Cucumber pollen low-temperature preservation method and application thereof

Abstract

The invention provides a cucumber pollen low-temperature preservation method and application thereof, and belongs to the technical field of agriculture. On one hand, the anti-oxidant and the osmotic regulator are used for pretreating the pollen, so that the stress resistance of the pollen in the subsequent low-temperature preservation process is improved; on the other hand, the preservation temperature of the cucumbers is controlled to be-20 DEG C to-40 DEG C, the metabolism rate of pollen is slowed down through a low-temperature environment, and the service life of the pollen is prolonged; then a low-oxygen and high-carbon-dioxide environment is created, the respiration of the pollen is reduced, the energy consumption is reduced, the activity of the pollen is further maintained, and the preservation time of the cucumber pollen can be prolonged to 6 months or above. Compared with an existing low-temperature preservation method, the pollination success rate reaches 80% or above, and the seed setting number of a single melon after pollination is increased by 30%-50%.

Description

Technical Field

[0001] The present invention relates to the technical field of agriculture, and particularly to a method for cryopreserving cucumber pollen and its application. Background Art

[0002] Cucumber is an important vegetable crop and is widely planted in agricultural production. During the cucumber breeding process, the opening times of male and female flowers of cucumber are inconsistent, with male flowers blooming earlier than female flowers, which leads to a dislocation between pollen supply and the receptive period of female flowers. Moreover, the natural pollination window period of cucumber is short, and it is easy to miss the best pollination time. Therefore, in the breeding and propagation process of cucumber, the preservation of pollen and effective pollination are key links.

[0003] Currently, traditional pollen preservation methods have many limitations. For example, the preservation time at room temperature is short, and the pollen viability decreases rapidly, resulting in difficult-to-guarantee pollen quality when pollination operations are required, affecting the pollination success rate and the quality of seed development. Some existing cryopreservation methods have deficiencies in aspects such as temperature control, humidity regulation, and preventing pollen damage, which may cause damage to the pollen cell membrane and cannot meet the requirements for long-term and stable preservation of pollen viability. Instead, it will reduce the germination rate of pollen, leading to unstable pollination effects and affecting the seed yield and quality.

[0004] Therefore, it is of great practical significance to develop a pollen preservation method that can effectively extend the preservation time of cucumber pollen, maintain pollen viability, and improve the pollination success rate. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for cryopreserving cucumber pollen and its application, which can effectively extend the preservation time of cucumber pollen, maintain pollen viability, and improve the pollination success rate and pollen germination rate.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for cryopreserving cucumber pollen, comprising the following steps:

[0008] (1) Pretreat and dry the pollen after collection;

[0009] (2) Place the pollen dried in step (1) in an environment with a temperature of -20°C to -40°C, a humidity of 30% to 50%, an oxygen concentration of 2% to 5%, and a carbon dioxide concentration of 5% to 10% for preservation.

[0010] Preferably, in step (1), the method of pretreatment is: mix an antioxidant and an osmotic regulator to obtain a mixed solution, and soak the pollen with the mixed solution.

[0011] Preferably, the concentration of the antioxidant is 0.1% - 0.5%, the concentration of the penetration regulator is 5% - 10%, the antioxidant includes one or more of vitamin C and vitamin E; the penetration regulator includes one or more of sucrose and mannitol.

[0012] Preferably, in step (1), the pollen needs to be dried to a water content of 5% - 8% during drying.

[0013] Preferably, in step (2), the temperature is -30°C, the humidity is 40%, the oxygen concentration is 3%, and the carbon dioxide concentration is 8%.

[0014] The present invention also provides an application of the above cucumber pollen low-temperature preservation method in cucumber pollen preservation.

[0015] The beneficial effects of the present invention compared with the prior art are as follows:

[0016] (1) On the one hand, the present invention uses an antioxidant and a penetration regulator to pretreat the pollen to improve the stress resistance of the pollen during subsequent low-temperature preservation; on the other hand, the temperature for cucumber preservation is controlled at -20°C to -40°C, and the low-temperature environment is used to slow down the metabolic rate of the pollen and extend the pollen lifespan; then a low-oxygen and high-carbon dioxide environment is created to reduce the respiration of the pollen and reduce energy consumption, further maintaining the pollen viability. The preservation time of cucumber pollen can be extended to more than 6 months, significantly longer than the traditional preservation method, providing sufficient pollen resources for cucumber cross-breeding and cross-season pollination.

[0017] (2) The cucumber pollen low-temperature preservation method of the present invention can reduce the consumption of nutrients and cell damage in the pollen, so that the pollen still maintains a high viability (the germination rate reaches more than 70%) after preservation. Compared with the existing low-temperature preservation method, the pollination success rate reaches more than 80%, and the number of seeds per single fruit after pollination increases by 30% - 50%. Detailed Embodiments

[0018] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0021] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0022] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0023] This invention provides a method for cryopreserving cucumber pollen, and the specific steps are as follows:

[0024] (1) Pretreat and dry the pollen after collection; wherein, the method of the pretreatment is: mix an antioxidant and an osmotic regulator to obtain a mixed solution, and soak the pollen with the mixed solution.

[0025] Preferably, the concentration of the antioxidant is 0.1% - 0.5%, the concentration of the osmotic regulator is 5% - 10%, the antioxidant includes one or more of vitamin C and vitamin E; the osmotic regulator includes one or more of sucrose and mannitol.

[0026] Preferably, in step (1), the pollen needs to be dried to a water content of 5% - 8% during drying.

[0027] (2) Store the pollen dried in step (1) in an environment with a temperature of -20°C to -40°C, a humidity of 30% - 50%, an oxygen concentration of 2% - 5%, and a carbon dioxide concentration of 5% - 10%; more preferably, the temperature is -30°C, the humidity is 40%, the oxygen concentration is 3%, and the carbon dioxide concentration is 8%.

[0028] This invention also provides a method for cucumber pollination, including the following steps:

[0029] (1) Preparation of pollen suspension:

[0030] Prepare a suspension according to the final concentration of gibberellin of 0.01 mg / L to 0.1 mg / L, the final concentration of auxin of 0.01 mg / L to 0.1 mg / L, and the final concentration of sodium carboxymethylcellulose of 0.5% to 1%. Obtain a pollen suspension according to the mass ratio of pollen: solution = 1:10 to 20, and stir with a magnetic stirrer for 10 minutes to evenly disperse the pollen in the solution.

[0031] (2) Spray pollination:

[0032] Load the pollen suspension into a high-pressure spray bottle. Spray each female flower 1 - 2 times to ensure that the stigma surface is evenly covered with the pollen suspension, improving the pollination efficiency and success rate.

[0033] Among them, the high-pressure spray bottle is purchased, and the nozzle aperture is 0.2 mm to 0.3 mm, with fine and uniform spraying.

[0034] Example 1

[0035] Example 1 of the present invention provides a method for low-temperature preservation of cucumber pollen, and the specific steps are as follows:

[0036] (1) Pollen collection:

[0037] During the full-bloom period of cucumbers, collect male flowers that are about to open or have just opened at 9 - 11 am on a sunny day and bring them back to the laboratory. Collect pollen under sterile conditions in the laboratory.

[0038] (2) Pollen pretreatment:

[0039] A. Preparation of the mixed solution: Prepare a mixed solution according to the final concentration of vitamin C of 0.3%, the final concentration of vitamin E of 0.2%, and the final concentration of sucrose of 8%.

[0040] B. Transfer the pollen collected in step (1) to the mixed solution, gently stir to allow the pollen to fully contact the solution, filter after 20 minutes of treatment to remove the excess solution, and dry at low temperature (4°C) until the water content is about 6%.

[0041] (3) Low-temperature preservation

[0042] Load the pollen pretreated in step (2) into a plastic bottle with good sealing performance and resistance to low temperature, and load 0.5 g to 1 g of pollen in each bottle. Place the plastic bottle in an environment with a temperature of -30°C, a humidity of 40%, an oxygen concentration of 3%, and a carbon dioxide concentration of 8%, and control the temperature fluctuation range within -30°C ± 0.5°C. Start the temperature control system, humidity adjustment system, and gas replacement system to make the preservation environment reach the set parameters and remain stable. Regularly check the operation of the device and the preservation status of the pollen.

[0043] Example 2

[0044] Embodiment 2 of the present invention provides a method for low-temperature preservation of cucumber pollen, and the specific steps are as follows:

[0045] (1) Pollen collection:

[0046] During the full-bloom period of cucumbers, collect the male flowers that are about to open or have just opened at 9-11 am on a sunny day and bring them back to the laboratory. Under sterile conditions in the laboratory, collect the pollen.

[0047] (2) Pollen pretreatment:

[0048] A. Prepare a mixed solution according to the final concentration of vitamin C being 0.1%, the final concentration of vitamin E being 0.4%, and the final concentration of sucrose being 5%.

[0049] B. Transfer the pollen collected in step (1) to the mixed solution, gently stir to make the pollen fully contact the solution, filter after treatment for 20 minutes to remove the excess solution, and dry it at low temperature (4°C) until the water content is about 8%.

[0050] (3) Low-temperature preservation

[0051] Put the pollen pretreated in step (2) into a plastic bottle with good sealing performance and resistance to low temperature, and the amount of pollen in each bottle is 0.5 g - 1 g. Place the plastic bottle in an environment with a temperature of -40°C, a humidity of 30%, an oxygen concentration of 2%, and a carbon dioxide concentration of 10%, and control the temperature fluctuation range within -40°C ± 0.5°C. Start the temperature control system, humidity adjustment system, and gas replacement system to make the preservation environment reach the set parameters and remain stable. Regularly check the operation of the device and the preservation status of the pollen.

[0052] Embodiment 3

[0053] Provides a method for low-temperature preservation of cucumber pollen, and the specific steps are as follows:

[0054] (1) Pollen collection:

[0055] During the full-bloom period of cucumbers, collect the male flowers that are about to open or have just opened at 9-11 am on a sunny day and bring them back to the laboratory. Under sterile conditions in the laboratory, collect the pollen.

[0056] (2) Pollen pretreatment:

[0057] A. Prepare a mixed solution according to the final concentration of vitamin C being 0.5% and the final concentration of sucrose being 10%.

[0058] B. Transfer the pollen collected in step (1) to the mixed solution, gently stir to make the pollen fully contact the solution, filter after treatment for 20 minutes to remove the excess solution, and dry it at low temperature (4°C) until the water content is about 5%.

[0059] (3) Low-temperature preservation

[0060] Load the pollen pretreated in step (2) into plastic bottles with good airtightness and resistance to low temperature, and load 0.5 g to 1 g of pollen into each bottle. Place the plastic bottles in an environment with a temperature of -20°C, a humidity of 50%, an oxygen concentration of 5%, and a carbon dioxide concentration of 5%, and control the temperature fluctuation range within -20°C ± 0.5°C. Start the temperature control system, humidity regulation system, and gas replacement system to make the storage environment reach the set parameters and remain stable. Regularly check the operation of the device and the storage status of the pollen.

[0061] Example 4

[0062] In Example 4 of the present invention, the pollen stored in Example 1 is used for pollination, and the specific steps are as follows:

[0063] (1) Preparation of pollen suspension:

[0064] Prepare a suspension according to a final concentration of gibberellin of 0.05 mg / L, a final concentration of auxin of 0.03 mg / L, and a final concentration of sodium carboxymethylcellulose of 0.8%, and obtain a pollen suspension according to a mass ratio of pollen: solution = 1:15. Stir with a magnetic stirrer for 10 minutes to evenly disperse the pollen in the solution.

[0065] (2) Spray pollination:

[0066] Load the pollen suspension into a high-pressure spray bottle and tighten the lid. Spray each female flower 1 - 2 times to ensure that the stigma surface is evenly covered with the pollen suspension.

[0067] Among them, the high-pressure spray bottle is purchased, and the nozzle aperture is 0.2 mm to 0.3 mm.

[0068] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for low temperature preservation of cucumber pollen, characterized in that: The steps include: (1) Pre-treatment and drying of pollen after collection; (2) The pollen dried in step (1) is stored in an environment with a temperature of -20°C to -40°C, a humidity of 30% to 50%, an oxygen concentration of 2% to 5%, and a carbon dioxide concentration of 5% to 10%.

2. The method for low temperature preservation of cucumber pollen according to claim 1, characterized in that: In step (1), the pretreatment method is: mixing an antioxidant and an osmotic regulator to obtain a mixed solution, and using the mixed solution to soak pollen.

3. The method for low temperature preservation of cucumber pollen according to claim 2, characterized in that: The concentration of the antioxidant is 0.1% to 0.5%, the concentration of the osmotic regulator is 5% to 10%, the antioxidant includes one or more of vitamin C and vitamin E; the osmotic regulator includes one or more of sucrose and mannitol.

4. The method for low temperature preservation of cucumber pollen according to claim 1, characterized in that: In step (1), the pollen needs to be dried to a moisture content of 5% to 8%.

5. The method for low temperature preservation of cucumber pollen according to claim 1, characterized in that: In step (2), the temperature is -30°C, the humidity is 40%, the oxygen concentration is 3%, and the carbon dioxide concentration is 8%.

6. Use of the cucumber pollen low-temperature preservation method according to any one of claims 1 to 5 in cucumber pollen preservation.