PH calibration kit based on plant cell adaptation and application method thereof

Abstract

The invention relates to the technical field of biological detection, in particular to a plant cell adaptation-based pH calibration kit, which comprises a multi-pH buffer solution set, a plant adaptation fluorescent probe, a cell wall enzymolysis reagent, a pH calibration solution and a negative control solution, the multi-pH buffer solution set comprises eight buffer solutions with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 6.8 and 7.0, and the plant adaptation fluorescent probe is used for detecting the pH of the plant cell adaptation-based pH calibration kit. A buffer solution in an extreme acid region with the pH value of 4.0-5.5 takes citrate as a main buffer agent, a buffer solution in a weak acid region with the pH value of 5.5-6.5 takes 2-morpholine ethanesulfonic acid as a main buffer agent, and a buffer solution in a neutral region with the pH value of 7.0 takes 4-hydroxyethyl piperazine ethanesulfonic acid as a main buffer agent; through broad-spectrum pH coverage, efficient probe loading and anti-interference design, three core values are provided for plant cell research, multiple technical problems in plant cell pH accurate detection are effectively solved, and a reliable tool is provided for researching intracellular pH dynamics of plants in the processes of photosynthesis regulation, stress response, ion transport and the like.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a pH calibration kit based on plant cell adaptation and an application method thereof. Background Art

[0002] In plant physiology research, the dynamic changes in pH values inside and outside cells are important factors in regulating various life activities. For example, photosynthesis, nutrient absorption, and the response to adverse stresses such as salt or heavy metals are all significantly affected by pH fluctuations. In order to further reveal the regulatory mechanisms of these processes, researchers urgently need detection technologies that can accurately measure the pH values in different subcellular compartments of plant cells.

[0003] However, currently available pH calibration systems are primarily designed for animal cells. Direct application to plant cells faces technical bottlenecks such as pH detection range mismatch, difficulty in probe entry into plant cells, and mismatch in the ionic environment of the calibration solution. Although some pH probes and calibration systems have been attempted for plant cell research, most existing technologies are developed based on animal cell platforms and have significant drawbacks and deficiencies when directly transplanted to plant systems. These are primarily reflected in the following aspects:

[0004] 1. pH detection range is not covered enough

[0005] Existing pH measurement systems are mostly focused on the pH range of 5.5–7.0, failing to effectively cover the extreme acidic and alkaline regions within plant cells. For example, the pH of the vacuole can drop as low as 4.0, while the chloroplast stroma often exceeds pH 7.5. Existing calibration buffers cannot accurately measure these compartments, resulting in distorted pH readings and limiting in-depth research on the mechanisms regulating photosynthesis, vacuolar energy storage, and stress responses.

[0006] 2. Low probe loading efficiency and poor signal quality

[0007] The dense cell wall of plants significantly hinders the penetration of fluorescent probes. Traditional probes (such as BCECF-AM and SNARF-1) in plant protoplasts have low loading rates, uneven signal distribution, and high background noise, resulting in poor detection sensitivity and data reproducibility, seriously affecting the accuracy and reliability of experimental results.

[0008] 3. The ion environment simulation is not accurate and the calibration error is large

[0009] Existing calibration buffers are mostly designed based on animal cell standard systems. + / Na + The ratio and total ion strength of plant cells deviate significantly from the actual internal environment of plant cells. For example, plant cytoplasm is usually high in potassium (K + / Na +The pH measurement error is as high as ±0.3 units, while the probe pKa value shifts due to the presence of a high ionic strength (≈10:1) and a high ionic strength (approximately 200 mM). However, conventional buffer designs favor a low potassium, high sodium environment. This mismatch causes the probe pKa value to shift, the calibration curve to delinear, and ultimately results in a pH measurement error of up to ±0.3 units.

[0010] 4. Complicated operation and poor system adaptability

[0011] Existing pH detection protocols often require complex operations such as multiple rounds of centrifugation, washing, and resuspension, which not only increase the risk of cell damage but also reduce experimental efficiency, making them difficult to adapt to high-throughput or complex sample processing requirements. Furthermore, the lack of probe loading optimization strategies tailored to the structural characteristics of plant cells leads to poor compatibility between different plant materials and significant application limitations.

[0012] 5. Buffer design lacks plant specificity

[0013] Many commercial pH test kits labeled "plant-specific" do not fully consider the actual osmotic pressure, ionic strength, and pH dynamic characteristics of plant cells in their actual design. They only adapt to plant samples by extending the probe incubation time or simply adjusting the buffer concentration, failing to fundamentally improve the probe signal quality and calibration accuracy.

[0014] Existing pH detection and calibration technologies in plant cell applications suffer from a wide range of limitations, including low probe loading efficiency, distorted ionic environments, complex operation, poor compatibility, and a lack of plant-specific buffer design. These limitations make it difficult to accurately study the complex compartmentalized structures and rapid, dynamic pH changes of plant cells. Therefore, there is an urgent need to develop a pH detection and calibration system truly based on plant cell physiological characteristics, with both high resolution and high compatibility, to enhance the accuracy and reproducibility of studies on plant physiology and stress responses. Summary of the Invention

[0015] In order to overcome the common problems of existing pH detection and calibration technologies in plant cell applications, such as insufficient detection range, low probe loading efficiency, distorted ion environment, complex operation, poor adaptability, and lack of plant-specific buffer design.

[0016] The technical solution of the present invention is: a pH calibration kit based on plant cell adaptation, comprising a multi-pH buffer set, a plant-adapted fluorescent probe, a cell wall enzymatic hydrolysis reagent, a pH calibration solution and a negative control solution.

[0017] The multi-pH buffer set contains eight buffers with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 6.8, and 7.0. The buffer in the extreme acidic region of pH 4.0-5.5 uses citrate as the main buffer at a concentration of 20-25 mM, the buffer in the weakly acidic region of pH 5.5-6.5 uses 2-morpholinoethanesulfonic acid (MES) as the main buffer at a concentration of 20 mM, and the buffer in the neutral region of pH 7.0 uses 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) as the main buffer at a concentration of 10 mM. The buffer also contains potassium chloride (KCl), mannitol, polyvinylpyrrolidone (PVP), and ethylenediaminetetraacetic acid (EDTA).

[0018] The plant aptamer fluorescent probe was 8-hydroxypyrene-1,3,6-trisulfonic acid acetylmethyl ester (HPTS-AM), with a molecular weight of 524 Da, dissolved in dimethyl sulfoxide (DMSO) containing 0.01% Pluronic F-127;

[0019] The cell wall enzymatic hydrolysis reagent contained 0.5% (w / v) cellulase and 0.2% (w / v) pectinase, and an enzymatic hydrolysis buffer, wherein the enzymatic hydrolysis buffer was 20 mM MES (pH 5.5) and 10 mM calcium chloride (CaCl2).

[0020] Preferably, by constructing a multi-point buffer system with a pH range of 4.0 to 7.0 (a total of 8 pH values), comprehensive coverage of different cell compartments is achieved, and the resolution of the acidic microenvironment is significantly improved. By using HPTS-AM as a fluorescent probe and matching DMSO + Pluronic F-127 as a dispersion system, combined with a special enzymatic hydrolysis reagent (cellulase + pectinase) for mild wall removal treatment, the probe entry rate and signal quality are significantly improved. The buffer components are optimized (such as KCl 150mM) to better simulate the intracellular ion environment of plants, ensure the accuracy and repeatability of pH calibration, and provide standardized protoplast preparation and probe incubation processes. The frozen mixed components are quickly reconstituted and ready for use, which greatly simplifies the experimental operation and is suitable for high-throughput detection of various plant materials.

[0021] Preferably, the concentration of potassium chloride (KCl) is 150 mM, which is used to simulate a high potassium ion environment; the concentration of mannitol is 150 mM, which is used to simulate and regulate the osmotic pressure of the plant cytoplasm (300-320 mOsm / kg) to prevent cell swelling or shrinkage; the concentration of polyvinylpyrrolidone (PVP) is 0.2% (w / v), which is used to adsorb phenolic substances released by plant cells, reduce fluorescence quenching, and improve the signal-to-noise ratio; the concentration of ethylenediaminetetraacetic acid (EDTA) is 0.1 mM, which is used to chelate metal ions and protect the stability of the fluorescent probe.

[0022] Preferably, the preparation steps of pH 4.0 buffer are as follows:

[0023] Step (1): accurately weighing sodium citrate, potassium chloride, mannitol, polyvinylpyrrolidone (PVP) and ethylenediaminetetraacetic acid (EDTA) for use;

[0024] Step (2): Dissolve in 800 mL of ultrapure water and stir magnetically until completely dissolved;

[0025] Step (3): Use 1 M hydrogen chloride (HCl) / sodium hydroxide (NaOH) to accurately adjust the pH (error ≤ ± 0.05);

[0026] Step (4): Add water to make the volume 1L;

[0027] Step (5): Filter and sterilize using a 0.22 μm PES membrane, dispense into 10 mL bottles, and freeze-dry for storage.

[0028] 4. The plant cell-adapted pH calibration kit according to claim 1, wherein the steps for preparing the plant-adapted fluorescent probe are as follows:

[0029] Step (1): Dissolve 1 mg of HPTS-AM in 1 mL of DMSO containing 0.01% Pluronic F-127;

[0030] Step (2): vortex mix for 5 minutes;

[0031] Step (3): Aliquot into 50 μL / tube and store at -20℃ in the dark.

[0032] Preferably, the steps for preparing the cell wall enzymatic hydrolysis reagent are as follows:

[0033] Step (1): determining cellulase activity by DNS method and determining pectinase activity by titration method;

[0034] Step (2): Mix with freeze-drying protective agent (5% trehalose + 1% BSA);

[0035] Step (3): Prefreeze, freeze-dry, divide into individual aluminum foil bags, and store at -20°C.

[0036] The application method of the pH calibration kit based on plant cell adaptation includes the pH calibration kit based on plant cell adaptation as described above, and the steps are as follows:

[0037] The first step is protoplast preparation. Plant tissue is enzymatically hydrolyzed using a cell wall enzymatic reagent at 25°C for 30 minutes, followed by centrifugation for 5 minutes to collect the protoplasts.

[0038] The second step is fluorescent probe loading. The protoplasts are incubated with plant-adapted fluorescent probes for 1 hour and washed to remove unloaded probes.

[0039] The third step is pH value detection and calibration. The protoplasts loaded with probes are placed in different pH buffers. Then, the fluorescence ratio (I450 / I400) is detected under the conditions of excitation wavelength 405 / 450nm and emission wavelength 510nm. The standard curve (R 2 =0.99), and the pH value inside the vacuole was calculated based on the standard curve.

[0040] Preferably, in the first step, the plant tissue is selected from one of leaves, root tips or algae cells.

[0041] Preferably, the fluorescence intensity detection in the third step is performed by a fluorescence spectrophotometer or a confocal microscope.

[0042] As a preference, in the third step, the software uses a cubic polynomial correction algorithm to control the calibration error within ±0.05 pH units and supports one-click import of fluorescence data (I 450 / I 400 ratio), automatically generates a standard curve and calculates the pH value, avoiding the subjective bias of manual fitting.

[0043] The pH calibration kit adapted for plant cells is used in plant research. The pH calibration kit is suitable for dynamic pH monitoring of rice protoplasts, tobacco mesophyll cells, and algae.

[0044] Beneficial effects of the present invention:

[0045] This pH calibration kit based on plant cell adaptation and its application method provide three core values for plant cell research through broad-spectrum pH coverage, efficient probe loading and anti-interference design. First, broad-spectrum adaptability breaks through the pH range limitations of traditional kits and designs 8 buffers with pH 4.0-7.0 to achieve precise monitoring across compartments. It can accurately detect vacuolar acidification, rapid response of the apoplast and chloroplast alkalinity changes, providing reliable indicators for plant salt-tolerance gene screening, immune mechanism and photosynthesis research; second, high signal-to-noise ratio. To address the problems of plant cell wall barriers and insufficient probe penetration, the HPTS-AM probe is used, whose molecular weight is 48% lower than that of BCECF-AM, and the sulfonic acid group enhances cell wall affinity, the fluorescence intensity is increased by 2.5 times, and combined with cellulase and pectinase treatment, the probe loading efficiency is increased from 28% to 72%, the signal-to-noise ratio is increased from 3.2 to 8.5, and the probe concentration is reduced at the same time. To reduce intracellular self-quenching and prolong photostability; thirdly, it has strong anti-interference ability. In order to solve the interference problems of phenolic compounds and ionic environment released by plant cells, a dual strategy is adopted to add 0.2% PVP to bind phenolic substances, weaken the fluorescence signal decay rate, optimize the buffer ion environment, simulate the real high potassium ion system in the cytoplasm, reduce the probe pKa shift to ±0.05, and maintain membrane integrity, avoid false pH fluctuations caused by ion imbalance, thereby effectively solving many technical difficulties in the precise detection of plant cell pH, and providing a reliable tool for studying the intracellular pH dynamics of plants during photosynthesis regulation, stress response and ion transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a comparison chart of the buffer system components and osmotic pressure under different pH conditions of the present invention;

[0047] Figure 2 This is a comparison chart of the effects of different treatment methods of the present invention on sample loading efficiency, fluorescence signal-to-noise ratio and half-life;

[0048] Figure 3 The present invention has different K + / Na + Comparison of pH errors in ratio buffers. DETAILED DESCRIPTION

[0049] The present invention will be further described below with reference to the accompanying drawings and examples.

[0050] Example 1

[0051] The specific experimental steps for accurate calibration of vacuolar pH in Arabidopsis mesophyll cells are as follows:

[0052] ① Cell pretreatment: Arabidopsis leaves were treated with cellulase (0.5% w / v) and pectinase (0.2% w / v) for 30 minutes to obtain intact mesophyll protoplasts;

[0053] ② Probe loading: incubate with HPTS-AM (10 μM) for 1 hour (25°C in the dark), and then centrifuge and wash to remove free probes;

[0054] ③ Cell lysis: Use Triton X-100 (0.1% v / v) to lyse the cell membrane and release the vacuolar contents;

[0055] ④ Fluorescence detection: The lysate was resuspended in pH 4.0, 4.5, 5.0, and 5.5 buffers, and fluorescence spectrophotometer was used to detect I 450 / I 400 ratio (excitation wavelength 405 / 450 nm, emission wavelength 510 nm);

[0056] ⑤ Curve fitting: The software automatically generates a standard curve (R 2 =0.99), and the vacuolar pH was inferred to be 5.2±0.1 (the traditional method was 5.0±0.3).

[0057] Example 2: Ion Compatibility Verification

[0058] Contrasting designs:

[0059] Traditional Group: K + / Na + =3:1 buffer (KCl 50 mM + NaCl 16.7 mM);

[0060] The present invention group: K + / Na + =10:1 buffer (KCl 150 mM + NaCl 15 mM);

[0061] Testing process:

[0062] ①Use the same probe (HPTS-AM) to load Arabidopsis root tip cells;

[0063] ② Resuspend the cells with two sets of buffers respectively, detect the fluorescence ratio and calculate the pH value;

[0064] ③ The true pH value measured by the microelectrode method (pH microsensor) was 6.5±0.05;

[0065] Results comparison:

[0066] Error of traditional buffer solution: 6.5→6.85±0.35 (Δ=+0.35);

[0067] Error of the buffer solution of the present invention: 6.5→6.58±0.08 (Δ=+0.08).

[0068] Experimental Example 1: Comparative experiment of buffer system composition and osmotic pressure under different pH conditions

[0069] (1) Buffer selection basis:

[0070] The pKa values of Citrate, MES, and HEPES respectively match the target pH range (4.0-7.0), which is consistent with the buffer selection principle (pKa close to the target pH).

[0071] The buffer concentration gradient (e.g., Citrate from 25 to 15 mM) achieves pH stability by adjusting the ratio of the conjugate acid-base pair, referring to the preparation method of phosphate buffer.

[0072] (2) Osmotic pressure calculation and verification

[0073] Osmotic pressure formula: Total osmotic pressure = electrolyte (KCl) contribution + mannitol contribution + buffer contribution.

[0074] KCl (150 mM) completely dissociates and contributes 300 mOsm / kg (150×2).

[0075] For every 10 mM increase in mannitol concentration, the osmotic pressure increases by 10 mOsm / kg (1 mM = 1 mOsm / kg).

[0076] The experimental verification uses an ice point osmometer and is calibrated by comparison with standard solutions (such as glucose and NaCl).

[0077]

[0078] Table 1: Comparison of buffer system composition and osmotic pressure under different pH conditions

[0079] Among them, Citrate buffer (pH 4.0-5.0): suitable for acidic environment, the buffer concentration decreases from 25mM to 15mM as the pH increases, MES buffer (pH 5.5-6.5): suitable for near-neutral acidic conditions, the concentration is fixed at 20mM to maintain buffering capacity, HEPES buffer (pH 7.0), used in neutral conditions, a lower concentration (10mM) can meet the stability requirements. From the data in Table 1 and Figure 1 It can be seen that pH is linearly correlated with osmotic pressure. For every 0.5 unit increase in pH, the osmotic pressure increases by about 5-10mOsm / kg: precise regulation can be achieved by reducing the buffer concentration (such as Citrate from 25→15mM) and increasing the mannitol concentration (150→200mM).

[0080] Experimental Example 2: Enzymatic hydrolysis and ultrasonic treatment experiments

[0081] (1) Enzymatic hydrolysis and ultrasonic treatment

[0082] Enzymatic hydrolysis conditions: Cellulase (0.5% w / v) and pectinase (0.2% w / v) were reacted at 25°C for 30 minutes to simulate the decomposition of plant cell walls.

[0083] Ultrasonic treatment: 40kHz ultrasonic treatment for 5 minutes to promote cell membrane permeability.

[0084] (2) Indicator measurement method

[0085] Loading efficiency: calculated by the uptake of fluorescent markers (such as FITC-dextran) and measuring the residual fluorescence intensity of the supernatant after centrifugation.

[0086] Fluorescence signal-to-noise ratio: The ratio of target signal to background noise is measured using a fluorescence microscope.

[0087] Half-life: The time required for the fluorescence intensity to decay to 50% over time.

[0088] (3) Significance analysis

[0089] One-way analysis of variance (ANOVA) was used, and significant differences were determined at p < 0.05 (marked with letters a, b, c).

[0090] Treatment method Loading efficiency (%) Fluorescence signal-to-noise ratio Half-life (minutes) No enzymatic hydrolysis 28±5 3.2±0.5 5±1 Cellulase (0.5% w / v) 58±7 6.8±0.8 8±2 Cellulase + Pectinase (0.2% w / v) 72±8 8.5±1.2 12±3 Ultrasonic assisted (40kHz, 5 minutes) 65±6 7.2±1.0 9±2

[0091] Table 2: Comparison of the effects of different treatment methods on sample loading efficiency, fluorescence signal-to-noise ratio and half-life

[0092] Table 2 compares the effects of no enzymatic hydrolysis, cellulase treatment, combined cellulase + pectinase treatment, and ultrasound-assisted treatment in sample preparation, covering three key indicators: loading efficiency, fluorescence signal-to-noise ratio, and half-life. It highlights the comprehensive advantages of combined enzymatic hydrolysis (loading efficiency 72%, signal-to-noise ratio 8.5, and half-life 12 minutes).

[0093] See also Figure 2 , Figure 2 The horizontal axis represents the different treatment methods, while the vertical axis represents the loading efficiency, fluorescence signal-to-noise ratio, and half-life of the sample preparation under different treatment methods. Orange represents no enzymatic digestion; purple represents cellulase treatment; gray represents cellulase + pectinase treatment; and green represents ultrasound-assisted digestion. Standard deviations (SD) were calculated from three independent experiments (n = 3). Figures were plotted using R programming language. Letters a, b, and c indicate significant differences (p < 0.05).

[0094] Experimental Example 3: K + / Na + Ratio optimization and experimentation

[0095] (1) Buffer preparation and simulated environment

[0096] Traditional group (K+ / Na + =3:1): Designed based on the ion ratio of conventional plant cell culture medium.

[0097] The present invention group (K + / Na + =10:1): By increasing K + Concentration simulates high potassium intracellular environment and reduces Na + Interference with pH probe.

[0098] (2) pH error determination

[0099] The pH value of the buffer solution was continuously monitored at 37 °C for 6 h using a high-precision pH meter (±0.01 accuracy), and the fluctuation range was calculated.

[0100] Error calculation: standard deviation (repeated experiments from three independent replicates).

[0101] (3) Optimization basis

[0102] High K + / Na + The ratio reduces the ion competition effect and stabilizes the fluorescence response of the pH probe.

[0103] Culture medium type buffer pH error Traditional Group <![CDATA[K + / That + =3:1]]> 0.35±0.05 The present invention group <![CDATA[K + / That + =10:1]]> 0.08±0.02

[0104] Table 3: Different K + / Na + Comparison of pH Errors in Ratio Buffers

[0105] Table 3 compares the traditional group (K + / Na + =3:1) and the present invention group (K + / Na + =10:1) buffer solution when simulating the intracellular environment of plant cells. The results show that the present invention significantly reduces the error (from ±0.35 to ±0.08 pH units) by optimizing the ion ratio.

[0106] See also Figure 3 , Figure 3 The horizontal axis represents different K + / Na + The ratio of the buffer solution, the vertical axis represents the pH measurement error of the buffer solution when simulating the environment inside the plant cell. + / Na + =10:1) buffer solution pH measurement error; purple represents the traditional group (K + / Na +=3:1) buffer pH measurement error. Standard deviation (SD) was calculated from three independent experiments (n=3). Plots were drawn using R language. Letters a and b represent significant differences (p<0.05).

[0107] The embodiments of the present invention are described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Various changes can be made within the scope of knowledge of those skilled in the art without departing from the spirit of the present invention.

Claims

1. A plant cell-adapted pH calibration kit comprising a multi-pH buffer set, characterized in that: It also includes plant-adapted fluorescent probes, cell wall enzymatic hydrolysis reagents, pH calibration solution and negative control solution. Among them, the multi-pH buffer set contains 8 buffers with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 6.8 and 7.

0. The buffer in the extremely acidic region of pH 4.0-5.5 uses citrate as the main buffer at a concentration of 20-25mM, the buffer in the weakly acidic region of pH 5.5-6.5 uses 2-morpholinoethanesulfonic acid as the main buffer at a concentration of 20mM, and the buffer in the neutral region of pH 7.0 uses 4-hydroxyethylpiperazineethanesulfonic acid as the main buffer at a concentration of 10mM. The buffer also contains potassium chloride, mannitol, polyvinylpyrrolidone and ethylenediaminetetraacetic acid. The plant-compatible fluorescent probe is 8-hydroxypyrene-1,3,6-trisulfonic acid acetyl methyl ester with a molecular weight of 524 Da, dissolved in dimethyl sulfoxide containing 0.01% Pluronic F-127; The cell wall enzymatic hydrolysis reagent contains 0.5% (w / v) cellulase and 0.2% (w / v) pectinase, and an enzymatic hydrolysis buffer, wherein the enzymatic hydrolysis buffer is 20 mM 2-morpholineethanesulfonic acid (pH 5.5) and 10 mM calcium chloride.

2. The plant cell-adapted pH calibration kit according to claim 1, characterized in that: The concentration of potassium chloride is 150mM, which is used to simulate a high potassium ion environment. The concentration of mannitol is 150mM, which is used to simulate and regulate the osmotic pressure of plant cytoplasm (300-320mOsm / kg) to prevent cell swelling or shrinkage. The concentration of ethylenediaminetetraacetic acid is 0.1mM, which is used to chelate metal ions and protect the stability of the fluorescent probe.

3. The pH calibration kit based on plant cell adaptation according to claim 1, characterized in that The steps for preparing pH 4.0 buffer are as follows: Step (1): accurately weighing sodium citrate, potassium chloride, mannitol, polyvinylpyrrolidone and ethylenediaminetetraacetic acid for use; Step (2): Dissolve in 800 mL of ultrapure water and stir magnetically until completely dissolved; Step (3): Use 1M hydrogen chloride / sodium hydroxide to accurately adjust the pH (error ≤ ± 0.05); Step (4): Add water to make the volume 1L; Step (5): Filter and sterilize using a 0.22 μm PES membrane, dispense into 10 mL bottles, and freeze-dry for storage.

4. The pH calibration kit based on plant cell adaptation according to claim 1, characterized in that The steps for preparing plant-adapted fluorescent probes are as follows: Step (1): Dissolve 1 mg of 8-hydroxypyrene-1,3,6-trisulfonic acid acetyl methyl ester in 1 mL of dimethyl sulfoxide containing 0.01% Pluronic F-127; Step (2): vortex mix for 5 minutes; Step (3): Aliquot into 50 μL / tube and store at -20℃ in the dark.

5. The pH calibration kit based on plant cell adaptation according to claim 1, characterized in that The steps for preparing the cell wall enzymatic hydrolysis reagent are as follows: Step (1): determining cellulase activity by DNS method and determining pectinase activity by titration method; Step (2): Mix with freeze-drying protective agent (5% trehalose + 1% acetamide); Step (3): Prefreeze, freeze-dry, divide into individual aluminum foil bags, and store at -20°C.

6. The application method of the pH calibration kit based on plant cell adaptation is characterized by: The method comprises the plant cell adaptation-based pH calibration kit according to any one of claims 1 to 7, wherein the steps are as follows: The first step is protoplast preparation. Plant tissue is enzymatically hydrolyzed using a cell wall enzymatic reagent at 25°C for 30 minutes, followed by centrifugation for 5 minutes to collect the protoplasts. The second step is fluorescent probe loading. The protoplasts are incubated with plant-adapted fluorescent probes for 1 hour and washed to remove unloaded probes. The third step is pH value detection and calibration. The protoplasts loaded with probes are placed in different pH buffers. Then, the fluorescence ratio (I450 / I400) is detected under the conditions of excitation wavelength 405 / 450nm and emission wavelength 510nm. The standard curve (R 2 =0.99), and the pH value inside the vacuole was calculated based on the standard curve.

7. The application method of the plant cell-adapted pH calibration kit according to claim 6, characterized in that: In the first step, the plant tissue is selected from one of leaves, root tips or algae cells.

8. The application method of the plant cell-adapted pH calibration kit according to claim 6, characterized in that: In the third step, fluorescence intensity detection is completed by fluorescence spectrophotometer or confocal microscope.

9. The application method of the plant cell-adapted pH calibration kit according to claim 6, characterized in that: In the third step, the software uses a cubic polynomial correction algorithm to control the calibration error within ±0.05pH units and supports one-click import of fluorescence data (I 450 / I 400 ratio), automatically generates a standard curve and calculates the pH value, avoiding the subjective bias of manual fitting.

10. Use of the plant cell-adapted pH calibration kit in plant research according to claim 1, characterized in that: The pH calibration kit is suitable for dynamic pH monitoring of rice protoplasts, tobacco mesophyll cells and algae.

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