty.
[0003] There are significant differences in structure between plant cells and animal cells (in addition to cell walls, there are chloroplasts and other organelles that interfere with subsequent experiments), and there are significant differences between different species and different parts of plants. Therefore, it is challenging to isolate high-quality cell nuclei from plant tissues. Isolating cell nuclei from protoplasts is a commonly used method, which can provide relatively pure cell nuclei. However, the preparation of protoplasts is time-consuming and labor-intensive, and can only be used for a limited number of species.
[0004] In order to solve the problems of difficult plant cell nucleus acquisition and high impurity content, the present application provides a method for separating plant tissue cell nuclei. SUMMARY
[0005] The present application provides a method for separating plant tissue cell nuclei, characterized by the following steps: homogenizing and standing the liquid nitrogen frozen and ground plant tissue coarse powder with separation liquid A, then filtering to obtain a first filtrate, centrifuging the first filtrate, collecting the precipitate, resuspending with separation liquid B, centrifuging, collecting the precipitate, resuspending with separation liquid C, filtering to obtain a second filtrate, adding separation liquid D and separation liquid E to the second filtrate in sequence to form a density gradient system, centrifuging, collecting the interfacial layer solution containing cell nuclei, washing the interfacial layer solution containing cell nuclei with separation liquid C, and obtaining purified cell nuclei; wherein the separation liquid A does not contain a surfactant, the separation liquid B contains a non-ionic surfactant, and the separation liquid C does not contain a surfactant.
[0006] The present application realizes efficient purification of plant cell nucleus by combining the use of surfactants and density gradient separation in stages. Firstly, the cell wall is broken by freezing and grinding with liquid nitrogen, and the cell nucleus structure is protected and large-particle impurities are preliminarily removed by using separation liquid A containing no surfactant; then, the cell membrane is selectively dissolved to release the cell nucleus and remove part of the organelles by using separation liquid B containing non-ionic surfactant; then, the structure of the nuclear membrane is stabilized and residual debris is removed by using separation liquid C containing no surfactant; finally, the density gradient system formed by separation liquid D and separation liquid E is used to separate the cell nucleus and other organelles (especially chloroplasts), so that high-purity enrichment is realized. The composition design of each separation liquid can prevent excessive damage to the nuclear membrane and ensure effective removal of impurities (especially chloroplasts), so that high-purity and high-integrity cell nucleus is obtained.
[0007] Further, the separation liquid A contains buffer MOPS, electrolyte, chelating agent and stabilizer; the separation liquid B contains osmotic regulator, buffer, divalent cation and non-ionic surfactant; the separation liquid C contains osmotic regulator, buffer and divalent cation; and the separation liquid D and the separation liquid E are mixed solutions of density medium with different concentrations. The separation liquid A uses a buffer system containing no surfactant, the MOPS buffer maintains a physiological pH environment, the electrolyte maintains osmotic pressure balance, the chelating agent inhibits nuclease activity by binding metal ions, and the stabilizer protects the integrity of the nuclear membrane structure, which together ensures the stability of the cell nucleus structure in the initial breaking stage. The separation liquid B introduces the synergistic effect of non-ionic surfactant and osmotic regulator, selectively dissolves the cell membrane without damaging the nuclear membrane, maintains an isotonic environment to avoid nuclear membrane rupture, stabilizes the separation environment, strengthens the stability of the nuclear membrane, and forms controllable membrane structure separation conditions. The separation liquid C removes the surfactant but retains the osmotic regulation function, which not only removes the residual membrane debris but also avoids interference of the surfactant on the subsequent steps. The concentration gradient design of the separation liquid D and E forms differential density medium layers, which utilize the difference in sedimentation coefficients of different cell components to realize physical separation of the cell nucleus and other organelles by centrifugation, and finally capture high-purity cell nucleus in the interface layer.
[0008] Further, the concentration of the buffer MOPS in the separation liquid A is 10-100 mM, the electrolyte is 20-100 mM NaCl and 50-200 mM KCl, the chelating agent is 0.5-5 mM EDTA and 0.5-5 mM EGTA, and the stabilizer is 0.1-1 mM spermidine and 0.1-1 mM arginine. The concentration matching system of the components can maximize the maintenance of cell nucleus integrity and genetic material stability while ensuring effective breaking of the cell wall through the synergistic effect of the components.
[0009] Further, the osmotic regulator in the separation solution B is 0.25-0.5M sucrose, the buffer is 5-40mM Tris-HCl, the divalent cation is 5-40mM MgCl2, and the non-ionic surfactant is Triton X-100 with a concentration of 0.2%-2%. The selection of the above components allows the cell nucleus to maintain its shape when it is separated from the cytoplasmic environment, and effectively removes the soluble impurities in the cytoplasmic matrix.
[0010] Further, the osmotic regulator in the separation solution C is 0.25-0.5M sucrose, the buffer is 5-40mM Tris-HCl (pH=8), the divalent cation is 5-40mM MgCl2, and the non-ionic surfactant is not included. The above formulation can better maintain the shape of the cell nucleus during washing and filtration.
[0011] Further, the separation solution D is composed of 10%-50% by volume of the separation solution C and 50%-90% by volume of Percoll (a medium for cell separation), and the separation solution E is composed of 0%-50% by volume of the separation solution C and 50%-100% by volume of Percoll. By controlling the proportion of Percoll in the separation solution D and the separation solution E, solutions with different densities can be formed, thereby optimizing the layering effect of the cell nucleus and other organelles.
[0012] Further, the separation solution A contains 60mM MOPS, 90mM NaCl, 0.1M KCl, 1mM EDTA, 1mM EGTA, 0.2mM spermidine, and 0.1mM arginine. The above proportion of the separation solution A has a better effect.
[0013] Further, the specific composition of the separation solution B is 0.4M sucrose, 30mM Tris-HCl (pH=8), 20mM MgCl2, and 1.5% Triton X-100. The above proportion of the separation solution B has a better effect.
[0014] Further, the centrifugation parameters of the first filtrate, the centrifugation parameters of the resuspended centrifugate B, and the centrifugation parameters of the interface layer solution are independently selected from 800-2000xg for 5-15min; and the centrifugation parameters of the density gradient system are 15000-25000xg for 10-30min. The combination of the above centrifugation parameters can reduce the probability of cell nucleus breakage and reduce the impurity content.
[0015] Further, the plant tissue is selected from at least one of a maize leaf cell and a pepper leaf cell. The maize leaf cell has a thick cell wall and abundant chloroplasts, and the pepper leaf contains secondary metabolites, and traditional separation methods are prone to cause cell nucleus breakage or impurity residues. The present scheme can be suitable for the extraction of these two types of plant cells through the special design of the separation liquid formula and the operation method, and of course, it can also be suitable for the extraction of other conventional plant cells. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 Figure 1 is a microscope image of the maize leaf cell nucleus of Example 1 of the present application.
[0017] Figure 2 Figure 2 is an ATAC library quality detection image of the maize leaf cell nucleus of Example 1 of the present application.
[0018] Figure 3 Figure 3 is a microscope image of the nucleus of Example 2 of the present application.
[0019] Figure 4 Figure 4 is an ATAC library quality detection image of Example 2 of the present application.
[0020] Figure 5 Figure 5 is a microscope image of the pepper leaf cell nucleus of Example 3 of the present application.
[0021] Figure 6 Figure 6 is an ATAC library quality detection image of the pepper leaf cell nucleus of Example 3 of the present application. Figure 7 Figure 7 is a microscope image of the maize leaf cell nucleus of Comparative Example 1 of the present application. Figure 8 Figure 8 is an ATAC library quality detection image of the maize leaf cell nucleus of Comparative Example 1 of the present application. DETAILED DESCRIPTION
[0022] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application can be implemented in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.
[0023] In addition, the terms "first", "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second" can be explicitly or implicitly included at least one of the features. In the description of the application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise explicitly and specifically limited. In the description of the present application, the meaning of "several" is at least one, such as one, two, etc., unless otherwise explicitly and specifically limited.
[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.
[0025] In the present application, the technical features described in an open way include both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features.
[0026] In the present application, when a numerical interval is involved, unless otherwise specified, the numerical interval is considered to be continuous and includes the minimum value and the maximum value of the range, as well as every value between the minimum value and the maximum value. Further, when the range refers to an integer, every integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.
[0027] In the present application, the percentage content involved, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.
[0028] In the present application, the percentage concentration involved, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added ingredient in the system after the ingredient is added.
[0029] In the present application, the temperature parameter, unless otherwise specified, allows both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.
[0030] In the present application, the "particles" mentioned or the substances with defined particle size distribution are not necessarily spherical in shape, but can also be irregular, and can be primary particles or secondary particles. The particle size of irregular particles is calculated as the average of the maximum diameter and the minimum diameter.
[0031] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. In the embodiments, the specific conditions are not specified, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased on the market.
[0032] Embodiment 1: The present embodiment provides a method for separating the nuclei of corn leaf tissue cells and a quality inspection method.
[0033] S1: Add appropriate amount of liquid nitrogen and plant leaves to a mortar and grind into coarse powder. After grinding, transfer the coarse powder to a pre-cooled 2 ml centrifuge tube for later use;
[0034] S2: Add 1 ml of ice-cold separation solution A to the centrifuge tube, mix with a handheld homogenizer for 3 seconds, place on ice, and let stand for 5 minutes;
[0035] S3: Filter the separation solution A containing the leaf coarse powder through a filter with a pore size of 40 μm and collect the filtrate into a pre-cooled 1.5 ml centrifuge tube;
[0036] S4: Place the centrifuge tube in a horizontal centrifuge and centrifuge at 4°C, 1500×g for 10 min.
[0037] S5: Carefully remove the supernatant and add 1 ml of ice-cold separation buffer B to the centrifuge tube. Use a pipette to slowly pipette to resuspend the pellet.
[0038] S6: Place the centrifuge tube in a horizontal centrifuge and centrifuge at 4°C, 1500×g for 10 min.
[0039] S7: Carefully remove the supernatant and add 0.5 ml of ice-cold separation buffer C to the centrifuge tube. Use a pipette to slowly pipette to resuspend the pellet.
[0040] S8: Filter the separation solution C containing the cell nuclei through a filter with a pore size of 20 μm and collect the filtrate into a pre-cooled 1.5 ml centrifuge tube;
[0041] S9: Slowly add 0.5 ml of ice-cold separation solution D to the bottom of the centrifuge tube; then slowly add 0.5 ml of ice-cold separation solution E to the bottom of the centrifuge tube;
[0042] S10: Place the centrifuge tube in a horizontal centrifuge and centrifuge at 4°C, 2500×g for 30 min.
[0043] S11: After taking out the centrifuge tube, you should be able to observe that the solution in the centrifuge tube is divided into three layers. The junction between the second and third layers is the nuclear layer. Pipette 0.2 ml of the nuclear layer solution and transfer it to a new 1.5 ml centrifuge tube;
[0044] S12: Add 0.5 ml of ice-cold separation solution C to the centrifuge tube and pipette slowly 10 times;
[0045] S13: Place the centrifuge tube in a horizontal centrifuge and centrifuge at 4°C, 1500×g for 10 min.
[0046] S14: Carefully remove the supernatant, add an appropriate amount of ice-cold separation buffer C to the centrifuge tube, slowly pipette to resuspend the pellet, and perform DAPI staining;
[0047] S15: Calculate the number of nuclei, and then take 20000 nuclei for ATAC library construction.
[0048] Wherein:
[0049] The composition of separation solution A in S2: 60mM MOPS, 90mM NaCl, 0.1M KCl, 1mM EDTA, 1mM EGTA, 0.2mM spermidine, 0.1mM arginine, PH 7.
[0050] The composition of separation solution B in S5: 0.4M sucrose, 30mM Tris-HCl (PH=8), 20mM MgCl2, 1.5% Triton X-100.
[0051] The composition of separation solution C in S7: 0.4M sucrose, 30mM Tris-HCl (PH=8), 20mM MgCl2
[0052] The composition of separation solution D in S9: 20% volume of separation solution C, 80% volume of Percoll.
[0053] The composition of separation solution E in S10: 100% Percoll.
[0054] The microscopic results are shown in Table 1. Figure 1 As shown in Table 1, the method of the present embodiment extracts the cell nuclei of corn leaves with clean background and less impurities, but part of the nuclei appears to have broken nuclear membranes.
[0055] The ATAC library quality inspection data is shown in Table 1:
[0056] Table 1: ATAC library quality inspection data of corn leaf extraction in Example 1.
[0057]
[0058]
[0059] Figure 2 As shown, through ATAC library quality inspection, there is a main peak at 200-400bp, the cDNA concentration is 27.7ng / μL after 14 cycles of amplification, and the total amount is 554ng, which is qualified.
[0060] Example 2: The present embodiment provides a method for separating corn leaf tissue nuclei and a quality inspection method. The difference from Example 1 is that the centrifugation step parameters of S4, S6 and S13 are adjusted to 1000xg, centrifugation time 5min; the centrifugation step of S10 is adjusted to 20000xg, centrifugation time 20min.
[0061] The microscopic results are shown in Table 1.Figure 3 As shown, the corn leaf cell nuclei extracted according to the method of the present embodiment have a clean background, less impurities, and good integrity, without swelling, rupture and other phenomena. This shows that the combination of the above centrifugation parameters can reduce the probability of cell nucleus rupture and reduce the impurity content.
[0062] The ATAC library quality inspection data is shown in Table 2:
[0063] Table 2: ATAC library quality inspection data of corn leaf extraction in Example 2.
[0064]
[0065]
[0066] Figure 4 As shown, through ATAC library quality inspection, there is a main peak at 200-400 bp, the cDNA concentration is 13.2 ng / μL after 12 cycles of amplification, and the total amount is 264 ng, which is qualified.
[0067] Example 3: The present embodiment provides a method for separating plant tissue cell nuclei and a quality inspection method.
[0068] The difference from Example 2 is that the corn leaves are replaced by pepper leaves, and the other experimental conditions and parameters are the same as those of Example 2.
[0069] The microscopic results are shown in Table 3: Figure 5 As shown, the corn leaf cell nuclei extracted according to the method of the present embodiment have a clean background, less impurities, and good integrity, without swelling, rupture and other phenomena.
[0070] The ATAC library quality inspection data is shown in Table 3:
[0071] Table 3: ATAC library quality inspection data of corn leaf extraction in Example 3.
[0072]
[0073]
[0074] Figure 6 As shown, through ATAC library quality inspection, there is a main peak at 200-400 bp, the cDNA concentration is 13.2 ng / μL after 12 cycles of amplification, and the total amount is 264 ng, which is qualified.
[0075] Example 4: This example provides a method for isolating corn leaf tissue nuclei and a quality control method. Compared with Example 1, the difference is that the composition of separation solution A is: 10 mM MOPS, 20 mM NaCl, 50 mM KCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.1 mM spermidine and 0.1 mM arginine. The ATAC library is qualified by quality control.
[0076] Example 5: This example provides a method for isolating corn leaf tissue nuclei and a quality control method. Compared with Example 1, the difference is that the concentration of Triton X-100 in separation solution B is adjusted to 2%. The ATAC library is qualified by quality control.
[0077] Example 6: This example provides a method for isolating corn leaf tissue nuclei and a quality control method. Compared with Example 1, the difference is that the formula of separation solution D is: 10% separation solution C + 90% Percoll, and the formula of separation solution E is: 50% separation solution C + 50% Percoll. The ATAC library is qualified by quality control.
[0078] Comparative Example 1: This comparative example provides a method for isolating corn leaf tissue nuclei and a quality control method.
[0079] S1: Add an appropriate amount of liquid nitrogen and plant leaves to a mortar and grind into coarse powder. After grinding, transfer the coarse powder to a pre-cooled 2 ml centrifuge tube for standby;
[0080] S2: Transfer the ground sample to 1 mL of pre-cooled NEB1 buffer and lyse for 10 min;
[0081] S3: 4°C, 1500g centrifugation for 10 min;
[0082] S4: Discard the supernatant, add 1 mL of NEB2 nuclear extraction solution to the precipitate, mix well, and stand on ice for 10 min;
[0083] S6: Filter with a 40 μm cell sieve and collect the liquid in a new centrifuge tube. 4°C, 5000 rpm low speed centrifugation for 10 min, quickly discard the supernatant;
[0084] S7: Resuspend the precipitate with 500 μL of NEB3 buffer and add it to 500 μL of pre-cooled NEB3 buffer. 1600g, 4°C centrifugation for 45 min, remove the supernatant;
[0085] S8: Resuspend the precipitate with 1 mL of pre-cooled WB buffer;
[0086] S9: DAPI stain the nuclei and count the nuclei under a microscope;
[0087] in:
[0088] The composition of NEB1 buffer in S2: 10 mM Tirs-HCl (pH=7.5), 200 mM KCl, 10 mM EDTA, 10 mM MgCl2, 50% glycerol, 0.4 M sucrose, 1 M DTT, 5 mM β-mercaptoethanol, 0.2 mM spermidine, 0.1 mM arginine.
[0089] The composition of NEB2 nuclear extraction solution in S4: 10 mM PBS (PH=7.4), 0.4 M sucrose, 1% paraformaldehyde, 1% Triton X-100, 5 mM β-mercaptoethanol.
[0090] The composition of NEB3 buffer in S7: 10 mM Tirs-HCl (pH=7.5), 1.7 M sucrose, 10 mM MgCl2, 1% triton X-100, 5 mM β-mercaptoethanol.
[0091] The composition of WB buffer in S8: 10 mM PBS (PH=7.4), 0.4 M sucrose, 1% Triton X-100.
[0092] Microscopic examination results Figure 7 As shown, the cell nucleus suspension extracted according to the method of this comparative example has a high impurity content, and most of the cell nuclei are damaged and enlarged.
[0093] Table 3 Quality inspection data of ATAC library extracted from corn leaf tissue in Comparative Example 1.
[0094]
[0095]
[0096] Figure 8 The results showed that the ATAC library quality inspection showed a main peak at 200-400bp. After 12 cycles of amplification, the cDNA concentration was 30.5ng / μL and the total amount was 610ng, which passed the quality inspection.
[0097] According to the experimental results, it can be seen that Examples 1-6 can all obtain intact cell nuclei and contain less impurities. This is because the present application realizes efficient purification of plant cell nuclei by combining phased regulation of surfactant use and density gradient separation. First, the cell wall is broken by freezing and grinding with liquid nitrogen, and the separation liquid A containing no surfactant is used to protect the integrity of the cell nucleus structure and preliminarily remove large-particle impurities; then the separation liquid B containing a non-ionic surfactant is used to selectively dissolve the cell membrane to release the cell nucleus and remove part of the organelles; then the separation liquid C containing no surfactant is used to stabilize the structure of the nuclear membrane, and the residual debris is removed by filtration; finally, the density gradient system constructed by the separation liquid D and the separation liquid E is used to separate the cell nucleus and other organelles (especially chloroplasts), and realize high-purity enrichment. The composition design of each separation liquid can prevent excessive damage to the nuclear membrane and ensure effective removal of impurities (especially chloroplasts), thereby obtaining high-purity and well-integrity cell nuclei.
[0098] The technical features of the above examples can be combined in any manner. In order to make the description concise, all possible combinations of the technical features in the above examples are not described, however, as long as the combinations of the technical features do not contradict, they should be considered as within the scope of the present disclosure.
[0099] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of the patent of the present application should be subject to the appended claims.
Claims
1. A method for isolating plant tissue cell nuclei, characterized in that: The method comprises the following steps: homogenizing plant tissue coarse powder obtained by liquid nitrogen freeze-grinding with separation solution A, allowing the mixture to stand, and then filtering to obtain a first filtrate; centrifuging the first filtrate, collecting the precipitate, resuspending the precipitate in separation solution B, and centrifuging the precipitate; collecting the precipitate, resuspending the precipitate in separation solution C, and filtering to obtain a second filtrate; sequentially adding separation solution D and separation solution E to the second filtrate to form a density gradient system; collecting an interface layer solution containing cell nuclei after centrifugation; and washing the interface layer solution containing cell nuclei with separation solution C to obtain purified cell nuclei; The separation liquid A does not contain a surfactant, the separation liquid B contains a nonionic surfactant, and the separation liquid C does not contain a surfactant.
2. The method according to claim 1, characterized in that The separation liquid A contains a buffer MOPS, an electrolyte, a chelating agent and a stabilizer; the separation liquid B contains an osmotic regulator, a buffer, a divalent cation and a nonionic surfactant; the separation liquid C contains an osmotic regulator, a buffer and a divalent cation; the separation liquids D and E are density medium mixtures of different concentrations.
3. The method according to claim 2, characterized in that The concentration of the buffer MOPS in the separation solution A is 10-100 mM, the electrolytes are 20-100 mM NaCl and 50-200 mM KCl, the chelating agents are 0.5-5 mM EDTA and 0.5-5 mM EGTA, and the stabilizers are 0.1-1 mM spermidine and 0.1-1 mM arginine.
4. The method according to claim 2, characterized in that The osmotic regulator in the separation solution B is 0.25-0.5M sucrose, the buffer is 5-40mM Tris-HCl, the divalent cation is 5-40mM MgCl2, and the non-ionic surfactant is Triton X-100 with a concentration of 0.2%-2%.
5. The method according to claim 2, characterized in that The separation solution C contains 0.25-0.5 M sucrose as an osmotic regulator, 5-40 mM Tris-HCl as a buffer, 5-40 mM MgCl 2 as a divalent cation, and does not contain a nonionic surfactant.
6. The method according to claim 2, characterized in that The separation solution D consists of 10%-50% by volume of the separation solution C and 50%-90% by volume of Percoll, and the separation solution E consists of 0%-50% by volume of the separation solution C and 50%-100% by volume of Percoll.
7. The method according to claim 3, characterized in that The separation solution A contains 60 mM MOPS, 90 mM NaCl, 0.1 M KCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM spermidine, and 0.1 mM arginine.
8. The method according to claim 4, characterized in that The specific composition of the separation solution B is: 0.4M sucrose, 30mM Tris-HCl, 20mM MgCl2 and 1.5% Triton X-100.
9. The method according to claim 5, characterized in that The centrifugation parameters for the first filtrate, the centrifugation parameters for resuspending the centrifuge solution B, and the centrifugation parameters for washing the interface layer solution are independently selected from: 800-2000×g, centrifugation time is 5-15min; the centrifugation parameters for the density gradient system are 15000-25000×g, centrifugation time is 10-30min.
10. The method according to any one of claims 1 to 9, characterized in that The plant tissue is selected from at least one of corn leaf cells and pepper leaf cells.