Rapid dyeing method for detecting cold injury degree of durian and application

Abstract

The invention relates to the technical field of durian cold damage detection, and provides a rapid dyeing method for detecting the cold damage degree of durian and application. The invention relates to a visual detection method, which comprises the steps of dyeing, decoloration and preservation, and specifically comprises the steps of cold treatment, cleaning, vacuumizing, dyeing, decoloration, photographing recording and preservation, and the cold damage degree of durian can be quickly judged by obtaining cold damage phenotypes such as leaf outlines and metabolic changes of durian leaves in a cold damage state. The cold damage state and damage degree of different varieties of durian leaves can be directly observed by treating the durian leaves with the staining solution, the decoloring solution and the preserving solution according to the formula, and the durian leaves can be easily preserved at room temperature for a long time. The detection method has the advantages of simplicity and convenience in operation, clear dyeing contrast, rapidness, intuition and low cost, and can be used for observing and judging the cold damage degree of malvaceae and other species with similar leaf structures. The method is expected to be applied to the fields of plant protection, crop cultivation and production and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of durian chilling injury detection, and relates to a rapid staining method for detecting the chilling injury degree of durian and an application thereof. Background Art

[0002] Durian, known as the "king of fruits", has strict requirements for the growing environment and is a typical representative of tropical fruits. As a pure tropical crop, durian is extremely sensitive to temperature conditions and needs to grow well in a climate with an average annual absolute low temperature above 0°C and an accumulated temperature of more than 7000°C. As the world's largest durian consumer market, China's annual trade volume with Southeast Asia is nearly 10 billion US dollars, which shows that the potential market for domestic durian cultivation is extremely broad. However, in the process of migrating durian to the north for cultivation, high altitudes and cold climates pose severe challenges. Therefore, monitoring whether durian suffers from cold damage and the degree of damage is crucial to ensuring the success of durian cultivation and increasing yields.

[0003] Hydrogen peroxide (H2O2), as a key molecule among reactive oxygen species, plays an important role in plant physiology. Under environmental stresses such as low temperature stress, high light stress, water stress, salt stress and heavy metal stress, the production and accumulation of H2O2 can significantly affect multiple physiological aspects of plant growth, reproductive development, aging and response to adverse stress. Therefore, the determination of H2O2 levels has become an important indicator for evaluating the physiological state of plants. Under the action of catalase, H2O2 reacts with diaminobenzidine dye to quickly form a brown product, thereby achieving the localization and quantification of H2O2 in tissues. This method provides an intuitive and efficient analytical method for studying the oxidative stress response of plants under different environmental pressures. However, durian leaves are not easy to be stained, mainly due to the combined effects of their structure, composition and physiological characteristics. The specific reasons are as follows: (1) Durian leaves have a special structure and a thick cuticle. This cuticle is like a dense protective film that can reduce water loss and the invasion of external substances, and also greatly hinders the penetration of dyes. Its epidermal cells are closely arranged and have small intercellular gaps, which further increases the difficulty for the dye to enter the cells, making it difficult for the dye to fully contact the target substance in the cells, thus affecting the dyeing effect; (2) Durian leaves contain a large amount of polysaccharides, phenols, tannins and other substances. These components will react chemically with the dye, reduce the activity of the dye, and cause the dye to be unable to bind normally to the target substance, affecting the dyeing effect. For example, phenolic substances will form quinone compounds after oxidation, which can bind to biological macromolecules such as proteins and nucleic acids, thereby interfering with the binding of dyes to these biological macromolecules. In addition, polysaccharides may wrap around the cell surface, hindering the approach of dyes; (3) As a tropical plant, durian leaves have vigorous physiological metabolism. During the dyeing process, leaf cells will activate self-protection mechanisms, actively transport and expel dyes that have entered the cells, or change the intracellular environment through their own metabolic activities, making it difficult for dyes to work. For example, cells may pump dye ions out of cells through ion pumps, or produce some substances through metabolism to change the pH value in the cells, affecting the ionization state of the dye, thereby affecting the dyeing effect.

[0004] Some existing dyeing methods make it difficult for the dyeing solution to enter the plant leaves due to the hydrophobicity and surface tension of the leaves, resulting in poor dyeing effect, often incomplete dyeing, and only a part of the leaves can be dyed. The dyeing of complete leaves usually requires the processing of the leaf veins, which causes mechanical damage to the leaves, resulting in unclear decolorization effect after dyeing and failure to completely remove the mottled colors on the leaves, and failure to intuitively and accurately reflect the H2O2 accumulation caused by stress; or the reaction rate of the dyeing solution with H2O2 is not high enough, resulting in a long dyeing time of up to 8-12 hours or even longer; or the reaction sensitivity of the dyeing solution with H2O2 is not high enough, and the accuracy and clarity are not high.

[0005] In the existing plant leaf decolorization technology, alcohol solution is a more commonly used decolorization reagent. However, when faced with durian leaves with thick leaves and high pigment content, the limitations of using alcohol for decolorization alone are exposed. Due to the special structure and rich pigments of durian leaves, decolorization using only alcohol is often time-consuming, often taking several hours or even longer. This undoubtedly greatly slowed down the experimental process, seriously affected the decolorization efficiency, and hindered the advancement of research work.

[0006] In addition, long-term immersion in high-concentration alcohol will gradually make the durian leaves brittle and their cell structures damaged to varying degrees. This makes the leaves very easy to break during subsequent operations, such as slicing and transferring, resulting in incomplete leaf morphology, making it difficult to make accurate observations and not conducive to long-term preservation of samples.

[0007] In the plant leaf dyeing experiment, the decolorization treatment effectively reduced the possible interaction between the natural pigments of the leaves and the dyes, and reduced the risk of color change or fading of the dyes due to environmental factors such as light and oxygen. However, when we expect the leaves to be stored for a long time at room temperature, we need to maintain their structural integrity, prevent breakage and mildew, and ensure that the dyes are fixed to extend the shelf life of the dyed samples and meet the needs of subsequent scientific research. Therefore, the study of the composition of the preservation solution is particularly critical.

[0008] At present, common preservation methods have their own advantages and disadvantages. Although the dry preservation method is easy to operate, it is very easy to cause the leaves to crack and change color; although the frozen preservation can maintain the physiological state of the leaves to a certain extent, it may damage the cell structure, causing the leaves to rupture cells and collapse tissues after thawing, which seriously affects the observation effect. If the thawing speed is not properly controlled, it will also cause freeze-thaw damage. In comparison, the liquid preservation method has significant advantages. It can maintain the softness of the leaves and stabilize the cell structure. It can not only prevent the dye from fading, but also fix the color.

[0009] In the prior art, formalin solution is often used for liquid preservation. However, it cannot be ignored that formalin is highly toxic and volatile, which not only poses a potential hazard to human health, but also has adverse effects on the environment. Therefore, it is particularly important to develop a preservation solution component that is both safe and effective and can meet the needs of long-term preservation.

[0010] Therefore, there is an urgent need to provide a complete set of rapid processing and dyeing technology systems that adapt to the special needs of durian leaves, including the formulations of dyeing solution, decolorizing solution and room temperature storage solution, so as to detect the degree of chilling damage to durian. Summary of the invention

[0011] The object of the invention is to detect the degree of damage of Malvaceae durian by cold injury, and to develop a set of brand-new and simple to operate, fast dyeing method and system. Durian blade is known for its leathery characteristics, smooth surface, leather-like texture, thick cell wall, and granular scale structure covered on the back of the leaf, which give durian blade stronger hydrophobicity and surface tension. Based on these unique physiological characteristics, the present invention has specially designed a complete set of processing and dyeing technology systems for durian blade, including the formula of dyeing solution, decolorizing solution and room temperature preservation solution, to adapt to the special needs of durian blade. The application of these technologies will help to more accurately assess the physiological state of durian blades of different varieties under cold stress, and provide powerful technical support for cold resistance research and cultivation management of durian.

[0012] In order to achieve the above object, the present invention provides the following technical solutions:

[0013] The first aspect of the present invention provides a dyeing solution for durian leaves, wherein the dyeing solution is a mixed solution containing 0.1 mg / mL natural alginic acid and 2 mg / mL diaminobenzidine. Natural alginic acid and diaminobenzidine reagent are prepared into a mixed solution at a specific concentration, and the two cooperate with each other to achieve effective dyeing of H2O2 in durian leaves. Natural alginic acid creates a good dyeing environment, helps diaminobenzidine to better exert its dyeing function, makes the dyeing result more accurate and clear, and is convenient for relevant technical personnel in the field to observe and analyze.

[0014] The second aspect of the present invention provides a decolorizing solution for durian leaves, wherein the decolorizing solution is prepared by mixing anhydrous ethanol, 0.01M fatty acid methyl ester sulfonate and 50% v / v glycerol in a volume ratio of 5:1:1. The three cooperate with each other: anhydrous ethanol is mainly responsible for dissolving chlorophyll, fatty acid methyl ester sulfonate accelerates the dissolution and diffusion of chlorophyll by anhydrous ethanol through emulsification and reducing surface tension, and glycerol stabilizes the system, maintains the leaf morphology and assists in dissolving related substances, thereby achieving the purpose of efficiently removing chlorophyll.

[0015] The third aspect of the present invention provides a room temperature preservation solution for durian leaves, wherein the room temperature preservation solution is a mixed solution composed of 100 mg / mL aminolevulinic acid, 100% v / v triethanolamine and 50% v / v glycerol in a volume ratio of 1:6:1. 100 mg / mL aminolevulinic acid, 100% v / v triethanolamine and 50% v / v glycerol are mixed in a volume ratio of 1:6:1, and the components cooperate with each other and work together. This can ensure that aminolevulinic acid regulates the permeability of the leaves, and allows triethanolamine to fully play the function of regulating acid-base balance and moisturizing, while allowing glycerol to better maintain the moisture and structural stability of the cells. At room temperature, the preservation solution can effectively extend the storage time of durian leaves, maintain the biological activity and structural integrity of the leaves, and facilitate subsequent research, observation and analysis.

[0016] The fourth aspect of the present invention provides a rapid dyeing method for detecting the degree of chilling injury of durian, the steps are as follows:

[0017] (1) Sampling: Select the leaves affected by chilling from the durian plants; carefully cut the selected durian leaves affected by chilling from the base of the leaves with scissors, try to ensure the integrity of the leaves, avoid cutting or tearing the leaves, and then pre-treat the leaves;

[0018] (2) dyeing: in a light-proof environment, the pretreated durian leaves are immersed and dyed with a mixed dyeing solution containing natural alginic acid and diaminobenzidine in a PE self-sealing bag, the mixed dyeing solution is used to immerse the durian leaves, and then vacuum-treated until a brown-yellow substance appears on the leaves;

[0019] (3) Decolorization: The dyed durian leaves were first rinsed with ddH2O, then transferred to a decolorization solution composed of anhydrous ethanol, fatty acid methyl ester sulfonate and glycerol, and placed in a water bath to remove chlorophyll until the leaves were completely bleached;

[0020] (4) Observation and photography: Use a SLR camera to take photos of the decolorized durian leaves and record the phenotype;

[0021] (5) Storage: Transfer the decolorized durian leaves to a new ziplock bag, add a preservation solution consisting of aminolevulinic acid, triethanolamine, and glycerol to the ziplock bag, and ensure that the durian leaves are completely immersed in the preservation solution. Finally, seal the ziplock bag and store it at room temperature.

[0022] Furthermore, in step (1), the pretreatment is specifically as follows: placing the cut leaves on an operating table, and gently wiping the surface of the leaves with a clean soft cloth or cotton ball soaked in ddH2O. The wiping process should be gentle to remove dust and impurities attached to the surface of the leaves, while preventing damage to the leaf tissue.

[0023] Furthermore, in step (2), the vacuum treatment is specifically as follows: using a vacuum device to extract the air from the PE ziplock bag containing the durian leaves and the mixed dyeing solution, and evacuating the bag until the vacuum gauge reading is -0.1 Kg / cm 2 , and last for 50 minutes.

[0024] Furthermore, in step (2), in the mixed staining solution, the final concentration of natural alginate is 0.1 mg / mL, and the final concentration of diaminobenzidine is 2 mg / mL.

[0025] Furthermore, in step (3), in the decolorization solution, the volume ratio of anhydrous ethanol, 0.01M fatty acid methyl ester sulfonate and 50% v / v glycerol is set to 5:1:1.

[0026] Furthermore, in step (3), the temperature of the water bath is set at 80-90° C., and the water bath time is set at 10 min.

[0027] Furthermore, in step (3), the decolorizing solution is replaced multiple times until the leaves are completely bleached.

[0028] Furthermore, in step (5), the volume ratio of 100 mg / mL aminolevulinic acid, 100% v / v triethanolamine and 50% v / v glycerol is set to 1:6:1.

[0029] Beneficial effects of the present invention:

[0030] The present invention provides a visual detection method, including dyeing, decoloring and preservation, and the specific steps include cold treatment, cleaning, vacuuming, dyeing, decoloring, photographing and recording, and preservation. By obtaining the chilling phenotypes of durian leaves such as leaf contours and metabolic changes under the chilling injury state, the degree of chilling injury of durian can be quickly judged ( Figure 1 ). The state and degree of chilling damage to durian leaves of different varieties can be directly observed by treating the durian leaves with the dyeing solution, decolorizing solution and preserving solution of the present invention. The dyeing solution provided by the present invention has high dyeing efficiency, and the dyeing process can be completed by combining only 50min vacuum treatment, which is suitable for the needs of large-scale rapid detection; the three components of the decolorizing solution are synergistically stable, and the fatty acid methyl ester sulfonate enhances the mutual solubility of anhydrous ethanol and glycerol, making the entire decolorizing solution system more stable, and ensuring the consistency and reliability of the decolorizing effect. This stability enables the decolorization operation to obtain relatively consistent results in the detection of different batches, which is conducive to the comparison and analysis of experimental data; the preserving solution enables the leaves to effectively extend the storage time of durian leaves at room temperature, maintain the structural integrity of the leaves, and prevent mildew. At the same time, the preserving solution can also fix the dyed leaf tissue, which is convenient for scientific researchers to carry out subsequent research, observation and analysis. The detection method has the advantages of simple operation, clear dyeing contrast, fast and intuitive, and low cost, and can be used for observing and determining the degree of chilling damage to Malvaceae and other species with similar leaf structures. At the same time, the method is also expected to be applied in the fields of plant protection, crop cultivation and production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are comparison photos of the dyed leaves of Durio fragrans after cold stress.

[0032] Figure 2 These are comparison photos of the leaves of Duriosira japonica subjected to different dyeing times after cold stress.

[0033] Figure 3 This is a photo of the dyed leaves of Musang King durian after cold stress.

[0034] Figure 4 This is a photo of dyed Musang King durian leaves at room temperature.

[0035] Figure 5 Photo of durian leaves fully dyed in a ziplock bag. DETAILED DESCRIPTION

[0036] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0037] Example 1: Observation of leaf staining of Durian chinensis under cold stress

[0038] 1. Treatment: The leaves of Durian chinensis were subjected to cold stress treatment;

[0039] 2. Sampling: Carefully cut the selected leaves from the base of the leaves with scissors, try to ensure the integrity of the leaves, and avoid cutting or tearing the leaves; Place the cut leaves on the operating table, and gently wipe the surface of the leaves with a clean soft cloth or cotton ball soaked in ddH2O. Be gentle during the wiping process to remove dust and impurities attached to the surface of the leaves, and prevent damage to the leaf tissue;

[0040] 3. Staining: In a light-proof environment, the durian leaves were immersed and stained with a mixed staining solution containing a final concentration of 0.1 mg / mL natural alginate and 2 mg / mL diaminobenzidine in a PE self-sealing bag. The mixed staining solution was used to immerse the durian leaves, and then vacuum-treated for 20 minutes and 50 minutes until brown-yellow substances appeared on the leaves. The vacuum treatment was specifically as follows: the air in the PE self-sealing bag containing the durian leaves and the mixed staining solution was extracted using a vacuum device, and the vacuum was evacuated until the vacuum gauge reading was -0.1 Kg / cm 2 ;

[0041] 4. Decolorization: The dyed durian leaves were first rinsed with ddH2O, then transferred to a decolorization solution made by mixing anhydrous ethanol, 0.01M fatty acid methyl ester sulfonate and 50% v / v glycerol in a ratio of 5:1:1, and placed in a water bath at 80-90℃ for 10 minutes to remove chlorophyll. The decolorization solution was changed several times until the leaves were completely bleached;

[0042] 5. Observation and photography: Use a SLR camera to take photos of the decolorized durian leaves and record the phenotype. It can be found that the leaves of the golden pillow durian are deeply dyed as the dyeing time increases ( Figure 2 );

[0043] 6. Storage: Transfer the decolorized durian leaves to a new ziplock bag, add a preservation solution composed of aminolevulinic acid, triethanolamine, and glycerol to the ziplock bag, wherein the volume ratio is set to 100 mg / mL aminolevulinic acid: 100% v / v triethanolamine: 50% v / v glycerol = 1:6:1, ensure that the durian leaves are completely immersed in the preservation solution, finally seal the ziplock bag, and store the sealed ziplock bag at room temperature in a dark environment.

[0044] Example 2: Observation of leaf staining of Musang King durian under cold stress

[0045] 1. Treatment: The leaves of Musang King durian were subjected to cold stress treatment;

[0046] 2. Sampling: Carefully cut the selected leaves from the base of the leaves with scissors, try to ensure the integrity of the leaves, and avoid cutting or tearing the leaves; Place the cut leaves on the operating table, and gently wipe the surface of the leaves with a clean soft cloth or cotton ball soaked in ddH2O. Be gentle during the wiping process to remove dust and impurities attached to the surface of the leaves, and prevent damage to the leaf tissue;

[0047] 3. Staining: In a light-proof environment, the durian leaves were immersed and stained with a mixed staining solution containing a final concentration of 0.1 mg / mL natural alginate and 2 mg / mL diaminobenzidine in a PE self-sealing bag. The mixed staining solution was used to immerse the durian leaves, and then vacuum-treated for 50 minutes until brown-yellow substances appeared on the leaves. The vacuum treatment was specifically as follows: the air in the PE self-sealing bag containing the durian leaves and the mixed staining solution was extracted using a vacuum device, and the vacuum was evacuated until the vacuum gauge reading was -0.1 Kg / cm 2 ;

[0048] 4. Decolorization: The dyed durian leaves were first rinsed with ddH2O, then transferred to a decolorization solution made by mixing anhydrous ethanol, 0.01M fatty acid methyl ester sulfonate and 50% v / v glycerol in a ratio of 5:1:1, and placed in a water bath at 80-90℃ for 10 minutes to remove chlorophyll. The decolorization solution was changed several times until the leaves were completely bleached;

[0049] 5. Observation and photography: Use a SLR camera to take photos of the decolorized durian leaves and record the phenotype. It can be found that the leaves of Musang King durian are deeply dyed ( Figure 3 );

[0050] 6. Storage: Transfer the decolorized durian leaves to a new ziplock bag, add a preservation solution composed of aminolevulinic acid, triethanolamine, and glycerol to the ziplock bag, wherein the volume ratio is set to 100 mg / mL aminolevulinic acid: 100% v / v triethanolamine: 50% v / v glycerol = 1:6:1, ensure that the durian leaves are completely immersed in the preservation solution, finally seal the ziplock bag, and store the sealed ziplock bag at room temperature in a dark environment.

[0051] Comparative Example 3: Observation of dyeing of leaves of Musang King durian in the control group

[0052] 1. Sampling: Use scissors to carefully cut off the leaves of the Musang King durian variety at room temperature from the base of the leaves, try to ensure the integrity of the leaves, and avoid cutting or tearing the leaves; Place the cut leaves on the operating table, and gently wipe the surface of the leaves with a clean soft cloth or cotton ball soaked in ddH2O. Be gentle during the wiping process to remove dust and impurities attached to the surface of the leaves, and prevent damage to the leaf tissue;

[0053] 2. Staining: In a light-proof environment, the durian leaves were immersed and stained with a mixed staining solution containing a final concentration of 0.1 mg / mL natural alginate and 2 mg / mL diaminobenzidine in a PE self-sealing bag. The mixed staining solution was used to immerse the durian leaves, and then vacuum-treated for 50 minutes. The vacuum treatment was specifically as follows: the air in the PE self-sealing bag containing the durian leaves and the mixed staining solution was extracted using a vacuum device until the vacuum gauge reading was -0.1 Kg / cm 2 ;

[0054] 3. Decolorization: The dyed durian leaves were first rinsed with ddH2O, then transferred to a decolorization solution made by mixing anhydrous ethanol, 0.01M fatty acid methyl ester sulfonate and 50% v / v glycerol in a ratio of 5:1:1, and placed in a water bath at 80-90℃ for 10 minutes to remove chlorophyll. The decolorization solution was changed several times until the leaves were completely bleached;

[0055] 4. Observation and photography: Use a SLR camera to take photos of the decolorized durian leaves and record the phenotype. It can be found that the durian leaves at room temperature are basically not dyed ( Figure 4 );

[0056] 5. Storage: Transfer the decolorized durian leaves to a new ziplock bag, add a preservation solution composed of aminolevulinic acid, triethanolamine and glycerol to the ziplock bag, wherein the volume ratio is set to 100 mg / mL aminolevulinic acid: 100% v / v triethanolamine: 50% v / v glycerol = 1:6:1, ensure that the durian leaves are completely immersed in the preservation solution, finally seal the ziplock bag, and store the sealed ziplock bag at room temperature in a dark environment.

[0057] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A rapid dyeing method for detecting the degree of chilling injury of durian, characterized in that, Here are the steps: (1) Sampling: Select the leaves affected by chilling from the durian plants; carefully cut the selected durian leaves affected by chilling from the base of the leaves with scissors, try to ensure the integrity of the leaves, avoid cutting or tearing the leaves, and then pre-treat the leaves; (2) dyeing: in a light-proof environment, the pretreated durian leaves are immersed and dyed with a mixed dyeing solution containing natural alginic acid and diaminobenzidine in a PE self-sealing bag, the mixed dyeing solution is used to immerse the durian leaves, and then vacuum-treated until a brown-yellow substance appears on the leaves; (3) Decolorization: The dyed durian leaves were first rinsed with ddH2O, then transferred to a decolorization solution composed of anhydrous ethanol, 0.01 M fatty acid methyl ester sulfonate, and 50% v / v glycerol, and placed in a water bath to remove chlorophyll until the leaves were completely bleached; (4) Observation and photography: Use a SLR camera to take photos of the decolorized durian leaves and record the phenotype; (5) Storage: Transfer the decolorized durian leaves to a new ziplock bag, add a preservation solution consisting of 100 mg / L aminolevulinic acid, 100% v / v triethanolamine, and 50% v / v glycerol to the ziplock bag, ensure that the durian leaves are completely immersed in the preservation solution, and finally seal the ziplock bag and store it at room temperature.

2. A rapid dyeing method for detecting the degree of chilling injury of durian according to claim 1, characterized in that, In step (1), the pretreatment is specifically as follows: placing the cut leaves on an operating table, and gently wiping the surface of the leaves with a clean soft cloth or cotton ball soaked in ddH2O. The wiping process should be gentle to remove dust and impurities attached to the surface of the leaves, while preventing damage to the leaf tissue.

3. A rapid dyeing method for detecting the degree of chilling injury of durian according to claim 1, characterized in that, In step (2), the vacuum treatment is specifically as follows: using a vacuum device to extract the air from the PE ziplock bag containing the durian leaves and the mixed dyeing solution, and evacuating the bag until the vacuum gauge reading is -0.1 Kg / cm 2 , and last for 50 minutes.

4. A rapid dyeing method for detecting the degree of chilling injury of durian according to claim 1, characterized in that, In step (2), in the mixed staining solution, the final concentration of natural alginate is 0.1 mg / mL, and the final concentration of diaminobenzidine is 2 mg / mL.

5. A rapid dyeing method for detecting the degree of chilling injury of durian according to claim 1, characterized in that, In step (3), in the decolorizing solution, the volume ratio of anhydrous ethanol, 0.01M fatty acid methyl ester sulfonate and 50% v / v glycerol is set to 5:1:

1.

6. A rapid dyeing method for detecting the degree of chilling injury of durian according to claim 1, characterized in that, In step (3), the temperature of the water bath is set at 80-90° C., and the water bath time is set at 10 min.

7. A rapid dyeing method for detecting the degree of chilling injury of durian according to claim 1, characterized in that, In step (3), the decolorizing solution is replaced several times until the leaves are completely bleached.

8. A rapid dyeing method for detecting the degree of chilling injury of durian according to claim 1, characterized in that, In step (5), the volume ratio of 100 mg / L aminolevulinic acid, 100% v / v triethanolamine and 50% v / v glycerol is set to 1:6:1.

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