A staining solution and fading / color separation solution for a simple whole-pollen methyl green staining method

Abstract

The present invention discloses a staining solution and a fading / color separation solution for a simple whole-pollen methyl green staining method. The staining solution is composed of methyl green, manganese chloride, manganese sulfate, ethanol, and acetic acid, and the fading / color separation solution is composed of triethanolamine, ethanol, and glycerol. Based on the selective binding of methyl green to DNA and the dual effects of triethanolamine in fading methyl green and transparentizing the pollen wall, the present invention simplifies the post-staining fading, color separation, and transparency processes into a single step. This method not only achieves clear contrast in the staining of pollen nuclei, but also provides an easily controllable staining effect with good repeatability. Furthermore, the method is simple and quick to operate, and has the advantages of safety, environmental protection, and low cost. The method can be used to observe and determine the developmental stage of pollen from cruciferous crops and other crops with similar structures and sizes. It is also expected to find applications in research fields such as plant classification, sterile line conversion, and gene expression during pollen development.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue and organ staining, and in particular to a staining solution and a fading / color separation solution for a simple whole-pollen methyl green staining method, which is mainly used for staining and observing the development or abortion process of the pollen of cruciferous crops and other crops with similar structures and sizes. Background Art

[0002] Normal pollen development in higher plants directly impacts the sustainability of sexual reproduction and fruit set. Whole-pollen staining plays an important role in research on plant systematics, pollen abortion mechanisms, sterile line conversion, pollen developmental gene expression, and pollen culture techniques. Currently, the main whole-pollen staining methods include acetic acid carmine staining, phenol fuchsin staining, iron alum hematoxylin staining, and fluorescent staining. Because the pollen wall is a specialized cell wall composed of sporopollenin and cellulose, it is thicker and more complex than typical cell walls and exhibits extremely poor permeability and light transparency. This is particularly true for pollen at the dinucleate and trinucleate stages. Traditional acetic acid carmine and phenol fuchsin staining methods lack effective fading and color separation methods, resulting in essentially identical colors and difficulty in distinguishing pollen nuclei. Therefore, these two staining methods are primarily used for staining and observing pollen during the early stages of development (from the pollen mother cell division stage to the mid-uninucleate stage), when pollen wall permeability and light transparency are high. Since these two methods do not require decolorization or color separation after pollen staining, they have the advantages of being simple and fast to operate. However, the background color of the staining seriously interferes, and the contrast between the cytoplasm and the nucleus is not clear enough, which often affects the analysis and judgment of the results. The iron alum hematoxylin staining method combined with the methyl salicylate transparency technique can stain and observe pollen throughout the pollen development period, and the cytoplasm and the nucleus have obvious contrast, but its fading / color separation time is long and difficult to control. The dehydration and transparency steps are cumbersome and time-consuming, generally taking several hours, and as long as 2-3 days. As a result, the staining effect of this method is unstable and difficult to control. Compared with the aforementioned traditional pollen staining method, the fluorescent staining method uses the characteristics of fluorescent dyes such as Hoechst33258 and DAPI that can specifically bind to DNA chains, and has a fast reaction speed. Therefore, the fluorescent staining method has the advantages of fast staining, strong specificity, and stable results. Chinese patent CN108680418B discloses a rapid fluorescent staining method for the pollen of cruciferous crops. This method uses a mixed staining solution [20-40 μg / L Hoechst 33258, 8-10 wt% sucrose in PBS mixed with 60-70% (v / v) ethanol, 10% (v / v) ethyl acetate, and 30-20% (v / v) glycerol solutions in a 1:1 volume ratio] to complete pollen staining in one step. The entire staining operation can be completed in 2-5 minutes, and the staining effect is stable. However, the fluorescent staining method requires an expensive fluorescence microscope, which is generally not available in ordinary laboratories.

[0003] Methyl green, a triphenylmethane basic dye, readily binds to highly polymerized DNA in cells, resulting in a green or blue color. Traditionally, due to its specific DNA binding, methyl green has been used in combination with pyronin (pyronine) in studies of cell proliferation and differentiation to visualize changes and distribution of DNA and RNA within cells. However, due to the permeability and light transmission of plant pollen walls, methyl green, like other conventional dyes such as fuchsin and carmine, has not been effectively decolorized or separated for pollen staining. Consequently, methyl green, a dye that selectively binds to DNA, has been unable to be used for whole-pollen staining.

[0004] Therefore, it is necessary to invent a staining solution and a fading / color separation solution for a simple whole pollen methyl green staining method to solve the above problems. Summary of the Invention

[0005] The present invention aims to provide a staining solution and a fading / color separation solution for a simple whole-pollen methyl green staining method. By utilizing the selective binding property of methyl green to DNA, using divalent manganese ions to enhance and stabilize the binding, and then utilizing the dual effects of triethanolamine in an alcohol medium to slowly fade methyl green and transparentize the pollen wall, the fading, color separation and transparency of the pollen after staining are completed in one step, achieving the effect of obvious contrast in the staining of the pollen nucleus and a clear background, thereby overcoming the above-mentioned shortcomings in the technology.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: a staining solution and a fading / color separation solution for a simple whole-pollen methyl green staining method, wherein the staining solution is composed of methyl green, manganese chloride, manganese sulfate, ethanol, and acetic acid; the staining solution is a mixture containing 0.25% (w / v) methyl green, 0.2-1.0% (w / v) manganese chloride, 0-0.5% (w / v) manganese sulfate, 30-40% (v / v) ethanol, and 20-30% (v / v) acetic acid;

[0007] The fading / color separation liquid is composed of triethanolamine, ethanol and glycerol; the volume ratio of the components in the fading / color separation liquid is set to triethanolamine: ethanol: glycerol = 50-75: 50: 0-10;

[0008] The specific steps are as follows

[0009] Step 1: Staining: Pipette the staining solution onto a glass slide, pick the open flowers or buds that need to be stained from the inflorescence, pick the anthers and place them directly into the staining solution on the glass slide, gently squeeze the anthers to release the pollen, remove the debris, smear for a while, and then let it stand at room temperature for staining;

[0010] Step 2: Fading / color separation: Add fading / color separation solution directly onto the staining solution spot, then cover with a cover glass and let it stand for fading, color separation and transparency;

[0011] Step 3: Observation and photographic recording: Take photos and record them using a MicroPublisher 5.0 RTV camera under an Olympus BX51 microscope. The objective lens magnification of the camera is 40 times, and the eyepiece magnification is 10 times.

[0012] As a preferred embodiment of the present invention, the amount of the dye solution added is set to 8-10 μL, the number of anthers picked is set to 1-2, and the removed debris includes the anther wall.

[0013] As a preferred embodiment of the present invention, the smearing time in step 1 is set to 10-20 seconds, so that the pollen and the staining solution form a circular spot with a diameter of 8-10 mm on the slide;

[0014] The static dyeing time is set to 20-30 minutes, until the dyeing liquid spot is completely dry.

[0015] As a preferred embodiment of the present invention, the amount of the fading / color separation liquid added is set to 8-10 μL.

[0016] As a preferred embodiment of the present invention, the staining step is configured according to two stages with significantly different pollen wall permeability and uses different staining solutions, wherein

[0017] When staining pollen at the binucleate stage and beyond, the staining solution is 0.25% (w / v) methyl green, 0.5-1.0% (w / v) manganese chloride, 0-0.2% (w / v) manganese sulfate, 30-40% (v / v) ethanol, and 20-30% (v / v) acetic acid.

[0018] When staining pollen at the uninucleate stage and earlier, use 0.25% (w / v) methyl green, 0.2-0.5 (w / v)% manganese chloride, 0.2-0.5% (w / v) manganese sulfate, 30-40% (v / v) ethanol, and 20-30% (v / v) acetic acid as the staining solution.

[0019] As a preferred embodiment of the present invention, the fading / color separation step is configured according to two stages with significantly different pollen wall permeability and uses different fading / color separation solutions, wherein

[0020] For pollen that has developed to the binucleate stage and beyond, the volume ratio of the fading / separation solution is set to triethanolamine: ethanol = 2-3:2;

[0021] When staining pollen at the uninucleate stage and earlier, the volume ratio of the fading / color separation solution mixture is set to triethanolamine:ethanol:glycerol=50-75:50:5-10.

[0022] In the above technical solution, the technical effects and advantages provided by the present invention are:

[0023] 1. By utilizing the dual effects of triethanolamine in fading methyl green and transparentizing pollen walls, the fading, color separation, and transparency processes after staining are simplified into one step. Compared with conventional staining methods such as acetic acid carmine staining, phenol fuchsin staining, and iron alum hematoxylin staining, not only is the contrast of nuclear staining clear, but the staining effect is also easy to control and has good reproducibility. At the same time, the operation is simple and fast, and the pollen staining observation process can usually be completed within 1 hour.

[0024] Compared with the fluorescent staining method, the present invention uses an ordinary optical microscope and low-toxic, inexpensive reagents, and the amount of pollen sample and reagents used is also small. Therefore, the staining method of the present invention is safe, environmentally friendly and low-cost.

[0025] The present invention can be used to observe and determine the developmental period of cruciferous crop pollen and other crops with similar structures and sizes. It is also expected to be applied in research fields such as plant classification, sterile line breeding, and gene expression during pollen development. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, similar results can be obtained for other crops such as cruciferae according to the technical methods provided by the present invention.

[0027] Figure 1 Mature trinuclear pollen of Chinese cabbage dyed using the method of the present invention (400x);

[0028] Figure 2 For the method of the present invention is used to dye the mature three-core pollen of Chinese cabbage (400x);

[0029] Figure 3 The radish mature trinuclear pollen (400x) was dyed using the method of the present invention;

[0030] Figure 4 This is a trinuclear pollen of broccoli at the developmental stage stained using the method of the present invention (400x);

[0031] Figure 5 This is the mature trinuclear pollen of Chinese kale dyed using the method of the present invention (400x);

[0032] Figure 6 This is the pollen of Chinese cabbage at the dinucleate stage stained using the method of the present invention (400x);

[0033] Figure 7 This is the uninucleate early pollen of Chinese kale stained using the method of the present invention (400x);

[0034] Figure 8 This is the mononuclear mid-stage pollen of Chinese cabbage stained using the method of the present invention (400x);

[0035] Figure 9 This is a sample of late mononuclear pollen from a small white flower stained using the method of the present invention (400x);

[0036] Figure 10 This is mature dinuclear pollen of pepper (400x) dyed using the method of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0038] The present invention provides a staining solution and a fading / color separation solution for a simple whole-pollen methyl green staining method. The staining solution is composed of methyl green, manganese chloride, manganese sulfate, ethanol, and acetic acid. The staining solution is a mixture containing 0.25% (w / v) methyl green, 0.2-1.0% (w / v) manganese chloride, 0-0.5% (w / v) manganese sulfate, 30-40% (v / v) ethanol, and 20-30% (v / v) acetic acid.

[0039] The fading / color separation liquid is composed of triethanolamine, ethanol and glycerol; the volume ratio of the components in the fading / color separation liquid is set to triethanolamine: ethanol: glycerol = 50-75: 50: 0-10;

[0040] The specific steps are as follows

[0041] Step 1: Staining: Pipette the staining solution onto a glass slide, pick the open flowers or buds that need to be stained from the inflorescence, pick the anthers and place them directly into the staining solution on the glass slide, gently squeeze the anthers to release the pollen, remove the debris, smear for a while, and then let it stand at room temperature for staining;

[0042] Step 2: Fading / color separation: Add fading / color separation solution directly onto the staining solution spot, then cover with a cover glass and let it stand for fading, color separation and transparency;

[0043] Step 3: Observation and photographic recording: Take photos and record them using a MicroPublisher 5.0 RTV camera under an Olympus BX51 microscope, with the objective lens magnification of the camera at 40 times and the eyepiece magnification of the camera at 10 times.

[0044] Furthermore, in the above technical solution, the amount of the dye solution added is set to 8-10 μL, the number of anthers picked is set to 1-2, and the removed debris includes the anther wall.

[0045] Furthermore, in the above technical solution, the smearing time in step 1 is set to 10-20 seconds, so that the pollen and the staining solution form a circular spot with a diameter of 8-10 mm on the slide;

[0046] The static dyeing time is set to 20-30 minutes, until the dyeing liquid spot is completely dry.

[0047] Furthermore, in the above technical solution, the amount of the fading / color separation liquid added is set to 8-10 μL.

[0048] Furthermore, in the above technical solution, the staining step is configured according to two stages with significantly different pollen wall permeability and uses different staining solutions, wherein

[0049] When staining pollen at the binucleate stage and beyond, the staining solution is 0.25% (w / v) methyl green, 0.5-1.0% (w / v) manganese chloride, 0-0.2% (w / v) manganese sulfate, 30-40% (v / v) ethanol, and 20-30% (v / v) acetic acid.

[0050] When staining pollen at the uninucleate stage and earlier, use 0.25% (w / v) methyl green, 0.2-0.5 (w / v)% manganese chloride, 0.2-0.5% (w / v) manganese sulfate, 30-40% (v / v) ethanol, and 20-30% (v / v) acetic acid as the staining solution.

[0051] Furthermore, in the above technical solution, the fading / color separation step is configured according to two stages with significantly different pollen wall permeability and uses different fading / color separation solutions, wherein

[0052] For pollen that has developed to the binucleate stage and beyond, the volume ratio of the fading / separation solution is set to triethanolamine: ethanol = 2-3:2;

[0053] When staining pollen at the uninucleate stage and earlier, the volume ratio of the fading / color separation solution mixture is set to triethanolamine:ethanol:glycerol=50-75:50:5-10.

[0054] Based on the above method, the following examples were made to observe the dyeing of pollen from different crops and at different times:

[0055] Example 1: Observation of Staining of Mature Chinese Cabbage Pollen

[0056] 1. Staining: Use a micropipette to draw 8 μL of a staining solution containing 0.25% (w / v) methyl green, 1.0% (w / v) manganese chloride, 30% (v / v) ethanol, and 30% (v / v) acetic acid onto a glass slide. Then, pick an open flower from the Chinese cabbage inflorescence, pick an anther with tweezers, and place it directly into the staining solution on the glass slide. Gently squeeze the anther to release an appropriate amount of pollen, remove debris such as broken anther walls, and smear for about 10 seconds to form a circular spot with a diameter of about 8 mm. Then, let it stand at room temperature for 22 minutes to stain.

[0057] 2. Fading / color separation: Use a micropipette to draw 8 μL of fading / color separation solution with triethanolamine:ethanol = 3:2 (volume ratio), add it directly to the center of the stained spot, immediately cover with a coverslip, and place it at room temperature for fading / color separation;

[0058] 3. Observation and Photo Recording: After 25 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a 40x objective lens magnification and a 10x eyepiece magnification.

[0059] The results are as follows Figure 1 As shown, the pollen cells have clear outlines, the vegetative nucleus and two sperm nuclei are stained blue-green, and the contrast between the cytoplasm and the nucleus is obvious.

[0060] Example 2: Observation of mature pollen staining of Chinese cabbage

[0061] 1. Staining: Use a micropipette to draw 10 μL of staining solution containing 0.25% (w / v) methyl green, 1.0% (w / v) manganese chloride, 0.1% (w / v) manganese sulfate, 40% (v / v) ethanol, and 30% (v / v) acetic acid onto a glass slide. Then, pick an open flower from the cabbage inflorescence, pick one anther with tweezers and place it directly into the staining solution on the glass slide. Gently squeeze the anther to release an appropriate amount of pollen, remove debris such as broken anther walls, and smear for about 15 seconds to form a circular spot with a diameter of about 10 mm. Then, let it stand at room temperature for 25 minutes to stain.

[0062] 2. Fading / color separation: Use a micropipette to draw 10 μL of fading / color separation solution with a volume ratio of triethanolamine to ethanol = 1:1, and drop it directly onto the center of the stained spot. Immediately cover with a coverslip and place at room temperature for fading / color separation.

[0063] 3. Observation and Photo Recording: After 30 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a 40x objective lens magnification and a 10x eyepiece magnification.

[0064] The results are as follows Figure 2As shown, the pollen cells have clear outlines, the pollen vegetative nucleus and two sperm nuclei are stained blue-green, and the contrast between the cytoplasm and the nucleus is obvious.

[0065] Example 3: Observation of dyeing of mature radish pollen

[0066] 1. Staining: Use a micropipette to draw 10 μL of staining solution containing 0.25% (w / v) methyl green, 0.8% (w / v) manganese chloride, 0.2% (w / v) manganese sulfate, 40% (v / v) ethanol, and 20% (v / v) acetic acid and drop it on a glass slide. Then pick an open flower from the radish inflorescence, use tweezers to pick one anther and place it directly into the staining solution on the glass slide. Gently squeeze the anther to release an appropriate amount of pollen, remove broken anther walls and other debris, and smear for about 20 seconds to form a circular spot with a diameter of about 10 mm. Then let it stand at room temperature for staining for 28 minutes.

[0067] Fading / color separation: Use a micropipette to draw 10 μL of fading / color separation solution with a volume ratio of triethanolamine:ethanol = 1:1, and add it directly to the center of the stained spot. Immediately cover with a coverslip and place at room temperature for fading / color separation.

[0068] Observation and photography: After 35 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a magnification of 40x for the objective lens and 10x for the eyepiece.

[0069] The results are as follows Figure 3 As shown, the pollen cells have clear outlines, the pollen vegetative nucleus and two sperm nuclei are stained blue-green, and the contrast between the cytoplasm and the nucleus is obvious.

[0070] Example 4: Observation of broccoli mature pollen staining

[0071] 1. Staining: Use a micropipette to draw 10 μL of staining solution containing 0.25% (w / v) methyl green, 0.5% (w / v) manganese chloride, 0.1% (w / v) manganese sulfate, 40% (v / v) ethanol, and 30% (v / v) acetic acid and drop it on a glass slide. Then pick an open flower from the broccoli inflorescence, use tweezers to pick one anther and place it directly into the staining solution on the glass slide. Gently squeeze the anther to release an appropriate amount of pollen, remove broken anther walls and other debris, and smear for about 15 seconds to form a circular spot with a diameter of about 9 mm. Then let it stand at room temperature for staining for 30 minutes.

[0072] Fading / color separation: Use a micropipette to draw 10 μL of fading / color separation solution with triethanolamine:ethanol = 1:1 (volume ratio), add it directly to the center of the stained spot, immediately cover with a coverslip, and place it at room temperature for fading / color separation.

[0073] Observation and photographic recording: After 30 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a 40x objective lens magnification and a 10x eyepiece magnification.

[0074] The results are as follows Figure 4 As shown, the pollen cells have clear outlines, the pollen vegetative nucleus and two sperm nuclei are stained blue-green, and the contrast between the cytoplasm and the nucleus is obvious.

[0075] Example 5: Staining and Observation of Mature Chinese Kale Pollen

[0076] 1. Staining: Use a micropipette to draw 10 μL of staining solution containing 0.25% (w / v) methyl green, 0.8% (w / v) manganese chloride, 0.2% (w / v) manganese sulfate, 40% (v / v) ethanol, and 20% (v / v) acetic acid and drop it on a glass slide. Then pick 8 mm long buds from the kale inflorescence, use tweezers to pick one anther and place it directly into the staining solution on the glass slide. Gently squeeze the anther to release an appropriate amount of pollen, remove broken anther walls and other debris, and smear for about 10 seconds to form a circular spot with a diameter of about 10 mm. Then let it stand at room temperature for staining for 27 minutes.

[0077] Fading / color separation: Use a micropipette to draw 10 μL of fading / color separation solution with triethanolamine:ethanol = 1:1 (volume ratio), add it directly to the center of the stained spot, immediately cover with a coverslip, and place it at room temperature for fading / color separation.

[0078] Observation and photographic recording: After 30 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a 40x objective lens magnification and a 10x eyepiece magnification.

[0079] The results are as follows Figure 5 As shown, the pollen cells have clear outlines, the pollen vegetative nucleus and two sperm nuclei are stained blue-green, and the contrast between the cytoplasm and the nucleus is obvious.

[0080] Example 6: Pollen staining observation at developmental stages of Chinese kale

[0081] 1. Staining: Use a micropipette to draw 10 μL of staining solution containing 0.25% (w / v) methyl green, 0.3% (w / v) manganese chloride, 0.5% (w / v) manganese sulfate, 40% (v / v) ethanol, and 30% (v / v) acetic acid and drop it on a glass slide. Then pick buds with a length of 2 mm from the kale inflorescence, pick two anthers with tweezers and place them directly into the staining solution on the glass slide. Gently squeeze the anthers to release an appropriate amount of pollen, remove broken anther walls and other debris, smear for about 10 seconds to form a circular spot with a diameter of about 10 mm, and then let it stand at room temperature for staining for 25 minutes.

[0082] Fading / color separation: Use a micropipette to draw 10 μL of fading / color separation solution with triethanolamine:ethanol:glycerol = 75:50:5 (volume ratio), add it directly to the center of the stained spot, cover it immediately with a coverslip, and place it at room temperature for fading / color separation.

[0083] Observation and photographic recording: After 10 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a magnification of 40x for the objective lens and 10x for the eyepiece.

[0084] The results are as follows Figure 6 As shown, the pollen cells have clear outlines, the pollen development is in the early uninucleate stage, the nuclear staining is blue-green, and the nuclear-cytoplasmic contrast is obvious.

[0085] Example 7: Pollen staining observation at the developmental stage of Chinese cabbage

[0086] 1. Staining: Use a micropipette to draw 10 μL of staining solution containing 0.25% (w / v) methyl green, 0.2% (w / v) manganese chloride, 0.5% (w / v) manganese sulfate, 40% (v / v) ethanol, and 30% (v / v) acetic acid and drop it on a glass slide. Then pick a bud with a length of 1.8 mm from the Chinese cabbage inflorescence. Use tweezers to pick two anthers and place them directly into the staining solution on the glass slide. Gently squeeze the anthers to release an appropriate amount of pollen, remove broken anther walls and other debris, and smear for about 15 seconds to form a circular spot with a diameter of about 8 mm. Then let it stand at room temperature for staining for 29 minutes.

[0087] Fading / color separation: Use a micropipette to draw 10 μL of fading / color separation solution with triethanolamine: ethanol: glycerol = 50:50:5 (volume ratio), add it directly to the center of the stained spot, cover it immediately with a coverslip, and place it at room temperature for fading / color separation.

[0088] Observation and photography: After 35 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a magnification of 40x for the objective lens and 10x for the eyepiece.

[0089] The results are as follows Figure 7 As shown, the pollen cells have clear outlines, the pollen development is in the middle uninucleate stage, the nuclear staining is blue-green, and the nuclear-cytoplasmic contrast is obvious.

[0090] Example 8: Pollen staining observation at the developmental stages of Chinese cabbage

[0091] 1. Staining: Use a micropipette to draw 8 μL of staining solution containing 0.25% (w / v) methyl green, 0.3% (w / v) manganese chloride, 0.5% (w / v) manganese sulfate, 40% (v / v) ethanol, and 20% (v / v) acetic acid and drop it on a glass slide. Then pick flower buds with a diameter of 2.5 mm from the inflorescence of Chinese cabbage, pick two anthers with tweezers and place them directly into the staining solution on the glass slide. Gently squeeze the anthers to release an appropriate amount of pollen, remove broken anther walls and other debris, and apply it for about 10 seconds to mix the pollen and staining solution evenly. The diameter of the staining solution spot is about 10 mm, and then place it at room temperature for staining for 29 minutes.

[0092] Fading / color separation: Use a micropipette to draw 10 μL of fading / color separation solution with triethanolamine:ethanol:glycerol = 75:50:10 (volume ratio), add it directly to the center of the stained spot, immediately cover with a coverslip, and place it at room temperature for fading / color separation.

[0093] Observation and photography: After 30 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a magnification of 40x for the objective lens and 10x for the eyepiece.

[0094] The results are as follows Figure 8 As shown, the pollen cells have clear outlines, and the pollen development is in the mononuclear marginal stage. The staining is blue-green, and the contrast between the cytoplasm and the nucleus is obvious.

[0095] Example 9: Pollen staining observation at the developmental stage of Chinese cabbage

[0096] 1. Staining: Use a micropipette to draw 9 μL of staining solution containing 0.25% (w / v) methyl green, 0.5% (w / v) manganese chloride, 0.2% (w / v) manganese sulfate, 40% (v / v) ethanol, and 30% (v / v) acetic acid and drop it on a glass slide. Then pick buds with a length of 3.5 mm from the Chinese cabbage inflorescence. Use tweezers to pick two anthers and place them directly into the staining solution on the glass slide. Gently squeeze the anthers to release an appropriate amount of pollen, remove broken anther walls and other debris, and smear for about 20 seconds to form a circular spot with a diameter of about 9 mm. Then let it stand at room temperature for staining for 27 minutes.

[0097] Fading / color separation: Use a micropipette to draw 9 μL of fading / color separation solution with a volume ratio of triethanolamine to ethanol of 3:2, and directly add it to the center of the stained spot. Immediately cover with a coverslip and place it at room temperature for fading / color separation.

[0098] Observation and photography: After 35 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a magnification of 40x for the objective lens and 10x for the eyepiece.

[0099] The results are as follows Figure 9 As shown, the pollen cells have clear outlines, the pollen development is in the binucleate stage, the pollen vegetative nucleus and reproductive nucleus are stained blue-green, and the contrast between the cytoplasm and the nucleus is obvious.

[0100] Example 10: Staining observation of mature pepper pollen

[0101] 1. Staining: Use a micropipette to draw 10 μL of staining solution containing 0.25% (w / v) methyl green, 0.7% (w / v) manganese chloride, 0.05% (w / v) manganese sulfate, 40% (v / v) ethanol, and 20% (v / v) acetic acid and drop it on a glass slide. Then use tweezers to pick an anther from a newly opened pepper flower and place it directly into the staining solution on the slide. Gently squeeze the anther to release an appropriate amount of pollen, remove broken anther walls and other debris, and apply it for about 15 seconds to form a circular spot with a diameter of about 10 mm. Let it stand at room temperature for staining for 28 minutes.

[0102] Fading / color separation: Use a micropipette to draw 10 μL of fading / color separation solution with triethanolamine:ethanol = 3:2 (volume ratio), directly add it to the center of the stained spot, immediately cover with a coverslip, and place it at room temperature for fading / color separation.

[0103] Observation and photographic recording: After 25 minutes, the cells were placed under a microscope (Olympus BX51) and photographed using a MicroPublisher5.0 RTV camera with a magnification of 40x for the objective lens and 10x for the eyepiece.

[0104] The results are as follows Figure 10 As shown, pepper pollen cells have clear outlines, the pollen is mature, and it is dinucleate pollen. The vegetative nucleus and reproductive nucleus of the pollen are stained blue-green, and the contrast between the cytoplasm and the nucleus is obvious.

[0105] Through the observation and verification of Examples 1-10, this method not only has clear contrast in nuclear staining, but also has easy-to-control staining effects and good reproducibility. It is also simple and quick to operate, and the pollen staining observation process can usually be completed within 1 hour. In addition, the amount of pollen sample and reagent used is small. This staining method has the advantages of safety, environmental protection, and low cost.

[0106] This method can be used to observe and determine the developmental period of cruciferous crop pollen and other crops with similar structure and size. It is also expected to be applied in research fields such as plant classification, sterile line breeding, and gene expression during pollen development.

[0107] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A dyeing solution and a fading / color separation solution for a simple whole pollen methyl green staining method, characterized in that: The staining solution is composed of methyl green, manganese chloride, manganese sulfate, ethanol and acetic acid; the staining solution is a mixture containing 0.25% (w / v) methyl green, 0.2-1.0% (w / v) manganese chloride, 0-0.5% (w / v) manganese sulfate, 30-40% (v / v) ethanol and 20-30% (v / v) acetic acid; The fading / color separation liquid is composed of triethanolamine, ethanol and glycerol; the volume ratio of the components in the fading / color separation liquid is set to triethanolamine: ethanol: glycerol = 50-75: 50: 0-10; The specific steps are as follows: Step 1: Staining: Pipette the staining solution onto a glass slide, pick the open flowers or buds that need to be stained from the inflorescence, pick the anthers and place them directly into the staining solution on the glass slide, gently squeeze the anthers to release the pollen, remove the debris, smear for a while, and then let it stand at room temperature for staining; Step 2: Fading / color separation: Add fading / color separation solution directly onto the staining solution spot, then cover with a cover glass and let it stand for fading, color separation and transparency; Step 3: Observation and photographic recording: Take photos and record them using a MicroPublisher 5.0 RTV camera under an Olympus BX51 microscope, with the objective lens magnification of the camera at 40 times and the eyepiece magnification of the camera at 10 times.

2. The dyeing solution and fading / color separation solution for the simple whole pollen methyl green staining method according to claim 1, characterized in that: The amount of the dye solution added was set to 8-10 μL, the number of anthers picked was set to 1-2, and the removed debris included the anther wall.

3. The dyeing solution and fading / color separation solution for the simple whole pollen methyl green staining method according to claim 1, characterized in that: In step 1, the smearing time is set to 10-20 seconds, so that the pollen and the staining solution form a circular spot with a diameter of 8-10 mm on the slide; The static dyeing time is set to 20-30 minutes, until the dyeing liquid spot is completely dry.

4. The dyeing solution and fading / color separation solution for the simple whole pollen methyl green staining method according to claim 1, characterized in that: The amount of the decolorizing / color separation solution added is set to 8-10 μL.

5. The dyeing solution and fading / color separation solution for the simple whole pollen methyl green staining method according to claim 1, characterized in that: Before the staining step, the pollen is observed to be in the development stage of binucleate stage or later or in the development stage of uninucleate stage or earlier, and different staining solutions are prepared and used according to different stages, wherein When staining pollen at the binucleate stage and beyond, the staining solution is 0.25% (w / v) methyl green, 0.5-1.0% (w / v) manganese chloride, 0-0.2% (w / v) manganese sulfate, 30-40% (v / v) ethanol, and 20-30% (v / v) acetic acid. When staining pollen at the uninucleate stage and earlier, use 0.25% (w / v) methyl green, 0.2-0.5 (w / v)% manganese chloride, 0.2-0.5% (w / v) manganese sulfate, 30-40% (v / v) ethanol, and 20-30% (v / v) acetic acid as the staining solution.

6. The dyeing solution and fading / color separation solution for the simple whole pollen methyl green staining method according to claim 1, characterized in that: Before the fading / color separation step, the pollen is observed to be in the development stage of binucleate stage or later or in the development stage of uninucleate stage or earlier, and different fading / color separation solutions are prepared and used according to different stages, wherein For pollen that has developed to the binucleate stage and beyond, the volume ratio of the fading / separation solution is set to triethanolamine: ethanol = 2-3:2; When staining pollen at the uninucleate stage and earlier, the volume ratio of the fading / color separation solution mixture is set to triethanolamine:ethanol:glycerol=50-75:50:5-10.

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