Method for collecting high-activity blueberry pollen

Abstract

The invention discloses a method for collecting high-activity blueberry pollen, which comprises the following steps of: 1, selecting blueberry flowering branches which are robust in growth vigor, more in large buds and middle buds and relatively consistent in growth vigor, knocking out sundries on the branches, and performing bagging anti-pollution treatment; secondly, before flowers bloom, the bagged blueberry flowering branches in the first step are sheared and subjected to moisturizing treatment; 3, pruning the blueberry flowering branches in the step 2, and only reserving large buds and middle buds; 4, performing sterile water culture on the pruned blueberry flowering branches in the step 3; 5, slightly knocking the blueberry flowering branches subjected to sterile water culture in the step 4, and collecting pollen; and 6, sealing and preserving the pollen collected in the step 5 at low temperature for later use. Aiming at the defects in the existing anther powder dispersing method and field vibration powder taking method, the invention provides a technology which does not need manual anther stripping and anther drying, so that the pollen activity is remarkably improved while intermediate links are reduced and the pollution risk is reduced.

Description

Technical Field

[0001] The present invention relates to the field of pollen collection, and specifically to a method for collecting highly active blueberry pollen. Background Art

[0002] Currently, there are mainly two methods for collecting blueberry pollen. One is to pick the flower buds about to bloom, take out the anthers, and place the anthers in a room temperature environment to dry naturally or by drying. After the anthers are fully dispersed, they are collected for standby. This method is called the anther powder scattering method. The other is the field vibration pollen collection method. A receiving container (such as a petri dish, centrifuge tube, etc.) is placed under the open flowers of the blueberry plant, and pollen is collected by knocking. The anther powder scattering method for collecting pollen in a blueberry laboratory is "flower bud picking - anther separation - artificial drying and powder scattering or natural air drying and powder scattering". The operation process includes the following steps: pick flower buds at the large bud stage and bring them back to the laboratory; then manually strip the anthers with tweezers, etc.; place the stripped anthers in a constant temperature drying oven at 25 - 30°C for drying or place them in the room for natural air drying. The above process is cumbersome. A skilled worker can only strip about 80 - 100 flowers per hour, and the labor cost is relatively high. Moreover, microorganisms are easily introduced during the process of manually stripping the anthers. Secondly, the quality of the pollen is unstable, and problems such as premature picking (the pollen is not fully mature and has low activity) or late picking (the flower has opened, resulting in contamination and pollen loss) are likely to occur. Finally, when drying and powder scattering or air drying at the same time, the pollen is easily dehydrated excessively and the activity decreases due to long-term drying. In addition, there are many residues such as anthers, filaments, and petals after drying, and screening is required.

[0003] The field vibration pollen collection method of pollen mainly relies on manual vibration to collect pollen. The typical operation method is during the full bloom period, manually pat or use an electric vibrator (the frequency is generally 50 - 100Hz) to act on the flower branches or the receiving container, so that the pollen naturally falls off and is collected in the receiving container below (such as a petri dish, sulfuric acid paper, etc.). When vibrating, sepals, corollas, bracts, insects, pathogens, dust, etc. fall off synchronously, and subsequent screening is required for microscopic observation. At the same time, due to the influence of wind, insects, etc. in the field, pollen of different varieties overflows with each other, easily causing problems such as pollen contamination and low purity. Finally, due to direct sunlight and temperature fluctuations in the field, the pollen viability is likely to decrease after collection. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for collecting highly active blueberry pollen to solve the problems raised in the above background art.

[0005] To solve the above technical problems, the present invention provides the following technical solutions:

[0006] A method for collecting highly active blueberry pollen includes the following steps

[0007] 1. Select strong and healthy blueberry flower branches with medium and large flower buds and relatively consistent growth. Knock off the debris on the branches and perform bagging for anti-pollution treatment;

[0008] 2. Before the flowers bloom, cut the bagged blueberry flower branches in step 1 and perform moisture preservation treatment;

[0009] 3. Prune the blueberry flower branches in step 2, and only retain large and medium flower buds;

[0010] 4. Perform aseptic hydroponics on the pruned blueberry flower branches in step 3;

[0011] 5. Gently tap the blueberry flower branches after aseptic hydroponics in step 4 to collect pollen;

[0012] 6. Seal and store the pollen collected in step 5 at low temperature for later use.

[0013] Preferably, in step 2, the length of the cut bagged blueberry flower branches is 15 - 20 cm.

[0014] Preferably, the temperature of aseptic hydroponics in step 4 is 20 - 25 °C.

[0015] Preferably, keep the light for 10 - 14 h during the aseptic hydroponics in step 4.

[0016] Preferably, the time of aseptic hydroponics in step 4 is 24 - 96 h.

[0017] Preferably, the storage temperature of pollen in step 6 is not higher than -20 °C.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. Aiming at the defects in the existing anther powder scattering method and field vibration pollen collection method, the present invention invents a technology that does not require manual stripping of anthers and drying of anthers. While reducing intermediate links and reducing the risk of pollution, it significantly improves the pollen activity.

[0020] 2. The method in the present invention can accurately collect the pollen naturally scattered within 1 - 4 days after flowering, ensuring the quality and activity of the pollen, and effectively avoiding problems such as low pollen activity, large differences, and incomplete development of the pollen collected by the existing pollen collection technology.

[0021] 3. The pollen collected by the method in the present invention has a relatively high purity and extremely few impurities, and also omits the links of collecting anthers and pollen screening, saving time and effort. Description of the Drawings

[0022] Figure 1 The pollen purity collected by using the method in the present invention;

[0023] Figure 2The pollen purity collected by the anther powder scattering method;

[0024] Figure 3 The pollen purity collected by the field vibration pollen collection method;

[0025] Figure 4 The pollen germination situation of different pollen collection methods for the "Sunshine Blue" blueberry variety;

[0026] Figure 5 The different developmental stages of flowers and the corresponding pollen amounts collected;

[0027] Figure 6 The determination of the activity of pollen at different opening stages of blueberry flowers;

[0028] Figure 7 The pollen tube germination situation of the "Sunshine Blue" blueberry variety at different flower development stages. Specific implementation manners

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1

[0031] This embodiment provides a method for collecting high-activity blueberry pollen, including the following steps:

[0032] Step 1: After the plant shows flower buds, select flower branches with strong growth, many large buds and medium buds (flowers about to bloom) and relatively consistent, knock on the flower branches to knock off sundries such as dust and insect eggs on the branches, and put on an isolation bag (sulfuric acid paper bag) to avoid pollen contamination.

[0033] Step 2: Before the flowers open after bagging, cut the bagged flower branches with scissors. The length of the flower branches is preferably 18 cm. Wrap the cut end directly with a wet towel or directly insert it into water and bring it back to the laboratory.

[0034] Step 3: Prune the flower branches brought back to the laboratory, cut off small buds, young buds, and abnormally developed flower buds, and remove the newly germinated inflorescences below the flower branches. Only large buds and medium buds are left on the flower branches.

[0035] Step 4: Make a fresh cut at the cut end of the pruned flower branches and insert them into a bottle filled with sterile water for hydroponics, or hydroponics in a light incubator at 22°C with 12 hours of light. Note: The sterile water should not submerge the flowers, and the sterile water should be changed every day.

[0036] Step 5: Collect pollen at 72 h after hydroponics of flower branches or at 60 h after the flower buds bloom. The specific method is to prepare a sterilized culture dish (receptacle), place the culture dish on the table, take out the flower branch and place it above the culture dish, and gently tap the flower branch with a pen or forceps, etc., so that the pollen naturally falls into the culture dish.

[0037] Step 6: Put the pollen collected in the culture dish into a centrifuge tube or pollen tube, seal it and store it at low temperature (≤ -20 °C) for standby.

[0038] Among them, the hydroponics conditions in Step 4 can be indoor room temperature cultivation, just place it near the window, and there are no requirements for temperature, light, etc.; the sterile water can be replaced with a sterilized sucrose aqueous solution (five per thousand) for hydroponics.

[0039] Among them, the receptacle in Step 5 can use a centrifuge tube, pollen tube, dish, sulfuric acid paper, etc.; for the pollen collection method, the flower can be picked, the corolla mouth is facing downwards towards the pollen tube or centrifuge tube, and the flower is gently tapped to naturally scatter the pollen into the pollen tube or centrifuge tube; when tapping, forceps, pen, fingers, etc. can be used, and just tap gently.

[0040] The present invention has great improvements compared with the existing two pollen collection methods (anther powder scattering method, field vibration pollen collection method) in terms of flower selection, powder scattering process, pollen purity, pollen activity, pollen contamination degree, and labor input, as shown in Table 1.

[0041] Table 1 Comparison between the present invention and the prior art

[0042]

[0043]

[0044] As Figures 1-3 shown, the impurities contained in the pollen collected by different pollen collection methods vary greatly. The main impurities in the pollen collected by the anther powder scattering method are the anther wall, filaments, etc. in the stamens, and it needs to be sieved; the main impurities in the pollen collected by the field vibration pollen collection method are the corolla, bracts, stigmas, dust, etc., and it also needs to be sieved; the pollen collected by the method of the present invention is relatively clean, with very few impurities, high cleanliness and purity, and does not need to be sieved.

[0045] As Figure 4 shown, from left to right in the figure are the anther powder scattering collection method, the field natural powder scattering collection method, and the pollen collection method shown in the present invention. When collecting pollen from the "Sunshine Blue" blueberry variety using each method, the germination rates of the pollen vary greatly. The germination rate of the pollen collected by the anther powder scattering method is 20.6%, the germination rate of the pollen collected by the field vibration pollen collection method is 22.5%, and the germination rate of the pollen collected by the present invention is 90.5%. The pollen activity is relatively high, which is more than 4 times the pollen activity collected by the current conventional methods.

[0046] As shown Figure 5 in the figure, from left to right at the upper part of the picture, the flower development stages are: large bud stage, flower budding, flower opening for 1 - 2 days, flower opening for 3 - 4 days, flower opening for 5 - 6 days; at the lower part of the picture are the amounts of pollen collected from every 4 flowers at different development stages. The flower development stages of the "Sunshine Blue" blueberry variety include young bud stage, small bud stage, medium bud stage, large bud stage, flower budding stage, flowering stage, and flower withering stage. The single flower of blueberry generally takes 10 days to open. Research shows that pollen begins to appear after the large bud stage and the amount of pollen is extremely small; when the flower just begins to bud, the amount of pollen gradually increases; when the flower opens for 1 - 2 days, the amount of pollen is the largest, accounting for more than 60% of the total pollen amount; when the flower opens for 3 - 4 days, the amount of pollen begins to gradually decrease, and significantly decreases at 5 - 6 days. Therefore, it is better to collect pollen within 1 - 4 days after the flower opens, and more than 60% of the pollen amount can be collected.

[0047] As shown Figure 6 in the figure, from left to right the flower development stages are: large bud stage, flower budding, flower opening for 1 - 2 days, flower opening for 3 - 4 days, flower opening for 5 - 6 days. Using the "Sunshine Blue" blueberry variety as the test material and collecting pollen with the present invention, when performing the TTC pollen staining method to determine pollen viability, the pollen viabilities at the large bud stage, just budding, flowering for 1 - 2 days, flowering for 3 - 4 days, and flowering for 5 - 6 days are 82.2%, 90.3%, 95.1%, 94.6%, and 94.9% respectively. It shows that within the first 6 days after opening, the pollen viability is very high, but at the large bud stage and 5 - 6 days after opening, the color of pollen grains is mostly between red and light red, with more false positives, and the counting is prone to misjudgment. Therefore, it is necessary to combine the pollen tube germination test to determine the best period for pollen viability and germination rate.

[0048] As shown Figure 7 in the figure, from left to right the flower development stages are: large bud stage, flower budding, flower opening for 1 - 2 days, flower opening for 3 - 4 days, flower opening for 5 - 6 days. Selecting the "Sunshine Blue" blueberry variety as the test material and using the pollen collected by the present invention, culturing the pollen in vitro for 3 hours and determining the pollen germination rates at the large bud stage, just budding, flowering for 1 - 2 days, flowering for 3 - 4 days, and flowering for 5 - 6 days, the results show that the pollen germination rates at different stages are 0%, 79.48%, 94.47%, 97.62%, and 60.61% respectively. It shows that the pollen germination rate is the highest on the 1 - 4th day after budding.

[0049] Example 2

[0050] The difference between this embodiment and Embodiment 1 is that the length of the flower branch in Step 2 is 15 cm; the temperature of the light incubator in Step 4 is 20 °C, and the light hydroponic duration is 10 h; in Step 5, pollen is collected when the flower branch is hydroponically cultured for 48 h or 24 h after the flower bud blooms. Other steps and effects are the same as those in Embodiment 1 and will not be elaborated here.

[0051] Embodiment 3

[0052] The difference between this embodiment and Embodiment 1 is that the length of the flower branch in Step 2 is 20 cm; the temperature of the light incubator in Step 4 is 25 °C, and the light hydroponic duration is 14 h; in Step 5, pollen is collected when the flower branch is hydroponically cultured for 96 h or 96 h after the flower bud blooms. Other steps and effects are the same as those in Embodiment 1 and will not be elaborated here.

[0053] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for collecting highly active blueberry pollen, comprising the following steps:

1. Select blueberry branches that are strong, have more large and medium buds, and have relatively consistent growth. Remove debris from the branches and bag them to prevent pollution.

2. Before the flowers bloom, cut the bagged blueberry branches in step 1 and moisturize them; 3. Prune the blueberry branches in step 2, leaving only the large and medium buds; 4. Carry out sterile hydroponics on the blueberry branches pruned in step 3; 5. Gently tap the blueberry branches grown in sterile hydroponic culture in step 4 to collect pollen; 6. Seal and store the pollen collected in step 5 at low temperature for future use.

2. The method for collecting highly active blueberry pollen according to claim 1, characterized in that: In step 2, the length of the bagged blueberry branches cut is 15-20 cm.

3. The method for collecting highly active blueberry pollen according to claim 1, characterized in that: In step 4, the sterile hydroponic temperature is 20-25°C.

4. The method for collecting highly active blueberry pollen according to claim 1, characterized in that: In step 4, the light is maintained for 10-14 hours during the sterile hydroponic process.

5. The method for collecting highly active blueberry pollen according to claim 1, characterized in that: The sterile hydroponic culture time of the blueberry flower branches in step 4 is 24-96 hours.

6. The method for collecting highly active blueberry pollen according to claim 1, characterized in that: In step 6, the pollen storage temperature is no higher than -20°C.

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