Method for rapidly determining vitality of blueberry pollen

Abstract

The invention provides a method for rapidly determining the vitality of blueberry pollen, and relates to the technical field of blueberry pollen vitality detection. The method for rapidly determining the vitality of the blueberry pollen comprises the following specific steps: step 1, pollen collection: when blueberry flowers are in a big flower bud stage, picking the flowers into a self-sealing bag, putting the self-sealing bag into a preservation box with ice cubes, taking the preservation box back to a laboratory, and performing detection on the day; and step 2, preparing a PBS solution, namely fixing the volume of 3.16 g of monopotassium phosphate to 100 ml, fixing the volume of 2.28 g of dipotassium phosphate to 100 ml, preparing a mixed solution, and adding an acidic titration solution or an alkaline titration solution to prepare the PBS solution with the pH value of 7.0. TTC (2, 3, 5-triphenyltetrazole chloride) is dissolved in a PBS solution, a 1% TTC solution is prepared for dyeing, the blueberry pollen suspension prepared by the method can fully absorb a reaction solution to avoid pollen viability errors caused by individual differences, meanwhile, the blueberry pollen dyeing time can be 20 min, and the blueberry pollen viability detection time is greatly shortened.

Description

Technical Field

[0001] This invention relates to the field of blueberry pollen viability detection technology, specifically a method for rapidly determining blueberry pollen viability. Background Technology

[0002] Pollen viability significantly affects the fruit set rate of blueberries. Blueberries are clump-forming shrubs belonging to the genus Vaccinium in the Ericaceae family, with bell-shaped flowers that do not easily shed pollen. Due to the large number of blueberry varieties and their inconsistent flowering periods, blueberry hybridization breeding is challenging. Therefore, rapid and accurate determination of blueberry pollen viability is crucial.

[0003] Currently, TTC staining is commonly used to detect blueberry pollen viability. This method is simple to operate but time-consuming, usually taking 2 hours. This new method can significantly shorten the detection time, providing results in just 20 minutes of dark reaction. Summary of the Invention

[0004] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for rapidly determining blueberry pollen viability, solving the problem of long reaction time during the detection of blueberry pollen viability.

[0005] (II) Technical Solution To achieve the above objectives, the present invention provides a method for rapidly determining blueberry pollen viability, comprising the following specific steps: Step 1, Pollen Collection: When the blueberry flowers are in the large bud stage, pick the flowers, put them in a resealable bag, place them in an ice-filled container, and bring them back to the laboratory for testing on the same day; Step 2, PBS solution preparation: Add 3.16g potassium dihydrogen phosphate to 100ml and 2.28g dipotassium hydrogen phosphate to 100ml to prepare a mixed solution. Then add acidic or alkaline titrant to adjust the pH of the PBS solution to 7.0. Step 3: TTC solution preparation. Dissolve 0.1g of 2,3,5-triphenyltetrazolium chloride in a small amount of alcohol, add PBS solution to make up to 100ml, and prepare a 1% TTC solution for later use. Step 4: Preparation of pollen suspension: Shake the collected pollen into a 2ml centrifuge tube, add 1ml of the TTC solution from step 3, shake well, and prepare a pollen suspension for later use.

[0006] Step 5: Constant temperature incubation: Place the pollen suspension in an incubator for incubation, and remove it after 20 minutes.

[0007] Step 6: Slide preparation: Take 20 μL of the pollen suspension from step 5 and drop it onto the slide. Then, slowly cover the slide with a coverslip from one side.

[0008] Step 7: Observation and Calculation: Observe the slide using a microscope and calculate the blueberry pollen viability according to the calculation formula to obtain the blueberry pollen viability.

[0009] Preferably, the blueberry pollen used in step one is Camellia, Emerald, or H5.

[0010] Preferably, the concentration of the TTC solution in step three is 1%.

[0011] Preferably, in step five, the incubator is equipped with a temperature control device to maintain the temperature inside the incubator at 35°C, and the incubation method is dark incubation.

[0012] Preferably, the method for observing and judging the blueberry pollen viability in step seven is to observe the color, wherein pollen grains that are stained red or pink are viable, and pollen grains that do not change color are inactive; the formula for calculating blueberry pollen viability is: pollen viability (%) = (number of stained pollen grains / total number of observed pollen grains) × 100%.

[0013] (III) Beneficial Effects This invention provides a rapid method for determining blueberry pollen viability. It has the following beneficial effects: This invention involves dissolving TTC (2,3,5-triphenyltetrazolium chloride) in PBS solution to prepare a 1% TTC solution for staining. This method prepares a blueberry pollen suspension that can fully absorb the reaction solution and avoid pollen viability errors caused by individual differences. At the same time, it can reduce the blueberry pollen staining time to 20 minutes, greatly shortening the blueberry pollen viability detection time. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1: This invention provides a method for rapidly determining blueberry pollen viability, comprising the following specific steps: Step 1, Pollen Collection: When the blueberry flowers are in the large bud stage, the flowers are picked and placed in a resealable bag, then put in an ice-filled container and brought back to the laboratory for testing on the same day. The varieties of blueberry pollen used in Step 1 are Camellia, Emerald, and H5. Step 2, PBS solution preparation: Add 3.16g potassium dihydrogen phosphate to 100ml and 2.28g dipotassium hydrogen phosphate to 100ml to prepare a mixed solution. Then add sodium hydroxide solution to adjust the pH of the PBS solution to 7.0. Step 3: TTC solution preparation. Dissolve 0.05g, 0.1g, and 0.15g of 2,3,5-triphenyltetrazolium chloride in a small amount of alcohol, add PBS solution to make up to 100ml, and prepare 0.5% TTC, 1% TTC, and 1.5% TTC solutions for later use. Step 4: Preparation of pollen suspension: Shake the collected pollen into a 2ml centrifuge tube, add 1ml of the TTC solution from step 3, shake well, and prepare a pollen suspension for later use.

[0016] Step 5: Constant temperature incubation: Place the pollen suspension in an incubator for incubation, and remove it after 20 minutes and 35 minutes.

[0017] Step 6: Slide preparation: Take 20 μL of the pollen suspension from step 5 and drop it onto the slide. Then, slowly cover the slide with a coverslip from one side.

[0018] Step 7: Observation and Calculation: Observe the slide using a microscope and calculate the blueberry pollen viability according to the calculation formula to obtain the blueberry pollen viability.

[0019] The method for observing and judging the viability of blueberry pollen is to observe the color. Pollen grains that are stained red or pink are viable, while pollen grains that do not change color are inactive. The formula for calculating blueberry pollen viability is: Pollen viability (%) = (Number of stained pollen grains / Total number of observed pollen grains) × 100%.

[0020] test: The effect of different TTC concentrations on blueberry pollen viability was investigated. Pollen from Camellia, Emerald, and H5 blueberries was collected at 9:00 AM and tested using the method described in Example 1. The pollen viability results are shown in Table 1. Table 1

[0021] Example 2: This invention provides a method for rapidly determining blueberry pollen viability, comprising the following specific steps: Step 1, Pollen Collection: When the blueberry flowers are in the large bud stage, pick the flowers and put them into a resealable bag, place them in an ice-filled container, and bring them back to the laboratory for testing on the same day. The blueberry pollen used in Step 1 is camellia. The picking times are 9:30 am, 11:30 am, 1:30 pm and 3:30 pm on the same day.

[0022] Step 2, PBS solution preparation: Add 3.16g potassium dihydrogen phosphate to 100ml and 2.28g dipotassium hydrogen phosphate to 100ml to prepare a mixed solution. Then add acidic or alkaline titrant to prepare a PBS solution with a pH of 7.0. Potassium dihydrogen phosphate is a key auxiliary reagent in pollen viability detection. Its core functions are to provide nutrition, regulate osmotic pressure, and protect cell structure, providing a physiological basis for pollen to maintain activity, color development, and germination, thus achieving the purpose of pollen protection. Step 3: TTC solution preparation. Dissolve 0.1g of 2,3,5-triphenyltetrazolium chloride (TTC) in a small amount of alcohol, add PBS solution to make up to 100ml, and prepare a 1% TTC solution for later use. The principle of TTC solution detection is that viable pollen grains contain dehydrogenase, which can reduce colorless TTC to insoluble red formazan, while inactive pollen grains will not change color. Step 4: Preparation of pollen suspension: Shake the collected pollen into a 2ml centrifuge tube, add 1ml of the TTC solution from Step 3, and shake well to prepare a pollen suspension for later use. Preparing the suspension ensures the pollen has sufficient contact with the reaction solution, eliminating the possibility of incomplete staining due to insufficient contact. Simultaneously, shaking all the pollen into the centrifuge tube ensures a more homogeneous sample, reducing errors caused by low pollen viability due to individual flower specimens.

[0023] Step 5: Constant Temperature Incubation: Place the pollen suspension in an incubator and incubate for 20 minutes, then remove it. The incubator temperature is set at 35℃, and the incubation method is dark incubation.

[0024] Step 6: Slide preparation: Take 20 μL of the pollen suspension from step 5 and drop it onto the slide. Then, slowly cover the slide with a coverslip from one side.

[0025] Step Seven: Observation and Calculation: Observe the slide using a microscope and calculate the blueberry pollen viability according to the formula. The method for observing and judging blueberry pollen viability in Step Eight is to observe the color. Pollen grains stained red or pink are viable, while those that do not change color are inactive. The formula for calculating blueberry pollen viability is: Pollen Viability (%) = (Number of stained pollen grains / Total number of observed pollen grains) × 100%.

[0026] Table 2

[0027] The data in Table 1 shows that, using Example 1, the pollen viability of all three blueberry pollen samples (Camellia and Emerald) reached over 90% at a TTC concentration of 1% and a dark reaction time of 20 min. At a dark reaction time of 20 min and a TTC concentration of 0.5%, the pollen viability was 0. Pollen viability gradually increased with prolonged dark reaction time. At a dark reaction time of 20 min and a TTC concentration of 1.5%, the blueberry pollen viability was around 20%, increasing with further dark reaction time. Table 1 indicates that a TTC concentration of 1% and a dark reaction time of 20 min resulted in optimal pollen viability. Using Example 2, Table 2 shows that using a TTC concentration of 1% and a dark reaction time of 20 min, pollen viability was good when flowers were collected at different times of the day. Therefore, it can be concluded that a TTC concentration of 1% and a dark reaction time of 20 min are beneficial for the rapid detection of blueberry pollen viability. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapidly determining blueberry pollen viability, characterized in that: The specific steps include the following: Step 1, Pollen Collection: When the blueberry flowers are in the large bud stage, pick the flowers, put them in a resealable bag, place them in an ice-filled container, and bring them back to the laboratory for testing on the same day; Step 2, PBS solution preparation: Add 3.16g potassium dihydrogen phosphate to 100ml and 2.28g dipotassium hydrogen phosphate to 100ml to prepare a mixed solution. Then add acidic or alkaline titrant to adjust the pH of the PBS solution to 7.

0. Step 3: TTC solution preparation. Dissolve 0.1g of 2,3,5-triphenyltetrazolium chloride in a small amount of alcohol, add PBS solution to make up to 100ml, and prepare a 1% TTC solution for later use. Step 4: Preparation of pollen suspension: Shake the collected pollen into a 2ml centrifuge tube, add 1ml of the TTC solution from step 3, shake well, and prepare a pollen suspension for later use. Step 5: Constant temperature incubation: Place the pollen suspension in an incubator for incubation, and remove it after 20 minutes. Step 6: Slide preparation: Take 20 μL of the pollen suspension from Step 5 and drop it onto the slide. Then, slowly cover the slide with a coverslip from one side. Step 7: Observation and Calculation: Observe the slide using a microscope and calculate the blueberry pollen viability according to the calculation formula to obtain the blueberry pollen viability.

2. The method for rapidly determining blueberry pollen viability according to claim 1, characterized in that: The blueberry pollen used in step one is Camellia, Emerald, and H5.

3. The method for rapidly determining blueberry pollen viability according to claim 1, characterized in that: The TTC concentration in step three is 1%.

4. The method for rapidly determining blueberry pollen viability according to claim 1, characterized in that: In step five, the incubator is equipped with a temperature control device to maintain the temperature inside the incubator at 35°C, and the incubation method is dark incubation.

5. The method for rapidly determining blueberry pollen viability according to claim 1, characterized in that: In step eight, the method for observing and judging the blueberry pollen viability is to observe the color. Pollen grains that are stained red or pink are viable, while pollen grains that do not change color are inactive. The formula for calculating blueberry pollen viability is: Pollen viability (%) = (Number of stained pollen grains / Total number of observed pollen grains) × 100%.

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