Method for nondestructively acquiring stomatal mode of plant leaf and observing stomatal density

Abstract

The invention discloses a method for non-destructively acquiring a plant leaf stomata mode and observing stomata density. The method comprises the following steps: preparing a pear leaf epidermis gel sample, preparing a leaf stomata imprinting film, preparing a stomata film temporary loading sheet, carrying out microscope observation and photographing, carrying out stomata density statistical analysis and the like. According to the method, the leaf glue sample is used for replacing the leaf, and the method has the advantages of nondestructive collection, rapid batch sampling, convenience in long-term sample preservation for observation and statistics and the like. The method focuses on collection of relevant information of the pear leaf stomata, is beneficial to analysis of distribution, form and quantity of the pear leaf stomata and research on relevant physiological activities of the stomata, and provides a method reference for investigation of stomata density and other relevant parameters of other plant leaves and tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant morphology, and specifically relates to a method for non-destructively acquiring the stomatal pattern of plant leaves and observing the stomatal density. The method comprises the following steps: using a silicone polymer-dental impression gel to prepare a gel sample with the epidermal structure of a plant leaf to replace the leaf for permanent preservation; using transparent nail polish to copy the gel sample to prepare a temporary stomatal film mount; observing and photographing the stomata under a microscope; and using software to count the number of stomata. This method provides a complete set of non-destructive methods for collecting, observing, and calculating the stomatal density of pear leaves. Background Art

[0002] Stomata in plant leaves are crucial channels for gas exchange (primarily carbon dioxide and water vapor) between plants and the environment. Their distribution and structural characteristics are closely linked to the intensity of photosynthesis and transpiration. Stomata density, morphological structure, and distribution vary significantly between plant leaves. Stomata density determines the rate at which a plant absorbs CO2. Plants with higher stomatal densities typically exhibit greater adaptability in photosynthesis, respiration, and transpiration, and are able to respond more quickly to environmental changes. Therefore, establishing an efficient method for collecting stomatal density in pear leaves is crucial for studying the characteristics of pear varieties and breeding drought-resistant varieties.

[0003] At present, there are three main traditional methods for observing stomatal density: (1) directly photographing leaves with an environmental scanning electron microscope; (2) treating leaves with a fixative and a dye and then photographing them under an optical microscope; (3) applying a thin film on the leaf epidermis with transparent nail polish and then photographing them under a microscope. Although all three methods can meet the requirements for observing and counting stomata on the leaf epidermis, they all have certain limitations. The observations of the first two are based on damaged leaves, which is not conducive to continuous observation of leaves. The third method uses nail polish, which contains irritating and volatile substances such as ethyl acetate and butyl acetate, which can cause a certain degree of damage to the epidermis of plant leaves. Moreover, none of the three methods can meet the requirements for high-throughput collection of a large number of samples in a short period of time at a specific stage of plant growth and development for a variety of plant samples to be processed. Therefore, for field sample collection work with tight time, heavy tasks and long distances, the above three methods alone cannot be achieved.

[0004] Therefore, developing a rapid, non-destructive, and high-throughput method for collecting and observing pear leaf stomata will facilitate continuous observation of stomatal density in pear leaves under different varieties, conditions, or treatments, thereby promoting research on stomatal morphology, photosynthesis, respiration, and transpiration, among other important physiological processes. This will provide key technical support for the breeding of stress-resistant pear varieties. Summary of the Invention

[0005] The present invention aims to provide a method for non-destructively acquiring stomatal patterns and observing stomatal density in plant leaves. This method utilizes a silicone polymer-dental impression gel to obtain silicone samples of leaf epidermal structure. Combined with microscopic imaging technology, this method enables high-precision observation of stomatal patterns and density statistics. This method is suitable for studying pear physiology and breeding stress-resistant varieties.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention claims a method for non-destructively obtaining the stomatal pattern of plant leaves and observing the stomatal density, the method comprising the following steps:

[0008] (1) Preparation of silicone samples for plant leaf epidermis: Assemble the silicone polymer-dental impression gel tube and the matching silicone gun, apply the silicone gel to the middle of the plant leaf epidermis avoiding the main veins, let it stand until the silicone gel is air-dried, and then remove it for use;

[0009] (2) Preparation of pore-marked film: After applying a thin layer of transparent nail polish evenly on the surface of the glue sample with the mark obtained in step (1), place it under a glue-baking lamp and irradiate it until a dry transparent film is formed on the surface of the glue sample;

[0010] (3) Preparation of a temporary film mount: tear off the transparent film on the surface of the gel sample in step (2), stick it on the center of a transparent glass slide, and gently press it to make it fit tightly to make a temporary film mount;

[0011] (4) Microscope observation and photography: The temporary film slide in step (3) is placed under a microscope (10× objective lens) for observation, the focus is adjusted until the pores are clearly visible, and multiple different fields of view are selected to take photos and save them; in the specific embodiment of the present invention, 5 different observation fields are selected to take photos and save them.

[0012] (5) Stomatal counting: Use the counting function of the microscope software to count the number of stomata in each photo;

[0013] (6) Calculation of stomatal density: Calculate the stomatal density of leaves according to the formula. The calculation formula is as follows:

[0014] Leaf stomatal density (pieces / mm 2 ) = Number of pores in a single photo (pieces) / Photo area (mm 2 ).

[0015] Furthermore, the silicone polymer-dental impression gel tube contains a non-toxic dental silicone rubber impression material comprising a base vinyl polysiloxane and a catalyst platinum silica. The silicone gel tube can be assembled with a matching silicone rubber gun. Pressure is applied to extrude the two materials in the tube in equal volumes, creating a plastic gel that can be applied to the surface of plant leaves to obtain epidermal stomata information.

[0016] Further, step (1) is to apply 1-2cm on the middle of the plant leaf epidermis avoiding the main vein. 2 Silicone gel, wait 1-5 minutes.

[0017] Furthermore, the transparent nail polish is a common commercially available glossy polish product, the main ingredients of which include butyl acetate, ethyl acetate, acrylic acid (ester) / VA copolymer and triethyl citrate, etc. The liquid is transparent, has strong fluidity, and is easy to air-dry or bake to form a film.

[0018] Furthermore, the glue curing lamp is a common LED glue curing lamp available on the market with a power of 120W and a wavelength of 400-500nm, which is used to accelerate the curing of nail polish.

[0019] Furthermore, the microscope is equipped with a 1360×1024 pixel camera, and the area of ​​a single photo taken is 1.2-1.5mm 2 , and has a scale function to facilitate the calculation of pore density.

[0020] Furthermore, the stomatal counting software is an image analysis software matched with a microscope, which has an automatic or manual marking and counting function and is used to quickly count the number of stomata.

[0021] Furthermore, the plant leaves are herbaceous or woody plant leaves. Still further, the plant leaves are leaves of fruit trees of the Rosaceae family, such as pear tree leaves, apple tree leaves, etc., but not limited thereto.

[0022] The key points of the technical solution of the present invention are:

[0023] Observation of pear leaf stomatal morphology and related parameters is crucial for studying photosynthesis and transpiration. However, conventional stomatal observation methods rely on direct observation of the leaves themselves, often involving damage. These methods, such as cutting leaves with knives, fixing leaf morphology with fixatives, staining cells with safranin dye, and applying nail polish to leaves, directly damage the leaves. These damaging sampling methods, along with the use of chemical reagents like fixatives, safranin dye, and nail polish, all pose a direct threat to the leaves. These methods make continuous observation of pear leaves, which require long-term observation, impossible during experiments. Furthermore, traditional methods make it difficult to sample and repeatedly observe large numbers of pear leaves within a specific timeframe. This is especially true given the long distances between the laboratory and field locations, making field sample collection difficult and preserving the leaves in their original condition challenging. Therefore, the key to observing the number of pear leaf epidermal stomata lies in overcoming the challenges of non-destructive sampling of large numbers of pear leaves in the field and long-term laboratory observation.

[0024] The present invention utilizes dental silicone rubber impression material, which is a non-toxic and harmless liquid with strong fluidity before solidification. It is composed of a matrix vinyl polysiloxane and a catalyst platinum silicon oxide and other substances. The equal volume mixture of the two can produce a plastic gel. The pear leaf epidermal structure is copied with the material to prepare a glue sample that can replace the leaf stomata, which can quickly and accurately record the stomatal structure information of the pear leaf epidermis. Compared with the traditional method of directly observing the leaf stomata, the production of leaf stomata glue samples has the characteristics of non-destructive and instantaneous collection, and the stomatal glue samples can be permanently preserved and repeatedly observed, allowing the laboratory to have a longer time to observe and collect images of the stomata at any time. Combined with the transparent nail polish copying technology, a transparent film of the pear leaf epidermal stomatal structure is produced, which can clearly show the number of stomata under a microscope, which is very convenient for collecting and counting large quantities of pear leaf stomata.

[0025] The implementation scheme of the technical solution of the present invention is as follows: a dental silicone rubber impression material is used to make a glue sample that replicates the epidermal structure of a pear leaf, a transparent nail polish glue is used to replicate the glue sample to make a transparent film for observing the stomata structure, the film is dried under LED light to make a temporary slide, the number of stomata in the pear leaf is observed under a microscope and photographed and saved, the stomata are counted in the picture using graphic analysis software, and finally the stomatal density of the pear leaf is calculated (see Figure 1 ).

[0026] Beneficial effects of the present invention:

[0027] (1) The present invention proposes the "imprinting method" for the first time to collect the stomatal density of pear leaves, a fruit tree of the Rosaceae family. It uses non-toxic and harmless dental silicone rubber impression materials to non-destructively collect the stomatal information of pear leaves and preserve it permanently. It is convenient for the unified collection of large quantities of pear leaf samples in the field in a short period of time and the subsequent long-term observation in the laboratory, which greatly facilitates the field stomatal collection work.

[0028] (2) The present invention summarizes a complete set of collection processes for the stomatal density of pear leaves, which can achieve standardization and unification of observations of all samples, enhance the stability of stomatal density collection and observation and the accuracy of comparison between samples.

[0029] (3) This method is not only applicable to the collection of stomata and the investigation of stomatal density in pear leaves, but also provides a reference for the statistics of other plant leaves or tissues and other stomatal-related parameters. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a simplified diagram of the non-destructive collection process of stomata from pear leaves.

[0031] Figure 2 Schematic diagram of the complete operation process for collecting stomata from pear leaves.

[0032] Figure 3This is a diagram showing common problems that occur when taking photos without detailed processing.

[0033] Figure 4 Schematic diagram of the photos of pores after the process of the present invention. DETAILED DESCRIPTION

[0034] The following examples further illustrate the present invention. In the following detailed description, certain exemplary embodiments of the present invention are described by way of illustration only. It is understood that those skilled in the art will recognize that the described embodiments may be modified in various ways without departing from the spirit and scope of the present invention. Therefore, the description is illustrative in nature and is not intended to limit the scope of the claims.

[0035] In this embodiment, leaves of potted Pyrus betulae seedlings in a greenhouse were used as experimental materials, and the method of the present invention was used to perform non-destructive stomatal sampling and observation on mature leaves of the seedlings and to calculate the stomatal density.

[0036] Example 1 Non-destructive sampling, observation and statistical analysis of stomatal density of mature leaves of Pyrus betula

[0037] 1. Non-destructive collection of pear leaf epidermal stomata and preparation of silica gel samples

[0038] Preparation of silicone sample for pear leaf epidermis: Assemble the silicone polymer-dental impression gel tube and the matching silicone gun, and apply 1-2 cm to the middle of the pear leaf epidermis, avoiding the main vein. 2 Apply silicone gel of different sizes to the leaf surface, wait for 2 minutes, and remove it after the silicone gel is air-dried and set aside.

[0039] The silicone polymer dental impression gel tube contains a common, commercially available dental silicone rubber impression material. This non-toxic material comprises a base of vinyl polysiloxane and a catalyst of platinum silica. The silicone gel tube can be assembled with a matching silicone rubber gun. Pressure is applied to extrude the two materials in the tube in equal volumes, creating a plastic gel that can be applied to the surface of plant leaves to obtain epidermal stomata information.

[0040] 2. Preparation and Observation of Stomatal Imprint Films

[0041] (1) Preparation of pore imprint film: Apply a thin layer of transparent nail polish evenly on the imprinted side of the prepared glue sample, and bake it under an LED light glue baking lamp with a power of 120W and a wavelength of 400-500nm for 2 minutes. A dry transparent film will form on the surface of the glue sample.

[0042] (2) Preparation of temporary film mount: Use tweezers to carefully tear off the transparent film on the surface of the gel sample and stick it on the center of a clean transparent slide. Use your fingertips to gently press the film so that it sticks tightly to the slide to make a temporary film mount.

[0043] (3) Microscope observation and photography: Place the film under a microscope, set the observation parameters to objective lens × 10, camera pixel 1360 × 1024, select a suitable field of view and adjust the coarse and fine focus knobs until the pore morphology in the field of view is clear, take a picture and save it. Select 5 different fields of view for each film, and the shooting area of ​​a single photo is about 1.4 mm 2 , and has a scale function to facilitate the calculation of pore density.

[0044] 3. Stomatal Counting and Density Calculation

[0045] (1) Stomatal counting: Use the counting function of the graphic analysis software that comes with the microscope to count the stomata in the pictures taken, and finally get the number of stomata in each picture.

[0046] (2) Stomatal density statistics: Count the number of stomata in each image and calculate the stomatal density. The formula is: Stomatal density of leaves (number / mm 2 ) = Number of pores in a single photo (pieces) / Area of ​​a single photo (mm 2 ) = Number of air holes in a single photo / 1.387876mm 2 .

[0047] The complete operation process of collecting stomata from pear leaves is as follows: Figure 2 As shown in the figure, the operation process is as follows: prepare the impression gel, assemble the glue gun, squeeze the gel out of the leaf epidermis and air-dry to prepare the glue sample, evenly apply nail polish on the imprint side of the glue sample, dry it under a glue baking lamp, tear off the film with tweezers, transfer the film to a glass slide to prepare a temporary mount, observe under a microscope, adjust the parameters to a clear field of view and take pictures, and count with software.

[0048] Comparative Example 1

[0049] If step 1 is omitted and transparent nail polish is directly applied to the pear leaf epidermis, the remaining steps are the same as those in Example 1. This makes it impossible to quickly collect a large number of pear leaf stomata and subsequently observe the samples in the laboratory for a long time.

[0050] If the step of drying the nail polish with LED light to form a film in step 2 is omitted, the remaining steps are the same as in Example 1. The observation results show that the drying speed is faster than that of the unbaked film, which can effectively save more than half of the observation time. In addition, the unbaked nail polish is slow to form a film by air drying alone, and it is easy for some residue to remain on the surface of the sample during the film removal process, causing "tearing" ( Figure 3 A) and "small hole" ( Figure 3 B) film; and, short-term air-dried film has high viscosity, which is prone to appearing and "bubble" film when preparing temporary film mounting ( Figure 3C), these abnormal membranes affect the observation and statistics of the final number of stomata to a greater or lesser extent. After the complete process of Example 1, a stable and clear stomatal morphology of the pear leaf epidermis can be obtained ( Figure 4 A,

[0051] B). Figure 4 A is a schematic diagram of the imprint on the front of a pear leaf. The tightly arranged fibrous network structure is the epidermal cells of the pear leaf. It can be seen that there are no obvious stomatal imprints on the front of the pear leaf. Figure 4 B is a schematic diagram of the imprint on the back of a pear leaf. The oval or circular structures with cavities in the figure are stomata. It can be seen that the stomata are concentrated on the back of the pear leaf.

Claims

1. A method for non-destructively obtaining the stomatal pattern of plant leaves and observing stomatal density, characterized in that: The method comprises the following steps: (1) Preparation of silicone samples for plant leaf epidermis: Assemble the silicone polymer-dental impression gel tube and the matching silicone gun, apply the silicone gel to the middle of the plant leaf epidermis avoiding the main veins, let it stand until the silicone gel is air-dried, and then remove it for use; (2) Preparation of pore-marked film: After applying a thin layer of transparent nail polish evenly on the surface of the glue sample with the mark obtained in step (1), place it under a glue-baking lamp and irradiate it until a dry transparent film is formed on the surface of the glue sample; (3) Preparation of a temporary film mount: tear off the transparent film on the surface of the gel sample in step (2), stick it on the center of a transparent glass slide, and gently press it to make it fit tightly to make a temporary film mount; (4) Microscope observation and photography: Place the temporary film slide prepared in step (3) under a microscope for observation, adjust the focus until the pores are clearly visible, and select multiple different fields of view to take photos and save them; (5) Stomatal counting: Use the counting function of the microscope software to count the number of stomata in each photo; (6) Calculation of stomatal density: Calculate the stomatal density of leaves according to the formula. The calculation formula is as follows: Leaf stomatal density (pieces / mm 2 ) = Number of pores in a single photo (pieces) / Photo area (mm 2 ).

2. The method according to claim 1, characterized in that The silicone polymer-dental impression gel tube contains a dental silicone rubber impression material, which contains a matrix of vinyl polysiloxane and a catalyst of platinum silicon oxide.

3. The method according to claim 1, characterized in that Step (1) Apply 1-2cm on the middle of the plant leaf epidermis, avoiding the main veins 2 Silicone gel, wait 1-5 minutes.

4. The method according to claim 1, wherein The main components of the transparent nail polish are butyl acetate, ethyl acetate, acrylic acid (ester) / VA copolymer and triethyl citrate.

5. The method according to claim 1, wherein The glue curing lamp is an LED glue curing lamp with a power of 120W and a wavelength of 400-500nm, and is used to accelerate the curing of nail polish.

6. The method according to claim 1, characterized in that The microscope is equipped with a 1360×1024 pixel camera, and the area of ​​a single photo taken is 1.2-1.5mm 2 , and has a scale function to facilitate the calculation of pore density.

7. The method according to claim 1, characterized in that The stoma counting software is an image analysis software matched with a microscope, which has an automatic or manual marking and counting function and is used to quickly count the number of stomata.

8. The method according to claim 1, characterized in that The plant leaves are herbaceous or woody plant leaves.

9. The method according to claim 8, characterized in that The plant leaves are leaves of Rosaceae fruit trees.

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