Fluorescent staining method for dendrobium stigma pollen germination

Abstract

The invention relates to the technical field of plant biology, in particular to a fluorescent staining method for dendrobium stigma pollen germination, which comprises the following five steps: collecting dendrobium flowers with stigmas, removing redundant parts such as sepals and the like, only reserving the stigmas, trimming two sides of each stigma into a horizontal shape, putting the stigmas into a precooled stationary liquid for fixing, and then taking out the stigmas; carrying out dehydration treatment by using finished alcohol; cutting the dehydrated stigma along the central line of the stigma by using a scalpel; treating the cut sample in a softening agent to soften the tissue of the sample; transferring the transparent sample into a staining solution containing a fluorescent dye, and staining under a dark condition; and after dyeing is completed, taking out the sample, fixing the sample on a glass slide, covering with a cover glass, and observing the germination condition of the dendrobium stigma pollen under a fluorescence microscope. By means of the method, the germination condition of the dendrobium stigma pollen can be clearly and accurately observed, and powerful technical support is provided for relevant research of dendrobium and plant materials with large stigmas.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to a fluorescent staining method for germination of stigma pollen of an orchidaceae plant material, Dendrobium. Background Art

[0002] Dendrobium is a plant of the genus Dendrobium in the family Orchid, which has extremely high ornamental and medicinal value. At present, the mainstream breeding method of Dendrobium is still hybrid breeding. The key to hybrid pollination is to determine whether the pollination parents are compatible. The germination of pollen on the stigma is the first step in pollination and fertilization. It is the key to our judgment of whether the parents are compatible and whether there are pre-fertilization obstacles. It can provide ideas and directions for improving and improving the parental affinity, and provide basic support for plant breeding. In the current breeding research and reproductive biology research of Dendrobium, most of the focus is on the use of the benzidine-hydrogen peroxide method to determine the receptivity of the stigma, as well as the fruiting rate and seed vitality after pollination. For example, the patent number CN201610221260.0, the invention name of the patent entitled Dendrobium officinale seed production method based on flower organ development and the corresponding acquisition method, mentions the use of the benzidine-hydrogen peroxide method to determine the receptivity of the stigma of small flowers with different flowering days throughout the flowering period. At present, this method has certain effects, but it also has some limitations. Whether there are other effective methods is what people in the field of this technology need to consider.

[0003] Ordinary optical microscope observation may not be able to clearly show the fine structure of the pollen tube and some dynamic changes during the germination process. The use of a fluorescence microscope can better determine the receptivity of the stigma and the growth of the pollen tube, and the results are extremely intuitive and accurate. For example, the patent with application number CN202210498650.8 and invention name is a rapid screening method and application of pollination male plants in a soft-fruited kiwifruit garden. It proposes a rapid screening technology for pollination male plants based on callose fluorescence staining, which is conducive to the scientific and reasonable configuration of soft-fruited kiwifruit pollination trees. However, the present invention does not disclose the specific method and steps of staining. In combination with the research of the present technical field and the applicant, the traditional fluorescent staining of the stigma of the flower is mainly carried out by direct staining and compression observation. For example, a soft-fruited kiwifruit flower disclosed in the above application has the following main characteristics: 1. Small stigmas (such as cruciferous plant materials, the stigma diameter is about 1 mm) are easy to penetrate by dyes, and clear pollen tubes can be observed by direct compression. 2. Pollen is powdery pollen grains, which are not easy to fall off in groups after hydration. Pollen tubes are independent and form a single plane, so the germination effect is easy to observe. 3. The stigma pollination position is the tip of the stigma. The pollen tube can be clearly observed by pressing the tablet directly without operating the stigma.

[0004] After searching by the applicant, no patent has been found so far that uses fluorescent staining method to observe the germination of pollen on the stigma of Dendrobium orchid. The applicant subjectively believes that this is related to the special organs of Dendrobium orchid (see attached Figure 1 ): 1. The stigma has a special structure, located below the column, and presents a groove shape; 2. The stigma is large, slightly lignified, and the style cell tissue is thick. The common diameter of the stigma of Dendrobium is about 3-6mm, and the column cell tissue is thick; 3. Pollen grains aggregate into three-dimensional pollen grains. Therefore, the specificity and clarity of the conventional stigma fluorescent staining treatment for the germination of Dendrobium stigma pollen are not ideal, and the following situations may occur: 1. The pollen tube growth process is blocked by the column tissue; 2. The pollen block falls and the pollen tube cannot be observed; 3. The pollen block is displaced during the pressing process, and the accurate growth position cannot be observed.

[0005] Therefore, it is difficult to accurately and efficiently study the germination process of Dendrobium stigma pollen with the current staining method. The applicant searched for related technologies and found that there is only one SCI article on fluorescent staining of Dendrobium stigma, and the staining steps in the paper are consistent with the current conventional stigma staining steps, and no detailed process is reported. Therefore, it is of great significance to develop a fluorescent staining method suitable for Dendrobium stigma pollen germination. Summary of the invention

[0006] The purpose of the present invention is to provide a fluorescent staining method for the germination of Dendrobium stigma pollen, which can clearly and accurately observe the germination of Dendrobium stigma pollen and provide strong technical support for the relevant research of Dendrobium and plant materials with larger stigmas.

[0007] The fluorescent staining method for the germination of Dendrobium stigma pollen of the present invention comprises the following steps: Step 1: Sample collection and fixation, specifically: According to the detection needs, the flowers of Dendrobium orchid with stigma were collected and pre-processed: the redundant parts such as sepals were removed to keep only the stigma, and the two sides of the stigma were trimmed into a horizontal shape, fixed in a pre-cooled fixative, and then dehydrated with gradient alcohol; The fixing time is 8 to 48 hours, preferably 24 hours; The fixing solution may be FAA fixing solution, which is: 89 ml of 80% alcohol, 6 ml of glacial acetic acid, and 5 ml of formalin; The gradient alcohol uses alcohol with concentrations of 30%, 50%, and 70%. When performing dehydration treatment, each concentration of alcohol is treated for 10 minutes respectively; When the Dendrobium orchid is Dendrobium chrysotoxum, the preferred stigma diameter is 5-6 mm; when the Dendrobium orchid is Dendrobium officinale, the preferred stigma diameter is 1-3 mm.

[0008] Step 2: Cutting, specifically: The dehydrated stigma was cut open along the midline with a scalpel.

[0009] Step 3: Softening and cutting, specifically: The cut specimens are placed in a softener to soften the tissue; The softening agent is 4 mol / L NaOH solution, and the softening is carried out at 40 degrees Celsius for 2-3 hours to soften the sample tissue for subsequent staining operations and observations. Different materials have different softening times, and special attention should be paid to the shedding of pollen blocks.

[0010] Step 4: Fluorescence staining, specifically: The transparent sample is transferred into a staining solution containing a fluorescent dye and stained in the dark; Before staining, the softened samples were washed three times with sterile water and treated in 0.1N phosphate buffer for 10 min; The dyeing time is 8 to 48 hours, preferably 24 hours; The fluorescent dye is water-soluble aniline blue, and the concentration of the dye solution is 0.1%.

[0011] Step 5: Preparation and observation, specifically: After staining, the sample was taken out, fixed on a glass slide, covered with a coverslip, and the germination of the stigma pollen of Dendrobium orchid was observed under a fluorescence microscope, including the growth direction and length of the pollen tube and the germination state of the pollen grains, and the images were collected and analyzed; When observing, select the excitation light wavelength of 350nm and the emission light wavelength of 450nm for observation; During fixation, the specimens are mounted on slides with glycerol to prepare non-permanent sections, or with neutral gum to prepare permanent sections.

[0012] Beneficial effects: 1. The fluorescent staining method of the present invention can specifically stain the pollen mass of the Dendrobium stigma, so that the pollen tube and the pollen mass present a clear fluorescent image under a fluorescence microscope. The sperm nucleus can also be observed in the pollen tube, as well as the position and speed of the pollen tube growth, which is convenient for accurately observing the various stages and details of pollen germination.

[0013] 2. By optimizing the conditions of fixation, softening, staining and other steps, the success rate and stability of staining are improved, and the probability of experimental failure caused by pollen block shedding is reduced.

[0014] 3. This method is relatively simple to operate, does not require complex instruments and equipment, and does not require cumbersome operating procedures. It is suitable for general laboratories to carry out research related to the reproductive biology of Dendrobium orchids, and will help promote the progress of Dendrobium orchid breeding and reproductive mechanism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Display picture of Dendrobium stigma.

[0016] Figure 2 : Photographs of stigma pretreatment before softening.

[0017] Figure 3 : In Example 1, the stigma of Dendrobium chrysotoxum was subjected to fluorescent staining effect diagram.

[0018] Figure 4 : The stigma of Dendrobium officinale was subjected to fluorescent staining effect diagram in Example 1.

[0019] Figure 5 : The stigma fluorescence staining effect diagram of Dendrobium chrysotoxum stained with alcohol-soluble aniline blue in Example 2.

[0020] Figure 6 : Schematic diagram of the fluorescent staining effect of the stigma of Dendrobium officinale stained with alcohol-soluble aniline blue in Example 2.

[0021] Figure 7 : The alcohol-soluble aniline blue in Example 2 shows the fluorescent staining of Dendrobium chrysotoxum stigma.

[0022] Figure 8 : The fluorescence staining of Dendrobium chrysotoxum stigma by water-soluble aniline blue in Example 2.

[0023] Fig. 9 : The alcohol-soluble aniline blue in Example 2 shows the fluorescent staining of Dendrobium officinale stigma.

[0024] Fig.10 : The fluorescence staining of Dendrobium officinale stigma by water-soluble aniline blue in Example 2.

[0025] Fig.11 : The effect diagram of not cutting along the midline of the stigma before dyeing of the stigma of Dendrobium chrysotoxum in Example 3.

[0026] Fig.12 : The effect diagram of the stigma of Dendrobium officinale in Example 3 before dyeing without cutting along the midline of the stigma.

[0027] Fig.13 : In Example 3, the stigma of Dendrobium chrysotoxum was not cut, and the effect diagram of fluorescent staining of Dendrobium stigma was shown.

[0028] Fig.14: The stigma of Dendrobium chrysotoxum in Example 3 is cut to show the effect of fluorescent staining on the stigma of Dendrobium orchid.

[0029] Fig.15 : The stigma of Dendrobium officinale was not cut in Example 3, which showed the effect of fluorescent staining on the stigma of Dendrobium officinale.

[0030] Fig.16 : The stigma of Dendrobium officinale was cut in Example 3 to show the effect of fluorescent staining on the stigma of Dendrobium officinale.

[0031] Fig.17 : Schematic diagram of the stigma dyeing effect of Dendrobium chrysotoxum when the NaOH concentration is too high during softening in Example 4.

[0032] Fig.18 : Schematic diagram of the stigma dyeing effect of Dendrobium officinale when the NaOH concentration is too high during softening in Example 4.

[0033] Fig.19 : 8 mol / L NaOH concentration in Example 4 has an effect on the stigma staining of Dendrobium chrysotoxum.

[0034] Fig. 20 : 4 mol / L NaOH concentration in Example 4 has an effect on the stigma staining of Dendrobium chrysotoxum.

[0035] Fig.21 : Graph showing the effect of 8 mol / L NaOH concentration on the stigma staining of Dendrobium officinale in Example 4.

[0036] Fig. 22 : 4 mol / L NaOH concentration in Example 4 has an effect on the stigma staining of Dendrobium officinale.

[0037] Fig.23 : Fluorescence staining of the cut Dendrobium chrysotoxum stigma before fixation in Example 5.

[0038] Fig.24 : Fluorescence staining of the cut Dendrobium chrysotoxum stigma after fixation and before softening in Example 5.

[0039] Fig.25 : Fluorescent staining of the cut Dendrobium chrysotoxum stigma after softening in Example 5.

[0040] Fig.26 : Fluorescence staining of the cut Dendrobium officinale stigma before fixation in Example 5.

[0041] Fig. 27 : Fluorescence staining of the cut Dendrobium officinale stigma after fixation and before softening in Example 5.

[0042] Fig.28 : Example 5 shows the fluorescent staining of the cut Dendrobium officinale stigma after softening. DETAILED DESCRIPTION

[0043] The stigma of Dendrobium plays an important role in pollination and reproduction. The stigmas of Dendrobium chrysotoxum and Dendrobium officinale are representative among Dendrobium orchids. The stigma morphology and structure of Dendrobium chrysotoxum and Dendrobium officinale are typical stigma morphology of Dendrobium orchids. The stigma of Dendrobium orchids is generally located at the top of the column, while the stigmas of Dendrobium chrysotoxum and Dendrobium officinale show the common characteristics of the genus in terms of color and texture, and can also represent other species of Dendrobium orchids with different stigmas. Dendrobium chrysotoxum has a relatively large stigma, with a diameter of up to 5mm. It is a commonly used breeding parent for ornamental Dendrobium orchid breeding, with excellent ornamental and application value, and the stigma size of most native Dendrobium species used for hybrid breeding is within this range; Dendrobium officinale is a famous medicinal plant in my country. Currently, it is mostly used as a parent to select offspring with medicinal value. Dendrobium officinale has small flowers, so the stigma is also small, with a diameter of about 3mm. Other Dendrobiums with this stigma size include Dendrobium slender-stemmed and Dendrobium tiangong, which have materials with high ornamental breeding value.

[0044] Therefore, the stigma of Dendrobium chrysotoxum and Dendrobium officinale were selected as the research objects in the embodiment of the present invention, which is representative and avoids the error caused by the difference in the size of the stigma of Dendrobium, so that most species of Dendrobium can be operated. The present invention will be further described in detail below in conjunction with specific embodiments.

[0045] Example 1: Step 1: Sample collection and fixation, specifically: Select healthy Dendrobium orchid plants, pollinate them at the best pollination period, collect flowers with stigmas 48 hours after pollination, trim them, and perform pretreatment: remove redundant parts such as sepals to keep only the stigmas, and trim the two sides of the stigmas into a horizontal shape, fix them in precooled fixative, and then dehydrate them with gradient alcohol; The fixed time is 24 hours; The fixative used is FAA fixative, which is: 89 ml of 80% alcohol, 6 ml of glacial acetic acid, and 5 ml of formalin; The gradient alcohol uses alcohol with concentrations of 30%, 50%, and 70%. When performing dehydration treatment, each concentration of alcohol is treated for 10 minutes respectively; When the Dendrobium orchid is Dendrobium chrysotoxum, the preferred stigma diameter is 5-6 mm; when the Dendrobium orchid is Dendrobium officinale, the preferred stigma diameter is 1-3 mm.

[0046] Step 2: Cutting, specifically: The dehydrated stigma was cut open along the midline with a scalpel.

[0047] Step 3: Softening and cutting, specifically: The cut specimens are placed in a softener to soften the tissue; The softening agent is 4 mol / L NaOH solution, and the softening is carried out at 40 degrees Celsius for 2 hours to soften the sample tissue, which is convenient for subsequent staining operations and observations. Different materials have different softening times, and special attention should be paid to the shedding of pollen blocks.

[0048] Step 4: Fluorescence staining, specifically: The transparent sample is transferred into a staining solution containing a fluorescent dye and stained in the dark; Before staining, the softened samples were washed three times with sterile water and treated in 0.1N phosphate buffer for 10 min; The staining time is 24 hours; The fluorescent dye is water-soluble aniline blue, and the concentration of the dye solution is 0.1%.

[0049] Step 5: Preparation and observation, specifically: After staining, the sample was taken out, fixed on a glass slide, covered with a coverslip, and the germination of the stigma pollen of Dendrobium orchid was observed under a fluorescence microscope, including the growth direction and length of the pollen tube and the germination state of the pollen grains, and the images were collected and analyzed; When observing, select the excitation light wavelength of 350nm and the emission light wavelength of 450nm for observation; During fixation, the specimens are mounted on slides with glycerol to prepare non-permanent sections, or with neutral gum to prepare permanent sections.

[0050] In this embodiment, the experiment was conducted on Dendrobium chrysotoxum with a stigma diameter of about 4 mm. The results are shown in the attached figure. Figure 3 As shown in the figure. The Dendrobium officinale with a stigma diameter of about 2 mm was collected for the experiment, and the results are shown in the attached figure. Figure 4 shown.

[0051] The results of both experiments showed that the germination of Dendrobium stigma pollen was clearly discernible, and the pollen tubes exhibited bright blue fluorescence, which enabled the pollen tube length to be accurately measured and the germination status to be observed.

[0052] Example 2: The operating steps of Example 1 were repeated, except that the water-soluble fluorescent dye aniline blue was replaced by alcohol-soluble aniline blue, and other conditions remained unchanged.

[0053] In this embodiment, the experiment was conducted on Dendrobium chrysotoxum with a stigma diameter of about 4 mm. The results are shown in the attached figure. Figure 5 As shown; Dendrobium officinale with a stigma diameter of about 2 mm was collected for experiment, and the results are shown in the attached Figure 6As shown; the basic conditions of pollen germination were not observed in either of them.

[0054] Compared with Example 1, the dyeing effect is not good. Figure 7 , Attachment Figure 8 , Attachment Fig. 9 , Attachment Fig.10 as shown in .

[0055] Figure 7 This is the staining of the stigma of Dendrobium chrysotoxum with alcohol-soluble aniline blue. Figure 8 This is the staining of the stigma of Dendrobium chrysotoxum with water-soluble aniline blue. Figure 7 and Figure 8 P stands for pollinia, PTB stands for pollen bundle, and ST stands for stigma.

[0056] Fig. 9 This is the staining of the stigma of Dendrobium officinale with alcohol-soluble aniline blue. Fig.10 This is the staining of Dendrobium officinale stigma with water-soluble aniline blue. Fig. 9 and Fig.10 P stands for pollen mass and PTB stands for pollen bundle.

[0057] According to the dyeing conditions in the above-mentioned figures, it can be known that alcohol-soluble aniline blue is not suitable for dyeing the stigma of Dendrobium.

[0058] Example 3: The operating steps of Example 1 were repeated, except that the column head was not cut in half before softening, and other conditions remained unchanged.

[0059] In this embodiment, the experiment was conducted on Dendrobium chrysotoxum with a stigma diameter of about 4 mm. The results are shown in the attached figure. Fig.11 As shown; Dendrobium officinale with a stigma diameter of about 2 mm was collected for experiment, and the results are shown in the attached Fig.12 shown.

[0060] It was observed that, in this embodiment, although the germination of some pollen tubes was clearly discernible, all pollen tubes could not be stained due to the large size of the stigma, and the pollen grains and pollen tubes would be displaced during the pressing process, making it impossible to accurately observe the germination state.

[0061] Compared with Example 1, the effect of splitting the column head in half is poor. Fig.13 , Attachment Fig.14 , Fig.15 , Fig.16 shown.

[0062] Fig.13 The stigma of Dendrobium chrysotoxum is not cut, and the effect of fluorescent staining on the stigma of Dendrobium orchid is shown. Fig.14 Cutting the stigma of Dendrobium chrysotoxum to obtain the fluorescent staining effect of the stigma of Dendrobium chrysotoxum. Fig.13 and Fig.14P stands for pollinia, PTB stands for pollen bundle, and ST stands for stigma.

[0063] Fig.15 This is the fluorescent staining effect of the stigma of Dendrobium officinale without cutting it. Fig.16 Cutting the stigma of Dendrobium officinale to achieve the fluorescent staining effect of the stigma of Dendrobium officinale. Fig.15 and Fig.16 P stands for pollen mass and PTB stands for pollen bundle.

[0064] It can be seen from the above figures that cutting the stigma in half before softening has a significant beneficial effect on dyeing.

[0065] Example 4: The operating steps of Example 1 were repeated, except that the NaOH concentration during softening was 8 mol / L, and other conditions remained unchanged.

[0066] In this embodiment, the experiment was conducted on Dendrobium chrysotoxum with a stigma diameter of about 4 mm. The results are shown in the attached figure. Fig.17 As shown; Dendrobium officinale with a stigma diameter of about 2 mm was collected for experiment, and the results are shown in the attached Fig.18 shown.

[0067] It was observed that in this embodiment, the pollen blocks were very easy to fall off during the softening process, and the pollen blocks that did not fall off were also very easy to move during tableting (such as the attached Fig.17 , Attachment Fig.18 As shown in the figure), although the pollen tube is clearly visible, it is not possible to observe the position of the pollen tube growing to the ovary at that time, and the germination state cannot be accurately observed, which is poorer than that in Example 1.

[0068] The dyeing conditions after softening with different NaOH concentrations are shown in the attached figure. Fig.19 , Attachment Fig. 20 , Attachment Fig.21 , Attachment Fig. 22 shown.

[0069] Fig.19 The staining of the stigma of Dendrobium chrysotoxum after softening with 8 mol / L NaOH. Fig. 20 The results of 4 mol / L NaOH softening treatment on the stigma of Dendrobium chrysotoxum. Fig.19 and Fig. 20 P stands for pollinia, PTB stands for pollen bundle, and ST stands for stigma.

[0070] Fig.21 The staining of Dendrobium officinale stigma after softening with 8 mol / L NaOH. Fig. 22 The staining of Dendrobium officinale stigma after softening with 4 mol / L NaOH. Fig.21 and Fig. 22P stands for pollinia, PTB stands for pollen bundle, and ST stands for stigma.

[0071] It can be seen from the above figures that the concentration of NaOH will affect the dyeing effect.

[0072] Example 5: Based on the operating method of Example 1, Dendrobium chrysotoxum and Dendrobium officinale were selected, and three stages were selected to cut the stigma: before fixation, after fixation and before softening, and after softening.

[0073] The dyeing conditions of two different kinds of Dendrobium are shown in the attached figure. Fig.23 -Attached Fig.28 As shown, P is a pollen mass, PTB is a pollen bundle, and ST is a stigma of Dendrobium.

[0074] When the stigma of Dendrobium chrysotoxum was used as the material, the stigma was cut before fixation, and the results of fluorescent staining showed that the ovary could be observed, but the pollen blocks fell off, and only a small amount of pollen and pollen tubes were observed on the stigma; when the stigma was cut after fixation but before softening, it was found that the growth status of the pollen blocks and pollen tube bundles could be clearly observed; when the stigma was cut after softening, the pollen tubes could be observed, but they were dislocated and disorderly, and the exact location of the pollen tube growth could not be found.

[0075] Dendrobium officinale was used as the material for treatment, and the results were found to be similar to those of the treatment of the stigma of Dendrobium chrysotoxum.

[0076] When the stigma is cut before fixation, the pollen blocks fall off and no pollen tubes are seen; when the stigma is cut after fixation but before softening, the growth status of the pollen blocks and pollen tube bundles can be clearly observed; when the stigma is cut after softening, the pollen blocks on the stigma fall off and no pollen tubes are seen, and because the stigma is softened and then cut, the stigma ovary is deformed in the pressed film.

[0077] As can be seen from the above figures, the order of cutting, softening and dyeing has a huge impact on the effect.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescent staining method for germination of Dendrobium stigma pollen, characterized in that: The steps include: Step 1: Sample collection and fixation, specifically: Collect the Dendrobium orchid flowers with stigma, remove the extra parts such as sepals and keep only the stigma, trim the two sides of the stigma into a horizontal shape, put it into a pre-cooled fixative, and then dehydrate it with graded alcohol; Step 2: Cutting, specifically: The dehydrated stigma was cut open along the midline of the stigma with a scalpel; Step 3: Softening and cutting, specifically: The cut specimens are placed in a softener to soften the tissue; Step 4: Fluorescence staining, specifically: The transparent sample is transferred into a staining solution containing a fluorescent dye and stained in the dark; Step 5: Preparation and observation, specifically: After staining, the samples were taken out, fixed on a glass slide, covered with a coverslip, and the germination of the stigma pollen of Dendrobium orchid was observed under a fluorescence microscope, and images were collected and analyzed; The softening agent is 4 mol / L NaOH solution, the softening is carried out at 40 degrees Celsius, and the treatment time is 2-3 hours; The fluorescent dye is water-soluble aniline blue, and the concentration of the dye solution is 0.1%.

2. The fluorescent staining method for germination of Dendrobium stigma pollen according to claim 1, characterized in that: The fixative used is FAA fixative, and the components of FAA fixative are: 89 ml of 80% alcohol, 6 ml of glacial acetic acid, and 5 ml of formalin.

3. The fluorescent staining method for germination of Dendrobium stigma pollen according to claim 2, characterized in that: The fixation time using the fixative is 8 to 48 hours.

4. The fluorescent staining method for germination of Dendrobium stigma pollen according to claim 2, characterized in that: The fixation time using the fixative is 24 hours.

5. The fluorescent staining method for germination of Dendrobium stigma pollen according to claim 1, characterized in that: The gradient alcohol uses alcohol with concentrations of 30%, 50%, and 70%. When performing the dehydration treatment, each concentration of alcohol is used for 10 minutes.

6. The fluorescent staining method for germination of Dendrobium stigma pollen according to claim 1, characterized in that: When the Dendrobium orchid is Dendrobium chrysotoxum, the selected stigma diameter is 5-6mm; when the Dendrobium orchid is Dendrobium officinale, the selected stigma diameter is 1-3mm.

7. The fluorescent staining method for germination of Dendrobium stigma pollen according to claim 1, characterized in that: Before staining, the softened samples were washed three times with sterile water and placed in 0.1N phosphate buffer for 10 minutes.

8. The fluorescent staining method for germination of Dendrobium stigma pollen according to claim 1, characterized in that: The staining time is 24 hours.

9. The fluorescent staining method for Dendrobium stigma pollen germination according to claim 1, characterized in that: When observing, select the excitation light wavelength of 350nm and the emission light wavelength of 450nm for observation.

10. The method for fluorescent staining of Dendrobium stigma pollen germination according to claim 1, characterized in that: During fixation, the specimens are mounted on slides with glycerol to prepare non-permanent sections, or with neutral gum to prepare permanent sections.

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