Leguminous plant pollen germination method
By preparing liquid culture medium with a specific formula and carrying out systematic pollen germination and culture treatment, the problems of complex germination and culture process and inaccurate germination rate statistics in the prior art are solved, and simple and rapid culture processing and accurate statistical processing are achieved.
Patent Information
- Application Number
- CN202411990165.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-03-28
AI Technical Summary
During the germination and cultivation of existing legume plants, the cultivation process is complex, and the germination rate statistical process is too random and general, so simple and rapid cultivation and accurate statistical processing cannot be achieved.
Pollen germination was cultured by preparing liquid culture medium, including a specific proportion of sucrose, H3BO3, PEG-6000, sodium hydrosulfide, CaCl2·2H2O, KNO3 and nutrient solution. Specific steps include weighing raw materials, preparing culture medium, sterilization equipment, sampling treatment, tableting treatment and germination rate calculation.
It realizes simple and rapid culture processing during the pollen germination and culture process, and conducts more accurate germination rate statistics through average statistical methods. The cultivation process is simple and reliable, and the statistical process is accurate and convincing, achieving the purpose of completing pollen germination and culture processing quickly and accurately.
Smart Images
Figure CN119709588A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant cultivation, in particular to a pollen germination method for leguminous plants. Background Art
[0002] Tropical legume grass, stylosanthes, is an excellent legume grass variety that can grow in tropical areas. It is also cultivated in many countries in the world. It has great development potential and a very broad development space. It can even play a certain role in improving the soil. The grass varieties cultivated in different environments in different places must be different. The planting time of tropical legume grass depends on the natural climate of each place. Generally, it should be sown in advance in southern cities and delayed in northern areas. It is planned according to natural climate conditions. Now most places have cultivation. Forage grass is a high-quality variety in breeding. Whether in the southwest, semi-nearby areas, Xinjiang, northern plateaus, Guizhou mountainous areas, or Yunnan and Guangxi, there are a lot of plantings, or used for direct grazing and storage. Forage grass is suitable for feeding cows, sheep, stallions, ducks and other ruminants or poultry and other grazing animals. Forage grass has high nutrients, appropriate fiber levels, and a high edible rate. Herbivores can fatten quickly, absorb more easily, and have strong disease resistance.
[0003] In the existing pollen germination culture process of leguminous plants, the culture process is complicated, and the statistical process of pollen germination rate is too arbitrary and general, which cannot achieve simple and rapid culture processing in the pollen germination culture process and more accurate statistical processing through average statistical methods. It is impossible to achieve the purpose of completing the pollen germination culture process quickly and accurately, which is very unfavorable for the research on pollen germination culture process. Summary of the invention
[0004] 1. Technical issues to be resolved In view of the shortcomings of the prior art, the present invention provides a method for germinating pollen of leguminous plants, which solves the problems that the culturing process is complicated, the statistical process of pollen germination rate is too arbitrary and general, and it is impossible to perform simple and rapid culturing process in the pollen germination culturing process and perform more accurate statistical processing through average statistical method, and it is impossible to achieve the purpose of completing pollen germination culturing process quickly and accurately.
[0005] (II) Technical solution To achieve the above objectives, the present invention is implemented through the following technical scheme: a pollen germination method for leguminous plants, specifically comprising the following steps: S1. Weighing of raw materials of liquid culture medium: First, 10%-20% sucrose, 0.01%-0.06% H3BO3, 10%-20% PEG-6000, 1%-2% sodium hydrosulfide, 0.01%-0.1% CaCl2·2H2O, 0.01%-0.06% KNO3 and 57.78%-78.93% nutrient solution are measured by weighing equipment in required weight percentages; S2. Preparation of liquid culture medium: Add the raw materials weighed in step S1 into a beaker in sequence, and stir slowly and evenly for 3-5 minutes with a stirring rod to obtain a liquid culture medium; S3. Pre-culture treatment: sterilize the culture dish, filter paper and glass slide. During culture, soak the filter paper in 70% alcohol for 3-8 minutes and then dry it. Wet it with distilled water and place it on the bottom of the culture dish. S4, sampling process: take pollen grains from 3-5 florets and mix the pollen grains on a glass slide; S5. Preparation and processing: Drop an appropriate amount of the liquid culture medium prepared in step S2 onto the glass slide with pollen grains placed in step S4, mix well with a glass rod, place the glass slide on wet filter paper in a culture dish, cover the culture dish with wet filter paper, and culture in a constant temperature box with moisturizing. After culturing for 10-12 minutes, examine under a microscope, take 5 fields of view for each preparation, 3 preparations each time, and take the average value; S6. Calculation of germination rate: observe the germination of pollen, take the pollen tube length exceeding the pollen grain diameter as the germination standard, and calculate the germination rate: germination rate = (number of germinated pollen / total number of observed pollen) × 100%.
[0006] Preferably, in step S1, 15% sucrose, 0.03% H3BO3, 15% PEG-6000, 1.5% sodium hydrosulfide, 0.01% CaCl2·2H2O, 0.03% KNO3 and 68.43% nutrient solution are measured in required weight percentages.
[0007] Preferably, the required weight percentages are 10% sucrose, 0.01% H3BO3, 10% PEG-6000, 1% sodium hydrosulfide, 0.05% CaCl2·2H2O, 0.01% KNO3 and 78.93% nutrient solution.
[0008] Preferably, the required weight percentages are 20% sucrose, 0.06% H3BO3, 20% PEG-6000, 2% sodium hydrosulfide, 0.1% CaCl2·2H2O, 0.06% KNO3 and 57.78% nutrient solution.
[0009] Preferably, the culture dish, filter paper and glass slide in step S3 are sterilized by irradiating with ultraviolet sterilization equipment for 5-8 minutes.
[0010] Preferably, the culture after slice preparation in step S5 is carried out under the conditions of pH 7.0 and 30°C.
[0011] Preferably, the constant temperature box in step S5 is a biochemical incubator of model SPX-50.
[0012] Preferably, the nutrient solution in step S1 is any combination of two or more of potassium nitrate, calcium nitrate, ammonium phosphate, magnesium sulfate, ferric chloride or potassium iodide.
[0013] (III) Beneficial effects The present invention provides a method for germinating pollen of legumes. Compared with the prior art, the method has the following beneficial effects: The method for germinating pollen of legumes comprises the following steps: S1, weighing the raw materials of the liquid culture medium: first, using a weighing device to weigh the required weight percentages of 10%-20% sucrose, 0.01%-0.06% H3BO3, 10%-20% PEG-6000, 1%-2% sodium hydrosulfide, 0.01%-0.1% CaCl2·2H2O, 0.01%-0.06% KNO3 and 57.78%-78.93% nutrient solution; S2, preparation of liquid culture medium: add the raw materials weighed in step S1 into a beaker in turn, and slowly and evenly stir with a stirring rod for 3-5 minutes to obtain a liquid culture medium; S3, pre-culture treatment: sterilize the culture dish, filter paper and glass slide. During culture, soak the filter paper in 70% alcohol for 3-8 minutes and then dry it, wet it with distilled water and place it on the bottom of the culture dish; S4, sampling treatment: take pollen grains from 3-5 small flowers and mix the pollen grains on a glass slide; S5, slide preparation treatment: Drop an appropriate amount of the liquid culture medium obtained in step S2 onto the glass slide on which the pollen grains are placed in step S4, mix well with a glass rod, place the glass slide on the moistened filter paper in a culture dish, cover the culture dish with the moistened filter paper, and culture in a constant temperature box to keep the moisture. After culturing for 10-12 minutes, examine under a microscope, take 5 fields of view for each preparation, 3 preparations each time, and take the average value; S6, calculation of germination rate: observe the germination of pollen, take the pollen tube length exceeding the pollen grain diameter as the germination standard, and calculate the germination rate: germination rate = (germinated pollen number / observed pollen total number) × 100%, which can realize simple and rapid culture processing in the pollen germination culture process and more accurate statistical processing through the average statistical method, the culture process is simple and reliable, and the statistical process of pollen germination rate is accurate and convincing, which well achieves the purpose of completing the pollen germination culture processing quickly and accurately, and is very beneficial to the research on pollen germination culture processing. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 Flow chart of the germination method of the present invention. DETAILED DESCRIPTION
[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0016] See also Figure 1 The embodiment of the present invention provides four technical solutions: a pollen germination method for leguminous plants, specifically including the following embodiments: Embodiment 1: A method for germinating pollen of leguminous plants, comprising the following steps: S1. Weighing of raw materials of liquid culture medium: First, 15% sucrose, 0.03% H3BO3, 15% PEG-6000, 1.5% sodium hydrosulfide, 0.01% CaCl2·2H2O, 0.03% KNO3 and 68.43% nutrient solution of required weight percentage are measured by weighing equipment, wherein the nutrient solution is a combination of potassium nitrate, calcium nitrate, ammonium phosphate, magnesium sulfate, ferric chloride and potassium iodide; S2. Preparation of liquid culture medium: Add the raw materials weighed in step S1 into a beaker in sequence, and stir slowly and evenly for 4 minutes with a stirring rod to obtain a liquid culture medium; S3. Pre-culture treatment: sterilize the culture dish, filter paper and glass slide. During culture, soak the filter paper in 70% alcohol for 6 minutes and then dry it. Wet it with distilled water and place it on the bottom of the culture dish. The culture dish, filter paper and glass slide are sterilized by irradiating them with ultraviolet sterilizer for 6 minutes. S4, sampling process: take pollen grains from 4 florets and mix the pollen grains on a glass slide; S5, slide preparation: drop an appropriate amount of the liquid culture medium prepared in step S2 onto the glass slide with pollen grains placed in step S4, mix well with a glass rod, place the glass slide on a moist filter paper in a culture dish, cover the culture dish with the moist filter paper, and culture in a constant temperature box with moisturizing, and examine under a microscope after 11 minutes of culture, take 5 fields of view for each slide, 3 slides each time, and take the average value, the culture base after the slide preparation is cultured under the conditions of pH 7.0 and 30°C, and the constant temperature box is a biochemical culture box of model SPX-50; S6. Calculation of germination rate: observe the germination of pollen, take the pollen tube length exceeding the pollen grain diameter as the germination standard, and calculate the germination rate: germination rate = (number of germinated pollen / total number of observed pollen) × 100%.
[0017] Embodiment 2: A method for germinating pollen of leguminous plants, comprising the following steps: S1. Weighing of raw materials of liquid culture medium: First, 10% sucrose, 0.01% H3BO3, 10% PEG-6000, 1% sodium hydrosulfide, 0.05% CaCl2·2H2O, 0.01% KNO3 and 78.93% nutrient solution of required weight percentage are measured by weighing equipment, wherein the nutrient solution is a combination of potassium nitrate, calcium nitrate and ammonium phosphate; S2. Preparation of liquid culture medium: Add the raw materials weighed in step S1 into a beaker in sequence, and stir slowly and evenly with a stirring rod for 3 minutes to obtain a liquid culture medium; S3. Pre-culture treatment: sterilize the culture dish, filter paper and slide glass. During culture, soak the filter paper in 70% alcohol for 3 minutes and then dry it. Wet it with distilled water and place it on the bottom of the culture dish. The culture dish, filter paper and slide glass are sterilized by irradiating them with ultraviolet sterilizer for 5 minutes. S4, sampling process: take pollen grains from 3 florets and mix the pollen grains on a glass slide; S5, slide preparation: drop an appropriate amount of the liquid culture medium prepared in step S2 onto the glass slide with pollen grains placed in step S4, mix well with a glass rod, place the glass slide on a moist filter paper in a culture dish, cover the culture dish with the moist filter paper, and culture in a constant temperature box for moisturizing. After 10 minutes of culture, examine under a microscope. Take 5 fields of view for each slide, 3 slides each time, and take the average value. The culture base after the slide preparation is cultured at a pH value of 7.0 and 30°C. The constant temperature box is a biochemical culture box of model SPX-50; S6. Calculation of germination rate: observe the germination of pollen, take the pollen tube length exceeding the pollen grain diameter as the germination standard, and calculate the germination rate: germination rate = (number of germinated pollen / total number of observed pollen) × 100%.
[0018] Embodiment 3: A method for germinating pollen of leguminous plants, comprising the following steps: S1. Weighing of raw materials of liquid culture medium: First, 20% sucrose, 0.06% H3BO3, 20% PEG-6000, 2% sodium hydrosulfide, 0.1% CaCl2·2H2O, 0.06% KNO3 and 57.78% nutrient solution of required weight percentage are measured by weighing equipment, wherein the nutrient solution is a combination of magnesium sulfate, ferric chloride and potassium iodide; S2. Preparation of liquid culture medium: Add the raw materials weighed in step S1 into a beaker in sequence, and stir slowly and evenly with a stirring rod for 5 minutes to obtain a liquid culture medium; S3. Pre-culture treatment: sterilize the culture dish, filter paper and slide glass. During culture, soak the filter paper in 70% alcohol for 8 minutes and then dry it. Wet it with distilled water and place it on the bottom of the culture dish. The culture dish, filter paper and slide glass are sterilized by irradiating them with ultraviolet sterilizer for 8 minutes. S4, sampling process: take pollen grains from 5 florets and mix the pollen grains on a glass slide; S5, slide preparation: drop an appropriate amount of the liquid culture medium prepared in step S2 onto the glass slide with pollen grains placed in step S4, mix well with a glass rod, place the glass slide on a moist filter paper in a culture dish, cover the culture dish with the moist filter paper, and culture in a constant temperature box with moisturizing, and examine under a microscope after 12 minutes of culture, take 5 fields of view for each slide, 3 slides each time, and take the average value, the culture base after the slide preparation is cultured under the conditions of pH 7.0 and 30°C, and the constant temperature box is a biochemical culture box of model SPX-50; S6. Calculation of germination rate: observe the germination of pollen, take the pollen tube length exceeding the pollen grain diameter as the germination standard, and calculate the germination rate: germination rate = (number of germinated pollen / total number of observed pollen) × 100%.
[0019] Embodiment 4: A method for germinating pollen of leguminous plants, comprising the following steps: S1. Weighing of raw materials of liquid culture medium: First, 11% sucrose, 0.02% H3BO3, 11% PEG-6000, 1.5% sodium hydrosulfide, 0.02% CaCl2·2H2O, 0.02% KNO3 and 76.44% nutrient solution of required weight percentage are measured by weighing equipment, wherein the nutrient solution is a combination of potassium nitrate, calcium nitrate and potassium iodide; S2. Preparation of liquid culture medium: Add the raw materials weighed in step S1 into a beaker in sequence, and stir slowly and evenly for 4 minutes with a stirring rod to obtain a liquid culture medium; S3. Pre-culture treatment: sterilize the culture dish, filter paper and glass slide. During culture, soak the filter paper in 70% alcohol for 4 minutes and then dry it. Wet it with distilled water and place it on the bottom of the culture dish. The culture dish, filter paper and glass slide are sterilized by irradiating them with ultraviolet sterilizer for 6 minutes. S4, sampling process: take pollen grains from 4 florets and mix the pollen grains on a glass slide; S5, slide preparation: drop an appropriate amount of the liquid culture medium prepared in step S2 onto the glass slide with pollen grains placed in step S4, mix well with a glass rod, place the glass slide on a moist filter paper in a culture dish, cover the culture dish with the moist filter paper, and culture in a constant temperature box with moisturizing, and examine under a microscope after 11 minutes of culture, take 5 fields of view for each slide, 3 slides each time, and take the average value, the culture base after the slide preparation is cultured under the conditions of pH 7.0 and 30°C, and the constant temperature box is a biochemical culture box of model SPX-50; S6. Calculation of germination rate: observe the germination of pollen, take the pollen tube length exceeding the pollen grain diameter as the germination standard, and calculate the germination rate: germination rate = (number of germinated pollen / total number of observed pollen) × 100%.
[0020] In summary, the present invention can realize simple and rapid cultivation treatment in the pollen germination cultivation process and more accurate statistical processing through the average statistical method. The cultivation process is simple and reliable, and the statistical process of pollen germination rate is accurate and convincing, which well achieves the purpose of completing the pollen germination cultivation treatment quickly and accurately, and is very beneficial to the research on pollen germination cultivation treatment.
[0021] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.
[0022] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.
[0023] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for germinating pollen of leguminous plants, characterized in that: The specific steps include: S1. Weighing of raw materials of liquid culture medium: firstly, using a weighing device, respectively weigh 10%-20% sucrose, 0.01%-0.06% H3BO3, 10%-20% PEG-6000, 1%-2% sodium hydrosulfide, 0.01%-0.1% CaCl2·2H2O, 0.01%-0.06% KNO3 and 57.78%-78.93% nutrient solution in the required weight percentage; S2. Preparation of liquid culture medium: Add the raw materials weighed in step S1 into a beaker in sequence, and stir slowly and evenly for 3-5 minutes with a stirring rod to obtain a liquid culture medium; S3. Pre-culture treatment: sterilize the culture dish, filter paper and glass slide. During culture, soak the filter paper in 70% alcohol for 3-8 minutes and then dry it. Wet it with distilled water and place it on the bottom of the culture dish. S4, sampling process: take pollen grains from 3-5 florets and mix the pollen grains on a glass slide; S5. Preparation and processing: Drop an appropriate amount of the liquid culture medium prepared in step S2 onto the glass slide with pollen grains placed in step S4, mix well with a glass rod, place the glass slide on wet filter paper in a culture dish, cover the culture dish with wet filter paper, and culture in a constant temperature box with moisturizing. After culturing for 10-12 minutes, examine under a microscope, take 5 fields of view for each preparation, 3 preparations each time, and take the average value; S6. Calculation of germination rate: observe the germination of pollen, take the pollen tube length exceeding the pollen grain diameter as the germination standard, and calculate the germination rate: germination rate = (number of germinated pollen / total number of observed pollen) × 100%.
2. A method for germinating pollen of leguminous plants according to claim 1, characterized in that: In the step S1, 15% sucrose, 0.03% H3BO3, 15% PEG-6000, 1.5% sodium hydrosulfide, 0.01% CaCl2·2H2O, 0.03% KNO3 and 68.43% nutrient solution are measured in required weight percentages.
3. A method for germinating pollen of leguminous plants according to claim 1, characterized in that: In the step S1, 10% sucrose, 0.01% H3BO3, 10% PEG-6000, 1% sodium hydrosulfide, 0.05% CaCl2·2H2O, 0.01% KNO3 and 78.93% nutrient solution are measured in required weight percentages.
4. A method for germinating pollen of leguminous plants according to claim 1, characterized in that: In the step S1, 20% sucrose, 0.06% H3BO3, 20% PEG-6000, 2% sodium hydrosulfide, 0.1% CaCl2·2H2O, 0.06% KNO3 and 57.78% nutrient solution are measured in required weight percentages.
5. The method for germinating pollen of leguminous plants according to claim 1, characterized in that: In step S3, the culture dish, filter paper and glass slide are sterilized by irradiating with ultraviolet sterilization equipment for 5-8 minutes.
6. A method for germinating pollen of leguminous plants according to claim 1, characterized in that: The culture after the slice preparation in step S5 is based on the conditions of pH 7.0 and 30°C.
7. A method for germinating pollen of leguminous plants according to claim 1, characterized in that: The constant temperature box in step S5 is a biochemical incubator of model SPX-50.
8. A method for germinating pollen of leguminous plants according to any one of claims 1 to 4, characterized in that: The nutrient solution in step S1 is any combination of two or more of potassium nitrate, calcium nitrate, ammonium phosphate, magnesium sulfate, ferric chloride or potassium iodide.