A method for rapid detection of kiwifruit pollen viability at an early stage

Abstract

The present application relates to the kiwi cultivation technical field, specifically to a method capable of early rapid detection of kiwi pollen activity, steps are as follows: S1, pollen sample is obtained at the early stage of kiwi male flower reproductive development; S2, anthers in pollen are soaked with Carnoy fixing solution; S3, DAPI staining solution is added to the soaked anthers, and a slice is prepared; S4, fluorescence microscope observation and activity determination are carried out.The present application has the beneficial effects that the present application can collect pollen and detect activity at the early stage of kiwi male flower reproductive development, and the activity detection result is consistent with that of the traditional method; subsequent accurate sampling is realized, work efficiency is improved, and comprehensive cost is reduced; pollination stability is ensured; pollen purity and collection efficiency are considered.

Description

Technical Field

[0001] This invention relates to the field of kiwifruit cultivation technology, and in particular to a method for rapidly detecting kiwifruit pollen viability at an early stage. Background Technology

[0002] Kiwifruit, belonging to the genus Actinidia of the family Actinidiaceae, is a perennial, dioecious, large, deciduous, woody vine. It possesses extremely high nutritional and economic value and is an important specialty fruit crop in my country. Due to its dioecious biological characteristics, it relies on insects for pollination under natural conditions. However, when the flowering periods of male and female plants do not coincide, when climatic conditions during the flowering period are harsh, or when the insect population in the field is insufficient, natural pollination cannot guarantee high-quality and high-yield results. Therefore, in kiwifruit research, breeding, and commercial production, artificial pollination is a core technical means to ensure pollination and fertilization of female plants, improve fruit set rate, and enhance fruit quality. Pollen viability is a key indicator that directly determines pollination effectiveness and ultimately affects yield and quality.

[0003] Currently, the mainstream methods for detecting kiwifruit pollen viability mainly include in vitro germination method, TTC staining method, I2-KI staining method, and inorganic acid determination method. The general operating procedure for the above methods is as follows: flower buds are collected in the late reproductive development stage of male kiwifruit plants (from the large bud stage to the stage when the flower is about to open). After the anthers are manually removed, they are naturally air-dried at room temperature or dried at a constant temperature for about 24 hours. After the anthers dehisce and release pollen, mature pollen is collected, and then the viability is measured using the above methods.

[0004] The current method of collecting flower buds and testing viability during the late reproductive development stage of male kiwifruit plants has the following problems: (1) The detection window period is seriously delayed and lacks early prediction ability. Because the existing methods all require waiting for the anthers to develop to the large bud stage and close to the pollen shedding state before sampling and testing can be performed, it is impossible to predict the pollen viability in the early stage of reproductive development, resulting in a lack of advance planning basis for breeding and production work. (2) Indiscriminate sampling is inefficient and labor-intensive. Since pollen viability cannot be known in advance, indiscriminate collection and pretreatment of large buds of all male plants are required. The sampling and pollen preparation of multiple groups and large quantities greatly increases the workload and significantly reduces work efficiency. (3) The flowering period is short and there is not enough room for emergency adjustment. The flowering period of kiwifruit is greatly affected by the weather and temperature. The male flowers only last for 5 to 8 days. If the pollen viability is found to be low after testing, the time to replace the male plants for sampling or purchase spare pollen is very short. If the pollen viability of the male plants in the park is generally poor, it will directly delay the pollination window period and cause great passivity to the breeding work and production management. (4) Sampling after pollen shedding results in a double loss of purity and yield; if pollen is collected after the flowers open, wind, insects and other media can easily cause pollen from different plants to mix in, resulting in mixed parent plants and unclear lineage; at the same time, the natural shedding of pollen will lead to a significant reduction in the amount of pollen that can be collected, which is not conducive to the reserve of pollen for artificial pollination. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for rapid detection of kiwi pollen viability in the early stages, which solves the problems of lack of early prediction, low work efficiency, possible delay in pollination, and contamination of pollen from other plants caused by the requirement that male kiwi plants must be in the later stages of reproductive development.

[0006] The objective of this invention is achieved through the following technical solution: a method for rapidly detecting kiwi pollen viability at an early stage, comprising the following steps: S1, Sampling; During the early reproductive development of male kiwifruit flowers, several robust and uniformly growing male kiwifruit plants were randomly selected. Multiple flower buds were randomly picked from each of the target male plants. Flower buds from the same male plant were stored as a group to form multiple groups of flower buds. The growth state of each flower bud picked was consistent. At this time, the anthers on the flower buds had not dehisced and the pollen was in the microspore development stage. S2, anther preparation; To obtain multiple groups of anthers, perform the following operations on each group of flower buds: Gently peel off the sepals and petals of the flower bud with tweezers to fully expose the anthers inside, and then gently peel off all the anthers; the degree of gentleness should be such that mechanical damage to the microspores inside the anthers is avoided. Immediately place the detached anthers into Carnoy fixative and mix thoroughly to completely submerge the anthers; Place the container containing the sample in a refrigerator overnight to fix it, thus completing the sample pretreatment; S3 and DAPI fluorescent staining preparation; The anthers that underwent sample pretreatment in S2 were subjected to the following operations: Randomly take an appropriate amount of anthers from Carnoy fixative, place them on sterile filter paper and quickly blot off any remaining Carnoy fixative on the surface; Place the anther in the center of a clean glass slide, gently puncture the anther wall with a sterile needle, and squeeze to fully release and evenly disperse the pollen microspores inside. Add ready-to-use DAPI staining solution to the sample area, gently cover with a coverslip to avoid air bubbles, and place the coverslip in a dark environment for staining. S4. Fluorescence microscopy observation and viability determination; (1) Data statistics In S3, multiple sample slides are obtained after staining and preparing slides of each group of anthers; several groups are randomly selected from the multiple sample slides of each group of anthers as microscopic observation sample slides, and the microscopic observation sample slides are operated as follows: The stained slides were observed under a fluorescence microscope. Multiple fields of view were randomly selected for each sample observed under the microscope. The number of normally developing cells and the total number of cells in each field of view were counted, and the rate of normally developing cells was calculated using the following formula. Normal development cell rate = (Number of normally developing cells / Total number of cells) × 100% Cells with a clear blue highlighted chromosome signal are considered to be normally developing cells, while cells without a clear chromosome fluorescence signal are considered to be abnormally developing cells. For the microscopic observation sample, multiple fields of view were randomly selected repeatedly, and the average rate of normally developing cells for each microscopic observation sample was calculated. (2) Criteria for determining normally developing cells; If the rate of normally developing cells is <40%, it is considered low-viability pollen. If 40% ≤ normal developing cell rate < 70%, then it is considered medium viability pollen; If the rate of normally developing cells is ≥ 70%, it is considered high-viability pollen.

[0007] Furthermore, in S2: when the peeled anthers are immediately placed into Carnoy fixative and thoroughly mixed, the volume ratio of fixative to anthers is 10:1 to ensure that the anthers are completely submerged.

[0008] Furthermore, in S2: the container containing the sample is placed in a 4°C refrigerator overnight to fix it, thus completing the sample pretreatment.

[0009] Further, in S3: 3-5 anthers are randomly selected from Carnoy fixative; 30 μL of ready-to-use DAPI staining solution is added to the sample area, a coverslip is gently placed on top to avoid air bubbles, and the slide is placed in a dark environment for staining for 10 min.

[0010] Furthermore, in S3: 10 fields of view are randomly selected for each sample observed under a microscope.

[0011] Furthermore, in S1: three healthy male kiwifruit plants with uniform growth are selected; 10 flower buds are picked from each male plant, and the 10 flower buds from each male plant are stored as a group.

[0012] Furthermore, the selected high-viability pollen will be used for pollination in hybridization breeding.

[0013] Definitions: Carnoy's fixative, also known as Carnoy's fixative, is a rapidly penetrating fixative widely used in cell biology and histology. This protocol uses Carnoy's fixative because it has strong penetrability, rapidly coagulates nucleic acids and proteins within cells, fixes the morphology and structure of chromosomes, prevents autolysis of early pollen microspores, and preserves the original state of cell development, providing a structural basis for subsequent staining and observation.

[0014] DAPI staining solution is a fluorescent dye commonly used in cell biology and molecular biology experiments; its full name is 4',6-diamidino-2-phenylindole. It is typically used to stain fixed anther cells to allow for clear observation of chromosomes under a fluorescence microscope. This protocol chooses DAPI staining solution because DAPI specifically binds to double-stranded DNA, emitting bright blue fluorescence under ultraviolet light excitation. By labeling chromosomes within the cell nucleus, it allows for a direct differentiation between normally developing cells with intact genetic material and aborted cells with abnormal development and missing chromosomes.

[0015] It should be noted that the principle behind this solution is: Based on the cellular mechanisms of pollen development: viable fertile pollen must undergo complete meiosis and cell development to form cells with complete chromosome structures; aborted pollen lacks complete genetic material and chromosome structure. By preserving the cellular structure of early pollen microspores with a fixative, and then utilizing the DNA-specific binding properties of DAPI fluorescent dye, chromosomes within the cell nucleus are labeled. By statistically analyzing the proportion of normal cells with clear chromosome signals, pollen viability can be quantitatively determined without waiting for anther shedding.

[0016] The innovative aspects of this solution are: I. Defining the Early Sampling Period: Specifically, for the first time, a sampling point for pollen viability testing in the early reproductive development of male kiwifruit flowers was established, advancing the detection time by about 12 days compared to the traditional large bud stage. This breaks through the time limitation of existing technologies that require waiting for the anthers to mature and shed pollen (which is the core prerequisite for achieving "early detection"). II. Early anther fixation-fluorescence staining combined technique; Specifically, Carnoy fixative is used to fix early anthers at low temperature, combined with DAPI fluorescence staining technology, to achieve visual detection of the developmental status of pollen microspores in the undispersed state, omitting the anther drying and dispersing step of more than 24 hours in the traditional method (which is the core technology combination for achieving rapid and early detection). III. A quantitative assessment system for pollen viability based on chromosome signals; a three-level grading standard for pollen viability was established, with "normally developing cell rate" as the core quantitative indicator and chromosome fluorescence signal under a fluorescence microscope as the judgment basis; this indicator is highly consistent with the traditional pollen germination rate, ensuring the accuracy and reliability of early detection results; IV. Rapid detection mode throughout the entire process; the entire detection process only requires "sampling - low temperature fixation overnight - staining in the dark for 10 minutes - microscopic examination and counting" to obtain results, which greatly shortens the detection cycle and enables early and rapid determination of kiwi pollen viability.

[0017] The present invention has the following advantages: (1) This method can collect pollen and detect viability in the early stage of male flower reproductive development of kiwifruit, and the viability test results are consistent with the viability test results of traditional methods. It should be noted that traditional pollen viability testing methods (such as in vitro germination method, TTC staining method, I2-KI staining method, inorganic acid determination method, etc.) all require pollen to be collected and the corresponding pollen viability to be tested only in the later stages of reproductive development of male kiwifruit flowers. In this scheme, viability testing can be completed in the early stage of male flower reproductive development in kiwifruit (the viability testing results are consistent with the traditional method); compared with the earliest detectable bud stage of existing technology, it is about 12 days earlier, which provides sufficient advance planning time for scientific research, breeding and production management; (2) To achieve accurate sampling in the future, improve work efficiency, and reduce overall costs; Traditional pollen collection requires significantly reducing indiscriminate sampling and then removing pollen with poor viability through viability testing; this method requires indiscriminate sampling and corresponding viability testing, which incurs significant labor and time costs. In this scheme, viability testing can be completed in the early stage of reproductive development of male kiwifruit flowers. This allows for the selection of high-viability male plants for pollen collection and the discarding of low-viability male plants during harvesting, which can significantly reduce the labor and time costs of indiscriminate sampling and improve work efficiency. (3) Ensure the stability of pollination work; Knowing the pollen viability level in advance allows for the development of pollination plans. When the male plants in the orchard lack viability, exogenous pollen can be prepared in a timely manner to avoid pollination delays caused by short flowering periods and unqualified viability, thus stabilizing fruit set rate and fruit quality. (4) Balancing pollen purity and collection efficiency; In this scheme, both detection and subsequent sampling can be completed in the bud stage before the anthers dehisce, which not only eliminates the mixing of pollen from different plants brought by insects and wind, but also meets the high purity requirements of parent plants in hybridization breeding; it also avoids yield loss caused by the natural scattering of pollen. (5) Improve the pollen viability detection technology system; This invention fills the technical gap in the detection of pollen viability in the early reproductive development of kiwifruit, provides new technical support and theoretical basis for kiwifruit breeding research and production practice, and is an important supplement to the existing detection method system. Attached Figure Description

[0018] Figure 1 The diagram shows kiwifruit flower buds at different developmental stages; (buds 1 and 2 are early reproductive development buds, which are the sampling periods of this invention; bud 7 is a large bud, which is the earliest sampling period detectable by existing technology, and the two developmental periods differ by about 12 days). Figure 2 This is a schematic diagram of the normal development process of kiwifruit pollen under a fluorescence microscope; (A is the tetrad stage, B is the uninucleate stage, and C is the two-celled pollen grain stage; the blue highlighted parts in the diagram are chromosomes, and the blue areas are cell outlines). Figure 3 The images show a comparison of pollen observation using different detection methods compared to the present invention; (A1, B1, and C1 represent the field of view for observing pollen cell development under a fluorescence microscope using the method of the present invention; A2, B2, and C2 represent the field of view for observing pollen germination under an optical microscope using the traditional in vitro germination method). Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0020] (Example) This embodiment provides a method for rapid detection of kiwi pollen viability at an early stage, the steps of which are as follows: S1. For the Hongyang kiwifruit variety, randomly select 3 healthy male Hongyang kiwifruit plants with uniform growth, number them A, B, and C respectively, and label them. When the male plants' flower buds develop to the early stages shown in Figure 1 (1 and 2), collect a sufficient number of flower buds (usually 10 flower buds) from the three male plants A, B, and C respectively. S2. Gently peel off the sepals and petals of the flower bud with tweezers to fully expose the anthers inside, and then gently peel off all the anthers; the degree of gentleness should be such that mechanical damage to the microspores inside the anthers is avoided. Place the peeled anthers into 50 mL sterile centrifuge tubes and immediately add Carnoy fixative II until the anthers are completely submerged (Carnoy fixative to anther volume ratio of 10:1); seal and fix overnight in a 4°C refrigerator. The corresponding samples are numbered A1, B1, and C1. S3 and DAPI fluorescent staining preparation; (1) Take 3 to 5 anthers from the centrifuge tube (any number within this range is acceptable, but 3 is preferred in this embodiment), blot the surface fixative with sterile filter paper, place them on a clean glass slide, and gently puncture the anthers with a sterile needle to release the pollen microspores and disperse them evenly; (2) Add 30 μL of ready-to-use DAPI solution, gently cover with a coverslip, and stain in the dark for 10 min; (3) Observe the prepared slides under a fluorescence microscope. Randomly select 10 fields of view for each sample, count the number of normally developing cells and the total number of cells, and calculate the rate of normally developing cells. Normal development cell rate = number of normal development cells / total number of cells × 100%. Cells with clear blue highlighted chromosome signals are considered normal development cells, while cells without clear chromosome fluorescence signals are considered abnormal development cells. Each sample was subjected to 3 biological replicates (i.e., the following actions were repeated 3 times: 10 fields of view were randomly selected, the number of normally developing cells and the total number of cells were counted, and the rate of normally developing cells was calculated), and the average value was taken as the final result (average rate of normally developing cells). It should be noted that: a. Field of view, also known as microscope field of view, is the area of ​​the slide sample that can be covered by a single observation through the eyepiece after the fluorescence microscope has been focused and is clear, corresponding to a complete microscopic image. Essentially, it is the fixed-size circular observation range that the microscope optical system can present in a single image. Its specific area is determined by the objective lens magnification and eyepiece parameters. The higher the magnification, the smaller the observation range of a single field of view and the clearer the cell details. b. In this invention, in the pollen viability detection scenario of this patent, a field of view is an independent statistical unit: in the image of this range, the DAPI fluorescence staining signal of each pollen microspore can be clearly distinguished, and the "number of normally developing cells with clear chromosome fluorescence" and the "total number of all cells" in this area can be counted to complete a single sampling count. c. The purpose of the design of "randomly selecting multiple fields of view" is that the distribution of pollen microspores on the slide cannot be absolutely uniform during the slide preparation process. The results of counting only a single field of view are easily affected by local distribution deviations, resulting in a large sampling error. Therefore, this method requires the random selection of multiple fields of view (10 in this scheme) for independent counting. By using multi-point sampling to cover sample areas at different locations on the slide, the statistical sample is more representative, and the final calculated "normal development cell rate" is closer to the true viability level of the entire pollen sample, ensuring the accuracy and repeatability of the test results. It should also be noted that: a. The "cells with clear, bright blue chromosome signals" mentioned here refer only to cells where the blue fluorescence intensity of the chromosome / nucleus region is significantly higher than the background nonspecific fluorescence of the cytoplasm. The difference in brightness between the two is clear, and the nuclear region can be visually distinguished from the cell background. There are no cases where the fluorescence signal is extremely weak and almost indistinguishable from the background; b. Abnormally developing cells are those that have no blue fluorescence signal at all, or only have diffuse, blurred, weak blue fluorescence, where the complete nucleus / chromosome morphology cannot be distinguished and there is no obvious difference from the background fluorescence; c. It can be understood that cells with clear, sharp nuclear morphological boundaries and significantly higher fluorescence intensity than the cell background are considered to have clear, bright blue chromosome signals; cells with no blue fluorescence signal, or only diffuse, blurred, weak blue fluorescence, where the complete nuclear morphology cannot be distinguished, are considered to be abnormally developing cells. S4. Determine the pollen viability level of each sample according to the grading standards; If the rate of normally developing cells is <40%, it is considered low-viability pollen. If 40% ≤ normal developing cell rate < 70%, then it is considered medium viability pollen; If the rate of normally developing cells is ≥ 70%, it is considered high-viability pollen.

[0021] It should be noted that the following precautions should be taken when operating this embodiment: (1) When peeling the anthers, the movements should be gentle to avoid prematurely piercing the anther wall and causing the microspores to be lost; (2) The entire process of DAPI staining and slide preparation must be kept away from light to prevent fluorescence quenching from affecting the observation results; (3) The field of view selected for microscopic examination should be random and uniform to avoid statistical bias caused by subjective selection of the field of view.

[0022] (Comparative Example) The traditional in vitro germination method was used as a comparative example: Approximately 12 days later, when the flower buds of the labeled male plants developed to the large bud stage (Figure 1, buds 6 and 7), flowers at the large bud stage were collected from three male plants (A, B, and C). The intact anthers were gently peeled off with tweezers. The anthers were evenly spread in a petri dish and dried at a constant temperature of 25°C for 24 hours. After the anthers naturally dehisced and released pollen, the pollen was collected into a brown glass bottle, sealed, and stored in a refrigerator at -20°C. The corresponding sample numbers were A2, B2, and C2.

[0023] Pollen viability was determined using the traditional in vitro germination method as a control: (1) Prepare solid germination medium: sucrose + boric acid + agar, adjust pH to 6.5, sterilize and pour into plates; (2) Spread the pollen evenly on the surface of the culture medium with a brush and incubate in a constant temperature incubator at 25℃ for 2 hours; (3) Observe the pollen germination under an optical microscope. Six fields of view are randomly selected for each sample, with no less than 50 pollen grains in each field of view. The pollen tube length is greater than the pollen diameter as the germination criterion. The pollen germination rate is calculated according to the following formula: Germination rate = (Number of germinating pollen grains in the field of view / Total number of pollen grains in the field of view) × 100%. The germination rate of the tested pollen is statistically analyzed.

[0024] (Test example) Low-viability pollen A1, medium-viability pollen B1, and high-viability pollen C1 were obtained using the detection method in this embodiment. Low-viability pollen A2, medium-viability pollen B2, and high-viability pollen C2 were obtained using a comparative detection method. A1, B1, C1, A2, B2, and C2 were then cross-pollinated under identical conditions, and the germination rate of the corresponding seeds was detected. The test results are shown in Table 1 and... Figure 3 As shown.

[0025] Table 1. Germination rates of seeds after pollination using low-viability, medium-viability, and high-viability pollen in this embodiment and comparative examples. pass Figure 3 It can be seen that: Figure 3 In this embodiment, the proportion of blue fluorescent signal cells in A1 under the fluorescence field of view corresponds highly to the proportion of germinating pollen in the optical field of comparison example A2. In this embodiment, the proportion of blue fluorescent signal cells in B1 under the fluorescence field of view corresponds highly to the proportion of germinating pollen in the optical field of comparison example B2. In this embodiment, the proportion of blue fluorescent signal cells in A3 under the fluorescence field of view corresponds highly to the proportion of germinating pollen in the optical field of comparison example A3. In layman's terms, the low, medium, and high viability results obtained by collecting flower buds in the early stages of male flower bud development and then testing them using appropriate methods are basically consistent with the low, medium, and high viability results obtained by collecting flower buds after male flower bud development is complete and then measuring pollen viability using the traditional in vitro germination method.

[0026] Furthermore, as shown in Table 1, the seed germination rate of low-viability pollen in this method after pollination is consistent with that of low-viability pollen measured by the traditional method. Similarly, the germination rate of medium-viability pollen in this method is consistent with that of the traditional method, as is the germination rate of high-viability pollen in this method.

[0027] In summary, this method collects pollen during the early reproductive development of male kiwifruit flowers and obtains pollen viability results through appropriate detection methods. The results are the same as those obtained by traditional methods, which collect pollen during the later reproductive development of male kiwifruit flowers and obtain pollen viability through appropriate detection methods (such as in vitro germination method, TTC staining method, I2-KI staining method, and inorganic acid determination method). In layman's terms, the early rapid detection method of this invention is completely consistent with the viability determination results of the traditional in vitro germination method, and the detection results are accurate and reliable; moreover, the detection point is advanced by about 12 days, which can effectively realize the early rapid detection of kiwifruit pollen viability.

[0028] The above embodiments only illustrate preferred implementation methods, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this invention, and these all fall within the protection scope of this invention.

Claims

1. A method for rapid detection of kiwifruit pollen viability at an early stage, characterized in that: Includes the following steps: S1, Sampling; During the early reproductive development of male kiwifruit flowers, several robust and uniformly growing male kiwifruit plants were randomly selected. Multiple flower buds were randomly picked from each of the target male plants. Flower buds from the same male plant were stored as a group to form multiple groups of flower buds. The growth state of each flower bud picked was consistent. At this time, the anthers on the flower buds had not dehisced and the pollen was in the microspore development stage. S2, anther preparation; To obtain multiple groups of anthers, perform the following operations on each group of flower buds: Gently peel off the sepals and petals of the flower bud with tweezers to fully expose the anthers inside, and then gently peel off all the anthers; the degree of gentleness should be such that mechanical damage to the microspores inside the anthers is avoided. Immediately place the detached anthers into Carnoy fixative and mix thoroughly to ensure the anthers are completely submerged; Place the container containing the sample in the refrigerator overnight to fix it, thus completing the sample pretreatment; S3 and DAPI fluorescent staining preparation; The anthers that underwent sample pretreatment in S2 were subjected to the following operations: Randomly take an appropriate amount of anthers from Carnoy fixative, place them on sterile filter paper and quickly blot off any remaining Carnoy fixative on the surface; Place the anther in the center of a clean glass slide, gently puncture the anther wall with a sterile needle, and squeeze to fully release and evenly disperse the pollen microspores inside. Add ready-to-use DAPI staining solution to the sample area, gently cover with a coverslip to avoid air bubbles, and place the coverslip in a dark environment for staining. S4. Fluorescence microscopy observation and viability determination; (1) Data statistics In S3, multiple sample slides are obtained after staining and preparing slides of each group of anthers; several groups are randomly selected from the multiple sample slides of each group of anthers as microscopic observation sample slides, and the microscopic observation sample slides are operated as follows: The stained slides were observed under a fluorescence microscope. Multiple fields of view were randomly selected for each sample observed under the microscope. The number of normally developing cells and the total number of cells in each field of view were counted, and the rate of normally developing cells was calculated using the following formula. Normal development cell rate = (Number of normally developing cells / Total number of cells) × 100% Cells with a clear blue highlighted chromosome signal are considered to be normally developing cells, while cells without a clear chromosome fluorescence signal are considered to be abnormally developing cells. For the microscopic observation sample, multiple fields of view were randomly selected repeatedly, and the average normal development cell rate of each microscopic observation sample was calculated. (2) Criteria for determining normally developing cells; If the rate of normally developing cells is less than 40%, it is considered low-viability pollen. If 40% ≤ normal developing cell rate < 70%, then it is considered medium viability pollen; If the rate of normally developing cells is ≥ 70%, it is considered high-viability pollen.

2. The method for rapid detection of kiwifruit pollen viability at an early stage according to claim 1, characterized in that: In S2: when the peeled anthers are immediately placed into Carnoy fixative and thoroughly mixed, the volume ratio of fixative to anthers is 10:1 to ensure that the anthers are completely submerged.

3. The method for rapid detection of kiwifruit pollen viability at an early stage according to claim 2, characterized in that: In step S2: the container containing the sample is placed in a 4°C refrigerator overnight to fix it, thus completing the sample pretreatment.

4. A method for rapid detection of kiwifruit pollen viability at an early stage, as described in any one of claims 1 to 3, characterized in that: In S3: Randomly select 3-5 anthers from the Carnoy fixative solution; Add 30 μL of ready-to-use DAPI staining solution to the sample area, gently cover with a coverslip to avoid air bubbles, and place the slide in a dark environment for staining for 10 min.

5. The method for rapid detection of kiwifruit pollen viability at an early stage according to claim 4, characterized in that: In S3, 10 fields of view are randomly selected for each sample observed under a microscope.

6. The method for rapid detection of kiwifruit pollen viability at an early stage according to claim 5, characterized in that: In S1: Three robust male Hongyang kiwifruit plants with uniform growth were selected. Ten flower buds were picked from each male plant, and the ten flower buds from each male plant were stored as a group.

7. The method for rapid detection of kiwifruit pollen viability at an early stage according to claim 1, characterized in that: The selected high-viability pollen was used for pollination in hybridization breeding.

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