Fluorescent staining method for pollen germination of dendrobium nobile column

Abstract

The present application relates to the field of plant biotechnology, and particularly relates to a fluorescence staining method for pollen germination of Dendrobium nobile Lindl, which comprises five steps: collecting Dendrobium nobile Lindl flowers with a stigma, removing extra parts such as sepals and only keeping the stigma, trimming the two sides of the stigma into a horizontal shape, placing it into a pre-cooled fixing solution for fixation, and then dehydrating it with gradient alcohol; cutting the dehydrated stigma along the middle line of the stigma with a scalpel; placing the cut sample into a softener for treatment to soften the tissue; transferring the transparent sample into a staining solution containing fluorescent dye for staining in dark conditions; after the completion of staining, taking out the sample, fixing it on a glass slide, covering it with a cover glass, and observing the pollen germination of the Dendrobium nobile Lindl stigma under a fluorescence microscope. The method can clearly and accurately observe the pollen germination of the Dendrobium nobile Lindl stigma, and provides strong technical support for the related research of Dendrobium nobile Lindl and other plant materials with large stigmas.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology, in particular to a fluorescence staining method for pollen germination of Dendrobium orchid. BACKGROUND

[0002] Dendrobium is an orchid plant of Dendrobium genus, which has high ornamental and medicinal value. At present, the mainstream breeding method of Dendrobium is still hybridization breeding, and the key of hybrid pollination is to determine whether the pollination parents are compatible. The pollen germination on the stigma is the first step of pollination and fertilization, which is the key to determine whether the parents are compatible and whether there is a pre-fertilization barrier, and can provide ideas and directions for improving and enhancing parental compatibility, and provide basic support for plant breeding. In the current breeding research and reproductive biology research of Dendrobium, the determination of stigma receptivity by benzidine-hydrogen peroxide method, and the seed setting rate and seed vigor after pollination are mainly focused on. For example, the patent number CN201610221260.0, the invention name is the Dendrobium officinale seed production method based on flower organ development and the corresponding obtaining method, which uses benzidine-hydrogen peroxide method to determine the receptivity of small flower stigma on different flowering days during the whole flowering period. This method has certain effect, but also has some limitations. Whether there is other effective method is considered by people in this technical field.

[0003] The fine structure of pollen tube and some dynamic changes in the germination process may not be clearly displayed by ordinary optical microscopy. Fluorescence microscopy can better determine the stigma receptivity and pollen tube growth condition, and the results are extremely intuitive and accurate. For example, the patent number CN202210498650.8, the invention name is a rapid screening method for pollination male plants in Actinidia deliciosa orchard and its application, which proposes a rapid screening technology for pollination male plants based on callose fluorescence staining, which is beneficial to the scientific and reasonable configuration of Actinidia deliciosa pollination trees. However, the specific method and steps of staining are not disclosed in the present application. Combined with the research of the present applicant and the present technical field, the traditional fluorescence staining of stigma of flowers mainly adopts direct staining and slice observation, such as the Actinidia deliciosa flower disclosed in the above application. These plant materials mainly have the following characteristics: 1. Small stigma (for example, the stigma diameter of Brassicaceae plant material is about 1mm) staining agent is easy to penetrate, and direct slice processing can observe clear pollen tubes. 2. The pollen is powdery pollen grain, which is not easy to form a group after hydration, and the pollen tube becomes a single plane independently, and the germination effect is easy to observe. 3. The pollination position of the stigma is the tip of the stigma, which does not need to be operated on the stigma and can clearly observe the pollen tube by direct slice.

[0004] According to the applicant's search, there is no patent found by using fluorescence staining method to observe the pollen germination of Dendrobium nobile Lindl. The applicant subjectively believes that this is related to the special flower organ of Dendrobium nobile (see the attached Figure 1 ): 1. The special structure of the stigma is located below the gynostegium and presents a groove shape; 2. The stigma has a large volume and is slightly lignified, and the style cell tissue is relatively thick. The diameter of the stigma of Dendrobium nobile is usually about 3-6 mm, and the gynostegium cell tissue is relatively thick; 3. Pollen grains aggregate to form stereoscopic pollen grains. Therefore, the conventional stigma fluorescence staining treatment is not ideal for the specificity and clarity of the pollen germination of Dendrobium nobile, and the following situations may occur: 1. The pollen tube growth process is blocked by the gynostegium tissue; 2. The pollen block falls off and cannot be observed; 3. The pollen block is translocated during pressing, and the accurate growth position cannot be observed.

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

[0006] The purpose of the present application is to provide a fluorescence staining method for the pollen germination of Dendrobium nobile stigma, which can clearly and accurately observe the pollen germination of Dendrobium nobile stigma, and provide strong technical support for the related research of Dendrobium nobile and other large-scale stigma plant materials.

[0007] The fluorescence staining method for the pollen germination of Dendrobium nobile stigma of the present application comprises the following steps:

[0008] Step 1: sample collection and fixation, specifically:

[0009] According to the detection needs, collect Dendrobium nobile flowers with stigmas, pretreat: remove the sepals and other unnecessary parts and only keep the stigmas, trim the two sides of the stigmas to be horizontal, and then fix them in pre-cooled fixing solution, and then dehydrate with gradient alcohol;

[0010] The fixing time is 8 to 48 hours, preferably 24 hours;

[0011] The fixing solution can use FAA fixing solution, and the FAA fixing solution is: 80% alcohol 89ml, glacial acetic acid 6ml, formalin 5ml;

[0012] The gradient alcohol uses alcohol with concentrations of 30%, 50% and 70%, and each concentration of alcohol is treated for 10 minutes during dehydration treatment;

[0013] When the dendrobium is Dendrobium chryseum, the column head with a diameter of 5-6 mm is preferentially selected; when the dendrobium is Dendrobium candidum, the column head with a diameter of 1-3 mm is preferentially selected.

[0014] Step two: cutting, specifically:

[0015] The dehydrated column head is cut open along the midline of the column head with a surgical knife.

[0016] Step three: softening treatment and cutting, specifically:

[0017] The cut sample is placed in a softening agent to soften the tissue;

[0018] The softening agent is a 4 mol / L NaOH solution, and the softening is carried out at 40 degrees Celsius, and the treatment time is 2-3 hours, so that the sample tissue is softened, facilitating subsequent staining operation and observation; different materials have different softening times, and special attention should be paid to the shedding of pollen blocks.

[0019] Step four: fluorescent staining, specifically:

[0020] The transparent sample is transferred to a staining solution containing a fluorescent dye and stained in the dark;

[0021] Before staining, the softened sample is washed with sterile water three times and placed in a 0.1N phosphate buffer solution for 10 minutes;

[0022] The staining time is 8 to 48 hours, preferably 24 hours;

[0023] The fluorescent dye is water-soluble aniline blue, and the staining solution concentration is 0.1%.

[0024] Step five: sectioning and observation, specifically:

[0025] After staining is complete, the sample is removed and fixed on a glass slide, covered with a cover glass, and observed under a fluorescence microscope to observe the germination of the dendrobium column head pollen, including the growth direction, length of the pollen tube, and the germination state of the pollen grain, and image acquisition and analysis are performed;

[0026] When observing, the excitation light wavelength 350nm and the emission light wavelength 450nm are selected for observation;

[0027] When fixing, the sample is fixed on a glass slide to prepare a non-permanent section; or neutral gum is used for fixation to prepare a permanent section.

[0028] Beneficial effects: 1. The fluorescent staining method of the present application can specifically stain the pollinia of Dendrobium, so that the pollen tube and pollinium present clear fluorescent images under the fluorescence microscope, the sperm nucleus can also be observed in the pollen tube, and the position and speed of pollen tube growth are observed, which is convenient for accurately observing each stage and detail of pollen germination.

[0029] 2. By optimizing the conditions of fixation, softening, staining and the like, the success rate and stability of staining are improved, and the probability of experimental failure caused by pollinium shedding is reduced.

[0030] 3. The method is relatively simple to operate, does not require complex instruments and equipment and tedious operation procedures, is suitable for general laboratories to carry out Dendrobium reproductive biology related research work, and is helpful to promote the progress of Dendrobium breeding and reproductive mechanism research. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 : Dendrobium column head display diagram.

[0032] Figure 2 : Pre-treatment photo of column head before softening.

[0033] Figure 3 : Fluorescent staining effect diagram of the column head of Dendrobium devonianum in Example 1.

[0034] Figure 4 : Fluorescent staining effect diagram of the column head of Dendrobium candidum in Example 1.

[0035] Figure 5 : Fluorescent staining effect diagram of the column head of Dendrobium devonianum dyed with alcohol-soluble aniline blue in Example 2.

[0036] Figure 6 : Fluorescent staining effect diagram of the column head of Dendrobium candidum dyed with alcohol-soluble aniline blue in Example 2.

[0037] Figure 7 : Fluorescent staining situation diagram of the column head of Dendrobium devonianum dyed with alcohol-soluble aniline blue in Example 2.

[0038] Figure 8 : Fluorescent staining situation diagram of the column head of Dendrobium devonianum dyed with water-soluble aniline blue in Example 2.

[0039] Figure 9 : Fluorescent staining situation diagram of the column head of Dendrobium candidum dyed with alcohol-soluble aniline blue in Example 2.

[0040] Figure 10 : Fluorescent staining situation diagram of the column head of Dendrobium candidum dyed with water-soluble aniline blue in Example 2.

[0041] Figure 11: Effect picture of Dendrobium chrysotoxum before stigma staining without cutting along the midline of the stigma in Example 3.

[0042] Figure 12 : Effect picture of Dendrobium candidum before stigma staining without cutting along the midline of the stigma in Example 3.

[0043] Figure 13 : Effect picture of Dendrobium chrysotoxum without cutting the stigma of Dendrobium chrysotoxum for fluorescence staining of the stigma of Dendrobium in Example 3.

[0044] Figure 14 : Effect picture of Dendrobium chrysotoxum with cutting the stigma of Dendrobium chrysotoxum for fluorescence staining of the stigma of Dendrobium in Example 3.

[0045] Figure 15 : Effect picture of Dendrobium candidum without cutting the stigma of Dendrobium candidum for fluorescence staining of the stigma of Dendrobium in Example 3.

[0046] Figure 16 : Effect picture of Dendrobium candidum with cutting the stigma of Dendrobium candidum for fluorescence staining of the stigma of Dendrobium in Example 3.

[0047] Figure 17 : Effect picture of Dendrobium chrysotoxum for stigma staining when the concentration of NaOH is too high during softening in Example 4.

[0048] Figure 18 : Effect picture of Dendrobium candidum for stigma staining when the concentration of NaOH is too high during softening in Example 4.

[0049] Figure 19 : Effect picture of Dendrobium chrysotoxum for stigma staining with 8 mol / L NaOH concentration in Example 4.

[0050] Figure 20 : Effect picture of Dendrobium chrysotoxum for stigma staining with 4 mol / L NaOH concentration in Example 4.

[0051] Figure 21 : Effect picture of Dendrobium candidum for stigma staining with 8 mol / L NaOH concentration in Example 4.

[0052] Figure 22 : Effect picture of Dendrobium candidum for stigma staining with 4 mol / L NaOH concentration in Example 4.

[0053] Figure 23 : Fluorescence staining of Dendrobium chrysotoxum after cutting before fixation of the stigma in Example 5.

[0054] Figure 24 : Fluorescence staining of Dendrobium chrysotoxum after cutting before softening after fixation of the stigma in Example 5.

[0055] Figure 25: Fluorescent dyeing condition of the cutting of the Dendrobium chryseum column after softening in Example 5.

[0056] Figure 26 : Fluorescent dyeing condition of the cutting of the Dendrobium candidum column before fixing in Example 5.

[0057] Figure 27 : Fluorescent dyeing condition of the cutting of the Dendrobium candidum column after fixing and before softening in Example 5.

[0058] Figure 28 : Fluorescent dyeing condition of the cutting of the Dendrobium candidum column after softening in Example 5. DETAILED DESCRIPTION

[0059] The column of Dendrobium plays an important role in pollination and reproduction, and the columns of Dendrobium chryseum and Dendrobium candidum are representative in Dendrobium. The column of Dendrobium chryseum and Dendrobium candidum is a typical Dendrobium column in morphology. The column of Dendrobium is generally located at the top of the gynostemium, and the columns of Dendrobium chryseum and Dendrobium candidum show the common characteristics of the plants in the genus in color and texture, and can also represent the columns of other species of Dendrobium with different sizes. Dendrobium chryseum is a large column with a diameter of up to 5 mm, which is a commonly used breeding parent in the breeding of ornamental Dendrobium, and has excellent ornamental and application value. Most of the original species of Dendrobium used for hybridization breeding have a column size in this range. Dendrobium candidum is a famous medicinal plant in China, and is currently used as a parent to select and breed offspring with medicinal value. Dendrobium candidum flowers are small, so the column is also small with a diameter of about 3 mm. The columns of Dendrobium candidum and Dendrobium tenuicaule, Dendrobium tiangongense and other materials with high ornamental and breeding value also have this size.

[0060] Therefore, in the embodiments of the present application, the columns of Dendrobium chryseum and Dendrobium candidum are selected as the research object, which is representative and avoids errors caused by the difference in the size of the column of Dendrobium, so as to be operable for most species of Dendrobium. The present application will be further described in detail in combination with specific embodiments.

[0061] Example 1: Step one: sample collection and fixing, specifically:

[0062] Healthy growing Dendrobium plants are selected, pollination is performed at the best pollination period, 48 hours after pollination, the flowers with columns are collected and trimmed, pretreatment is performed: removing the sepals and other unnecessary parts and only keeping the columns, trimming the two sides of the column into a horizontal shape, and then placing them in a pre-cooled fixing solution for fixing, and then dehydrating them with gradient alcohol;

[0063] The fixing time is 24 hours;

[0064] The fixing liquid is FAA fixing liquid, and the FAA fixing liquid is 80% alcohol 89ml, glacial acetic acid 6ml and formaldehyde 5ml.

[0065] The gradient alcohol is alcohol with a concentration of 30%, 50% and 70%, and each concentration of alcohol is treated for 10 minutes during dehydration treatment.

[0066] When the dendrobium is Dendrobium chrysostachyum, the preferred column head diameter is 5-6mm; when the dendrobium is Dendrobium candidum, the preferred column head diameter is 1-3mm.

[0067] Step two: cutting, specifically:

[0068] The dehydrated column head is cut open along the midline of the column head with a scalpel.

[0069] Step three: softening treatment and cutting, specifically:

[0070] The cut sample is placed in a softener to soften the tissue;

[0071] The softener is 4mol / L NaOH solution, and the softening is carried out at 40 degrees Celsius for 2 hours to soften the sample tissue, facilitate subsequent staining operation and observation; different materials have different softening times, and special attention should be paid to the shedding of pollen blocks.

[0072] Step four: fluorescent staining, specifically:

[0073] The transparent sample is transferred to a staining solution containing fluorescent dye and stained in the dark;

[0074] The softened sample is washed with sterile water three times before staining, and is placed in 0.1N phosphate buffer for 10 minutes;

[0075] The staining time is 24 hours;

[0076] The fluorescent dye is water-soluble aniline blue, and the staining solution concentration is 0.1%.

[0077] Step five: slide preparation and observation, specifically:

[0078] After staining, the sample is taken out, fixed on a glass slide, covered with a cover glass, and observed under a fluorescence microscope to observe the germination of dendrobium column head pollen, including the growth direction and length of pollen tube and the germination state of pollen grain, and image acquisition and analysis are carried out;

[0079] During observation, the excitation wavelength is 350nm and the emission wavelength is 450nm;

[0080] Fixed, non-permanent sections are prepared by fixing the sample on a slide with glycerol; or permanent sections are prepared by fixing with neutral resin.

[0081] In this embodiment, the Dendrobium swinhoii with the column head diameter of about 4mm is collected for experiment, and the result is shown in the following Figure 3 In this embodiment, the Dendrobium candidum with the column head diameter of about 2mm is collected for experiment, and the result is shown in the following Figure 4

[0082] Both of the two experimental results show that the pollen germination of the Dendrobium column head is clear, the pollen tube presents bright blue fluorescence, and the pollen tube length can be accurately measured and the germination state can be observed.

[0083] In this embodiment, the Dendrobium swinhoii with the column head diameter of about 4mm is collected for experiment, and the result is shown in the following

[0084] In this embodiment, the Dendrobium swinhoii with the column head diameter of about 4mm is collected for experiment, and the result is shown in the following Figure 5 In this embodiment, the Dendrobium swinhoii with the column head diameter of about 4mm is collected for experiment, and the result is shown in the following Figure 6

[0085] In this embodiment, the Dendrobium swinhoii with the column head diameter of about 4mm is collected for experiment, and the result is shown in the following Figure 7 , the following Figure 8 , the following Figure 9 , the following Figure 10 .

[0086] Figure 7 The following Figure 8 is the staining condition of the Dendrobium swinhoii column head by the alcohol-soluble aniline blue, and the following Figure 7 is the staining condition of the Dendrobium swinhoii column head by the water-soluble aniline blue. Figure 8 In the above figures, P is the pollen mass, PTB is the pollen tube, and ST is the Dendrobium column head.

[0087] Figure 9 The following Figure 10 is the staining condition of the Dendrobium swinhoii column head by the alcohol-soluble aniline blue, and the following Figure 9 is the staining condition of the Dendrobium swinhoii column head by the water-soluble aniline blue. Figure 10 In the above figures, P is the pollen mass, PTB is the pollen tube, and ST is the Dendrobium column head.

[0088] According to the staining conditions in the above figures, it can be known that the alcohol-soluble aniline blue is not suitable for staining the Dendrobium column head.

[0089] In this embodiment, the Dendrobium swinhoii with the column head diameter of about 4mm is collected for experiment, and the result is shown in the following

[0090] ​​In this embodiment, the drumstick Dendrobium with the stigma diameter of about 4mm was collected for experiment, and the results are shown in the attached Figure 11 In this embodiment, the drumstick Dendrobium with the stigma diameter of about 4mm was collected for experiment, and the results are shown in the attached Figure 12 In this embodiment, the drumstick Dendrobium with the stigma diameter of about 4mm was collected for experiment, and the results are shown in the attached

[0091] It was found in observation that, in this embodiment, although part of the pollen tube germination was clear, the whole pollen tube could not be dyed due to the too large size of the stigma, and the pollen grains and pollen tubes were displaced when pressed into a sheet, so that the germination state could not be observed accurately.

[0092] Compared with the effect of cutting the stigma in half in embodiment 1, the effect was poor, as shown in the attached Figure 13 , the attached Figure 14 , Figure 15 , Figure 16 In this embodiment, the drumstick Dendrobium with the stigma diameter of about 4mm was collected for experiment, and the results are shown in the attached

[0093] Figure 13 is a fluorescence staining effect diagram of the stigma of Dendrobium without cutting, Figure 14 is a fluorescence staining effect diagram of the stigma of Dendrobium with cutting, Figure 13 and P in the attached Figure 14 is a pollen block, PTB is a pollen bundle, and ST is a Dendrobium stigma.

[0094] Figure 15 is a fluorescence staining effect diagram of the stigma of Dendrobium without cutting, Figure 16 is a fluorescence staining effect diagram of the stigma of Dendrobium with cutting, Figure 15 and P in the attached Figure 16 is a pollen block, and PTB is a pollen bundle.

[0095] It can be known from the above figures that cutting the stigma in half before softening has a significant beneficial effect on staining.

[0096] In this embodiment, the drumstick Dendrobium with the stigma diameter of about 4mm was collected for experiment, and the results are shown in the attached

[0097] In this embodiment, the drumstick Dendrobium with the stigma diameter of about 4mm was collected for experiment, and the results are shown in the attached Figure 17 In this embodiment, the drumstick Dendrobium with the stigma diameter of about 4mm was collected for experiment, and the results are shown in the attached Figure 18 In this embodiment, the drumstick Dendrobium with the stigma diameter of about 4mm was collected for experiment, and the results are shown in the attached

[0098] It was found in observation that, in this embodiment, the pollen blocks were extremely easy to fall off during the softening process, and the pollen blocks that did not fall off were also extremely easy to be displaced when pressed into a sheet (as shown in the attached Figure 17 , the attached Figure 18As shown in FIG. 6, although the pollen tube was clearly visible, the position of the pollen tube growing into the ovary at that time could not be observed, and the germination state could not be accurately observed, which was poorer than the effect in Example 1.

[0099] The dyeing conditions after softening treatment with different concentrations of NaOH are shown in FIGS. 8-11. Figure 19 , FIG. 9 Figure 20 , FIG. 10 Figure 21 , FIG. 11 Figure 22 .

[0100] Figure 19 FIG. 8 shows the dyeing conditions of Dendrobium nobile Lindl. stigma after 8 mol / L NaOH softening treatment. Figure 20 FIG. 9 shows the dyeing conditions of Dendrobium nobile Lindl. stigma after 4 mol / L NaOH softening treatment. Figure 19 FIG. 10 shows the dyeing conditions of Dendrobium candidum Wall. ex Lindl. stigma after 8 mol / L NaOH softening treatment. Figure 20 FIG. 11 shows the dyeing conditions of Dendrobium candidum Wall. ex Lindl. stigma after 4 mol / L NaOH softening treatment.

[0101] Figure 21 FIG. 8 shows the dyeing conditions of Dendrobium nobile Lindl. stigma after 8 mol / L NaOH softening treatment. Figure 22 FIG. 9 shows the dyeing conditions of Dendrobium nobile Lindl. stigma after 4 mol / L NaOH softening treatment. Figure 21 FIG. 10 shows the dyeing conditions of Dendrobium candidum Wall. ex Lindl. stigma after 8 mol / L NaOH softening treatment. Figure 22 FIG. 11 shows the dyeing conditions of Dendrobium candidum Wall. ex Lindl. stigma after 4 mol / L NaOH softening treatment.

[0102] It can be seen from the above figures that the concentration of NaOH affects the dyeing effect.

[0103] Example 5: Based on the operation method of Example 1, Dendrobium nobile Lindl. and Dendrobium candidum Wall. ex Lindl. were selected, and the stigma was cut at three stages: before fixation, after fixation and before softening, and after softening.

[0104] The dyeing conditions of the two different Dendrobium are shown in FIGS. 12-15. Figure 23 -FIG. 13 Figure 28 , FIG. 14 , FIG. 15

[0105] When Dendrobium nobile Lindl. stigma was used as the material, the fluorescence dyeing result showed that the ovary could be observed, but the pollen block fell off, and only a small amount of pollen and pollen tube was observed on the stigma. After cutting the stigma before fixation, it was found that the growth state of the pollen block and the pollen tube bundle could be clearly observed. After cutting after softening, the pollen tube could be observed, but the pollen tube translocation was disordered and could not be accurately determined.

[0106] When Dendrobium candidum Wall. ex Lindl. was used as the material for treatment, it was found that the treatment result was similar to that of Dendrobium nobile Lindl. stigma.

[0107] The pollen grains fall off the fixed stigma before cutting, and no pollen tubes are observed. The growth of the pollen grains and pollen tube bundles can be clearly observed by cutting the stigma before softening. The pollen grains fall off the fixed stigma after softening, and no pollen tubes are observed. The stigma is modified into a fruit after softening and cutting.

[0108] It can be seen from the above figures that the order of cutting, softening and dyeing has a great influence on the effect.

[0109] The above only describes the preferred embodiments of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for fluorescent staining of pollen germination from the stigma of Dendrobium orchids, characterized in that: Includes the following steps: Step 1: Sample collection and fixation, specifically: Collect Dendrobium orchid flowers with stigmas attached, remove sepals and other excess parts, leaving only the stigma, and trim the sides of the stigma horizontally. It was fixed in a pre-cooled fixative and then dehydrated using a gradient of alcohols. Step 2: Cutting, specifically: cut the dehydrated stigma along the midline of the stigma with a scalpel; Step 3: Softening treatment after cutting, specifically: placing the cut sample in a softening agent to soften the tissue; Step 4: Fluorescent staining, specifically: transfer the cleared sample into a staining solution containing fluorescent dye and stain in the dark; Step 5: Film preparation and observation, specifically: After staining, the sample was removed, 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 acquired and analyzed. The softening agent is a 4 mol / L NaOH solution, and the softening is carried out at 40 degrees Celsius for 2-3 hours. The fluorescent dye used is water-soluble aniline blue, and the concentration of the staining solution is 0.1%. Before staining, the softened sample was washed three times with sterile water and then placed in 0.1N phosphate buffer for 10 minutes. When the Dendrobium orchid is Dendrobium chrysanthum, the stigma diameter is 5-6 mm; when the Dendrobium orchid is Dendrobium officinale, the stigma diameter is 1-3 mm.

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

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

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

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

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

7. The fluorescent staining method for pollen germination of Dendrobium stigma according to claim 1, characterized in that: During observation, an excitation wavelength of 350 nm and an emission wavelength of 450 nm were selected.

8. The fluorescent staining method for the germination of Dendrobium orchid stigma pollen according to claim 1, characterized in that: For fixation, glycerol is used to fix the sample onto a glass slide to prepare non-permanent sections; or neutral resin is used to fix the sample to prepare permanent sections.

Images