An improved formula and preparation method of a plant physiological vigor qualitative detection solution

Abstract

The application discloses a formula and a preparation method of an improved plant physiological activity qualitative detection solution, and relates to the technical field of biological technology detection, and comprises the following raw materials: 2,3,5-chlorinated triphenyl tetrazolium, a phosphate buffer, a succinic acid buffer and a modified sodium sulfite solution; the application adds the modified sodium sulfite solution, the sodium sulfite in the modified sodium sulfite solution provides a strong reducing group to inhibit the oxidative decomposition of 2,3,5-chlorinated triphenyl tetrazolium, proline protects the activity of the sodium sulfite by weak coordination and slows down the failure speed of the sodium sulfite, and trehalose anchors each component to build a stable structure through a hydrogen bond network, and the three components are used in cooperation to significantly prolong the storage shelf life of the solution; the succinic acid buffer is added, the succinic acid buffer can provide a specific action target point for dehydrogenase, significantly enhances the enzymatic reaction efficiency, makes the reduction coloration of 2,3,5-chlorinated triphenyl tetrazolium faster and more uniform, and avoids the coloration light or local blank caused by insufficient enzyme activity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology detection technology, and in particular to a formulation and preparation method of an improved qualitative detection solution for plant physiological vitality. Background Technology

[0002] The qualitative detection solution for plant physiological vitality is a chemical reagent used to assess the life activity of plant tissues. Its core principle is to use colorless triphenyltetrazolium chloride as a substrate. Under weakly acidic conditions, it can be reduced by dehydrogenases in living cells to produce red triphenylmethylhydrazone, which is insoluble in water.

[0003] Traditional modified plant physiological activity qualitative detection solutions have extremely short shelf lives, only suitable for storage at 4°C in the dark for 3 days, requiring frequent fresh preparation and leading to cumbersome operations. The core components are prone to oxidation and decomposition, exhibiting poor stability and thus causing deviations in test results. pH fluctuations during storage and freeze-thaw cycles can easily degrade active ingredients. Non-specific reactions are also common, resulting in uneven color development and affecting observation and judgment. Furthermore, some related materials may pose toxicity or interference risks. Frequent preparation also increases experimental time and operational costs. Therefore, this invention provides an improved formulation and preparation method for a plant physiological activity qualitative detection solution. Summary of the Invention

[0004] The main objective of this invention is to provide a formulation for an improved qualitative detection solution for plant physiological activity with a long shelf life and high detection accuracy, which is applied to an improved formulation and preparation method for a qualitative detection solution for plant physiological activity.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a formulation for an improved qualitative detection solution for plant physiological activity, the improved plant physiological activity qualitative detection solution comprising the following raw materials: 0.08-0.12 parts of 2,3,5-triphenyltetrazolium chloride, 48-52 parts of phosphate buffer, 48-52 parts of succinate buffer, and 0.09-0.11 parts of modified sodium sulfite solution; The succinate buffer solution is a mixture of one of sodium succinate hexahydrate and anhydrous sodium succinate with deionized water; The mass ratio of sodium succinate hexahydrate to deionized water is 10.81:100; The mass ratio of anhydrous sodium succinate to deionized water is 6.48:100.

[0006] Furthermore, the phosphate buffer solution is prepared by mixing one of materials A and B with deionized water and adding a pH adjuster.

[0007] Furthermore, the mass ratio of material A to deionized water is 1.8623:98.1377.

[0008] Furthermore, material A is a mixture of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate in a mass ratio of 0.4056:1.4567.

[0009] Furthermore, the mass ratio of material B to deionized water is 0.9362:99.0638.

[0010] Furthermore, material B is composed of sodium dihydrogen phosphate monohydrate and anhydrous disodium hydrogen phosphate mixed in a mass ratio of 0.3588:0.5774.

[0011] Furthermore, the pH adjuster is one of a 0.1M phosphate solution and a 0.1M sodium hydroxide solution; The pH of the phosphate buffer solution is adjusted to 7 by adding a pH adjuster.

[0012] Furthermore, the phosphate solution is a mixture of one of sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, anhydrous sodium dihydrogen phosphate, sodium dihydrogen phosphate monohydrate, and anhydrous disodium hydrogen phosphate with deionized water.

[0013] Furthermore, the preparation of the modified sodium sulfite solution includes the following steps: adding sodium sulfite, proline and trehalose to deionized water and stirring at a speed of 250 rpm for 3 minutes to obtain the modified sodium sulfite solution; The mass ratio of sodium sulfite, proline, trehalose, and deionized water is 0.005:0.01:0.02:10.

[0014] Proline has a purity of ≥98% and is food grade. It binds to sodium sulfite through weak coordination, encapsulating its reducing groups, slowing down its oxidation by oxygen, and prolonging its antioxidant effect.

[0015] Trehalose with a purity of ≥99% protects the structure of 2,3,5-triphenyltetrazolium chloride from being destroyed by freeze-thaw cycles, allowing the solution to maintain its activity after freezing and thawing at -20℃, thus improving storage flexibility. It also maintains the stability of plant tissue cell membranes, reducing the unnatural decline in sample activity caused by dehydration or environmental changes during the detection process, and improving detection repeatability. In synergy with sodium sulfite and proline, it further extends the shelf life of the detection solution without affecting the core detection reaction.

[0016] Secondly, the present invention provides a method for preparing an improved qualitative detection solution for plant physiological activity, the preparation of which includes the following steps: mixing 2,3,5-triphenyltetrazolium chloride, phosphate buffer and succinate buffer evenly, adding modified sodium sulfite solution and mixing evenly to obtain the improved qualitative detection solution for plant physiological activity.

[0017] The present invention has the following beneficial effects: 1. In this invention, a phosphate buffer is added, which can precisely stabilize the pH of the solution within the neutral range through the synergistic effect of conjugate acid-base pairs, perfectly matching the optimal reaction environment of dehydrogenases and avoiding the decrease or inactivation of enzyme activity caused by pH fluctuations. At the same time, the mild ionic strength it provides can maintain the integrity of plant tissue cells, prevent cells from rupturing and releasing impurity proteins due to osmotic pressure imbalance, reduce non-specific reactions, and protect the 2,3,5-triphenyltetrazolium chloride structure from hydrolysis or oxidation, further improving the stability and repeatability of the detection system. Moreover, the raw materials are readily available and inexpensive, making it a preferred material for routine laboratory buffer systems.

[0018] 2. In this invention, succinate buffer is added. Succinate buffer provides a specific target site for dehydrogenase, significantly enhancing the efficiency of enzymatic reactions. This allows for faster and more uniform reduction and color development of 2,3,5-triphenyltetrazolium chloride, avoiding faint color development or local blank areas due to insufficient enzyme activity. It is also highly stable, not reacting with 2,3,5-triphenyltetrazolium chloride, phosphates, or other components in a neutral environment. Furthermore, its concentration is easily and precisely controlled, adapting to the detection needs of different plant tissues. This improves the effectiveness of the detection solution and ensures the consistency of the results.

[0019] 3. In this invention, a modified sodium sulfite solution is added. The sodium sulfite provides a strong reducing group to inhibit the oxidative decomposition of 2,3,5-triphenyltetrazolium chloride. Proline protects the activity of sodium sulfite and slows down its decomposition through weak coordination. Trehalose anchors each component to build a stable structure through a hydrogen bond network. The three work synergistically to significantly extend the shelf life of the solution while completely not interfering with the core detection reaction—it does not change the neutral pH environment of the solution, does not inhibit the activity of plant dehydrogenases, and does not react with 2,3,5-triphenyltetrazolium chloride or plant tissues. There is no background color interference. Moreover, the raw materials are all low-toxicity / food-grade reagents, which are highly safe, easy to dissolve and mix, and suitable for simple laboratory preparation procedures. It can also enhance the solution's freeze-thaw resistance and concentration uniformity, further improving the repeatability and reliability of the detection results. It fundamentally solves the pain points of traditional detection solutions that require frequent preparation and have poor storage stability. Attached Figure Description

[0020] Figure 1This is an instantaneous bioactivity test image of pollen after soaking in a conventional plant physiological bioactivity qualitative test solution for 15 minutes. Figure 2 This is an instantaneous bioactivity test image of pollen after soaking in a modified plant physiological bioactivity qualitative detection solution for 15 minutes. Figure 3 This is an instantaneous vitality test image of peanuts after soaking in a modified qualitative detection solution for plant physiological vitality for 15 minutes. Figure 4 This is an instantaneous vitality test image of peanuts after soaking in a modified plant physiological vitality qualitative detection solution for 1 hour. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] It should be noted that all raw materials used in the following experiments are commercially available.

[0023] Example 1, see Figures 1-4 A formulation of an improved qualitative detection solution for plant physiological activity, comprising the following ingredients: 0.08 parts of 2,3,5-triphenyltetrazolium chloride, 48 parts of phosphate buffer, 48 parts of succinate buffer, and 0.09 parts of modified sodium sulfite solution; Succinate buffer is a mixture of sodium succinate hexahydrate and deionized water; The mass ratio of sodium succinate hexahydrate to deionized water is 10.81:100.

[0024] Phosphate buffer is prepared by mixing material A with deionized water, adding a pH adjuster and mixing thoroughly, and then filtering with qualitative filter paper to remove impurities.

[0025] The mass ratio of material A to deionized water is 1.8623:98.1377.

[0026] Material A is a mixture of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate in a mass ratio of 0.4056:1.4567.

[0027] The pH adjuster is one of a 0.1M phosphate solution and a 0.1M sodium hydroxide solution; Adjust the pH of the phosphate buffer to 7 by adding a pH adjuster.

[0028] The phosphate solution is a mixture of sodium dihydrogen phosphate dihydrate and deionized water.

[0029] The preparation of modified sodium sulfite solution includes the following steps: adding sodium sulfite, proline and trehalose into deionized water and stirring at 250 rpm for 3 minutes to obtain modified sodium sulfite solution; The mass ratio of sodium sulfite, proline, trehalose, and deionized water is 0.005:0.01:0.02:10.

[0030] A method for preparing an improved qualitative detection solution for plant physiological activity includes the following steps: mixing 2,3,5-triphenyltetrazolium chloride, phosphate buffer, and succinate buffer evenly, adding modified sodium sulfite solution and mixing evenly to obtain the improved qualitative detection solution for plant physiological activity.

[0031] Take fresh, mature flowers that are about to open, carefully remove the petals and pistils, place the pollen on a glass slide, add 1-2 drops of a pollen-modified plant physiological activity qualitative detection solution, and cover with a coverslip. Incubate at 37℃ (away from light) for 15 minutes, and observe under a low-power microscope, taking 5 fields of view for each slide.

[0032] Example 2, see Figures 1-4 A formulation of an improved qualitative detection solution for plant physiological activity, comprising the following ingredients: 0.1 parts of 2,3,5-triphenyltetrazolium chloride, 50 parts of phosphate buffer, 50 parts of succinate buffer, and 0.1 parts of modified sodium sulfite solution; Succinate buffer is a mixture of anhydrous sodium succinate and deionized water; The mass ratio of anhydrous sodium succinate to deionized water is 6.48:100.

[0033] Phosphate buffer is prepared by mixing material B with deionized water, adding a pH adjuster and mixing thoroughly, and then filtering with qualitative filter paper to remove impurities.

[0034] The mass ratio of material B to deionized water is 0.9362:99.0638.

[0035] Material B is a mixture of sodium dihydrogen phosphate monohydrate and anhydrous disodium hydrogen phosphate in a mass ratio of 0.3588:0.5774.

[0036] The pH adjuster is one of a 0.1M phosphate solution and a 0.1M sodium hydroxide solution; Adjust the pH of the phosphate buffer to 7 by adding a pH adjuster.

[0037] The phosphate solution is a mixture of disodium hydrogen phosphate dodecahydrate and deionized water.

[0038] The preparation of modified sodium sulfite solution includes the following steps: adding sodium sulfite, proline and trehalose into deionized water and stirring at 250 rpm for 3 minutes to obtain modified sodium sulfite solution; The mass ratio of sodium sulfite, proline, trehalose, and deionized water is 0.005:0.01:0.02:10.

[0039] A method for preparing an improved qualitative detection solution for plant physiological activity includes the following steps: mixing 2,3,5-triphenyltetrazolium chloride, phosphate buffer, and succinate buffer evenly, adding modified sodium sulfite solution and mixing evenly to obtain the improved qualitative detection solution for plant physiological activity.

[0040] Take 0.5g of peanuts, remove the shells and seed coats, immerse the peanuts in a modified plant physiological activity qualitative detection solution, and incubate at 37℃ in the dark for 15min; observe the coloring.

[0041] Example 3, see Figures 1-4 A formulation of an improved qualitative detection solution for plant physiological activity, comprising the following ingredients: 0.12 parts of 2,3,5-triphenyltetrazolium chloride, 52 parts of phosphate buffer, 52 parts of succinate buffer, and 0.11 parts of modified sodium sulfite solution. Succinate buffer is a mixture of sodium succinate hexahydrate and deionized water; The mass ratio of sodium succinate hexahydrate to deionized water is 10.81:100.

[0042] Phosphate buffer is prepared by mixing material B with deionized water, adding a pH adjuster and mixing thoroughly, and then filtering with qualitative filter paper to remove impurities.

[0043] The mass ratio of material B to deionized water is 0.9362:99.0638.

[0044] Material B is a mixture of sodium dihydrogen phosphate monohydrate and anhydrous disodium hydrogen phosphate in a mass ratio of 0.3588:0.5774.

[0045] The pH adjuster is one of a 0.1M phosphate solution and a 0.1M sodium hydroxide solution; Adjust the pH of the phosphate buffer to 7 by adding a pH adjuster.

[0046] The phosphate solution is a mixture of anhydrous sodium dihydrogen phosphate and deionized water.

[0047] The preparation of modified sodium sulfite solution includes the following steps: adding sodium sulfite, proline and trehalose into deionized water and stirring at 250 rpm for 3 minutes to obtain modified sodium sulfite solution; The mass ratio of sodium sulfite, proline, trehalose, and deionized water is 0.005:0.01:0.02:10.

[0048] A method for preparing an improved qualitative detection solution for plant physiological activity includes the following steps: mixing 2,3,5-triphenyltetrazolium chloride, phosphate buffer, and succinate buffer evenly, adding modified sodium sulfite solution and mixing evenly to obtain the improved qualitative detection solution for plant physiological activity.

[0049] Take 0.5g of peanuts, remove the shells and seed coats, then immerse the peanuts in a modified plant physiological activity qualitative detection solution and incubate at 37℃ in the dark for 1 hour; observe the coloring.

[0050] Example 4, the preparation of a conventional qualitative detection solution for plant physiological activity includes the following steps: B1. Weigh 0.5g of 2,3,5-triphenyltetrazolium chloride, add 1mL of 95% ethanol and mix well, then add deionized water to make up to 100mL to obtain a 2,3,5-triphenyltetrazolium chloride solution. B2. Mix 0.4g of 2,3,5-triphenyltetrazolium chloride, 0.4056g of sodium dihydrogen phosphate dihydrate, 1.4567g of disodium hydrogen phosphate dodecahydrate and 80mL of deionized water until homogeneous, then add deionized water to bring the volume to 100mL to obtain a mixed solution. B3. Mix the 2,3,5-triphenyltetrazolium chloride solution and the mixed solution thoroughly to obtain a conventional qualitative detection solution for plant physiological activity.

[0051] Take fresh, mature flowers that are about to open, carefully remove the petals and pistils, place the pollen material on a glass slide, add 1-2 drops of a standard plant physiological activity qualitative test solution, and cover with a coverslip. Incubate at 37℃ (away from light) for 15 minutes, and observe under a low-power microscope, taking 5 fields of view for each slide.

[0052] Comparative Example 1: The difference between this comparative example and Example 1 is that: Modified sodium sulfite solution was not used in this comparative example.

[0053] Comparative Example 2: The difference between this comparative example and Example 1 is that: Phosphate buffer was not used in this comparative example.

[0054] Comparative Example 3 differs from Example 1 in that: In this comparative example, qualitative filter paper was not used to remove impurities.

[0055] Performance testing: The improved plant physiological activity qualitative detection solutions prepared in Examples 1, 2, 3, Comparative Examples 1, 2, and 3 were tested.

[0056] Performance testing: The relevant properties of the improved plant physiological activity qualitative detection solution provided in Examples 1-3 and Comparative Examples 1-3 were tested, and the test data are recorded in Table 1 below:

[0057] Based on the above data, the following conclusions can be drawn: Among them, the modified plant physiological activity qualitative detection solution prepared according to the test method in GB15346-2012 in Examples 1, 2, 3, Comparative Examples 1, 2, and 3 was tested for storage time at 4°C in the dark. The staining vigor deviation rate of the modified plant physiological vigor qualitative detection solution prepared according to the test methods in GB / T3543.9-2025 (Examples 1, 2, 3, Comparative Examples 1, 2, and 3) was tested. The content of humic acid in the modified plant physiological activity qualitative detection solutions prepared according to the test methods in GB / T23738-2009 (Examples 1, 2, 3, Comparative Examples 1, 2, and 3) was tested.

[0058] Through the above demonstrations, the present invention is significantly superior to the control group in terms of storage time at 4℃ in the dark, staining vitality deviation rate and humic acid content in the solution, thus verifying the advanced nature and rationality of the preparation process.

[0059] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0060] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A formulation for an improved qualitative detection solution of plant physiological activity, characterized in that, The improved qualitative detection solution for plant physiological activity comprises the following raw materials: 0.08-0.12 parts of 2,3,5-triphenyltetrazolium chloride, 48-52 parts of phosphate buffer, 48-52 parts of succinate buffer, and 0.09-0.11 parts of modified sodium sulfite solution; The succinate buffer solution is prepared by mixing one of sodium succinate hexahydrate and anhydrous sodium succinate with deionized water; The mass ratio of sodium succinate hexahydrate to deionized water is 10.81:100; The mass ratio of anhydrous sodium succinate to deionized water is 6.48:

100.

2. The improved plant physiological activity qualitative detection solution according to claim 1, characterized in that, The phosphate buffer solution is prepared by mixing one of materials A and B with deionized water and adding a pH adjuster.

3. The improved plant physiological activity qualitative detection solution according to claim 2, characterized in that, The mass ratio of material A to deionized water is 1.8623:98.1377.

4. The improved plant physiological activity qualitative detection solution according to claim 3, characterized in that, Material A is a mixture of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate in a mass ratio of 0.4056:1.4567.

5. The improved plant physiological activity qualitative detection solution according to claim 2, characterized in that, The mass ratio of material B to deionized water is 0.9362:99.0638.

6. The improved plant physiological activity qualitative detection solution according to claim 5, characterized in that, Material B is a mixture of sodium dihydrogen phosphate monohydrate and anhydrous disodium hydrogen phosphate in a mass ratio of 0.3588:0.5774.

7. The improved plant physiological activity qualitative detection solution according to claim 2, characterized in that, The pH adjuster is one of a 0.1M phosphate solution and a 0.1M sodium hydroxide solution.

8. The improved plant physiological activity qualitative detection solution according to claim 7, characterized in that, The phosphate solution is a mixture of one of sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, anhydrous sodium dihydrogen phosphate, sodium dihydrogen phosphate monohydrate, and anhydrous disodium hydrogen phosphate with deionized water.

9. The improved plant physiological activity qualitative detection solution according to claim 1, characterized in that, The preparation of the modified sodium sulfite solution includes the following steps: adding sodium sulfite, proline and trehalose to deionized water and mixing them evenly to obtain the modified sodium sulfite solution; The mass ratio of sodium sulfite, proline, trehalose, and deionized water is 0.005:0.01:0.02:

10.

10. A method for preparing a modified qualitative detection solution for plant physiological activity according to any one of claims 1-9, characterized in that, The preparation of the improved qualitative detection solution for plant physiological activity includes the following steps: mixing 2,3,5-triphenyltetrazolium chloride, phosphate buffer, and succinate buffer evenly, adding modified sodium sulfite solution and mixing evenly to obtain the improved qualitative detection solution for plant physiological activity.

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