Fluorescence microscopic observation method for attachment condition of hibiscus pollen on stigma

Abstract

The invention discloses a fluorescent microscopic observation method for the attachment condition of hibiscus pollen on stigmas, which comprises the following steps: firstly, selecting hibiscus flowers which bloom completely, picking off pistils, and quickly cutting stigmas to obtain stigma samples; putting the column head sample into the stationary liquid, and standing for more than 24 hours; taking out the column head sample from the stationary liquid, longitudinally cutting from the top end of the column head, and averagely dividing the column head into a plurality of parts to obtain a column head tissue; the stigma tissue is soaked in a sodium hydroxide solution for standing, and the standing time does not exceed 3 hours; taking a column head sample, washing with distilled water, and dyeing with a water-soluble aniline blue solution; placing the stigma sample in a glass slide, and gently rolling the stigma sample into a slice by using a cover glass, so that the tissue is semitransparent; and placing the glass slide on which the column head sample is placed under a fluorescence microscope for observation. The method provided by the invention can effectively observe the attachment condition of the hibiscus pollen on the stigma, and provides support for evaluation of the stigma pollen attachment condition and research of hibiscus pollination biology.

Description

Technical Field

[0001] The invention belongs to the technical field of plant microscopy, and particularly relates to a fluorescence microscopic observation method for the attachment of hibiscus pollen to a stigma. Background Art

[0002] Hibiscus tiliaceus (scientific name: Hibiscus rosa-sinensis Hibiscus sibiricum is a woody flower with important ornamental value in my country, blooming year-round in a rich variety of colors. The reproduction and evolution of plant populations require sexual reproduction, which includes flowering, pollination, fertilization, fruiting, and seed formation, with pollination being a crucial step. Pollen entry into the stigma of the pistil marks the completion of this pollination process. Therefore, assessing pollen adhesion within the stigma of Hibiscus sibiricum is a crucial tool for studying its pollination biology. This is of great significance for studying the reproduction and evolution of Hibiscus populations. It not only reveals key aspects of Hibiscus's reproductive mechanism but also guides horticultural breeding, identifying optimal pollination timing and conditions to improve hybridization success rates. It also plays a crucial role in biodiversity conservation. Pollen adhesion rate is a key indicator for assessing the natural reproductive capacity of rare and endangered plant populations in the wild, directly reflecting their reproductive potential. However, due to the small size of Hibiscus pollen grains, pollen adhesion within the stigma is often impossible to observe with the naked eye.

[0003] In the existing technology, the pollen vitality of hibiscus is mainly judged by the method of in vitro pollen culture, which is to remove the pollen on the stamen and culture it on a specific culture medium for germination, thereby judging the vitality of the pollen. In plant hybridization affinity research, the growth of pollen tubes in the style is mainly observed under a fluorescence microscope. The interaction between pollen and stigma is the first step in the successful pollination of plants. The above two methods all lack the observation of the attachment of pollen to the inside of the stigma and cannot be used to study the interaction between stigma and pollen. Therefore, finding a method for observing the attachment of hibiscus pollen to the stigma to evaluate the interaction between hibiscus stigma and pollen is very necessary in the pollination biology of hibiscus. Summary of the Invention

[0004] In response to the above-mentioned shortcomings, the present invention discloses a fluorescence microscopic observation method for the attachment of hibiscus pollen to the stigma, which can effectively observe the attachment of hibiscus pollen to the stigma, providing support for the evaluation of the attachment of stigma pollen and the study of hibiscus pollination biology.

[0005] The present invention is achieved by adopting the following technical solutions: A fluorescence microscopic observation method for observing the attachment of hibiscus pollen to stigma comprises the following steps: (1) Sampling: Select fully bloomed hibiscus flowers, remove the pistils, and quickly cut the stigma to obtain the stigma sample; (2) Fixation: Place the stigma sample obtained in step (1) into a fixative solution and then place it at 4-25°C for more than 24 hours; the fixative solution is obtained by ultrasonically mixing formaldehyde solution, 30-50% ethanol solution, glacial acetic acid and glycerol; the volume ratio of the formaldehyde solution, ethanol solution, glacial acetic acid and glycerol is 1:18:1:1; the ethanol concentration should not be higher than 50% to avoid dehydration and deformation of the pollen attached to the stigma; (3) Sampling: Use tweezers to clamp the style part and remove the stigma sample from the fixative. Then, according to the splitting of the stigma of Hibiscus hibiscus, cut the stigma tissue from the split part to obtain several pieces. Each piece of stigma tissue contains 1 to 3 lobes. (4) Softening: Soak the stigma tissue in sodium hydroxide solution at 25°C for 2 to 3 hours to avoid damage and separation of the tissue sample; (5) Tissue staining: The stigma sample treated in step (4) is rinsed with distilled water to remove the sodium hydroxide solution, and then stained with a 1% water-soluble aniline blue solution. The stigma sample is then allowed to stand for 3 to 5 minutes, and then rinsed with distilled water at a low flow rate to remove excess staining solution. (6) Tissue preparation: Place the stigma sample processed in step (5) on a glass slide and gently crush the stigma sample into a thin slice with a cover glass to make the tissue translucent; (7) Pollen observation: Place the slide containing the stigma sample under a fluorescence microscope, and then you can clearly observe the attachment of pollen grains to the stigma.

[0006] Furthermore, in step (1), when the stigma is quickly sheared after the pistil is removed, the shearing point is located on the style, and the distance between the connection between the stigma and the style and the shearing point is 1 to 2 cm. This can avoid damage to the pollen and stigma tissue during sample processing in subsequent experiments, thereby obtaining complete stigma tissue with pollen attached.

[0007] Furthermore, in step (2), a 40% formaldehyde solution by volume is used to prepare a fixative. Based on the special circumstances of the stigma and the hibiscus pollen attached thereto, the present invention adds appropriate amounts of glacial acetic acid and propylene glycol to the fixative. This effectively neutralizes alkalinity and prevents swelling, enhances permeability, and buffers dehydration pressure, reducing sample brittleness. Using a fixative with an unbalanced ratio can cause cells to swell and deform, and can also easily cause the sample to dehydrate too quickly, becoming brittle and ultimately affecting observation.

[0008] Furthermore, the concentration of the sodium hydroxide solution in step (4) is 8 mol / L.

[0009] Furthermore, in step (5), the column sample is rinsed with distilled water at a low flow rate, and the flow rate of the distilled water is controlled at 1 to 2 drops per second.

[0010] Furthermore, in step (6), filter paper is inserted into the gap between the cover glass and the slide to absorb excess moisture around the column sample.

[0011] Compared with the existing technology, this technical solution has the following beneficial effects: 1. The present invention addresses the problem that existing technologies cannot effectively observe the interaction between hibiscus pollen and stigma. After cutting hibiscus pollen-attached stigma from hibiscus flowers, the stigma is sampled and placed in a specially prepared fixative. The fixative is a mixture of formaldehyde solution, 30-50% ethanol solution, glacial acetic acid, and glycerol in a uniform proportion. This fixative not only kills cells, maintains the original morphology and structure, and prevents sample decay and deformation, but also reduces sample brittleness. The stigma sample treated with the fixative is cut to obtain suitable stigma tissue, which is then soaked in sodium hydroxide solution for treatment. The stigma tissue is then stained with a water-soluble aniline blue solution, rolled onto a glass slide into thin slices, and finally observed under a fluorescence microscope. The interaction between hibiscus pollen and stigma can be clearly observed, providing support for the evaluation of stigma pollen attachment and the study of hibiscus pollination biology.

[0012] 2. Since hibiscus pollen is relatively sticky, compared with direct stereomicroscopic examination, the method of the present invention can carefully observe the size and morphological characteristics of pollen grains, quickly evaluate pollination efficiency, and predict the fertilization success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a picture of hibiscus pollen attached to the stigma observed under a fluorescence microscope according to the method of the present invention in Example 1.

[0014] Figure 2 This is a picture of hibiscus pollen attached to the stigma observed under a fluorescence microscope according to the method of the present invention in Example 2.

[0015] Figure 3 This is a photograph of hibiscus pistil. DETAILED DESCRIPTION

[0016] The present invention is further illustrated by the following examples, which are not intended to limit the present invention. Specific experimental conditions and methods not specified in the following examples are conventional methods well known to those skilled in the art.

[0017] Example 1: A fluorescence microscopic observation method for the attachment of hibiscus pollen to the stigma, comprising the following steps: (1) Sampling: At 10:00 am, pollen from the male parent Hibiscus is applied to the stigma of the female parent Hibiscus. Then at 5:00 pm on the same day, the pistil (such as Figure 3 After the pistil is removed, the stigma is quickly sheared to obtain a stigma sample; when the stigma is quickly sheared after the pistil is removed, the shearing point is located on the style, and the distance between the connection between the stigma and the style and the shearing point is 1 cm; (2) Fixation: Use a 15 mL centrifuge tube filled with fixative in advance, quickly place the stigma sample obtained in step (1) into the fixative, and then place it at 20°C for 24 hours; the fixative is obtained by ultrasonically mixing a 40% formaldehyde solution, a 40% ethanol solution, glacial acetic acid, and glycerol; the volume ratio of the formaldehyde solution, ethanol solution, glacial acetic acid, and glycerol is 1:18:1:1; (3) Sampling: Use tweezers to clamp the style part and remove the stigma sample from the fixative. Then, according to the 5-lobed split of the hibiscus stigma, unfold the style and cut the stigma tissue from the split part to obtain two stigma tissues, one of which contains two lobes and the other contains three lobes. (4) Softening: Soak the stigma tissue in 8 mol / L sodium hydroxide solution at 25°C for 3 hours; (5) Tissue staining: Use a rubber-tipped dropper to draw distilled water, then rinse the stigma sample treated in step (4) with distilled water to remove the sodium hydroxide solution, then stain with a 1% water-soluble aniline blue solution, then let it stand for 5 minutes, then draw distilled water with a rubber-tipped dropper again, and rinse the stigma sample with distilled water at a low flow rate to remove excess staining solution. The flow rate of distilled water is controlled at 1 drop per second; (6) Tissue preparation: Place the stigma sample processed in step (5) on the center of the slide, and gently roll the stigma sample into a thin slice with a cover glass to make the tissue translucent; insert filter paper into the gap between the cover glass and the slide to absorb excess water around the stigma sample; (7) Pollen observation: Place the slide containing the stigma sample under a fluorescence microscope for observation. Figure 1 As shown, the attachment of pollen grains to the stigma can be clearly observed.

[0018] Example 2: A fluorescence microscopic observation method for the attachment of hibiscus pollen to the stigma, comprising the following steps: (1) Sampling: At 10:30 a.m., pollen from the male parent Hibiscus chinensis was applied to the stigma of the female parent Hibiscus chinensis. Then, at 11:30 a.m. on the same day, the pistil was removed and its stigma was quickly sheared to obtain a stigma sample. When the stigma was quickly sheared after the pistil was removed, the shearing point was located on the style, and the distance between the connection between the stigma and the style and the shearing point was 1 cm; (2) Fixation: Use a 15 mL centrifuge tube filled with fixative in advance, place the stigma sample obtained in step (1) into the fixative, and then place it at 25°C for 24 hours; the fixative is obtained by ultrasonically mixing a 40% formaldehyde solution, a 50% ethanol solution, glacial acetic acid, and glycerol; the volume ratio of the formaldehyde solution, ethanol solution, glacial acetic acid, and glycerol is 1:18:1:1; (3) Sampling: Use tweezers to clamp the style part and remove the stigma sample from the fixative. Then, according to the 5-lobed split of the hibiscus stigma, unfold the style and cut the stigma tissue from the split part to obtain two stigma tissues, one of which contains two lobes and the other contains three lobes. (4) Softening: Soak the stigma tissue in 8 mol / L sodium hydroxide solution at 25°C for 2 hours; (5) Tissue staining: Use a rubber-tipped dropper to draw distilled water, then rinse the stigma sample treated in step (4) with distilled water to remove the sodium hydroxide solution, then stain with a 1% water-soluble aniline blue solution, then let it stand for 5 minutes, then draw distilled water with a rubber-tipped dropper again, and rinse the stigma sample with distilled water at a low flow rate to remove excess staining solution. The flow rate of distilled water is controlled at 2 drops per second; (6) Tissue preparation: Place the stigma sample processed in step (5) on the center of the slide, and gently roll the stigma sample into a thin slice with a cover glass to make the tissue translucent; insert filter paper into the gap between the cover glass and the slide to absorb excess water around the stigma sample; (7) Pollen observation: Place the slide containing the stigma sample under a fluorescence microscope for observation. Figure 2 As shown, the attachment of pollen grains to the stigma can be clearly observed.

[0019] Example 3: A fluorescence microscopic observation method for the attachment of hibiscus pollen to the stigma, comprising the following steps: (1) Sampling: At 10:00 a.m., pollen from the male parent Hibiscus chinensis was applied to the stigma of the female parent Hibiscus chinensis. Then, at 1:00 p.m. on the same day, the pistil was removed and its stigma was quickly sheared to obtain a stigma sample. When the stigma was quickly sheared after the pistil was removed, the shearing point was located on the style, and the distance between the connection between the stigma and the style and the shearing point was 2 cm; (2) Fixation: The stigma sample obtained in step (1) is placed in a fixative solution and then allowed to stand at 10°C for 28 hours; the fixative solution is obtained by ultrasonically mixing a 40% formaldehyde solution, a 30% ethanol solution, glacial acetic acid, and glycerol; the volume ratio of the formaldehyde solution, ethanol solution, glacial acetic acid, and glycerol is 1:18:1:1; (3) Sampling: Use tweezers to hold the style part and take the stigma sample out of the fixative. Then, according to the 5-lobed split of the stigma of Hibiscus hibiscus, unfold the style and cut from the split of the stigma to obtain three stigma tissues, one of which contains one lobe, and the other two contain two lobes each. (4) Softening: Soak the stigma tissue in 8 mol / L sodium hydroxide solution at 25°C for 2.5 hours; (5) Tissue staining: Use a rubber-tipped dropper to draw distilled water, then rinse the stigma sample treated in step (4) with distilled water to remove the sodium hydroxide solution, then stain with a 1% water-soluble aniline blue solution, then let it stand for 3 minutes, then draw distilled water with a rubber-tipped dropper again, and rinse the stigma sample with distilled water at a low flow rate to remove excess staining solution. The flow rate of distilled water is controlled at 2 drops per second; (6) Tissue preparation: Place the stigma sample processed in step (5) on the center of the slide, and gently roll the stigma sample into a thin slice with a cover glass to make the tissue translucent; insert filter paper into the gap between the cover glass and the slide to absorb excess water around the stigma sample; (7) Pollen observation: Place the slide containing the stigma sample under a fluorescence microscope, and then you can clearly observe the attachment of pollen grains to the stigma.

[0020] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A fluorescence microscopic observation method for the attachment of hibiscus pollen to stigma, characterized by: The following steps are involved: (1) Sampling: Select fully bloomed hibiscus flowers, remove the pistils, and quickly cut the stigma to obtain the stigma sample; (2) Fixation: The stigma sample obtained in step (1) is placed in a fixative solution, and then placed at 4 to 25°C for more than 24 hours; the fixative solution is obtained by ultrasonically mixing formaldehyde solution, 30 to 50% ethanol solution, glacial acetic acid, and glycerol; the volume ratio of the formaldehyde solution, ethanol solution, glacial acetic acid, and glycerol is 1:18:1:1; (3) Sampling: Use tweezers to clamp the style part and remove the stigma sample from the fixative. Then, according to the splitting of the stigma of Hibiscus hibiscus, cut the stigma tissue from the split part to obtain several pieces. Each piece of stigma tissue contains 1 to 3 lobes. (4) Softening: Soak the stigma tissue in sodium hydroxide solution at 25°C for 2 to 3 hours; (5) Tissue staining: The stigma sample treated in step (4) is rinsed with distilled water to remove the sodium hydroxide solution, and then stained with a 1% water-soluble aniline blue solution. The stigma sample is then allowed to stand for 3 to 5 minutes, and then rinsed with distilled water at a low flow rate to remove excess staining solution. (6) Tissue preparation: Place the stigma sample processed in step (5) on a glass slide and gently crush the stigma sample into a thin slice with a cover glass to make the tissue translucent; (7) Pollen observation: Place the slide containing the stigma sample under a fluorescence microscope for observation.

2. The fluorescence microscopic observation method for the attachment of hibiscus pollen to stigma according to claim 1, characterized in that: In step (1), when the stigma is quickly cut after the pistil is removed, the cutting point is located on the style, and the distance between the connection between the stigma and the style and the cutting point is 1 to 2 cm.

3. The fluorescence microscopic observation method for the attachment of hibiscus pollen to stigma according to claim 1, characterized in that: In step (2), a formaldehyde solution with a volume fraction of 40% is used to prepare the fixative.

4. The fluorescence microscopic observation method for the attachment of hibiscus pollen to stigma according to claim 1, characterized in that: The concentration of the sodium hydroxide solution in step (4) is 8 mol / L.

5. The fluorescence microscopic observation method for the attachment of hibiscus pollen to stigma according to claim 1, characterized in that: In step (5), the column sample is rinsed with distilled water at a low flow rate, and the flow rate of the distilled water is controlled at 1 to 2 drops per second.

6. The fluorescence microscopic observation method for the attachment of hibiscus pollen to stigma according to claim 1, characterized in that: In step (6), filter paper is inserted into the gap between the cover glass and the slide to absorb excess moisture around the stigma sample.

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