Method for rapidly detecting vitality of annona squamosa pollen

Abstract

The invention belongs to the field of annona squamosa pollen detection, and particularly relates to a method for rapidly detecting the vitality of annona squamosa pollen. Compared with traditional FDA-PI double staining, the mixed solution for pollen viability detection has longer fluorescence lifetime, the pollen maintains the form without bursting within a certain time, the in-vitro germination process can be continuously observed, the verification of the detection result of pollen viability is facilitated, and the accuracy of the detection result is improved. The mixed solution can count the number of pollen cell nucleuses, helps to determine the pollen development stage, can mutually verify the pollen activity detection result, is convenient to comprehensively judge the pollen activity, is higher in dyeing speed, does not have processes such as centrifugation, and is simpler and more convenient to operate.

Description

Technical Field

[0001] The invention belongs to the field of sugar apple pollen detection, and particularly relates to a method for quickly detecting the vitality of sugar apple pollen. Background Art

[0002] Sugar apple, also known as sugar apple, is a fruit of the genus Annona in the family Annonaceae. Annona ) is a perennial semi-deciduous small tree with a cultivation history of more than 400 years in tropical and subtropical regions of my country (Fujian, Guangdong, Guangxi, Yunnan, Hainan, etc.). In recent years, as the cultivation of traditional tropical crops / fruit trees such as bananas, mangoes, and sugarcane has become saturated, sugar apples have been sought after year by year and have become a new growth point. However, its related cultivation technology is still in its infancy in my country, and there are still many aspects to be optimized in theoretical research and actual production and planting. In this regard, it is of great significance to carry out targeted exploration of some key issues in sugar apple production and scientific research.

[0003] Artificial pollination refers to the technical measure of artificially transferring plant pollen to the stigma to complete fertilization. In production, it is an important means to improve the fruit setting rate and fruit quality of sugar apple varieties. At the same time, for the innovation of sugar apple germplasm resources, it is also one of the important technologies for the creation of new germplasm. There are two main methods of sugar apple pollination: 1. Natural pollination (wind pollination, insect pollination, etc.): Use air flow and natural behaviors such as insects to complete pollination. Advantages: labor saving in the early stage; Disadvantages: For the main varieties of AP soursop, pineapple sugar apple, etc., the yield is extremely low, the fruit shape is irregular, the fruit ripening period is scattered, the harvesting is inconvenient, and the low input and low output.

[0004] 2. Artificial pollination method: Collect pollen manually and perform artificial pollination on the pistils that are in a pollinable state. Specifically: Prune in batches (calculated according to market demand and fruit development period), check the female flowers for fertility before flowering (whether the environmental temperature and humidity are suitable, whether the stigma is normally developed, and whether it is moist and covered with mucus), collect the mature flowers from the pollination trees about 4-6 hours before flowering, store them in batches in a cool place, and collect pollen after the stamens and anthers burst naturally The flowers that are in the pistil mature state one day before flowering are smeared for pollination. Advantages: Pollination can be centralized according to the market batch, centralized optimization management, synchronized fruit maturity, regular fruit, high quality, and higher input-output than the first two methods. Disadvantages: Pollen collection needs to be carried out in advance on each pollination day, which requires labor. In addition, there are obvious fluctuations in pollen vitality, and even pollen abortion occurs, resulting in poor pollination results and time-consuming and labor-intensive problems.

[0005] From the above comparison, it can be seen that artificial pollination of sugar apple flowers is the best production solution at present. For artificial pollination, sufficient pollination and fruit setting rate are highly dependent on sufficient pollen vitality. The accurate vitality judgment of pollen in storage or near maturity is of great significance to the smooth development of scientific research on sugar apple hybridization experiments or artificial pollination in production.

[0006] Most sugar apples have the characteristic of blooming after a fixed number of days after pruning. Therefore, artificial differentiated pruning in the appropriate season can synchronize the flowering period of different sugar apples. However, this alone cannot achieve ideal pollination results. The interaction between the pistil and the stamen is complex when flowering is approaching. Sugar apple flowers are complete flowers (each flower includes a fully functional pistil group and stamen group), with clear female and male periods. Most varieties have pistils that mature first. The flowering process includes petal cracking and rapid opening (pistil maturity period), disappearance of stigma mucus, browning of pistil group (pistil aging, loss of receptivity), anther bursting, and pollen release (stamen maturity period). Such characteristics are destined to have a natural time difference in the optimal pollination period of pollen and stigma, and they cannot be synchronized. The pollen of the main cultivated varieties AP sugar apple and pineapple sugar apple generally matures in the afternoon or evening, and the pollination time in production is 8-11 pm.

[0007] Pollen vitality mainly depends on the environmental temperature, humidity, pollen state (water content, maturity, contents) and the combination of the above factors. Pollen of Annonaceae plants is sensitive to dryness due to its high water content, and cannot survive for a long time after cracking. The temperature and humidity during the flowering period have a significant impact on the development process and the vitality of the stamens and pistils. Drought and high temperature can cause abnormal flower development and pollen abortion, while excessive humidity can also affect the cracking and release of pollen from anthers. Therefore, the judgment and rapidity of pollen vitality are of great significance to improving the efficiency of artificial pollination of Annona. Summary of the invention

[0008] The present invention provides a staining method for rapidly testing the vitality and developmental stage of sugar apple pollen, which can not only detect the vitality of pollen, but also further trace its developmental period and cell nucleus dynamics, providing convenience for pollen vitality detection in production and theoretical research on pollen germination process.

[0009] The technical solution of the present invention is achieved in this way: A mixed solution for dyeing custard apple pollen comprises the following components in mass concentrations: sucrose 6-16%, coconut milk (CCM) 0.1-1%, calcium nitrate (Ca(NO3)2·4H2O) 250μg / mL, magnesium sulfate (MgSO4·7H2O) 200μg / mL, potassium nitrate (KNO3) 100μg / mL, boric acid (H3BO3) 100μg / mL, PEG8000 5%, fluorescein diacetate (FDA) 20-50μg / mL, and propidium iodide (PI) 3-10μg / mL.

[0010] Furthermore, the mixed solution for dyeing custard apple pollen includes the following components in mass concentrations: sucrose 6-16%, coconut milk 0.1-1%, calcium nitrate 250 μg / mL, magnesium sulfate 200 μg / mL, potassium nitrate 100 μg / mL, boric acid 100 μg / mL, PEG8000 5%, fluorescein diacetate 20-50 μg / mL, and propidium iodide 3-5 μg / mL.

[0011] Furthermore, the mixed solution for dyeing custard apple pollen includes the following components in mass concentrations: sucrose 6-16%, coconut milk 0.1-1%, calcium nitrate 250 μg / mL, magnesium sulfate 200 μg / mL, potassium nitrate 100 μg / mL, boric acid 100 μg / mL, PEG8000 5%, fluorescein diacetate 20 μg / mL, and propidium iodide 3 μg / mL.

[0012] The above-mentioned method for preparing a mixed solution for dyeing custard apple pollen comprises the following steps: (1) Take the formulated amount of fluorescein diacetate and dissolve it completely in a small amount of acetone, with the upper limit being just enough to dissolve all the solute, and set aside; (2) Take the formulated amount of propidium iodide and dissolve it in deionized water; add sucrose, coconut milk, calcium nitrate, magnesium sulfate, potassium nitrate, boric acid and PEG8000 according to the ratio, add deionized water to dissolve sucrose, coconut milk, calcium nitrate, magnesium sulfate, potassium nitrate, boric acid and PEG8000 until the volume is about 95% of the total volume, add the fluorescein diacetate dissolved in step (1), and make up to the same volume.

[0013] A method for rapidly testing the viability of sugar apple pollen comprises the following steps: (1) Stamen pretreatment Disperse the anthers for later use; (2) Reagent mixing and incubation Transfer the pollen to be tested to the sample slot of the silica gel staining plate, add the above-mentioned mixed solution, repeatedly suck and spit with a pipette to fully react, keep it in a dark place for 30 seconds, then add it to the slide and cover it with a cover glass; (3) Fluorescence microscopy Wide-field fluorescence microscopy: Select BF, WG and WB channels for bright field, red fluorescence and green fluorescence observation and image acquisition respectively. Bright field is used for pollen appearance and aggregation detection, red fluorescence is used for pollen cell nucleus number detection, and green fluorescence is used for pollen vitality detection. Laser confocal microscopy: select 488nm laser beam as excitation light, collect green fluorescence of fluorescein diacetate at 520nm wavelength and red fluorescence of propidium iodide at 647nm; collect ESID bright field signal to observe the appearance of pollen; collect images of the above three signals; (4) Data statistics The software was used to perform statistical calculations on the images of different channels and calculate the pollen vitality.

[0014] Furthermore, in step (1), if the anther is about to bloom and the pollen is about to burst (exploding), or if the anther is stored, the anther can be directly taken out for the next step of processing; if it is an earlier anther that has not been separated from the flower, it is necessary to use tweezers to break up the anther so as to separate it from the flower.

[0015] Furthermore, in the step (2), 9-20 μL of the above-mentioned mixed solution is added to every 10 anthers.

[0016] Furthermore, in the step (2), the pipette is repeatedly aspirated 5 times; the solution is kept in a dark place for 30 seconds; and the cover glass is a 6 mm diameter round cover glass.

[0017] Further, in step (4), the pollen vitality is calculated as follows: pollen vitality = number of pollens producing green fluorescence / number of pollens * 100%, and / or pollen vitality = number of pollens not colored in the pollen / number of pollens * 100% (for comprehensive determination) Furthermore, the step (4) is calculated and statistically analyzed using ImageJ software.

[0018] Beneficial effects of the present invention: The mixed solution and detection method of the present invention have the following advantages: The mixed solution of the present invention is used to detect pollen vitality, which has a longer fluorescence life than the traditional FDA-PI double staining. The pollen maintains its shape without bursting for a certain period of time, which can be maintained for up to 10 hours. The in vitro germination process can be continuously observed, which is conducive to verifying the test results of pollen vitality and improving the accuracy of the test results.

[0019] The amount of mixed solution used each time can be as low as 10μL (reagent concentration FDA 20μL / mg and PI 3.0μL / mg). The amount of fluorescent reagent used is greatly reduced, the toxicity is reduced, and the total consumption can be greatly reduced compared to the conventional 100μL each time.

[0020] In addition to supporting the FDA's identification results on cell viability, it can also partially play the role of cell nucleus staining. The staining ability of this mixture for cell nuclei can partially replace DAPI. For samples with low viability, directly stain the WG channel or 647nm to observe and count the cell nuclei. For samples with high viability, only an additional inactivation step is required to count the number of pollen nuclei, which helps determine the stage of pollen development. It can be mutually verified with the pollen viability test results, which is convenient for comprehensive judgment of pollen viability.

[0021] Compared with the appearance inspection of water dispersion, TTC, and conventional iodine-potassium iodide staining, the results are more reliable.

[0022] The dyeing speed is faster, and there is no centrifugation process. It can be used to dye cherimoya, yellow dragon cherimoya, African pride, pineapple sugar apple, Thai cherimoya, etc., and the operation is easier. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 Effect of different sucrose concentrations on staining effect.

[0025] Figure 2 Effects of different FDA concentrations on staining effects.

[0026] Figure 3 The staining effect was achieved when the PI concentration was 3 μg / mL. DETAILED DESCRIPTION

[0027] 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. Example 1

[0028] A mixed solution for dyeing custard apple pollen comprises the following components in mass concentrations: 6% sucrose, 0.1% coconut milk (CCM), 250 μg / mL calcium nitrate (Ca(NO3)2·4H2O), 200 μg / mL magnesium sulfate (MgSO4·7H2O), 100 μg / mL potassium nitrate (KNO3), 100 μg / mL boric acid (H3BO3), 5% PEG8000, 50 μg / mL fluorescein diacetate (FDA), and 10 μg / mL propidium iodide (PI); wherein FDA and PI are improved fluorescent dyes, and the other components are components required for maintaining the vitality and osmotic pressure of in vitro pollen.

[0029] A method for preparing a mixed solution for dyeing custard apple pollen: (1) Take the formulated amount of FDA and dissolve it completely in a small amount of acetone, with the upper limit being just enough to dissolve all the solute, and set aside; (2) Take the formulated amount of PI and dissolve it in deionized water; add sucrose, coconut milk, calcium nitrate, magnesium sulfate, potassium nitrate, boric acid and PEG8000 according to the ratio, add deionized water to dissolve sucrose, coconut milk, calcium nitrate, magnesium sulfate, potassium nitrate, boric acid and PEG8000 until the volume is about 95% of the total volume, add the FDA and PI dissolved in step (1) and make up to the same volume. Example 2

[0030] A mixed solution for dyeing custard apple pollen comprises the following components in mass concentrations: 8% sucrose, 1% coconut milk (CCM), 250 μg / mL calcium nitrate (Ca(NO3 )2·4H2O), 200 μg / mL magnesium sulfate (MgSO4·7H2O), 100 μg / mL potassium nitrate (KNO3), 100 μg / mL boric acid (H3BO3), 5% PEG8000, 20 μg / mL fluorescein diacetate (FDA), and 3 μg / mL propidium iodide (PI).

[0031] The preparation method is the same as that in Example 1. Example 3

[0032] A mixed solution for dyeing custard apple pollen comprises the following components in mass concentrations: 10% sucrose, 0.5% coconut milk (CCM), 250 μg / mL calcium nitrate (Ca(NO3 )2·4H2O), 200 μg / mL magnesium sulfate (MgSO4·7H2O), 100 μg / mL potassium nitrate (KNO3), 100 μg / mL boric acid (H3BO3), 5% PEG8000, 20 μg / mL fluorescein diacetate (FDA), and 5 μg / mL propidium iodide (PI).

[0033] The preparation method is the same as that in Example 1. Example 4

[0034] A mixed solution for dyeing custard apple pollen comprises the following components in mass concentrations: 16% sucrose, 0.1% coconut milk (CCM), 250 μg / mL calcium nitrate (Ca(NO3 )2·4H2O), 200 μg / mL magnesium sulfate (MgSO4·7H2O), 100 μg / mL potassium nitrate (KNO3), 100 μg / mL boric acid (H3BO3), 5% PEG8000, 22 μg / mL fluorescein diacetate (FDA), and 3 μg / mL propidium iodide (PI).

[0035] The preparation method is the same as that in Example 1. Example 5

[0036] A method for rapidly testing the viability of sugar apple pollen comprises the following steps: (1) Stamen pretreatment Disperse the anthers for later use; if they are about to bloom (explode), or if they are stored anthers, just take them out for the next step; if they are older anthers that have not been separated, use tweezers to break them apart so that they can be separated from the flower; (2) Reagent mixing and incubation Use flat-mouthed tweezers to pinch and transfer the pollen and anthers to be tested to the sample slot of the silica gel staining plate, and drip the mixed solution described in any one of Examples 1-4, and drip 9-20 μL of the mixed solution for every 10 anthers; use a pipette to repeatedly suck and spit 5 times for sufficient reaction, keep it away from light for 30 seconds, then drip it onto the slide, and cover it with a 6 mm diameter round cover glass; (3) Fluorescence microscopy Wide-field fluorescence microscopy: Select BF, WG and WB channels for bright field (pollen appearance, aggregation detection), red fluorescence (pollen cell nucleus number detection) and green fluorescence (pollen vitality detection) observation and image acquisition respectively; Laser confocal microscopy: The excitation light is a 488nm laser beam, and the detection light wavelengths are collected near the peaks of 520nm and 647nm, respectively corresponding to the green fluorescence of fluorescein diacetate and the red fluorescence of propidium iodide; at the same time, the bright field signal of ESID (pollen appearance) is collected; the above three signals are imaged; (4) Data statistics The images of different channels were calculated and statistically analyzed using ImageJ and other software to calculate pollen vitality.

[0037] The pollen vitality is calculated as follows: pollen vitality = number of pollens producing green fluorescence / number of pollens * 100%, or pollen vitality = pollens not colored in the pollen / number of pollens * 100% for comprehensive determination. The two are mutually verified to improve the accuracy of detection.

[0038] 1. Comparison of the effects of different dyeing methods on the dyeing of Annona pollen The stamens of AP sugar apples were collected at different times and randomly divided into 6 groups. The pollen viability was tested using the methods in Table 1. The test results were statistically analyzed and compared with the different test methods. The results of staining and live pollen viability test are shown in Table 2.

[0039] Table 1 Comparison of different staining methods

[0040] Table 2 Statistics of different staining methods

[0041] It can be seen from Table 1 and Table 2 that the improved FDA-PI staining method described in the present invention not only has stable results and strong repeatability, but also has higher accuracy. It can stain the pollen of multiple varieties of sugar apples, such as custard apple, yellow dragon sugar apple, African pride, pineapple sugar apple, Thai sugar apple, etc., and can accurately judge the pollen vitality, and the pollen detection effect is significantly improved.

[0042] 2. Effects of different dyeing conditions on the dyeing effect of Annona pollen In order to compare the effects of different staining conditions on the staining effect, AP custard apple pollen collected at the same time was taken, and different concentrations of sucrose, FDA and PI were selected to prepare mixed solutions. Then, the method of Example 5 was used to detect pollen vitality, and the green fluorescence, red fluorescence and pollen morphology retention time were measured respectively. The specific results are shown in Table 3.

[0043] Table 3 Comparison of dyeing effects under different dyeing conditions Serial number Sucrose concentration (%) FDA concentration (μg / mL) PI concentration (μg / mL) Dyeing effect 1 6 20 3 Green fluorescence: + Red fluorescence: + Holding time: +++ 2 8 20 3 Green fluorescence: + Red fluorescence: + Holding time: +++++ 3 10 20 3 Green fluorescence: + Red fluorescence: + Holding time: +++++ 4 6 50 3 Green fluorescence: ++Red fluorescence: +Retention time: +++ 5 8 50 3 Green fluorescence: ++Red fluorescence: +Retention time: +++ 6 10 50 3 Green fluorescence: ++Red fluorescence: +Retention time: +++ 7 6 20 5 Green fluorescence: + Red fluorescence: +++ Holding time: +++ 8 8 20 5 Green fluorescence: + Red fluorescence: ++ Holding time: ++++ 9 10 20 5 Green fluorescence: + Red fluorescence: ++ Holding time: +++ 10 6 20 1 Green fluorescence: + Red fluorescence: Cell nucleus cannot be displayed Holding time: ++++ 11 8 20 1 Green fluorescence: + Red fluorescence: Cell nucleus cannot be displayed Holding time: +++++ 12 10 20 1 Green fluorescence: + Red fluorescence: Cell nucleus cannot be displayed Holding time: ++++ 13 6 10 3 Green fluorescence: - (barely distinguishable) Red fluorescence: + Holding time: ++++ 14 8 10 3 Green fluorescence: confused with autofluorescence Red fluorescence: + Holding time: +++++ 15 10 10 3 Green fluorescence: confused with autofluorescence Red fluorescence: + Holding time: ++++ As can be seen from Table 3, under the concentration ratio conditions of treatment groups 1-9, both green fluorescence and red fluorescence can be observed, and pollen can be maintained for a certain period of time, but under the concentration ratio conditions of treatment groups 8-15, the observation of green fluorescence and red fluorescence is affected to varying degrees. Therefore, the concentration ratio of sucrose, FAD and PI has a great influence on the retention time of fluorescence and pollen morphology. In order to better determine the appropriate concentration, the staining condition determination experiment was further carried out based on the above results.

[0044] 3. Determination of FDA-PI staining conditions The pollen used in this experiment was from Annona mume collected at the same time.

[0045] 1. Sucrose concentration Mixed solutions (FDA 22 μg / mL, PI 3 μg / mL) were prepared at sucrose concentrations of 0%, 4%, 8%, 16%, and 25%, respectively, and pollen viability was tested according to the method of Example 5. The specific results are as follows.

[0046] (1) 0% sucrose-free component, with the strongest fluorescence reaction at 1-3 min, but the pollen outer wall was quickly damaged ( Figure 1 1 arrow), cannot maintain integrity for a long time ( Figure 1 4).

[0047] After the sucrose component was added, the fluorescence was not obvious in the first 2 minutes, and then slowly increased.

[0048] (2) 4% sucrose: normal for 3 min, then the pollen outer wall began to rupture ( Figure 1 2 arrows).

[0049] (3) 8% sucrose, most of them remained normal ( Figure 1 3) until some pollen tubes germinate, which indicates that this concentration should be the relative optimal state ( Figure 1 The 6 arrows show the germination of pollen tubes).

[0050] (4) 16% sucrose, initial normal fluorescence color development, followed by pollen cytoplasm shrinkage 5 minutes later ( Figure 1 shown in 5) (5) 25% sucrose, same as 16% sucrose.

[0051] 2.FDA concentration FDA prepared mixed solutions according to the concentration gradient of 2, 20, 200, and 2000 μg / mL, respectively, and performed pollen viability detection according to the method of Example 5. The results are as follows.

[0052] 2μg / mL is almost indistinguishable from autofluorescence ( Figure 2 1), green fluorescence can be seen at 20μg / mL ( Figure 2 2), 200μg / mL green fluorescence intensity increased. ( Figure 2 3) But it will dry within 5 minutes. 2000μg / mL will dry within 3 minutes, and the pollen will be inactivated quickly. It is not suitable for both dosage and vitality maintenance ( Figure 2 4).

[0053] Figure 2 The dosage of PI in the experiment was 3 μg / mL. At the same time, within the experimental concentration range of 3-10 μg / mL, the dosage of PI had no effect on the green fluorescence detection results.

[0054] Therefore, FDA 20 μg / mL is the lower limit of concentration.

[0055] 3. Effect of PI concentration The 10μg / mL concentration of PI can be further optimized (especially for AP and indehiscent soursop). The final concentration as low as 3μg / mL is the concentration at which nuclear energy can be displayed. Figure 3 As shown. FDA has no effect on red fluorescence detection in the concentration range of 20-50μg / mL and sucrose 6-10%. Figure 3 The FDA dosage is 20.μg / mL.

[0056] 4. Amount of dye used The mixed solutions were prepared according to the ratios in Example 2, and then 10 anthers were taken from each sample. Pollen viability was tested according to the method in Example 5. The results are shown in Table 4.

[0057] Table 4 Effect of mixed solution dosage on detection results Serial number Mixed solution volume (μL) Test results 1 3 The pollen cannot be dispersed and cannot be released from the anther normally. Even if the pollen can be sucked out and transferred to the slide during the loosening stage, it will quickly dry and die due to the low amount of liquid. 2 5 Pollen is not easy to come out, and the amount of pollen is too small to be counted. If there are too many anthers, it will not be diluted enough, and the pollen will dry quickly and cannot be observed. 3 8 The activity is not affected, but the liquid dries quickly. Without a 6mm coverslip, the time required for observation and fluorescence microscopy photography cannot be met. 4 9-10 The dosage is moderate. 9μL with a coverslip will have a more lasting effect. 10μL can maintain a stable color state for 10 minutes even without a coverslip, and complete repeated observations and photography of more than three fields of view. 5 20 The effect is the same as 9-10μL, but the amount of mixed solution is increased, and the cost is increased. It can be seen from Table 3 that adding 9-20 μL of the mixed solution to every 10 anthers can achieve a good staining effect and meet the detection requirements, but the increase in the amount of the mixed solution will increase the experimental cost. Therefore, it is better to add 9-10 μL of the mixed solution to every 10 anthers.

[0058] 5. Stamen detection results at different developmental stages Stamens at different developmental stages and in vitro were taken respectively, and mixed solutions were prepared according to the ratio in Example 2. Then, pollen viability was tested according to the detection method in Example 5. The comparative test results are shown in Table 5.

[0059] Table 5 Detection results of stamens at different developmental stages

[0060] It can be seen from Table 5 that the stamens that were not separated from the mother plant and the stamens that were loosely opened during the male flowering period can achieve good test results. Stamens preserved in vitro can still show activity, but are difficult to germinate.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A mixed solution for dyeing custard apple pollen, characterized in that: The mixed liquid includes the following components in mass concentrations: sucrose 6-16%, coconut milk 0.1-1%, calcium nitrate 250 μg / mL, magnesium sulfate 200 μg / mL, potassium nitrate 100 μg / mL, boric acid 100 μg / mL, PEG8000 5%, fluorescein diacetate 20-50 μg / mL, and propidium iodide 3-10 μg / mL.

2. A mixed solution for dyeing sugar apple pollen as claimed in claim 1, characterized in that: The mixed solution for dyeing custard apple pollen includes the following components in mass concentrations: 8-10% sucrose, 1% coconut milk, 250 μg / mL calcium nitrate, 200 μg / mL magnesium sulfate, 100 μg / mL potassium nitrate, 100 μg / mL boric acid, 5% PEG8000, 20-50 μg / mL fluorescein diacetate, and 3-5 μg / mL propidium iodide.

3. A mixed solution for dyeing sugar apple pollen as claimed in claim 1, characterized in that: The mixed solution for dyeing custard apple pollen includes the following components in mass concentrations: 8% sucrose, 1% coconut milk, 250 μg / mL calcium nitrate, 200 μg / mL magnesium sulfate, 100 μg / mL potassium nitrate, 100 μg / mL boric acid, 5% PEG8000, 20 μg / mL fluorescein diacetate, and 3 μg / mL propidium iodide.

4. A method for preparing a mixed solution for dyeing custard apple pollen according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Take the formulated amount of fluorescein diacetate and dissolve it completely in a small amount of acetone, with the upper limit being just enough to dissolve all the solute, and set aside; (2) Dissolve the formulated amount of propidium iodide in deionized water; add sucrose, coconut milk, calcium nitrate, magnesium sulfate, potassium nitrate, boric acid and PEG8000 according to the ratio; add deionized water to dissolve sucrose, coconut milk, calcium nitrate, magnesium sulfate, potassium nitrate, boric acid and PEG8000 until the volume is 95% of the total volume; add the fluorescein diacetate dissolved in step (1) and make up to the same volume.

5. A method for rapidly testing the viability of sugar apple pollen, characterized in that: The following steps are involved: (1) Stamen pretreatment Disperse the anthers for later use; (2) Reagent mixing and incubation Use flat-mouthed tweezers to pinch and transfer the pollen and anther to be tested to the sample slot of the silica gel staining plate, add the mixed solution described in any one of claims 1 to 3, repeatedly suck and spit with a pipette to fully react, keep it away from light for 30 seconds, then add it dropwise to the slide glass, and cover it with a cover glass; (3) Fluorescence microscopy Wide-field fluorescence microscopy: Select BF, WG and WB channels for bright field, red fluorescence and green fluorescence observation and image acquisition respectively. Bright field is used for pollen appearance and aggregation detection, red fluorescence is used for pollen cell nucleus number detection, and green fluorescence is used for pollen vitality detection. Laser confocal microscopy: select 488nm laser beam as excitation light, collect green fluorescence of fluorescein diacetate at 520nm wavelength and red fluorescence of propidium iodide at 647nm; collect ESID bright field signal to observe the appearance of pollen; collect images of the above three signals; (4) Data statistics The software was used to perform statistical calculations on the images of different channels and calculate the pollen vitality.

6. The method according to claim 5, characterized in that If the pollen is about to burst out of flowering, or it is a stored anther, you can directly take out the anther and proceed to the next step; if it is an earlier anther that has not been separated from the flower, you need to use tweezers to break up the anther so that it can be separated from the flower.

7. The method according to claim 5, characterized in that In the step (1), the stamens to be dispersed include stamens that have not been separated from the mother plant, stamens that have just entered the male flowering period and are loosely bursting, and stamens that are preserved in vitro.

8. The method according to claim 5, characterized in that In the step (2), 9-20 μL of the above-mentioned mixed solution is added to every 10 anthers; the liquid is repeatedly sucked and discharged by a pipette for 5 times; the mixture is kept in a dark place for 30 seconds; and the cover glass is a 6 mm diameter round cover glass.

9. The method according to claim 5, characterized in that In the step (4), the pollen vitality is calculated as follows: pollen vitality = number of pollens producing green fluorescence / number of pollens * 100%, and / or pollen vitality = number of pollens that are not colored in the pollen / number of pollens * 100%.

10. The method according to claim 5, characterized in that The step (4) is calculated and statistically analyzed using ImageJ software.

Images