A method for rapidly determining the viability of watermelon pollen

Abstract

A method for rapidly determining the viability of watermelon pollen, comprising the following steps: preparing a stock solution: adding 0.9 - 1.2 g of anhydrous CaCl<subgt;2< / subgt>, 0.9 - 1.0 g of KNO<subgt;3< / subgt>, 0.3 - 0.5 g of MgSO<subgt;4< / subgt>·7H<subgt;2< / subgt>O, 0.1 - 0.3 g of KCl, and 0.1 - 0.12 g of H<subgt;3BO<subgt;3 to 600 - 800 mL of water, completely dissolving and then making up the volume to 1 L to obtain the stock solution, storing it at room temperature; preparing the viability detection culture medium: adding 1.0 - 1.5 g / 10 g of sucrose to the above-prepared stock solution, and obtaining the viability detection culture medium after the sucrose is completely dissolved; viability detection: laying a filter paper moistened with water in a petri dish, placing a glass slide on the filter paper, dropping 1 - 2 drops of the viability detection culture medium on the glass slide, dipping the fresh watermelon flowers to be detected in the culture medium several times, at this time the number of pollen grains can be observed, covering the petri dish lid, and culturing at room temperature for 20 - 30 min; observing the pollen tube germination situation and calculating the pollen viability percentage. Using the culture medium of the present invention to culture pollen, the result can be observed in only 20 - 30 min, and it only needs to be placed at room temperature during the culture without special requirements.

Description

Technical Field

[0001] The present invention relates to a method for rapidly determining the viability of watermelon pollen, belonging to the technical field of plant cultivation. Background Art

[0002] In traditional seed production, male flowers of the male parent are directly picked and used for pollination. The daily pollen quantity and pollen viability vary. The temperature, ventilation, growth vigor, pruning situation in the greenhouse, and the node position of the flower will all affect the quality of male flowers. When pollinating, the amount of pollinated flowers is allocated according to the number of male flowers. When the quantity is sufficient, one male flower pollinates one female flower; when the quantity is insufficient, one male flower pollinates two female flowers. For those that fail to set fruits after pollination, female flowers are left for secondary pollination. This process not only has a high labor cost but also reduces the number of seeds in the melon due to insufficient viable pollen quantity, resulting in a reduction in seed production yield.

[0003] Pollen viability detection technology can effectively solve the above-mentioned technical problems existing in traditional seed production. Monitor the quality of male flowers a few days before pollination and timely adjust the planting method. Before pollination, detect the pollen viability in advance and adjust the amount of male flowers to ensure that the quantity of viable pollen required for pollination meets the standard, thereby increasing the fruit setting rate, reducing the labor cost, effectively increasing the number of seeds per melon, and doubling the overall seed production yield compared to before.

[0004] Among the existing pollen viability detection methods, a solid medium is used. When preparing, it needs to be heated first. The medium contains plant hormones that need to be dissolved in organic solvents, the preparation is cumbersome, the cultivation time is long, requiring 4 - 5 hours, and it is not suitable for long-term storage, which is not applicable to actual production. In addition, the existing cultivation using the existing medium requires dark cultivation and the cultivation temperature is required to be 25 - 28°C. Summary of the Invention

[0005] In order to overcome the drawbacks of the prior art, the present invention provides a method for rapidly determining the viability of watermelon pollen. Using a liquid medium without hormones, the prepared liquid stock solution can be stored for more than one year. Using this culture solution to culture pollen, the result can be observed in only 20 minutes, and it only needs to be placed at room temperature during cultivation without special requirements.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] A method for rapidly determining the viability of watermelon pollen, the method comprising the following steps:

[0008] Step a, prepare a stock solution: Add 0.9 - 1.2 g of anhydrous CaCl2, 0.9 - 1.0 g of KNO3, 0.3 - 0.5 g of MgSO4·7H2O, 0.1 - 0.3 g of KCl, and 0.1 - 0.12 g of H3BO3 to 600 - 800 mL of water. After completely dissolving, make up the volume to 1 L to obtain the stock solution, and store it at room temperature;

[0009] Step b, Preparation of the vitality detection culture medium: Add 1.0 - 1.5 g of sucrose per 10 g to the above-prepared stock solution. After the sucrose is completely dissolved, the vitality detection culture medium is obtained.

[0010] Step c, Vitality detection: Lay a piece of filter paper moistened with water in a petri dish. Place a glass slide on the filter paper and drop 1 - 2 drops of the vitality detection culture medium on the glass slide. Dip the watermelon fresh flowers to be detected a few times in the culture medium. At this time, the number of pollen grains can be observed. Cover the petri dish and incubate at room temperature for 20 - 30 min; Randomly detect 3 - 5 male flowers of the same batch using the same detection method.

[0011] Step d, Observe the germination of pollen tubes and calculate the percentage of pollen vitality: Observe the growth of pollen tubes under a 40×10 microscope. Pollen grains with pollen tube lengths exceeding the pollen diameter are viable pollen grains. The percentage of pollen vitality is the number of viable pollen grains divided by the total number of pollen grains. Take the average value after calculating the percentage of pollen vitality in 3 - 5 fields of view.

[0012] In the above method for rapidly determining the vitality of watermelon pollen, in step a, in the stock solution, it includes 1 g / L of CaCl2, 0.95 g / L of KNO3, 0.37 g / L of MgSO4·7H2O, 0.175 g / L of KCl, and 0.1 g / L of H3BO3.

[0013] In the above method for rapidly determining the vitality of watermelon pollen, the preferred amount of sucrose added to the stock solution in step b is 1.2 g per 10 g.

[0014] In the above method for rapidly determining the vitality of watermelon pollen, the vitality detection culture medium prepared in step b can be directly used for pollen vitality detection or stored refrigerated for less than 7 days before pollen vitality detection.

[0015] The beneficial effects of the present invention are:

[0016] In the liquid medium used for detecting the vitality of watermelon pollen in the present invention, there is no hormone. The prepared stock solution can be stored for more than one year. During vitality detection, only sucrose needs to be added to the stock solution for pollen culture, which is more suitable for use in production bases.

[0017] Using the liquid medium of the present invention for pollen germination culture, the culture results can be observed in only 20 - 30 min, with higher culture efficiency and more practical application value; and the culture process can be carried out at room temperature without special requirements. The germination rate of pollen tubes cultured with the liquid medium of the present invention is as high as 90% or more. Description of the Drawings

[0018] Figure 1 It is a microscopic examination diagram before the germination of watermelon pollen in Example 1 of the present invention;

[0019] Figure 2 Microscopic detection after the germination of watermelon flowers in Example 1;

[0020] Figure 3 Microscopic detection after the germination of watermelon flowers in Comparative Example 1;

[0021] Figure 4 Microscopic detection after the germination of watermelon flowers in Comparative Example 2;

[0022] Figure 5 Microscopic detection after the germination of watermelon flowers in Comparative Example 3. Detailed implementation mode

[0023] The liquid medium of the present invention contains 0.9 - 1.2 g / L of CaCl2, 0.9 - 1.0 g / L of KNO3, 0.3 - 0.5 g / L of MgSO4·7H2O, 0.1 - 0.3 g / L of KCl, 0.1 - 0.12 g / L of H3BO3, and 1.0 - 1.5 g / 10 g of sucrose. Calcium, boron, and sucrose are essential nutrients for pollen tube germination. Sucrose, as the carbon source for pollen tube germination, provides energy for pollen germination. At the same time, its addition amount must be controlled to ensure the osmotic pressure of the solution and the germination rate, that is, the percentage of pollen viability. The amount of calcium and boron also affects the germination of watermelon pollen. Through a large number of experimental explorations, the addition amounts of calcium and boron in the present invention are obtained. If the addition amount exceeds this range, the percentage of watermelon pollen viability obtained by cultivation will decrease.

[0024] The present invention will be further described below in conjunction with examples.

[0025] Example 1

[0026] A method for quickly measuring the viability of watermelon pollen includes the following steps:

[0027] Step a: Prepare the stock solution: Add 1 g of anhydrous CaCl2, 0.95 g of KNO3, 0.37 g of MgSO4·7H2O, 0.175 g of KCl, and 0.1 g of H3BO3 to 800 mL of water. After complete dissolution, make up the volume to 1 L to obtain the stock solution, and store it at room temperature;

[0028] Step b: Prepare the viability detection culture solution: Add 1.2 g / 10 g of sucrose to the stock solution prepared above. After complete dissolution of the sucrose, the viability detection culture solution is obtained;

[0029] Step c: Viability detection: Lay a piece of filter paper moistened with water in a petri dish. Place a glass slide on the filter paper. Drop 1 - 2 drops of the viability detection culture solution on the glass slide. Dip the watermelon fresh flower to be detected a few times in the culture solution. At this time, the number of pollen grains can be observed. Cover the petri dish and incubate at room temperature for 20 min;

[0030] Step d, observe the pollen tube germination situation and calculate the pollen viability percentage: Observe the growth of pollen tubes under a microscope with a magnification of 40×10. Pollen grains with a pollen tube length exceeding the pollen diameter are viable pollen grains. The ratio of viable pollen grains to the total number of pollen grains is the pollen viability percentage. Calculate the pollen viability percentage in 3 - 5 fields of view and then take the average value. The obtained pollen viability percentage is 95%. As Figure 1 and Figure 2 , respectively, are the microscopic examination pictures of watermelon pollen before and after germination.

[0031] Example 2

[0032] A method for rapidly determining the viability of watermelon pollen, comprising the following steps:

[0033] Step a, prepare the stock solution: Add 0.9 g of anhydrous CaCl2, 1 g of KNO3, 0.4 g of MgSO4·7H2O, 0.2 g of KCl, and 0.11 g of H3BO3 to 700 mL of water. After complete dissolution, make up the volume to 1 L to obtain the stock solution, and store it at room temperature;

[0034] Step b, prepare the viability detection culture solution: Add 1.3 g / 10 g of sucrose to the above-prepared stock solution. After complete dissolution of the sucrose, obtain the viability detection culture solution;

[0035] Step c, viability detection: Lay a filter paper moistened with water in a petri dish, place a glass slide on the filter paper, drop 1 - 2 drops of the viability detection culture solution on the glass slide, dip the fresh watermelon flowers to be detected a few times in the culture solution. At this time, the number of pollen grains can be observed. Cover the petri dish and incubate at room temperature for 25 min;

[0036] Step d, observe the pollen tube germination situation and calculate the pollen viability percentage: Observe the growth of pollen tubes under a microscope with a magnification of 40×10. Pollen grains with a pollen tube length exceeding the pollen diameter are viable pollen grains. The ratio of viable pollen grains to the total number of pollen grains is the pollen viability percentage. Calculate the pollen viability percentage in 3 - 5 fields of view and then take the average value. The pollen viability percentage is 92%.

[0037] Example 3

[0038] A method for rapidly determining the viability of watermelon pollen, comprising the following steps:

[0039] Step a, prepare the stock solution: Add 1.1 g of anhydrous CaCl2, 0.9 g of KNO3, 0.5 g of MgSO4·7H2O, 0.3 g of KCl, and 0.12 g of H3BO3 to 600 mL of water. After complete dissolution, make up the volume to 1 L to obtain the stock solution, and store it at room temperature;

[0040] Step b, Preparation of the viability detection culture medium: Add 1.5 g of sucrose per 10 g to the above-prepared stock solution. After the sucrose is completely dissolved, the viability detection culture medium is obtained.

[0041] Step c, Viability detection: Line a petri dish with a filter paper moistened with water. Place a glass slide on the filter paper and drop 1 - 2 drops of the viability detection culture medium on the slide. Dip the watermelon fresh flowers to be detected a few times in the culture medium. At this time, the number of pollen grains can be observed. Cover the petri dish and incubate at room temperature for 25 min.

[0042] Step d, Observe the pollen tube germination situation and calculate the pollen viability percentage: Observe the growth of pollen tubes under a 40×10 microscope. Pollen grains with a pollen tube length exceeding the pollen diameter are viable pollen grains. The viable pollen grains divided by the total number of pollen grains is the pollen viability percentage. Take the average value after calculating the pollen viability percentage in 3 - 5 fields of view. The pollen viability percentage is 90%.

[0043] Comparative Example 1

[0044] A method for rapidly determining the viability of watermelon pollen, comprising the following steps:

[0045] Step a, Preparation of the stock solution: Add 1 g of anhydrous CaCl2, 0.95 g of KNO3, 0.37 g of MgSO4·7H2O, 0.175 g of KCl, and 0.01 g of H3BO3 to 800 mL of water. After complete dissolution, make up the volume to 1 L to obtain the stock solution and store it at room temperature.

[0046] Step b, Preparation of the viability detection culture medium: Add 1.2 g of sucrose per 10 g to the above-prepared stock solution. After the sucrose is completely dissolved, the viability detection culture medium is obtained.

[0047] Step c, Viability detection: Line a petri dish with a filter paper moistened with water. Place a glass slide on the filter paper and drop 1 - 2 drops of the viability detection culture medium on the slide. Dip the watermelon fresh flowers to be detected a few times in the culture medium. At this time, the number of pollen grains can be observed. Cover the petri dish and incubate at room temperature for 20 min.

[0048] Step d, Observe the pollen tube germination situation (see Figure 3 ), calculate the pollen viability percentage: Observe the growth of pollen tubes under a 40×10 microscope. Pollen grains with a pollen tube length exceeding the pollen diameter are viable pollen grains. The viable pollen grains divided by the total number of pollen grains is the pollen viability percentage. Take the average value after calculating the pollen viability percentage in 3 - 5 fields of view. The pollen viability is 52%.

[0049] Boron is one of the essential elements in the growth process of pollen tubes. Too low boric acid content leads to a significant decrease in pollen germination rate.

[0050] Comparative Example 2

[0051] A method for rapidly determining the viability of watermelon pollen includes the following steps:

[0052] Step a: Prepare the stock solution: Add 1 g of anhydrous CaCl2, 0.95 g of KNO3, 0.37 g of MgSO4·7H2O, 0.175 g of KCl, 0.1 g of H3BO3, and 150 g of PEG to 800 mL of water. After complete dissolution, make up the volume to 1 L to obtain the stock solution, and store it at room temperature.

[0053] Step b: Prepare the viability detection culture medium: Add 1.2 g / 10 g of sucrose to the above-prepared stock solution. After complete dissolution of the sucrose, the viability detection culture medium is obtained.

[0054] Step c: Viability detection: Lay a piece of filter paper moistened with water in a petri dish, place a glass slide on the filter paper, drop 1 - 2 drops of the viability detection culture medium on the glass slide, dip the fresh watermelon flowers to be detected in the culture medium a few times. At this time, the number of pollen grains can be observed. Cover the petri dish and incubate at room temperature for 20 min.

[0055] Step d: Observe the germination of pollen tubes (see Figure 4 ), calculate the percentage of pollen viability: Observe the growth of pollen tubes under a 40×10 microscope. Pollen grains with a pollen tube length exceeding the pollen diameter are viable pollen grains. The percentage of pollen viability is calculated by dividing the number of viable pollen grains by the total number of pollen grains. Take the average value after calculating the percentage of pollen viability in 3 - 5 fields of view. The pollen viability is 0.

[0056] PEG is one of the essential drugs for the germination of Compositae pollen, but adding it to the watermelon pollen culture medium will cause the watermelon pollen to fail to germinate.

[0057] Comparative Example 3

[0058] A method for rapidly determining the viability of watermelon pollen includes the following steps:

[0059] Step a: Prepare the stock solution: Add 1 g of anhydrous CaCl2, 0.95 g of KNO3, 0.37 g of MgSO4·7H2O, 0.175 g of KCl, 0.1 g of H3BO3 to 800 mL of water. After complete dissolution, make up the volume to 1 L to obtain the stock solution, and store it at room temperature.

[0060] Step b: Prepare the viability detection culture medium: Add 0.5 g / 10 g of sucrose to the above-prepared stock solution. After complete dissolution of the sucrose, the viability detection culture medium is obtained.

[0061] Step c, viability detection: Line a culture dish with a filter paper moistened with water. Place a glass slide on the filter paper and add 1 - 2 drops of viability detection culture solution on the glass slide. Dip the watermelon fresh flowers to be detected into the culture solution a few times. At this time, the pollen quantity can be observed. Cover the culture dish and incubate at room temperature for 20 min;

[0062] Step d, observe the pollen tube germination situation (see Figure 5 ), calculate the pollen viability percentage: Observe the growth of pollen tubes under a 40×10 microscope. Pollen grains with pollen tube lengths exceeding the pollen diameter are viable pollen grains. The number of viable pollen grains divided by the total number of pollen grains is the pollen viability percentage. Calculate the pollen viability percentage for 3 - 5 fields of view and then take the average. The pollen viability percentage is only 30%.

[0063] Sucrose provides energy and osmotic pressure for pollen germination. Reducing the sucrose content will lead to a decrease in the pollen germination rate.

Claims

1. A method for rapidly determining the viability of watermelon pollen, characterized in that: The method includes the following steps: Step a, preparing a stock solution: Add 0.9 - 1.2 g of anhydrous CaCl2, 0.9 - 1.0 g of KNO3, 0.3 - 0.5 g of MgSO4·7H2O, 0.1 - 0.3 g of KCl, and 0.1 - 0.12 g of H3BO3 to 600 - 800 mL of water. After complete dissolution, make up the volume to 1 L to obtain the stock solution, and store it at room temperature. Step b, preparing a culture medium for viability detection: Add 1.0 - 1.5 g / 10 g of sucrose to the stock solution prepared above. After the sucrose is completely dissolved, a culture medium for viability detection is obtained. Step c, viability detection: Lay a filter paper moistened with water in a petri dish, place a glass slide on the filter paper, drop 1 - 2 drops of the culture medium for viability detection on the glass slide, dip the watermelon fresh flowers to be detected in the culture medium a few times. At this time, the number of pollen grains can be observed. Cover the petri dish and culture at room temperature for 20 - 30 min; randomly detect 3 - 5 male flowers of the same batch by the same detection method. Step d, observing the germination of pollen tubes and calculating the percentage of pollen viability: Observe the growth of pollen tubes under a 40×10 microscope. Pollen grains with pollen tube length exceeding the pollen diameter are viable pollen grains. The percentage of pollen viability is the number of viable pollen grains divided by the total number of pollen grains. Calculate the percentage of pollen viability in 3 - 5 fields of view and then take the average value.

2. The method for rapidly determining the viability of watermelon pollen according to claim 1, wherein: In step a, in the stock solution, it includes 1 g / L of CaCl2, 0.95 g / L of KNO3, 0.37 g / L of MgSO4·7H2O, 0.175 g / L of KCl, and 0.1 g / L of H3BO3.

3. The method for rapidly determining the viability of watermelon pollen according to claim 2, characterized in that: In step b, the preferably added amount of sucrose in the stock solution is 1.2 g / 10 g.

4. The method for rapidly determining the viability of watermelon pollen according to claim 3, wherein: The culture medium for viability detection prepared in step b is directly used for pollen viability detection or stored refrigerated for less than 7 days before pollen viability detection.

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