Accurate metering method for mature hairy bodies on surface of cotton leaf surface

Abstract

The invention relates to the technical field of plant detection, in particular to a method for accurately measuring mature hairy bodies on the surface of a cotton leaf surface, which is characterized in that fresh cotton leaves are used as test materials, the optimal parameters of the test environment are that the temperature is 23 DEG C, the humidity is 30%, and different treatment schemes are used according to the difference of the shape, length and density of the hairy bodies. Interference between the back face and leaf margin hair is removed firstly, then liquid nitrogen soaking low-temperature treatment is carried out, water molecules in air are evenly attached to the surface of the leaf hairy body and crystallized, and therefore a clear leaf hairy body image is obtained. The method has the advantages of no need of reagent preparation, short sample preparation time, clear imaging, clear leaf hairy number and length characteristics, and substantial improvement of the metering efficiency of the mature hairy bodies on the surfaces of the cotton leaves. An efficient and reliable metering means is provided for related breeding work such as morphological research, insect-resistant mechanism analysis and developmental regulation pathway function verification related to cotton leaves, and the problem that leaf hairy bodies, especially dense hairy bodies, cannot be accurately metered is solved.

Description

Technical Field

[0001] This invention relates to the field of plant detection technology, and in particular to a method for accurately measuring mature trichomes on the surface of cotton leaves. Background Technology

[0002] Cotton leaf trichomes are single-celled or multi-celled protrusions formed from the epidermal cells of cotton leaves, also known as leaf hairs. Based on their density, they can exhibit three phenotypes: smooth, hairy, and trichome-like. Leaf trichomes play multiple crucial roles in the physiological activities and stress resistance of cotton. They regulate leaf transpiration, photosynthesis, and surface temperature, enhancing the plant's drought and UV resistance. They also enhance resistance to pests through physical barriers and chemical defenses, hindering pest feeding and oviposition. Their developmental gene network shares pathways with seed fiber differentiation. Studying cotton leaf trichomes is a key direction for elucidating its stress and pest resistance mechanisms, developmental regulatory pathways, and their correlation with fiber quality, possessing both basic research value and promising agricultural applications. However, cotton leaf trichomes are characterized by their tiny size, semi-transparent or fully transparent morphology, and crisscrossing structure. While they can be observed directly using low-magnification optical microscopy, it is difficult to obtain clear images that can be used to statistically analyze the length, number, and distribution patterns of the trichomes. Although scanning electron microscopy and transmission electron microscopy can achieve fine structural observation, they cannot accurately characterize the morphology and number of mature leaf trichomes. Furthermore, they suffer from drawbacks such as cumbersome sample preparation procedures, high experimental costs, and long detection cycles, which cannot meet the needs of rapid screening of large-scale samples.

[0003] Current technologies for observing the surface structure of cotton leaves primarily rely on optical and electron microscopy, focusing on cellular and subcellular structures, and lack systematic methods for observing the macroscopic characteristics of leaf trichomes. Specifically, existing technologies struggle to efficiently obtain trichome density parameters, density distribution differences in different leaf regions (near the midrib, leaf margin, and mesophyll region), and cannot directly analyze the arrangement patterns of trichomes, such as whether they are parallel to the veins, whether they are randomly distributed, and the differences in trichome density and distribution between the upper and lower surfaces of the leaf. Furthermore, the operational procedures for low-magnification microscopic observation have not yet been standardized, making it difficult to balance observation efficiency and data accuracy, thus limiting the large-scale acquisition and subsequent analysis of cotton trichome-related phenotypic data. Therefore, in order to overcome the problem of inaccurate measurement of leaf trichomes, especially dense trichomes, it is necessary to develop an experimental method that is simple to use, requires no reagents, has a short sample preparation time, and whose imaging results can clearly present leaf trichomes and precise measurement parameters such as trichome length and density. This method can significantly improve the measurement efficiency of mature trichomes on the surface of cotton leaves and provide an efficient and reliable technical means for morphological research on cotton leaves, analysis of insect resistance mechanisms, and verification of developmental regulatory pathways. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a precise measurement method for mature trichomes on the surface of cotton leaves. This method requires no reagent preparation, has a short sample preparation time, and the imaging results can clearly present the leaf trichomes and precise measurement parameters such as trichome length and density, which can significantly improve the measurement efficiency of mature trichomes on the surface of cotton leaves.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for accurately measuring mature trichomes on the surface of cotton leaves, the specific steps of which are as follows:

[0007] Step S1: Select fresh cotton leaves, cut the part to be observed into square samples with a side length of 2-5 mm, examine them under a stereomicroscope, and determine the subsequent processing plan based on the microscopic examination results.

[0008] Step S2: For samples where microscopic examination shows that the density of trichomes is sparse or moderate and the leaf surface can be clearly or relatively clearly observed, proceed directly to step S3.

[0009] Step S3: According to the dosage of 0.5 μL per 1 mm of sample side length, add deionized water to the surface of the glass slide, place the sample with the observation side facing up in the water droplet area, gently press the sample to make the water spread evenly to the back of the sample, use filter paper or dust-free paper to absorb the excess water on the surface of the glass slide and the sample, take another glass slide to cover the sample surface and press it down appropriately, put the assembled sample vertically into liquid nitrogen, so that the sample is submerged 2 cm below the liquid surface, soak for 7 seconds, take it out, and immediately examine it under a stereomicroscope with a magnification of 1.5 to 2 times;

[0010] Step S4: For samples where microscopic examination shows a high density of trichomes and the leaf surface cannot be clearly observed, first cut off the leaf margin of the sample and remove the trichomes on the back of the observation surface to eliminate interference.

[0011] Step S5: Samples treated with liquid nitrogen immersion need to stand for a preset time before microscopic examination to obtain ideal observation results; among them, the standing time for samples with moderate trichome density is 5 seconds, and the standing time for samples with short and sparse trichomes or extremely high density is no less than 10 seconds.

[0012] Step S6: During microscopic examination, a stereomicroscope with a camera function is used to photograph the hair-like structure of the sample, and the observation data is recorded. To improve the imaging quality, the imaging parameters of the microscopic imaging can be optimized and adjusted based on the parameters such as hair-like morphology, length, and density obtained from the preliminary experimental results, and the experiment can be repeated based on the adjusted parameters to obtain the best imaging effect.

[0013] Preferably, step S4 includes the following method:

[0014] Step S4.1, the method for removing the hair-like structures on the back is as follows: Add 0.5 μL of deionized water to the surface of the slide according to the dosage of 1 mm of sample side length. Place the sample with the back side facing up in the water droplet area, gently press the sample to spread the water evenly to the observation surface, and use filter paper to absorb the excess water from the slide and sample surface. Take another slide to cover the sample surface and press it down appropriately. Immerse the assembled sample in liquid nitrogen for 3 seconds and then take it out. Prepare a container with liquid nitrogen inside, and set a platform in the container that is higher than the liquid nitrogen level. Place the slide carrying the sample on the platform, and use a brush dipped in liquid nitrogen to gently brush the sample surface. Repeat the operation until the hair-like structures are completely removed. Mark the back side of the sample with a marker pen.

[0015] Step S4.2: Cut a 1mm×2mm subsample from the sample treated above, and then divide the subsample into multiple segments with a length of 1mm and a width of less than 0.5mm. Cover the upper and lower surfaces of the segments with dry glass slides, press the glass slides lightly and shake them slightly from side to side to compact them appropriately. Place the assembled glass slides vertically into liquid nitrogen, immersing the segments 2cm below the liquid surface. After soaking for 7 seconds, remove the slides and immediately examine them under a stereomicroscope. Repeat this step until all segments are examined under a microscope. The magnification is 3 to 4 times.

[0016] Preferably, the baseline parameters for liquid nitrogen immersion treatment are a temperature of 20–25°C and a humidity of 20–35%. When the ambient humidity is below 30%, the minimum immersion time of liquid nitrogen is extended by 1 second for every 5% decrease in humidity. For cotton leaf samples with extremely sparse or extremely high trichomes, the immersion time can be appropriately extended to improve the observation effect.

[0017] Preferably, the baseline parameters for liquid nitrogen immersion treatment are an ambient temperature of 23°C and a humidity of 30%.

[0018] Preferably, the minimum immersion time in liquid nitrogen is 7 seconds, and the actual immersion time can be extended but not shortened.

[0019] Preferably, in step S5, the sample after liquid nitrogen immersion treatment needs to be left to stand before microscopic examination. The standing time is determined according to the density of the hairs in the sample, and is 5 to 10 seconds or longer.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. This invention innovatively provides a precise measurement method for mature trichomes on the surface of cotton leaves, which is a novel method for enhancing the development of cotton leaf trichomes. It has the advantages of simple sample preparation and low cost.

[0022] 2. This invention does not require the use of high-purity liquid nitrogen; liquid nitrogen recovered from routine experiments can meet the requirements. The required experimental equipment and facilities are low-threshold, which greatly reduces the cost of sample preparation and observation.

[0023] 3. The operation process of this invention is simple and time-saving, which can efficiently shorten the observation cycle and make it convenient and clear to observe the phenotypic characteristics of cotton leaf trichomes. It can realize the rapid collection of trichome phenotypic data and overcome the problem of inaccurate measurement of leaf trichomes, especially dense trichomes. Attached Figure Description

[0024] Figure 1 These are images showing the effects of treating cotton leaves with and without liquid nitrogen, and a comparison of the treatment effects on the back side of hairy leaves. Figure 1 The image shown is a micrograph of the leaf without liquid nitrogen treatment. Figure 1 be is a micrograph of the leaf after liquid nitrogen treatment. Figure 1 c is a comparison image showing the liquid nitrogen treatment of the underside of the leaf with high trichome density, without the use of a brush to remove the trichomes. Figure 1 f is the effect image after removing the trichomes on the back of the surface to be observed;

[0025] Figure 2 The diagram shows the effects of using leaves with different trichome densities and different liquid nitrogen treatment times according to the present invention. Figure 2 Image a shows the effect of treating leaves with high trichome density with liquid nitrogen. Figure 2 b is an image showing the effect of treating leaves with moderate trichome density with liquid nitrogen. Figure 2 Image c shows the effect of liquid nitrogen treatment on leaves with low trichome density. Figure 2 Image d shows the effect of treating micro-fragments obtained after treating leaves with excessively high trichome density with liquid nitrogen. Figure 2 The image shows the effect after insufficient liquid nitrogen treatment time. Figure 2 f is the effect after liquid nitrogen treatment for too long;

[0026] Figure 3 This is a schematic diagram of the equipment required for removing the hair-like structures on the back of the sample according to the present invention;

[0027] Figure 4 This is a schematic diagram of the assembled double-layer glass slide containing the sample to be tested according to the present invention. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Preparation of low-density tuft metering material in cotton leaves

[0030] 1. Experimental Methods

[0031] (1) Take fresh cotton leaves.

[0032] (2) Avoid the veins when taking samples to obtain a 5mm square sample, and perform preliminary microscopic examination under a stereomicroscope.

[0033] (3) The density of the trichomes is moderate in the stereoscopic field of view, and they can be directly frozen with liquid nitrogen.

[0034] (4) Take a glass slide as the base, add 2.5 μL of water between the glass slide and the base, place the sample with the observation side facing up in the water droplet area, press the sample lightly to make the water spread evenly to the back of the sample, use filter paper or dust-free paper to absorb the excess water on the glass slide and sample surface, take another glass slide to cover the sample surface and press it down appropriately.

[0035] (5) Observe the ambient humidity. If the current humidity is 25%, the liquid nitrogen treatment time is 8 seconds.

[0036] (6) Place the assembled sample vertically into liquid nitrogen, immersing it 2 cm below the liquid surface. After soaking for 8 seconds, remove the sample and immediately examine it under a stereomicroscope.

[0037] (7) If the sample is found to be not frozen enough or too frozen, the freezing time can be extended or shortened accordingly.

[0038] 2. Experimental Results

[0039] The experimental results are as follows: Microscopic examination and imaging were performed using the Langzi E3IS PM stereoscope. Figure 1 As shown in b, the trichomes on the leaf surface were obtained.

[0040] The images of the hair follicles were processed using ImageJ software to obtain parameters such as the number and length of the hair follicles.

[0041] Example 2: Preparation of high-density capillary metering material from cotton leaves

[0042] 1. Experimental Methods

[0043] (1) In the stereoscopic field of view, the density of the trichomes is too high, so they need to be cut into multiple small fragments before being treated with liquid nitrogen.

[0044] (2) Take a glass slide as the base, add 2.5 μL of water between the slide and the sample, place the sample with the observation side facing down in the water droplet area, press the sample gently to spread the water evenly to the front of the sample, and use filter paper or dust-free paper to absorb the excess water on the surface of the glass slide and the sample.

[0045] (3) According to Figure 3 Set up the operating environment by preparing a container filled with liquid nitrogen and constructing a platform inside the container that is above the liquid nitrogen level. Figure 4 Assemble the glass slide, immerse the assembled sample in liquid nitrogen for 3 seconds, then remove it. Place the glass slide carrying the sample on the platform, and use a brush dipped in liquid nitrogen to gently brush the sample leaf surface. Repeat the operation until the hairs are completely removed. Mark the back of the sample with a marker pen for easy subsequent operations.

[0046] (4) Cut a 1mm×2mm subsample from the processed sample, and then divide the subsample into multiple segments with a length of 1mm and a width of 0.5mm. Take a dry glass slide and cover the upper and lower surfaces of the segments to be inspected. Press the glass slide lightly and shake it slightly from side to side to compact it appropriately. Place the assembled glass slide vertically into liquid nitrogen so that the segments to be inspected are submerged 2cm below the liquid surface. After soaking for 7 seconds, take it out and immediately examine it under a stereomicroscope. Repeat this step until all segments to be inspected are examined under a microscope. Since the sample volume is small, the entire operation process should be completed quickly to prevent the sample from drying out.

[0047] 2. Experimental Results

[0048] The experimental results are as follows: Microscopic examination and imaging were performed using the Langzi E3IS PM stereoscope. Figure 2 As shown in d, the image of the trichomes on the leaf surface was obtained.

[0049] The images of the hair follicles were processed using ImageJ software to obtain parameters such as the number and length of the hair follicles.

[0050] Verification Example 1: The effect of trichomorph density and liquid nitrogen treatment time on the effectiveness of this method.

[0051] 1. Experimental Methods

[0052] To demonstrate the effect of different liquid nitrogen treatment times on imaging results, the following experimental treatment was conducted: influencing factors were set as follows: liquid nitrogen treatment for 5s, 8s, and 12s, respectively, followed by microscopic examination.

[0053] The remaining steps are the same as in Example 1.

[0054] 2. Experimental Results

[0055] Experimental results are as follows Figure 2 As shown in b, liquid nitrogen treatment for 8 seconds yielded the best results, with clearer hairline edges; as... Figure 2 As shown in e, the 5s liquid nitrogen treatment was insufficient, resulting in blurred edges of the trichomes; as... Figure 2 As shown in f, the liquid nitrogen treatment for 12 seconds was too long, resulting in excessive ice crystal growth on the hair follicles, making it impossible to identify the edges of the hair follicles.

[0056] In summary, the present invention has a simple operation process, requires no reagent preparation, has a short sample preparation time, and the imaging can clearly show the number and length characteristics of leaf hairs. It can significantly improve the measurement efficiency of mature hairs on the surface of cotton leaves, and provide an efficient and reliable measurement method for related breeding work such as morphological research on cotton leaves, analysis of insect resistance mechanisms, and verification of developmental regulatory pathways. It overcomes the problem of inaccurate measurement of leaf hairs, especially dense hairs.

[0057] The descriptions and practices disclosed in this invention are readily apparent and understandable to those skilled in the art, and various modifications and refinements can be made without departing from the principles of this invention. Therefore, any modifications or improvements made without departing from the spirit of this invention should also be considered within the scope of protection of this invention.

Claims

1. A method for accurately measuring mature trichomes on the surface of cotton leaves, characterized in that, The specific steps are as follows: Step S1: Select fresh cotton leaves, cut the part to be observed into square samples with a side length of 2-5 mm, examine them under a stereomicroscope, and determine the subsequent processing plan based on the microscopic examination results. Step S2: For samples where microscopic examination shows sparse or moderate trichome density and clear leaf surface observation, proceed directly to step S3; Step S3: According to the dosage of 0.5 μL per 1 mm of sample side length, add deionized water to the surface of the glass slide, place the sample with the observation side facing up in the water droplet area, gently press the sample to make the water spread evenly to the back of the sample, use filter paper or dust-free paper to absorb the excess water on the surface of the glass slide and the sample, take another glass slide to cover the sample surface and press it down appropriately, put the assembled sample vertically into liquid nitrogen, so that the sample is submerged 2 cm below the liquid surface, soak for 7 seconds, take it out, and immediately examine it under a stereomicroscope with a magnification of 1.5 to 2 times; Step S4: For samples where microscopic examination shows a high density of trichomes and the leaf surface cannot be clearly observed, first cut off the leaf margin of the sample and remove the trichomes on the back of the observation surface to eliminate interference. Step S5: After being soaked in liquid nitrogen, the sample needs to be left to stand for a preset time before being examined under a microscope to obtain ideal observation results. Step S6: During the microscopic examination, a stereomicroscope with a camera function is used to photograph the hair-like structure of the sample and retain the observation data.

2. The method for accurately measuring mature trichomes on the surface of cotton leaves according to claim 1, characterized in that, Step S4 includes the following methods: Step S4.1, the method for removing the hair-like structures on the back is as follows: Add 0.5 μL of deionized water to the surface of the slide according to the dosage of 1 mm of sample side length. Place the sample with the back side facing up in the water droplet area, gently press the sample to spread the water evenly to the observation surface, and use filter paper to absorb the excess water from the slide and sample surface. Take another slide to cover the sample surface and press it down appropriately. Immerse the assembled sample in liquid nitrogen for 3 seconds and then take it out. Prepare a container with liquid nitrogen inside, and set a platform in the container that is higher than the liquid nitrogen level. Place the slide carrying the sample on the platform, and use a brush dipped in liquid nitrogen to gently brush the sample surface. Repeat the operation until the hair-like structures are completely removed. Mark the back side of the sample with a marker pen. Step S4.2: Cut a 1mm×2mm subsample from the sample treated above, and then divide the subsample into multiple segments with a length of 1mm and a width of less than 0.5mm. Cover the upper and lower surfaces of the segments with dry glass slides, press the glass slides lightly and shake them slightly from side to side to compact them appropriately. Place the assembled glass slides vertically into liquid nitrogen, immersing the segments 2cm below the liquid surface. After soaking for 7 seconds, remove the slides and immediately examine them under a stereomicroscope. Repeat this step until all segments are examined under a microscope. The magnification is 3 to 4 times.

3. The method for accurately measuring mature trichomes on the surface of cotton leaves according to claim 2, characterized in that, The baseline parameters for liquid nitrogen immersion treatment are a temperature of 20–25°C and a humidity of 20–35%. When the ambient humidity is below 30%, the minimum liquid nitrogen immersion time is extended by 1 second for every 5% decrease in humidity.

4. The method for accurately measuring mature trichomes on the surface of cotton leaves according to claim 3, characterized in that, The baseline parameters for liquid nitrogen immersion treatment are an ambient temperature of 23°C and a humidity of 30%.

5. The method for accurately measuring mature trichomes on the surface of cotton leaves according to claim 2, characterized in that, The minimum immersion time in liquid nitrogen is 7 seconds.

6. The method for accurately measuring mature trichomes on the surface of cotton leaves according to claim 2, characterized in that, In step S5, the sample after liquid nitrogen immersion needs to be left to stand before microscopic examination. The standing time is determined according to the density of the hairs in the sample and is 5 to 10 seconds.

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