A rapid dyeing method for detecting the degree of cold damage of durian and application thereof

Abstract

The present application relates to the technical field of detection of durian cold injury, and provides a rapid dyeing method for detecting the degree of durian cold injury and application.The present application is a visual detection method, which comprises dyeing, decoloring and preservation, and the specific steps comprise cold treatment, cleaning, vacuumizing, dyeing, decoloring, photographing and recording and preservation process, the degree of durian cold injury is rapidly judged by obtaining the leaf profile and metabolic change of durian leaf under the state of cold injury, so as to obtain the cold injury phenotype.The dyeing solution, the decoloring solution and the preservation solution of the present application are used to treat durian leaves, the state and the damage degree of the cold injury of the leaves of different varieties of durian can be directly observed, and the leaves are easy to be stored for a long time at room temperature.The present detection method has the advantages of simple operation, clear dyeing contrast, rapid and intuitive and low cost, and can be used for observing and judging the degree of cold injury of malvaceae and other species with similar leaf structure.The method is expected to be applied in the fields of plant protection, crop cultivation and production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection of durian cold damage, and relates to a rapid dyeing method for detecting the degree of durian cold damage and application. BACKGROUND

[0002] Durian, known as the "King of Fruits", has strict requirements for 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 climate conditions with an annual absolute low temperature higher than 0℃ and an accumulated temperature exceeding 7000℃. China, as the world's largest durian consumption market, has an annual trade volume of nearly 10 billion US dollars with Southeast Asia, which indicates that the potential market for domestic durian planting is extremely broad. However, during the process of durian migration and planting to the north, high altitude and cold climate constitute a severe challenge. Therefore, monitoring whether durian suffers from cold damage and the damage degree is crucial for guaranteeing the success of durian planting and improving yield.

[0003] Hydrogen peroxide (H2O2) as a key molecule in 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 the growth and development, senescence and response to stress of plants at multiple physiological levels. Therefore, the determination of H2O2 level has become an important indicator for evaluating the physiological state of plants. H2O2 reacts with diaminobenzidine to form a brown product under the action of catalase, thereby realizing the localization and quantification of H2O2 in tissues. This method provides an intuitive and efficient analysis means 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 structure, composition and physiological characteristics. The specific reasons are as follows: (1) The structure of durian leaves is special, with a thick cuticle layer that acts like a dense protective film, reducing water loss and invasion of external substances, while also greatly hindering the penetration of the staining agent. The epidermal cells are closely arranged with small intercellular spaces, further increasing the difficulty of the staining agent entering the cell interior, making it difficult for the staining agent to fully contact with the target substance inside the cell, thereby affecting the staining effect; (2) The durian leaf contains a large amount of polysaccharides, phenolic compounds and tannins and other substances. These components can react with the staining agent, reducing the activity of the staining agent, preventing the staining agent from normally combining with the target substance, and affecting the staining effect. For example, phenolic compounds can form quinone compounds after oxidation, which can bind to biological macromolecules such as proteins and nucleic acids, thereby interfering with the binding of the staining agent to these biological macromolecules. In addition, polysaccharides may be wrapped on the cell surface, hindering the approach of the staining agent; (3) As a tropical plant, durian leaves have a vigorous physiological metabolism. During the staining process, the cells of the leaves will initiate self-protection mechanisms, actively transporting the staining agent out of the cells, or changing the intracellular environment through metabolic activities, making it difficult for the staining agent to function. For example, cells may pump out the staining agent through ion pumps, or produce substances through metabolism to change the pH of the cell, affecting the ionization state of the staining agent, and thus affecting the staining effect.

[0004] Some existing staining methods are not easy for the staining solution to enter the plant leaves due to the hydrophobicity and surface tension of the leaves, resulting in poor staining effect and incomplete staining. Only a part of the leaf can be stained, and the complete leaf staining usually requires processing of the leaf veins, causing mechanical damage to the leaf, resulting in poor decolorization effect after staining and incomplete removal of the stains on the leaf, which cannot intuitively and accurately reflect the accumulation of H2O2 caused by stress; or the reaction rate of the staining solution with H2O2 is not high enough, resulting in a long staining time of 8-12 hours or even more; or the reaction sensitivity of the staining solution with H2O2 is not high enough, resulting in low accuracy and clarity.

[0005] In the existing plant leaf decolorization technology, alcohol solution is a commonly used decolorizing reagent. However, when facing durian leaves with thick and high pigment content, the limitations of alcohol decolorization alone are exposed. Due to the special structure and rich pigments of durian leaves, only alcohol decolorization often takes a long time, often several hours or even longer. This undoubtedly greatly slows down the experimental process, seriously affects the decolorization efficiency, and hinders the progress of research work.

[0006] In addition, long-term soaking in high-concentration alcohol will gradually make the texture of durian leaves brittle, and the cell structure will be damaged to varying degrees. This makes the leaves easily broken during subsequent operations such as slicing, transferring, etc., resulting in incomplete leaf shape, making it difficult to observe accurately, and not conducive to long-term sample preservation.

[0007] In plant leaf staining experiments, decolorization effectively reduces the interaction between natural pigments and staining agents, reducing the risk of color change or fading of the staining agent caused by environmental factors such as light and oxygen. However, when we expect the leaves to be stored at room temperature for a long time, it is crucial to maintain their structural integrity, prevent fragmentation and mold, and ensure dye fixation to extend the shelf life of the stained sample and meet the needs of subsequent scientific research.

[0008] Currently, common preservation methods have their own advantages and disadvantages. Dry preservation is simple to operate, but it is easy to cause leaf brittle fracture and color change; frozen preservation can maintain the physiological state of the leaves to a certain extent, but it may damage the cell structure, causing cell rupture and tissue collapse after thawing, which seriously affects the observation effect, and improper thawing speed can also cause freeze-thaw damage. Compared with the above methods, liquid preservation has obvious advantages, as it can maintain the softness of the leaves and stabilize the cell structure, preventing the fading of the staining agent and fixing the color.

[0009] In the existing technology, formalin solution is commonly used for liquid preservation. However, it is not to be ignored that formalin is highly toxic and volatile, which not only poses potential harm to human health but also has adverse effects on the environment. Therefore, it is particularly important to develop a safe and effective preservation solution that meets the long-term preservation needs.

[0010] Therefore, it is urgent to provide a complete set of rapid processing and staining technology system suitable for the special needs of durian leaves, including the formula of staining solution, decolorizing solution and room temperature preservation solution, to realize the detection of the degree of cold damage of durian. SUMMARY

[0011] The purpose of the present application is to detect the damage degree of Malvaceae durian under cold damage, and to develop a new and simple, rapid dyeing method and system. Durian leaves are known for their leather-like characteristics, with a smooth surface, a texture similar to leather, thick cell walls, and a fine granular scale structure on the back of the leaf. These characteristics give durian leaves strong hydrophobicity and surface tension. Based on these unique physiological characteristics, the present application specifically designs a set of processing and dyeing technology system for durian leaves, including the formula of dyeing solution, decolorizing solution and room temperature preservation solution, to adapt to the special needs of durian leaves. The application of these technologies will help to more accurately assess the physiological state of durian leaves of different varieties under cold stress, and provide strong technical support for the cold resistance research and cultivation management of durian.

[0012] To achieve the above purpose, the present application provides the following technical solutions:

[0013] The first aspect of the present application provides a dyeing solution for durian leaves, which is a mixed solution containing 0.1 mg / mL natural alginate and 2 mg / mL diaminobenzidine. The natural alginate and diaminobenzidine reagents are prepared into a mixed solution with specific concentrations, and they work together to achieve effective dyeing of H2O2 in durian leaves. Natural alginate creates a good dyeing environment, which helps diaminobenzidine to better perform its dyeing function, making the dyeing result more accurate and clear, and facilitating observation and analysis by relevant technical personnel in the field.

[0014] The second aspect of the present application provides a decolorizing solution for durian leaves, which is a decolorizing solution mixed by anhydrous ethanol, 0.01 M fatty acid methyl sulfonate and 50% v / v glycerol in a volume ratio of 5:1:1. The three components work together: anhydrous ethanol is mainly responsible for dissolving chlorophyll, fatty acid methyl sulfonate accelerates the dissolution and diffusion of anhydrous ethanol to chlorophyll by emulsification and reducing surface tension, and glycerol stabilizes the system, maintains the shape of the leaves and assists in dissolving related substances, together achieving the purpose of efficient removal of chlorophyll.

[0015] The third aspect of the present application provides a room temperature preservation solution for durian leaves, which 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. The 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 work together. This not only ensures that aminolevulinic acid regulates the permeability of the leaves, but also allows triethanolamine to fully play its role in regulating acid-base balance and moisturizing, while glycerol better maintains the water content and structural stability of the cells. At room temperature, the preservation solution can effectively prolong the storage time of durian leaves, maintain the biological activity and structural integrity of the leaves, and facilitate subsequent research, observation and analysis work.

[0016] The fourth aspect of the present application provides a rapid dyeing method for detecting the degree of cold damage of durians, comprising the following steps:

[0017] (1) Sampling: selecting a cold-damaged leaf from a durian plant; carefully cutting the selected cold-damaged durian leaf from the base of the leaf with scissors, trying to ensure the integrity of the leaf, avoiding cutting or tearing the leaf, and then pretreating the leaf;

[0018] (2) Dyeing: immersing and dyeing the pretreated durian leaf in a PE self-sealing bag in a dark environment with a mixed dyeing solution containing natural alginic acid and diamino-benzidine, the mixed dyeing solution immersing the durian leaf, and then vacuum treatment until brown-yellow substances appear on the leaf;

[0019] (3) Decolorization: rinsing the dyed durian leaf with ddH2O first, then transferring it to a decolorization solution composed of anhydrous ethanol, fatty acid methyl ester sulfonate and glycerol, and placing it in a water bath to remove chlorophyll until the leaf is completely bleached;

[0020] (4) Observation and photography: photographing the bleached durian leaf using a single-lens reflex camera and recording the phenotype;

[0021] (5) Preservation: transferring the bleached durian leaf to a new self-sealing bag, adding a preservation solution composed of aminolevulinic acid, triethanolamine and glycerol to the self-sealing bag, ensuring that the durian leaf is completely immersed in the preservation solution, finally sealing the self-sealing bag, and placing the sealed self-sealing bag in a room temperature environment for preservation.

[0022] Further, in step (1), the pretreatment specifically comprises: placing the cut leaf on an operating table, wiping the leaf surface with a clean soft cloth or cotton ball soaked with ddH2O, and gently wiping during the process to remove dust and impurities attached to the leaf surface, while preventing damage to the leaf tissue.

[0023] Further, in step (2), the vacuum treatment specifically comprises: using a vacuum device to extract the air in the PE self-sealing bag containing the durian leaf and the mixed dyeing solution, vacuuming to a vacuum table reading of -0.1 Kg / cm 2 for 50 min.

[0024] Further, in step (2), the final concentration of natural alginic acid in the mixed dyeing solution is 0.1 mg / mL, and the final concentration of diamino-benzidine is 2 mg / mL.

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

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

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

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

[0029] The beneficial effects of the present application are:

[0030] The present application provides a visual detection method, including dyeing, decolorizing and preservation, and the specific steps include cold treatment, cleaning, vacuumizing, dyeing, decolorizing, photographing and recording, and preservation process, through obtaining the leaf profile and metabolic changes of durian leaves under cold damage state, the degree of durian cold damage is quickly judged. Figure 1 The dyeing liquid, decolorizing liquid and preservation liquid of the present application can be used to treat durian leaves, and the state and degree of cold damage of durian leaves of different varieties can be directly observed. The dyeing liquid provided by the present application has high dyeing efficiency, and the dyeing process can be completed only by combining 50 min vacuum treatment, which is suitable for the demand of large-scale rapid detection; the three components of the decolorizing liquid are stable in cooperation, the fatty acid methyl ester sulfonate enhances the mutual solubility of anhydrous ethanol and glycerol, so that the whole decolorizing liquid system is more stable, and the consistency and reliability of the decolorizing effect are ensured. This stability enables the decolorizing operation to obtain relatively consistent results in different batches of detection, which is conducive to the comparison and analysis of experimental data; the preservation liquid can effectively prolong the preservation time of durian leaves at room temperature, maintain the structural integrity of the leaves, and prevent mold growth. At the same time, the preservation liquid can also fix the dyed leaf tissue, which is convenient for researchers to carry out subsequent research, observation and analysis work. The detection method has the advantages of simple operation, clear dyeing contrast, rapid and intuitive, and low cost, and can be used for observing and judging the cold damage degree of malvaceae and other species with similar leaf structure. At the same time, the method is expected to be applied in the fields of plant protection, crop cultivation and production. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a dyeing contrast photo of durian leaves after cold stress.

[0032] Figure 2 It is a dyeing contrast photo of durian leaves after cold stress.

[0033] Figure 3 It is a dyeing photo of durian leaves after cold stress.

[0034] Figure 4 Figure 1 is a photograph of cat's claw fig leaf blade staining at room temperature.

[0035] Figure 5 Figure 2 is a photograph of cat's claw fig leaf blade fully stained in a self-sealing bag. DETAILED DESCRIPTION

[0036] The specific embodiments of the present application are described below to enable those skilled in the art to understand the present application, but it should be clear that the present application is not limited to the scope of the specific embodiments, and that all the inventions utilizing the concept of the present application are within the scope of the present application as long as various changes are obvious to those skilled in the art within the spirit and scope of the present application as defined by the appended claims.

[0037] Example 1: Cold stress treatment of cat's claw fig leaf blade staining observation

[0038] 1. Treatment: Cold stress treatment of cat's claw fig leaf blade;

[0039] 2. Sampling: Carefully cut the selected leaf blade from the base of the leaf blade with scissors, try to ensure the integrity of the leaf blade, avoid cutting or tearing the leaf blade; place the cut leaf blade on the operating table, use a clean soft cloth or cotton ball soaked with ddH2O to gently wipe the surface of the leaf blade, the wiping process should be gentle to remove dust and impurities attached to the surface of the leaf blade, while preventing damage to the leaf blade tissue;

[0040] 3. Staining: In a light-proof environment, immerse the fig leaf blade in a mixed staining solution containing a final concentration of 0.1 mg / mL natural alginic acid and 2 mg / mL diaminobenzidine in a PE self-sealing bag, the mixed staining solution immerses the fig leaf blade, then vacuum treatment for 20 min and 50 min to appear brown-yellow material on the leaf blade, the vacuum treatment is as follows: using a vacuum device to extract the air in the PE self-sealing bag containing fig leaf blade and mixed staining solution, vacuum extraction to a vacuum table reading of -0.1 Kg / cm 2 ;

[0041] 4. Decolorization: After staining, the fig leaf blade is first rinsed with ddH2O, then transferred to a decolorization solution mixed by anhydrous ethanol, 0.01 M fatty acid methyl sulfonate and 50% v / v glycerol in a ratio of 5:1:1, and placed in a water bath at 80-90°C for 10 min to remove chlorophyll, and the decolorization solution is replaced multiple times until the leaf blade is completely bleached;

[0042] 5. Observation and photography: Take a photograph of the decolorized fig leaf blade using a single-lens reflex camera and record the phenotype, it can be found that the cat's claw fig leaf blade is deeply stained with increasing staining time Figure 2 );

[0043] 6. Preservation: After decolorization, the durian leaves were transferred to a new ziplock bag, and a preservation solution composed of amino levulinic acid, triethanolamine, and glycerol was added to the ziplock bag, wherein the volume ratio was set to 100 mg / mL amino levulinic acid: 100% v / v triethanolamine: 50% v / v glycerol = 1:6:1, ensuring that the durian leaves were completely immersed in the preservation solution, and finally the ziplock bag was sealed and placed in a room temperature, light-proof environment for preservation.

[0044] Example 2: Cold stress treatment of cat mountain king durian leaf staining observation

[0045] 1. Treatment: The leaves of cat mountain king durian were subjected to cold stress treatment;

[0046] 2. Sampling: The selected leaves were carefully cut from the base of the leaves with scissors, and the integrity of the leaves was ensured as much as possible to avoid cutting or tearing the leaves; the cut leaves were placed on the operating table, and a clean soft cloth or cotton ball soaked with ddH2O was used to gently wipe the surface of the leaves, and the action was gentle during the wiping process to remove dust and impurities attached to the surface of the leaves, while preventing damage to the leaf tissue;

[0047] 3. Staining: In a light-proof environment, the durian leaves were immersed in a mixed staining solution containing a final concentration of 0.1 mg / mL natural alginic acid and 2 mg / mL diaminobenzidine in a PE material ziplock bag, the mixed staining solution immersed the durian leaves, and then vacuum treatment was performed for 50 min until brown-yellow substances appeared on the leaves, the vacuum treatment was as follows: using a vacuum device to extract the air in the PE ziplock bag containing durian leaves and mixed staining solution, vacuum extraction was performed 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 composed of anhydrous ethanol and 0.01 M fatty acid methyl sulfonate and 50% v / v glycerol mixed in a ratio of 5:1:1, and placed in a water bath at 80-90°C for 10 min to remove chlorophyll, and the decolorization solution was replaced multiple times until the leaves were completely bleached;

[0049] 5. Observation and photography: The decolorized durian leaves were photographed using a single-lens reflex camera, and the phenotype was recorded, and it was found that the cat mountain king durian leaves were deeply dyed Figure 3 );

[0050] 6. Preservation: After bleaching, the durian leaf pieces are transferred to a new ziplock bag, and a preservation solution composed of amino levulinic acid, triethanolamine, and glycerol is added to the ziplock bag, with a volume ratio of 100 mg / mL amino levulinic acid: 100% v / v triethanolamine: 50% v / v glycerol = 1:6:1. The durian leaf pieces are completely immersed in the preservation solution, and the ziplock bag is finally sealed and stored in a room temperature, light-proof environment.

[0051] Comparative Example 3: Control group of cat's mountain durian leaf pieces for staining observation

[0052] 1. Sampling: Durian leaf pieces of the cat's mountain variety at room temperature are carefully cut from the base of the leaf pieces using scissors, and the integrity of the leaf pieces is ensured to avoid cutting or tearing the leaf pieces; the cut leaf pieces are placed on an operating table, and a clean soft cloth or cotton ball soaked with ddH2O is used to gently wipe the surface of the leaf pieces, with gentle movements to remove dust and impurities attached to the surface of the leaf pieces, while preventing damage to the leaf tissue;

[0053] 2. Staining: In a light-proof environment, the durian leaf pieces are immersed in a mixed staining solution containing a final concentration of 0.1 mg / mL natural alginic acid and 2 mg / mL diaminobenzidine in a PE ziplock bag, which immerses the durian leaf pieces, and then vacuum treatment for 50 min, the vacuum treatment is as follows: using a vacuum device to extract the air in the PE ziplock bag containing durian leaf pieces and mixed staining solution, vacuum extraction to a vacuum table reading of -0.1 Kg / cm 2 ;

[0054] 3. Bleaching: After staining, the durian leaf pieces are first rinsed with ddH2O, then transferred to a bleaching solution composed of anhydrous ethanol and 0.01 M fatty acid methyl sulfonate and 50% v / v glycerol mixed in a ratio of 5:1:1, and placed in a water bath at 80-90°C for 10 min to remove chlorophyll, and the bleaching solution is replaced multiple times until the leaf pieces are completely bleached;

[0055] 4. Observation and photography: The bleached durian leaf pieces are photographed using a single-lens reflex camera, and the phenotype is recorded. It is found that the durian leaf pieces at room temperature are basically not stained Figure 4 ) ;

[0056] 5. Preservation: After bleaching, the durian leaf pieces are transferred to a new ziplock bag, and a preservation solution composed of amino levulinic acid, triethanolamine, and glycerol is added to the ziplock bag, with a volume ratio of 100 mg / mL amino levulinic acid: 100% v / v triethanolamine: 50% v / v glycerol = 1:6:1. The durian leaf pieces are completely immersed in the preservation solution, and the ziplock bag is finally sealed and stored in a room temperature, light-proof environment.

[0057] The foregoing merely illustrates some exemplary embodiments of the application, and no doubt numerous modifications and alterations thereto will be apparent to those skilled in the art. Accordingly, the above description is intended for purposes of illustration only and should not be construed as limiting the scope of the application.

Claims

1. A rapid staining method for detecting the degree of chilling injury of durians, characterized by, The steps are as follows: (1) Sampling: selecting the cold-damaged leaves on the durian plant; carefully cutting the selected cold-damaged durian leaves from the base of the leaves with scissors, trying to ensure the integrity of the leaves, avoiding cutting or tearing the leaves, and then pretreating the leaves; (2) Staining: in a light-proof environment, immersing and staining the pretreated durian leaves in a mixed staining solution containing natural alginic acid and diamino benzidine in a PE material self-sealing bag, the mixed staining solution immerses the durian leaves, and then vacuum treatment is performed until brown-yellow substances appear on the leaves; in the mixed staining solution, the final concentration of natural alginic acid is 0.1 mg / mL, and the final concentration of diamino benzidine is 2 mg / mL; (3) Decolorization: the dyed durian leaves are first rinsed with ddH2O, then transferred to a decolorization solution mixed by anhydrous ethanol, 0.01M fatty acid methyl sulfonate and 50% v / v glycerol, and placed in a water bath to remove chlorophyll until the leaves are completely bleached; in the decolorization solution, the volume ratio of anhydrous ethanol, 0.01M fatty acid methyl sulfonate and 50% v / v glycerol is set to 5:1:1; the temperature of the water bath is set to 80-90℃, and the water bath time is set to 10 min; (4) Observation and photography: taking photographs of the decolorized durian leaves using a single-lens reflex camera and recording the phenotype; (5) Preservation: transferring the decolorized durian leaves to a new self-sealing bag, adding a preservation solution composed of 100 mg / L aminolevulinic acid, 100% v / v triethanolamine and 50% v / v glycerol to the self-sealing bag, ensuring that the durian leaves are completely immersed in the preservation solution, finally sealing the self-sealing bag, and placing the sealed self-sealing bag in a room temperature environment for preservation; the volume ratio of 100 mg / L aminolevulinic acid, 100% v / v triethanolamine and 50% v / v glycerol is set to 1:6:

1.

2. The rapid staining method for detecting the degree of cold injury of durians according to claim 1, characterized in that, In step (1), the pretreatment is specifically: placing the cut leaves on the operating table, wiping the leaf surface with a clean soft cloth or cotton ball soaked with ddH2O, and gently wiping during the wiping process to remove dust and other impurities attached to the leaf surface, while preventing damage to the leaf tissue.

3. The rapid staining method for detecting the degree of cold injury of durians according to claim 1, characterized in that, In step (2), the vacuum treatment is specifically: using a vacuum device to extract the air in the PE self-sealing bag containing the durian leaves and the mixed dyeing solution, vacuumizing to -0.1 Kg / cm 2 for 50 min.

4. The rapid staining method for detecting the degree of cold injury of durians according to claim 1, wherein, In step (3), the decolorization solution is replaced several times until the leaves are completely bleached.

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