Method for rapidly identifying kiwifruit pollen viability

Abstract

The invention relates to a method for rapidly identifying the activity of kiwifruit pollen, and belongs to the technical field of plants. The method solves the problem of long time consumption of a culture medium in-vitro germination method. The method provided by the invention can be used for the activity of kiwi pollen, has the advantages of simplicity, convenience, rapidness and accuracy, and can be used for dyeing pollen grains by using 5% of red ink, so that the pollen dyeing condition can be immediately observed under a microscope, and then the pollen activity can be calculated. Reagents and instruments used for detection are easy to operate, short in time consumption, low in cost and good in effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant physiology and agricultural science, and in particular to a method for rapidly identifying kiwifruit pollen viability. BACKGROUND

[0002] Kiwifruit belongs to the Actinidiaceae family and the Actinidia genus, and is a large perennial dioecious deciduous liana. Kiwifruit has a wide range of ploidy levels, including diploid (2n = 2x = 58), tetraploid, hexaploid, octoploid, and higher ploidy levels. The polyploid kiwifruit maintains a more abundant genetic diversity. China is the world's largest producer of kiwifruit, and the commercially grown species are mainly diploid or tetraploid Actinidia chinensis var. chinensis and hexaploid Actinidia chinensis var. deliciosa. In the Qin-Ba region, the kiwifruit industry has become one of the main sources of income for farmers. Xi'an Zhouzhi County and Baoji Meixian County have been planting kiwifruit as an important industry to promote county economic development. Zhouzhi County, located in the Qin-Chuan hinterland, is the origin of kiwifruit, and the climate is very suitable for the growth and development of kiwifruit.

[0003] Kiwifruit is a typical dioecious plant. Due to different flowering periods, uneven male-to-female ratio, and adverse weather conditions, poor fertilization may occur. Good pollination and fertilization is the key to normal fruiting and high yield of kiwifruit. Pollen viability refers to the ability of pollen grains to germinate and form pollen tubes under suitable conditions. For fruit trees such as kiwifruit that rely on cross-pollination, the level of pollen viability directly affects the success of the pollination and fertilization process, and thus affects the yield and quality of the fruit. Therefore, accurately determining the viability of pollen is of great significance for improving the management level of kiwifruit cultivation and the efficiency of breeding. SUMMARY

[0004] The present application aims to provide a detection method for rapidly determining the viability of kiwifruit pollen, to quickly and accurately detect the viability of kiwifruit pollen, and to provide technical support for the collection, storage, and utilization of pollen.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solution: a method for rapidly identifying the viability of kiwifruit pollen, comprising the following steps:

[0006] Step 1: Collecting pollen: collect kiwifruit flower buds at the large bud stage, and manually peel or dry to release the pollen in the anthers;

[0007] Step 2: Preparing a staining agent: dilute the concentration of red ink and place it in a bottle for use;

[0008] Step three: pollen staining: three slides are prepared for each batch, each slide is cleaned, 1-2 drops of prepared staining agent is dropped on each concave slide, a small amount of pollen is evenly scattered in the staining agent using a capillary or spotter, the pollen particles and the staining agent are fully contacted, a cover glass is covered, and microscopic examination is immediately performed;

[0009] Step four: pollen viability observation: the stained slide is placed under a microscope for observation, the focus and brightness of the microscope are adjusted to obtain a clear field of view, and the staining condition of the pollen particles is observed under the microscope;

[0010] Step five: pollen viability statistics: viable pollen grains remain normal in shape and color before and after staining due to selective permeability, that is, colorless, while non-viable pollen may appear abnormal staining or pollen grain structure damage phenomenon is dyed red, the number of pollen in the field of view and the number of dyed pollen are counted, and the viability of the pollen is calculated, the calculation formula is: the viability of each slide pollen % = (the number of each field of view unstained pollen grain / the total number of each field of view pollen grain) * 100.

[0011] Specifically, collecting pollen includes the following steps:

[0012] Step one: bud collection: in April to May, the kiwi flower buds with full and no damage, large bud stage are cut and packed with a sealing bag for standby;

[0013] Step two: pollen degranulation: the petals are torn at the front end, the anthers are gently peeled off with tweezers, the anthers are placed in a culture dish and dried in a constant temperature drying box, the pollen is scattered, and the pollen is separated with a sieve after drying, and the pollen of different varieties is classified and stored.

[0014] Specifically, the three slides are concave slides.

[0015] Specifically, the concentration of the staining agent is 3%, 5%, or 8%.

[0016] Specifically, the kiwi is diploid, tetraploid or hexaploid kiwi.

[0017] The principle and beneficial effects of the technical solution are as follows:

[0018] The principle of the application is based on the selective permeability of pollen cells. Viable pollen grains have complete cell membranes, which can prevent the pigment in red ink from entering the cells, thereby maintaining colorless; and the cell membrane integrity of non-viable pollen grains is damaged, and red ink can penetrate into the cells, making them dyed red. The staining condition of the pollen grains is observed under a microscope, which can quickly and accurately determine the viability of the pollen.

[0019] The present application is simple and fast in operation, only needs to contact the pollen with the diluted red ink and then perform microscopic examination, does not need complicated culture medium preparation and long time culture, greatly shortens the detection time, and is shortened from several hours or even several days of traditional culture medium in vitro germination method to several minutes. Meanwhile, the present application is low in cost, the red ink is easy to obtain and cheap, and the experimental equipment only needs an ordinary microscope, and is suitable for large-scale popularization and application. In addition, the present application is strong in adaptability to kiwifruit varieties, is suitable for kiwifruit of various ploidy levels such as diploid, tetraploid and hexaploid, the detection result is stable and reliable, is similar to the germination rate of the traditional culture medium in vitro germination method, and avoids the false positive problem that may occur in the traditional method. The present application provides efficient technical support for collection, preservation and utilization of kiwifruit pollen, and has important practical significance for improving kiwifruit planting management level and breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0020] Fig. 1 It is a microscope photograph of the present application 5% red ink staining method for detecting diploid kiwifruit pollen;

[0021] Fig. 2 It is a microscope photograph of the present application 5% red ink staining method for detecting tetraploid kiwifruit pollen;

[0022] Fig. 3 It is a microscope photograph of the present application 5% red ink staining method for detecting hexaploid kiwifruit pollen;

[0023] In the figure, 1 is uncolored viable pollen grain, and 2 is colored non-viable pollen grain. DETAILED DESCRIPTION

[0024] The present application will be further described in detail in combination with the drawings and embodiments:

[0025] Example 1

[0026] A method for rapidly identifying kiwifruit pollen viability, the method comprises:

[0027] 1. Pollen collection

[0028] 1.1 Bud collection

[0029] The collection of kiwifruit pollen needs to be carried out when the bud develops to the large bud stage, at which time the pollen grain is mature but has not yet scattered out, and the quality is best. The specific operation is as follows:

[0030] Time selection: From April to May every year, it is the season of blooming of male flowers of kiwifruit. The collection is carried out on a sunny and windless day to avoid pollen from being damp or blown away by the wind.

[0031] Bud selection: Select excellent diploid, tetraploid or hexaploid kiwifruit male plants. Cut the flower buds that are full, have no defects, and are in the large bud stage. The size and shape of the flower bud are important indicators of its maturity. Mature flower buds are usually bright in color, full in bud, free of pests and mechanical damage.

[0032] Collection tool: Use sterilized scissors or pruners to avoid contamination of the flower buds by bacteria or viruses on the scissors. When cutting the flower buds, try to preserve the integrity of the flower bud structure and avoid damaging the anthers.

[0033] Labeling and storage: Package the collected flower buds in a sealed bag and label them with the collection number, collection time, kiwifruit variety, and ploidy information. The collected flower buds should be taken to the laboratory as soon as possible to avoid exposure to the external environment for a long time, which may cause pollen viability to decrease.

[0034] 1.2 Pollen dehiscence

[0035] Pollen dehiscence is the process of separating pollen grains from anthers. The specific steps are as follows:

[0036] Anther stripping: In the laboratory, place the collected flower buds in a clean culture dish. Use tweezers to gently tear the petals at the tip to expose the anthers. Use tweezers to gently strip the anthers, avoiding damage to the pollen grains. The stripped anthers should be kept intact and the pollen grains should not be contaminated.

[0037] Drying treatment: Place the stripped anthers in a culture dish and dry them in a constant temperature drying oven. The drying temperature is controlled at about 30°C, and the drying time is 12 hours. During the drying process, the drying condition of the anthers should be checked regularly to avoid excessive drying that may cause pollen grains to break.

[0038] Pollen separation: The pollen grains in the dried anthers will naturally scatter. Use an 800-mesh sieve to separate the pollen, removing anther residues and other impurities. The separated pollen should be in uniform powder form without clumping.

[0039] Pollen storage: Store the separated pollen by variety and ploidy. Package it in centrifuge tubes and place it in a sealed bag, noting the collection number, collection time, kiwifruit variety, and ploidy information. For short-term storage, place it in a -20°C freezer, and for long-term storage, place it in a -80°C ultra-low temperature freezer to maintain pollen viability.

[0040] 2. Pollen viability detection

[0041] 2.1 Red ink staining method

[0042] The red ink staining method is a quick and simple method for detecting pollen viability. The specific operation steps are as follows:

[0043] Slide preparation: Choose clean concave slides, wash them with distilled water and dry them to ensure that the surface of the slides is free of impurities and moisture.

[0044] Stain preparation: Dilute red ink to a concentration of 5% and place it in a clean dropper bottle for use. The concentration of red ink has a significant impact on the test results, and a 5% concentration of red ink can accurately distinguish between viable and non-viable pollen grains.

[0045] Pollen staining: Prepare three slides for each batch. Drop 1-2 drops of prepared red ink stain on each concave slide, and use a capillary tube or spotter to evenly distribute a small amount of pollen in the stain, ensuring that the pollen particles are in full contact with the stain. Cover with a cover glass and gently press to ensure even distribution of pollen grains in the stain. Prepare three slides for each batch to ensure the reliability of the test results.

[0046] Microscopic observation: Place the stained slides under a 10x optical microscope immediately after staining. Adjust the focus and brightness of the microscope to obtain a clear field of view. Observe the staining of pollen grains, with viable pollen grains remaining colorless and non-viable pollen grains stained red. The results are shown in Figs. 1-3 .

[0047] Statistical analysis: Randomly select 3-6 non-overlapping fields of view from each slide and take photos. Count the total number of pollen grains and the number of stained pollen grains in each field of view. Calculate the pollen viability using the formula: % Pollen Viability per slide = (∑ Number of unstained pollen grains per field of view / ∑ Total number of pollen grains per field of view) x 100.

[0048] 2.2 Comparative Example 1: In vitro germination method

[0049] The in vitro germination method is a traditional method for detecting pollen viability, which can directly observe pollen germination, but the operation is complex and time-consuming. The specific steps are as follows:

[0050] Medium preparation: The components of the medium include 10% sucrose, 10 mg / L boric acid and 0.5% agar powder. Mix the components evenly and heat until the agar powder completely melts. Pour the medium into 9 petri dishes before it solidifies, and pour an appropriate amount of medium into each petri dish to evenly cover the bottom of the petri dish. Cover the petri dish with a lid and cool it to room temperature naturally.

[0051] Pollen inoculation: Dip a small amount of pollen with a brush and gently flick it onto the petri dish to evenly distribute the pollen. Label the basic information such as pollen variety, experimental time and number on the petri dish with a marker pen.

[0052] Incubation and observation: Place the inoculated petri dishes in a 27°C incubator for 2 hours. After incubation, remove the petri dishes and observe the pollen germination under a microscope. Take 3 biological replicates for each material, and select 3 random fields of view for each biological replicate to take photos and count. Calculate the germination rate using the formula: Germination rate (%) = 100% x number of germinated / total number.

[0053] 2.3 Comparative Example 2: Methylene blue staining method

[0054] Methylene blue staining is also a commonly used method for detecting pollen viability, but its application in kiwifruit is not satisfactory. The specific steps are as follows:

[0055] Stain preparation: Select 0.1% methylene blue as the staining agent and place it in a clean dropper bottle for use.

[0056] Pollen staining: Add 1-2 drops of methylene blue staining agent to the slide, and use a capillary tube or spotter to evenly distribute a small amount of pollen in the staining agent, ensuring that the pollen grains are in full contact with the staining agent. Cover with a cover glass and gently press to ensure even distribution of pollen grains in the staining agent.

[0057] Microscopic observation: Allow the stained slide to stand for 1 minute to allow the pollen to fully stain. Then place it under a 10x optical microscope and observe the staining of the pollen grains. Viable pollen grains stain light blue, while non-viable pollen grains stain dark blue.

[0058] Statistical analysis: Randomly select 3-6 non-overlapping fields of view for each slide to take photos, and count the total number of pollen grains and the number of stained pollen grains in each field of view. Calculate the pollen viability using the formula: % pollen viability per slide = (∑ number of unstained pollen grains per field / ∑ total number of pollen grains per field) x 100.

[0059] 3. Test results

[0060] 3.1 Screening results of staining agent concentration

[0061] In order to determine the optimal concentration of red ink staining agent, this experiment used 3%, 5% and 8% concentrations of red ink staining agent to detect diploid, tetraploid and hexaploid kiwifruit pollen. The specific results are shown in Table 1:

[0062] Table 1 Test results of red ink staining method for different ploidy kiwifruit pollen

[0063]

[0064] The results show that the pollen viability measured by 5% concentration of red ink staining agent is closest to the viability measured by in vitro germination method, and the data is the most stable.

[0065] 3.2 Different ploidy kiwi pollen vitality detection results

[0066] In this experiment, diploid, tetraploid and hexaploid kiwi pollen were detected by 5% red ink staining method, medium in vitro germination method and methylene blue staining method. The specific results are shown in Table 2:

[0067] Table 2 Different methods for detecting kiwi pollen vitality results

[0068]

[0069] The results show that the pollen vitality determination results of the present application example 1 are most similar to the germination rate of the medium in vitro germination method, and the data are most stable. Most of the pollen is dyed yellow by I2-KI staining method, and the pollen without activity is yellow-brown and not easy to distinguish. Reducing the concentration of the staining solution and prolonging the staining time do not change the staining effect, so it is not suitable for kiwi pollen vitality determination method. The pollen vitality detected by 0.1% methylene blue staining method is very close to the liquid germination method, but the use of methylene blue staining may cause false positive, resulting in inaccurate data, and the similar colors are not easy to distinguish.

[0070] In the staining method, red ink, I2-KI, methylene blue and gentian violet staining are mainly used, but they are not suitable for all species. In kiwi, I2-KI staining method, methylene blue staining method and gentian violet staining method have poor staining effect, while red ink staining method and pollen in vitro culture method have good effect.

[0071] 3.3 Different ploidy kiwi pollen vitality detection results

[0072] In this experiment, diploid, tetraploid and hexaploid kiwi pollen were detected by 5% red ink staining method, medium in vitro germination method and methylene blue staining method. The specific results are shown in Table 3:

[0073] Table 3 Different methods for detecting kiwi pollen vitality results

[0074]

[0075] The results show that the pollen vitality determination results of the present application example 1 are most similar to the germination rate of the medium in vitro germination method, and the data are most stable. In kiwi production, it is very important to detect the pollen vitality in advance when using stored pollen for artificial pollination. The 5% red ink detection method is similar to the medium in vitro germination method, and this detection method does not need complex medium preparation and culture process, and the operation is simpler, which can obtain the detection results in a short time, and provides convenience for timely judging the vitality of kiwi pollen in production.

[0076] 3.4 Conclusion

[0077] The experimental results show that the 5% red ink staining method is a rapid, simple and accurate kiwi pollen viability detection method. Compared with the traditional medium in vitro germination method and methylene blue staining method, the 5% red ink staining method has the following advantages:

[0078] Simple operation: without complex medium preparation and long-term culture, only need to contact the pollen with red ink and then observe under microscope, simple and fast operation.

[0079] Fast detection: from pollen staining to microscopic observation, the whole process only takes a few minutes, greatly shortening the detection time.

[0080] Low cost: red ink is easy to get and cheap, and the experimental equipment only needs an ordinary optical microscope, which is suitable for large-scale popularization and application.

[0081] Stable results: the detection results are stable and reliable, the germination rate is most similar to that of the medium in vitro germination method, and the false positive problem in the methylene blue staining method is avoided.

[0082] The present application provides efficient technical support for the collection, preservation and utilization of kiwi pollen, and has important practical significance for improving the management level and breeding efficiency of kiwi planting.

[0083] The above is only an embodiment of the present application, and the specific technical solutions or characteristics of the scheme are not described in detail. For those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should be regarded as the protection scope of the present application. The scope of protection of the present application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. A method for rapidly identifying kiwi pollen viability, characterized in that: The following steps are involved: Step 1: Collect pollen: Collect kiwifruit buds at the large bud stage, peel them manually or dry them as required to release the pollen in the anthers; Step 2: Prepare the dye: dilute the red ink and place it in a bottle for later use; Step 3: Pollen staining: Prepare three slides for each batch. Clean each slide and place 1-2 drops of the prepared stain on each concave slide. Use a capillary or spotter to evenly sprinkle a small amount of pollen into the stain, ensuring full contact between the pollen grains and the stain. Cover with a coverslip and immediately examine under a microscope. Step 4: Pollen Vitality Observation: Place the stained slide under a microscope for observation. Adjust the focus and brightness of the microscope to obtain a clear field of view and observe the staining of the pollen grains under the microscope. Step 5: Pollen viability statistics: Viable pollen grains will maintain their normal shape and color before and after staining due to their selective permeability, that is, they will be colorless. In contrast, inactive pollen may be stained abnormally or have structural damage, and may be stained red. The number of pollen in the field of view and the number of stained pollen are counted, and the pollen viability is calculated using the following formula: Pollen viability per slide (% = (∑ number of unstained pollen grains per field of view / ∑ total number of pollen grains per field of view) × 100.

2. The method for rapidly identifying kiwifruit pollen viability according to claim 1, wherein: Collecting pollen involves the following steps: Step 1: Collect flower buds: From April to May, cut off the kiwi buds that are full, intact, and in the large bud stage, put them in a sealed bag and set aside; Step 2: Pollen threshing: Tear open the tip of the petals, gently peel off the anthers with tweezers, place the anthers in a culture dish, and put it in a constant temperature drying oven to dry so that the pollen is released. After the pollen is dry, use a sieve to separate the pollen and classify and store different varieties of pollen.

3. The method for rapidly identifying kiwifruit pollen viability according to claim 1, wherein: The three slides are all concave slides.

4. The method for rapidly identifying kiwifruit pollen viability according to claim 1, wherein: The concentration of the dye is 3%, 5% and 8%.

5. The method for rapidly identifying kiwifruit pollen viability according to claim 1, wherein: The kiwi fruit is a diploid, tetraploid or hexaploid kiwi fruit.

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