A method for rubbing and sampling the morphology of stomatal apparatuses on plant leaves

Abstract

The present invention discloses a method for rubbing and sampling the morphology of stomatal apparatuses on plant leaves, which includes: Step 1, collecting the leaves of a plant sample; Step 2, making an epidermal specimen by rubbing with UV glue: putting on ultraviolet protection glasses, dropping 1-2 drops of UV glue on the front of a smooth leaf and covering it with a glass slide, pressing with fingers, and irradiating the UV glue through the glass slide with an ultraviolet lamp for 10-15 seconds for fixation; gently removing the leaf to ensure that the fixed UV colloid does not detach from the glass slide, and the UV glue on the glass slide has rubbed the epidermal pattern of the leaf as the upper epidermal specimen of the sample leaf. The technical effects of the present invention are: extremely fast sampling speed, simple materials used, simple operation, labor saving, economical and environmentally friendly, clear and accurate imaging, large observable area of the specimen, and no damage to the sample.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant ecological detection, and particularly relates to a sampling method for the morphology of plant leaf stomata Background Art

[0002] Plant leaf stomata are key devices connecting the soil-plant body-atmosphere, and are one of the most important structures for plant life activities, controlling the transpiration and photosynthesis of plants. According to the literature "A stomatal safety-efficiency trade-off constrains responses to leaf dehydration[J]", Henry C, John G P, Pan R, et al., Nature communications, 2019, 10(1): 3398 ("Stomatal safety-efficiency trade-off constrains responses to leaf dehydration" [J], Henry C, John G P, Pan R, et al., Nature Communications, 2019, 10(1): 3398), it is recorded that due to the morphological characteristics of stomata and the opening and closing functions of stomata, it has a major impact on plant growth, ecosystem productivity, and the global water cycle. Observation data of stomata in many research fields of botany show that the research on stomata is playing a central role in the development of global ecology, plant physiology, and evolutionary science

[0003] Under the influence of the global warming trend, extreme high-temperature events occur frequently. As the main cooling source of the climate environment, forest vegetation transpiration and water evaporation are regulated and controlled by plant stomata. And the observation of stomata is an essential part of studying the physiological mechanisms of plant growth, high-temperature resistance, drought resistance, and water conservation. Investigating and understanding the morphological characteristics of stomata of various forest plants has become a key link in understanding plant adaptation to extreme high-temperature climates

[0004] Currently, for the production of plant stomata observation specimens, conventional methods such as the hand-tearing method, electron microscope scanning method, nail polish method, hot melt adhesive method, and fixation method are commonly used in teaching and research. However, although there are many methods for obtaining the lower epidermis of plants, these methods all have some defects more or less, and it is difficult to adapt to the acquisition of stomata sample data of a large number of different species of plants. The specific description is as follows

[0005] The disadvantages of the most common manual tearing method are as follows: 1. It requires fine manual movements, with high precision requirements, consuming energy and eyesight. Many participants become fatigued after making 5 specimens. It is time-consuming and laborious to make specimens of batch plant samples and difficult to bear. 2. The leaf thickness and the adhesion degree between the lower epidermis and mesophyll cells of different species are different. It is extremely difficult to tear the lower epidermis of some thin-leaf plant species. For example, the epidermis of tea tree leaves cannot be torn. Therefore, this method is only applicable to plant leaves with loose adhesion between the lower epidermis and mesophyll cells. 3. The torn epidermis usually adheres to mesophyll cells, affecting the clarity and quality of the specimen. 4. It is difficult to grasp the size and position of the obtained lower epidermis, restricting the integrity and wholeness of the specimen.

[0006] The disadvantages of the hot melt adhesive method are: there are defects such as the generation of pores and shrinkage and deformation of epidermal cells during the high-temperature sol film covering process in the operation process, and it requires electricity and can only be operated in the laboratory, which is not conducive to observing the changes in the opening and closing behavior of stomatal apparatuses in the wild in real time. The disadvantages of the electron microscope scanning method are: it requires treatment with more than two chemical reagents for at least 4 hours or more. The long reagent treatment time is likely to cause potential cell deformation and affect the data accuracy. The cost of the electron microscope is high, and the operation is time-consuming and complex. The disadvantages of the nail polish method are: it requires waiting for at least 10 minutes or more for material treatment, the finished product image is not clear, certain operation techniques are required, and the effect on irregularly shaped tissues is not good. The disadvantages of the fixed adhesive method are: it also requires tearing the leaf epidermis, with high requirements for the quality of the adhesive and the application technique, a long specimen preparation time, and the same specimen preparation problems as those encountered in the manual tearing method. The disadvantages of the infiltration method are: it requires the use of volatile toxic and harmful infiltration reagents such as benzene and xylene, which are harmful to the environment and human body, and such methods should be avoided.

[0007] In summary, the currently used methods for observing stomatal apparatuses generally have disadvantages such as a large variety of reagent uses, being toxic and harmful, complex procedures, high precision requirements for operation, time-consuming, energy-consuming, low efficiency, strong dependence on instrument equipment, and high costs, thus affecting the batch research on stomatal apparatuses. Therefore, finding a method for observing stomatal apparatuses of leaves of different species of forest plants in batches, which is simple in operation, convenient in material use, is an urgent technical problem to be solved at present. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for rubbing and sampling the morphology of plant leaf stomatal apparatuses, which has extremely fast sampling speed, simple materials, simple operation, labor saving, economic and environmental protection, clear and accurate imaging, a large observable area of the specimen, and does not damage the sample, and can investigate the morphological characteristics of stomatal apparatuses of leaves of plant species in large quantities.

[0009] To solve the above technical problems,

[0010] A method for rubbing and sampling the morphology of plant leaf stomatal apparatuses provided by the present invention comprises the following steps:

[0011] Step 1: Collect plant sample leaves

[0012] Select a plant with a strong plant type and normal development in the wild, and cut off healthy leaves as sample leaves; or quickly put the cut leaves into a sealed plastic bottle with FAA fixative to submerge the sample leaves;

[0013] Step 2: Produce epidermal specimens by rubbing with UV glue for the sample leaves

[0014] Produce upper epidermal specimens: Wear ultraviolet protection glasses, drop 1-2 drops of UV glue on the front of the smooth leaf and cover it with a glass slide, press with fingers, and irradiate the UV glue through the glass slide with a UV lamp for 10-15 seconds for fixation; gently remove the leaf to ensure that the fixed UV colloid does not come off the glass slide. The UV glue on the glass slide has a rubbed leaf epidermal pattern, which serves as the upper epidermal specimen of the sample leaf;

[0015] Or / and produce lower epidermal specimens: Turn over the sample leaf, drop 1-2 drops of UV glue on the back of the smooth leaf, and repeat the production to obtain the lower epidermal specimen of the sample leaf.

[0016] Compared with the existing method for producing specimens for observing plant stomatal apparatuses, the technical effects of the present invention are as follows:

[0017] 1. Improve the production efficiency: From taking out the sample leaves to completing the production of specimens in the present invention, it only takes about 10-30 seconds, and it takes less than one hour to complete the production of 30 specimens. The present invention does not require fine operation by the experimenter's hands, and technicians can complete the production of stomatal apparatus specimens in a state of low energy. Compared with the common method, it saves more than 85% of the time and saves the energy of the researchers.

[0018] 2. Improve the stability and reliability of production: The production process of the present invention can sample when the leaf is alive, and does not require scraping off mesophyll cells, so the epidermis will not be damaged, and the epidermis of thin-leaved plants will not shrink, etc. The influence on the transverse diameter and longitudinal diameter of the stomatal apparatus is minimized, thus ensuring the accuracy and authenticity of the specimens. When observing under a microscope, the morphology of the stomatal apparatus is clear, there is no interference from mesophyll cells in the specimen field of view, and the success rate is as high as 98.9%. It has the advantages of high clarity of the prepared slides, less chemical treatment, little deformation of cells, and high data accuracy. This is very beneficial for observing important parameters such as the size of the stomatal apparatus and calculating the density of the stomatal apparatus.

[0019] 3. Have portability: The reagents and tools involved in the present invention are all light and portable. Coupled with the advantages of efficient and fast sampling, there is no need to limit the operation to the laboratory, and a large amount of sampling can be carried out on-site in the wild.

[0020] 4. Reproducibility: The manufacturing process of the present invention causes little or no damage to the leaf samples, ensuring the integrity of the leaves. Therefore, the same sample can be sampled repeatedly for many times, greatly reducing the operation difficulty and the requirement for fineness. At the same time, the required number of sampled samples is reduced, and the field sampling workload of researchers is decreased.

[0021] 5. Can ensure the integrity of organs: The present invention can be applied to the upper and lower epidermis of leaves, and the area can reach 90% - 100% of the leaf, with a large coverage range. The morphology of the stomatal apparatus is accurately and stably displayed, and the success rate can reach more than 98%. This is very important for observing characteristics such as the distribution of the stomatal apparatus on the leaf.

[0022] 6. Environmentally friendly and pollution-free: The tools and reagents used in the present invention are small in quantity and variety, and all have low volatility, are harmless and pollution-free, being friendly to both the environment and the health of operators.

[0023] 7. Economical and practical: The tools and reagents involved in the present invention have relatively low costs. The price of the ultraviolet lamp is 5 - 50 yuan, the price of the ultraviolet protection glasses is 15 - 30 yuan, and the consumable costs such as UV glue do not exceed 0.02 yuan per sample, significantly reducing the sampling cost. Description of the Drawings

[0024] The drawings of the present invention are described as follows:

[0025] Figure 1 The image of the stomatal apparatus on the lower epidermis of Beilschmiedia yaanensis leaves taken by a microscope for the specimen of Example 1;

[0026] Figure 2 The image of the stomatal apparatus on the lower epidermis of Melastoma normale D. Don var. strigillosum (DC.) C. Chen leaves taken by a microscope for the specimen of Example 2. Detailed Embodiment

[0027] The present invention will be further described below in conjunction with the drawings and embodiments:

[0028] The tools and materials used in the present invention include: several sealed plastic bottles of FAA fixing solution, several double-sided or single-sided blades, several glass slides, one high-branch shear, several flower shears; several pairs of tweezers for rubbing tools, UV glue in a plastic bottle with a pointed nozzle, one ultraviolet lamp, one pair of ultraviolet protection glasses, several packs of medical gauze and paper towels.

[0029] The UV glue mentioned above is a one-component ultraviolet or visible light-curing modified acrylate glue, with the specification of a high-hardness thin glue for coating and sealing (coating and sealing is a common means in the electronics and chemical industries, using colloids or protective paints to cover objects to form a physical isolation seal layer to play roles such as dust prevention, moisture prevention, and anti-aging), and the viscosity is 1500 - 3000 cps (25 °C, rotational viscometer).

[0030] The ultraviolet lamp is a device that can emit ultraviolet rays and can rapidly cure the UV glue. It is an LED ultraviolet lamp with a specification of a wavelength not less than 395 nm and a power of 3 - 40 W. Among them, the handheld LED ultraviolet lamp powered by ordinary dry batteries is suitable for field operations, and the desktop LED ultraviolet lamp powered by the power supply is suitable for indoor operations.

[0031] The ultraviolet protective glasses are protective equipment to prevent the direct strong ultraviolet rays from directly irradiating both eyes due to improper operation. However, short-term irradiation of the skin by ultraviolet rays and indirect non-direct viewing of purple light have no health hazards.

[0032] The present invention includes the following steps:

[0033] Step 1: Collect plant sample leaves

[0034] Select healthy and normal-growing plant plants in the wild, cut off the healthy leaves as sample leaves; if sampling indoors, put the cut leaves into a sealed plastic bottle with FAA fixative quickly and submerge the sample leaves.

[0035] Step 2: Make epidermal specimens by stamping the sample leaves with UV glue

[0036] When sampling indoors, take out the sample leaves from the FAA fixative with forceps, place them at room temperature to dry or press them dry with a tissue; when sampling in the wild, directly use the sample leaves;

[0037] Specifically, for sample leaves with epidermal hairs, wipe the hairs in a single direction with medical gauze until the hairs fall off; for sample leaves that are difficult to wipe with gauze, drop 1 - 2 drops of UV glue on the back of the leaf and cover it with a glass slide, press it with fingers and irradiate the UV glue through the glass slide with an ultraviolet lamp for 5 - 10 seconds to fix it, then use a double-sided or single-sided blade to gently scrape the leaf surface in a single direction forward, backward, left or right at an angle of 75 - 90° with the leaf until the epidermal hairs fall off. Do not scrape back and forth to avoid damaging the epidermis. This operation is not required for sample leaves without epidermal hairs.

[0038] Make upper epidermal specimens

[0039] Wear ultraviolet protective glasses, drop 1 - 2 drops of UV glue on the front of the smooth leaf and cover it with a glass slide, press it with fingers, and irradiate the UV glue through the glass slide with an ultraviolet lamp for 10 - 15 seconds to fix it;

[0040] Gently lift the leaf, ensure that the fixed UV colloid does not separate from the glass slide. The UV glue on the glass slide has stamped the leaf epidermis pattern, which serves as the upper epidermal specimen of the sample leaf; mark the upper epidermis and the plant name on the glass slide;

[0041] If the colloid has separated from the glass slide, it is necessary to repeat the production.

[0042] Make lower epidermal specimens

[0043] Turn the sample leaf over and drop 1-2 drops of UV glue on the smooth back of the leaf. Repeat the process to obtain the lower epidermis specimen of the sample leaf.

[0044] According to experimental needs, choose to make upper epidermis specimens or lower epidermis specimens.

[0045] Place the sample leaf after rubbing in step 2 back into the sealed plastic bottle of FAA fixative for reuse.

[0046] Example 1: Take the stomatal specimens from the lower epidermis of the leaves of Beilschmiedia yaanica

[0047] Step 1: Collect plant sample leaves

[0048] Select the Yaan Qiongnan with a healthy plant shape and normal development in the wild, cut the leaves near the main veins of the healthy leaves with high-branch shears, and quickly put them into a sealed plastic bottle with FAA fixative to submerge the leaves (3-5 leaves can be put into each bottle);

[0049] Step 2: UV glue rubbing of sample leaves to make epidermal specimens

[0050] Use tweezers to take out the sample leaf in the FAA fixative and press and dry the leaf with a paper towel;

[0051] Make upper epidermis specimens: Wear UV protective glasses, drop 1-2 drops of UV glue on the front of the smooth leaf and cover it with a glass slide, press with your fingers, irradiate with a UV lamp for 10-15 seconds to fix, gently peel off the leaf, make sure the fixed UV colloid does not fall off the glass slide, the UV glue on the glass slide has a leaf epidermis pattern, as the sample leaf upper epidermis specimen; mark upper epidermis and Ya'an Qiongnan on the glass slide;

[0052] Preparation of lower epidermis specimens: Turn the sample leaf over, drop 1-2 drops of UV glue on the back of the smooth leaf, repeat the process to obtain the lower epidermis specimen of the sample leaf, and take a photo with a microscope. Figure 1 The stomatal image of the lower epidermis of the leaf is shown. The image of the stomatal apparatus is clear, which is conducive to the determination and calculation of stomatal apparatus parameters.

[0053] Example 2: Taking Stomatal Organs from the Lower Epidermis of Leaves of Melastoma malabathricum

[0054] Step 1: Collect plant sample leaves

[0055] Select a healthy and normally developed wild Paeonia pubescens, cut off the leaves near the main veins of the healthy leaves with a pruning shears, and quickly put them into a sealed plastic bottle with FAA fixative to submerge the leaves;

[0056] Step 2: UV glue rubbing of sample leaves to make epidermal specimens

[0057] Use tweezers to take out the sample leaf in the FAA fixative and press and dry the leaf with a paper towel;

[0058] Wear UV protective glasses and wipe the fur in one direction with medical gauze until at least 1 cm of fur is removed. 2 .

[0059] Make upper epidermis specimens: drop 1-2 drops of UV glue on the front of the smooth leaf and cover it with a glass slide, press with your fingers, irradiate with a UV lamp for 10-15 seconds to fix, gently peel off the leaf, make sure the fixed UV colloid does not fall off the glass slide, the UV glue on the glass slide has a leaf epidermis pattern, which is used as the upper epidermis specimen of the sample leaf; mark the upper epidermis and wild peony on the glass slide;

[0060] Preparation of lower epidermis specimens: Turn the sample leaf over, drop 1-2 drops of UV glue on the back of the smooth leaf, repeat the process to obtain the lower epidermis specimen of the sample leaf, and take a photo with a microscope. Figure 2 The image of the stomata on the lower epidermis of a leaf is shown. Figure 2 In the image, the density of stomatal apparatus is relatively high, but it is still relatively clear, and there is no residual leaf back hair, which can be used for the determination and calculation of stomatal apparatus parameters.

[0061] The advantages and disadvantages of the present invention and the existing commonly used stomatal specimen preparation methods are compared, see Table 1:

[0062] Table 1

[0063]

[0064] Note: In Table 1, the "cost column" is the average price of reagents and consumables required to complete a specimen, and the brackets are the average common prices of non-consumables and instruments required for specimen preparation. The "time consumption column" is calculated from the time from taking out the leaf to the time of completing a specimen (the data can be found in the literature "A method of observing stomata by sticking leaf epidermis with transparent tape", Chen Baihong, Li Xinsheng, Cao Ziyi, Acta Physiologica Sinica [J], 2004, 40: 215-218; the literature "A simple method for observing the stomatal apparatus of wax leaf specimens", Xu Hao, China Science and Technology Information [J], 2008, 254-254; the literature "Comparison and improvement of cotton leaf stomatal preparation methods", Ye Chunxiu, Zhuang Zhengang, Li Youzhong, Molecular Plant Breeding [J], 2014, 000: 543-546).

Claims

1. A method for imprint sampling of the morphological structure of stomata on plant leaves, characterized in that, It includes the following steps: Step 1: Collect plant sample leaves In the wild, select plant plants with strong plant types and normal development, and cut off healthy leaves as sample leaves; or quickly put the cut leaves into a sealed plastic bottle of FAA fixing solution to submerge the sample leaves; Step 2: Make epidermal specimens by UV glue rubbing of sample leaves Make upper epidermal specimens: Wear ultraviolet protective glasses, drop 1-2 drops of UV glue on the smooth front of the leaf and cover it with a glass slide, press with fingers, and irradiate the UV glue through the glass slide with a UV lamp for 10-15 seconds to fix; Gently remove the leaf to ensure that the fixed UV colloid does not detach from the glass slide. The UV glue on the glass slide rubs the leaf epidermal pattern, which is used as the upper epidermal specimen of the sample leaf; Or / and make lower epidermal specimens: Turn the sample leaf over, drop 1-2 drops of UV glue on the smooth back of the leaf, and repeat the production to obtain the lower epidermal specimen of the sample leaf; The UV glue is a one-component photocurable modified acrylate glue with a viscosity of 1500-3000 cps; the UV lamp is an LED ultraviolet lamp with a wavelength of not less than 395 nm and a power of 3-40 W.

2. The method for rubbing and sampling the morphology of plant leaf stomata according to claim 1 is characterized in that: at Before Step 2, it also includes the pretreatment of sample leaves: Use tweezers to take out the sample leaves in the FAA fixing solution, and place them at room temperature to dry or press them dry with a paper towel.

3. The method for rubbing and sampling the morphology of stomatal apparatus of plant leaves according to claim 2, characterized in that: The pretreatment of sample leaves also includes removing epidermal hairs from sample leaves. Wipe the hairy part in a single direction with medical gauze until the hairs fall off; or, drop 1-2 drops of UV glue on the back of the leaf and cover it with a glass slide, press with fingers and irradiate the UV glue through the glass slide with a UV lamp for 5-10 seconds to fix, and then gently scrape the front of the leaf with a double-sided or single-sided blade at an angle of 75-90° to the leaf in a single direction until the epidermal hairs fall off.

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