Storage and fresh-keeping method and application of golden camellia pollen

Abstract

The invention discloses a storage and preservation method of golden camellia pollen. The method comprises the following steps: (1) collecting the pollen; (2) pretreating the pollen; (3) drying the pollen; according to the method, the convenience of collecting and storing the golden camellia pollen can be improved, the pollen is kept dry, and meanwhile the activity of the pollen is kept.

Description

Technical Field

[0001] The present invention relates to the technical field of pollen preservation, and particularly relates to a method for storing and preserving Camellia nitidissima pollen and its application. Background Art

[0002] Artificial cross-pollination is a traditional means for breeding new varieties of camellia. The main method is to use a pollination stick to transfer the pollen collected from the male parent to the stigma of the female parent to complete pollination. The breeding of new varieties of Camellia nitidissima is an important direction in camellia research. Camellia nitidissima species such as Camellia nitidissima Chi var. chuangtsoensis and Camellia ptilosperma, which have the characteristic of blooming throughout the year, are valuable species resources for breeding four-season camellias. In breeding work, they are often used as parent materials to cross with other Theaceae plants in order to breed new varieties of camellias with the characteristics of blooming in summer or having yellow flowers. To achieve this goal, it is necessary to collect and process the pollen of Camellia nitidissima and then store it to solve problems such as the non-coincidence of the flowering periods of different tea plants and the low pollination survival rate in cross-pollination. The pollen viability will continuously decrease over time, which is an important problem restricting cross-breeding. Only high-viability pollen can ensure the success of cross-breeding. Therefore, how to keep the pollen still have a high viability after cryopreservation is extremely crucial in variety cultivation.

[0003] The current storage method of Camellia nitidissima pollen is mainly cryopreservation. The main method is to collect the pollen, put it into a centrifuge tube of about 2 ml, and store it in a -20°C refrigerator. However, existing research shows that the traditional freezing method (-20°C storage) is prone to cause damage to cell membranes, and the germination rate after thawing is less than 30%; in addition, traditional methods also include the desiccant sealing method (such as silica gel drying), but the survival rate of this method decreases rapidly with the storage time, and the germination rate is lower than 10% after 60 days; based on this, existing preservation methods include improvements on the above basis, using vacuum packaging combined with cryopreservation, but the equipment cost is high and the operation is inconvenient, making it difficult to popularize in daily breeding work. Summary of the Invention

[0004] In view of this, the present invention provides a method for storing and preserving Camellia nitidissima pollen, which can improve the convenience of collecting and storing Camellia nitidissima pollen, keep the pollen dry, and at the same time maintain the viability of the pollen.

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

[0006] A method for storing and preserving Camellia nitidissima pollen, the method comprising the following steps:

[0007] (1) Pollen collection: Select Camellia chrysantha with healthy and pest-free trees, and take slightly opened flowers from the branches outside the crown; pick the flowers between 9.30 and 10.30 in the morning, put them in a sealed bag, bring them back to the laboratory operating table, use tweezers to peel off the sepals and petals of the flowers in turn, and then shake off the pollen on the anthers and collect them on weighing paper;

[0008] (2) Pretreatment of pollen: immerse the pollen in a composite preservative solution and allow to stand at 3-5°C for 25-35 minutes to obtain a pollen soaking solution;

[0009] (3) Pollen drying: Gradient drying method is used. First, the culture dish containing pollen soaking solution is placed on a sterile clean bench. The working conditions are set as follows: temperature 22-28°C, humidity 40-50%, wind speed 0.2-0.4 m / s, and time: 85-95 min to remove free surface moisture and reduce the moisture content to 12-14%. Then, deep drying is performed: the culture dish is placed in a constant temperature drying oven, set at 33-38°C, and dried to a moisture content of 8-10%.

[0010] (4) Pollen storage: Place the dried pollen in an aluminum foil composite film bag and evacuate to -0.08 to -0.1 MPa. Then place the aluminum foil composite film bag in an environment with a temperature of -2 to -1°C and store for 25-35 days. Then adjust the storage environment temperature to -4°C and the storage humidity to ≤15%.

[0011] Further, the flowers were collected at 10 am.

[0012] Furthermore, the composite fresh-keeping solution in step (2) contains 0.1 wt% trehalose, 0.05 wt% ascorbic acid and 50 ppm boric acid, and the pH value of the composite fresh-keeping solution is 6.0.

[0013] Furthermore, in the step (4), silica gel desiccant and activated carbon powder are placed in the aluminum foil composite film bag in a mass ratio of 3:1.

[0014] Furthermore, the storage in step (4) is performed in a light-proof manner throughout the entire process.

[0015] The present invention also provides a method for artificial pollination using pollen stored by the above method, the method comprising the following steps:

[0016] 1) Pollen resuscitation and activation: Using a gradient temperature recovery method, first place the stored pollen in an environment of 3-5℃ for 2 hours, and then transfer it to an environment of 22-28℃ for slow release for 1 hour;

[0017] 2) Use of pollen: After the revived and activated pollen is tested and its germination rate meets the requirements, it can be used for artificial pollination.

[0018] The method for detecting pollen activity is as follows:

[0019] Activation solution treatment: Use a solution containing 15% sucrose + 100 ppm CaCl 2 + 10 mg / L boric acid to suspend the pollen. After culturing at 26 °C for 90 minutes, observe the pollen germination under a microscope.

[0020] The present invention has the following beneficial effects compared with the prior art:

[0021] The present invention provides a method for storing and preserving Camellia nitidissima pollen, which is different from the traditional cold storage and silica gel drying preservation methods. Firstly, the flowers are collected at a suitable collection time to ensure the vitality of the pollen. Then, the pollen is soaked in a pretreatment solution, and the pollen soaking solution is dried by combining gradient drying, which can reduce the rupture of cell walls and the leakage of cell contents, maintain the integrity of the pollen, effectively reduce nutrient loss, and maintain the pollen vitality. Then, through packaging and low-temperature storage, the effective storage and preservation of Camellia nitidissima pollen are realized. The low-temperature storage in this application is -4 °C. Compared with the conventional low-temperature storage method, its energy consumption is greatly reduced, the cost is lower, and the benefit is improved. In addition, under the storage method described in this application, after a long-term storage, the germination rate of the pollen still remains at a high level. It realizes the utilization of pollen resources across years, solves the problem of non-meeting of flowering periods, does not require professional equipment such as liquid nitrogen tanks, reduces the energy consumption cost of storage, and is particularly suitable for popularization and use by scientific and technological personnel and Camellia lovers in actual work, providing a germplasm resource guarantee for the distant hybridization of Camellia nitidissima and accelerating the progress of the breeding work of new Camellia nitidissima varieties. Specific embodiments

[0022] The following examples can help those skilled in the art to understand the present invention more comprehensively, but cannot limit the present invention in any way.

[0023] Example 1

[0024] This example provides a method for storing and preserving Camellia nitidissima pollen, and the method includes the following steps:

[0025] (1) Collection of pollen: Select Camellia nitidissima with strong tree bodies and no pests and diseases, and pick the slightly blooming flowers on the outer branches of the tree crown; pick the flowers at 9:30 - 10:30 in the morning, put them into a sealed bag, bring them back to the laboratory workbench, use tweezers to remove all the sepals and petals of the flowers in turn, and then shake the pollen on the anthers and collect it on a weighing paper;

[0026] (2) Pretreatment of pollen: Immerse the pollen in the composite preservation solution and let it stand at 3°C for 25 minutes to obtain the pollen immersion solution; among them, the composite preservation solution contains 0.1 wt% trehalose, 0.05 wt% ascorbic acid and 50 ppm boric acid, and the pH value of the composite preservation solution is 6.0;

[0027] (3) Drying of pollen: Treat it by the gradient drying method. First, place the culture dish containing the pollen immersion solution in a sterile ultra-clean workbench, and set the working conditions as: temperature 22°C, humidity 40%, wind speed 0.2 m / s, time: 95 minutes to remove the surface free moisture and reduce the water content to 12%. Then carry out deep drying: Place the culture dish in a constant temperature drying oven (equipped with a silica gel dynamic dehumidification system), set the temperature at 33°C, and dry until the water content is 8%.

[0028] (4) Storage of pollen: Put the dried pollen into an aluminum foil composite film bag. There is a silica gel desiccant (granular) and activated carbon powder with a mass ratio of 3:1 placed in the aluminum foil composite film bag. Vacuumize to -0.08 MPa, then place the aluminum foil composite film bag in an environment with a temperature of -2°C and store for 25 days. Then adjust the storage environment temperature to -4°C and the storage humidity ≤ 15%. Keep it away from light throughout the storage process.

[0029] Example 2

[0030] This example provides a method for storing and preserving camellia nitidissima pollen, and this method includes the following steps:

[0031] (1) Collection of pollen: Select camellia nitidissima with strong tree trunks and no pests and diseases, and pick the slightly blooming flowers on the outer branches of the tree crown; pick the flowers at 10:00 am, put them into a sealed bag, bring them back to the laboratory workbench, and use tweezers to remove all the sepals and petals of the flowers in sequence. Then shake off the pollen on the anthers and collect it on a weighing paper;

[0032] (2) Pretreatment of pollen: Immerse the pollen in the composite preservation solution and let it stand at 4°C for 30 minutes to obtain the pollen immersion solution; among them, the composite preservation solution contains 0.1 wt% trehalose, 0.05 wt% ascorbic acid and 50 ppm boric acid, and the pH value of the composite preservation solution is 6.0;

[0033] (3) Drying of pollen: Treat it by the gradient drying method. First, place the culture dish containing the pollen immersion solution in a sterile ultra-clean workbench, and set the working conditions as: temperature 25°C, humidity 45%, wind speed 0.3 m / s, time: 90 minutes to remove the surface free moisture and reduce the water content to 13%. Then carry out deep drying: Place the culture dish in a constant temperature drying oven (equipped with a silica gel dynamic dehumidification system), set the temperature at 35°C, and dry until the water content is 9%.

[0034] (4) Storage of pollen: Put the dried pollen into an aluminum foil composite film bag, in which there is a silica gel desiccant (granular) and activated carbon powder with a mass ratio of 3:1. Vacuumize to -0.09 MPa, then place the aluminum foil composite film bag in an environment with a temperature of -1°C and store for 30 days. Then adjust the storage environment temperature to -4°C, the storage humidity ≤ 15%, and keep it away from light throughout the storage process.

[0035] Example 3

[0036] This example provides a method for storing and preserving Camellia nitidissima pollen, and this method includes the following steps:

[0037] (1) Collection of pollen: Select Camellia nitidissima with strong tree bodies and no pests and diseases, and pick slightly blooming flowers on the outer branches of the tree crown. Pick the flowers at 10:30 in the morning, put them into a sealed bag, bring them back to the laboratory workbench, and use tweezers to remove all the sepals and petals of the flowers in turn. Then shake the pollen on the anthers and collect it on a weighing paper.

[0038] (2) Pretreatment of pollen: Immerse the pollen in the composite preservative solution and let it stand at 5°C for 35 min to obtain a pollen soaking solution. Among them, the composite preservative solution contains 0.1 wt% trehalose, 0.05 wt% ascorbic acid and 50 ppm boric acid, and the pH value of the composite preservative solution is 6.0.

[0039] (3) Drying of pollen: Use the gradient drying method. First, place the culture dish containing the pollen soaking solution in a sterile ultra-clean workbench, and set the working conditions as: temperature 28°C, humidity 50%, wind speed 0.4 m / s, time: 85 min to remove the surface free water and reduce the water content to 14%. Then carry out deep drying: Place the culture dish in a constant temperature drying oven (equipped with a silica gel dynamic dehumidification system), set the temperature at 38°C, and dry until the water content is 10%.

[0040] (4) Storage of pollen: Put the dried pollen into an aluminum foil composite film bag, in which there is a silica gel desiccant (granular) and activated carbon powder with a mass ratio of 3:1. Vacuumize to -0.1 MPa, then place the aluminum foil composite film bag in an environment with a temperature of -1°C and store for 35 days. Then adjust the storage environment temperature to -4°C, the storage humidity ≤ 15%, and keep it away from light throughout the storage process.

[0041] Test example:

[0042] To illustrate the practical effect of the pollen storage of this application, the applicant conducts the following tests:

[0043] Test 1: Compare the influence of different collection times on pollen activity

[0044] As shown in Table 1, it is the comparison of pollen activity values at different collection times in a day:

[0045] Table 1 Comparison of pollen viability at different collection times

[0046]

[0047] According to the results in Table 1, it can be seen that the pollen viability of pollen collected at different times of the day is different. The viability value of Camellia nitidissima pollen at 10:00 is the highest, which is the best collection time.

[0048] Experiment 2: Compare the preservation effects of Camellia nitidissima pollen in each group under different storage treatment methods

[0049] The grouping is as follows:

[0050] The first group: Store the pollen in the manner described in Example 2;

[0051] The second group: Traditional refrigeration method, store the collected pollen at -20°C;

[0052] The third group: Traditional desiccant sealing method, place the petri dish containing pollen in a silica gel desiccator and seal it for storage.

[0053] The fourth group: Remove the pretreatment operation of the pollen, and the other methods are the same as those in the first group;

[0054] The fifth group: The drying method of the pollen is to place the petri dish in a constant temperature drying oven (equipped with a silica gel dynamic dehumidification system), set the temperature at 35°C, and dry it until the water content is 9%, and the other methods are the same as those in the first group.

[0055] Take the Camellia nitidissima pollen obtained from the storage of each group above for artificial pollination. The pollination steps include the following:

[0056] (1) Recovery and activation method:

[0057] Gradient temperature recovery: Place the stored pollen in an environment of 4°C for 2 hours, and then transfer it to an environment of 25°C for slow release for 1 hour;

[0058] (2) Pollen viability detection:

[0059] Treatment with activation solution: Suspend the pollen with a solution containing 15% sucrose + 100 ppm CaCl 2 + 10 mg / L boric acid, incubate at 26°C for 90 minutes, and then observe the pollen germination under a microscope.

[0060] (3) Use of pollen:

[0061] After the pollen is recovered and activated, and the germination rate meets the requirements after detection, it can be used for artificial pollination and other work.

[0062] Compare the germination rate and cross - fruit - setting rate after artificial pollination using the pollen of each of the above groups when the storage period is 60 days. The recorded data is shown in Table 2 as follows:

[0063] Table 2 Germination rate and cross - fruit - setting rate after artificial pollination with pollen stored for 60 days under each group's method

[0064] Pollen germination rate (%) The first group 91％ The second group 35％ The third group 10％ The fourth group 50％ The fifth group 53％

[0065] According to the results in Table 2, it can be seen that when storing pollen by the method of this application and then conducting artificial pollination on the stored pollen, the germination rate of the pollen is relatively high. However, for the traditional method, after 60 days of storage, it is obvious that the pollen activity can no longer be maintained. For the fourth and fifth groups, without pollen pretreatment or by changing the pollen drying method, the effects of this application cannot be achieved. This shows that each step of this application coordinates with each other, effectively stores Camellia nitidissima pollen, realizes the utilization of pollen resources across years, solves the problem of non - overlapping flowering periods, does not require professional equipment such as liquid nitrogen tanks, reduces the storage energy consumption cost, is especially suitable for popularization and use by scientific and technological personnel and Camellia enthusiasts in actual work, provides germplasm resource guarantee for distant hybridization of Camellia nitidissima, and accelerates the progress of the breeding work of new Camellia nitidissima varieties.

[0066] The above description is a detailed description of the preferred and feasible embodiments of the present invention. However, the embodiments are not used to limit the scope of the patent application of the present invention. Any equivalent changes or modifications made under the technical spirit disclosed by the present invention should fall within the scope of the patent covered by the present invention.

Claims

1. A method for storing and preserving Camellia chrysantha pollen, characterized in that: The method comprises the following steps: (1) Pollen collection: Select Camellia chrysantha with healthy and pest-free trees, and take slightly opened flowers from the branches outside the crown; pick the flowers between 9.30 and 10.30 in the morning, put them in a sealed bag, bring them back to the laboratory operating table, use tweezers to peel off the sepals and petals of the flowers in turn, and then shake off the pollen on the anthers and collect them on weighing paper; (2) Pretreatment of pollen: immerse the pollen in a composite preservative solution and allow to stand at 3-5°C for 25-35 minutes to obtain a pollen soaking solution; (3) Pollen drying: Gradient drying method is used. First, the culture dish containing pollen soaking solution is placed on a sterile clean bench. The working conditions are set as follows: temperature 22-28°C, humidity 40-50%, wind speed 0.2-0.4 m / s, and time: 85-95 min to remove free surface moisture and reduce the moisture content to 12-14%. Then, deep drying is performed: the culture dish is placed in a constant temperature drying oven, set at 33-38°C, and dried to a moisture content of 8-10%. (4) Pollen storage: Place the dried pollen in an aluminum foil composite film bag and evacuate to -0.08 to -0.1 MPa. Then place the aluminum foil composite film bag in an environment with a temperature of -2 to -1°C and store for 25-35 days. Then adjust the storage environment temperature to -4°C and the storage humidity to ≤15%.

2. The method according to claim 1, characterized in that The composite fresh-keeping solution in step (2) contains 0.1wt% trehalose, 0.05wt% ascorbic acid and 50ppm boric acid, and the pH value of the composite fresh-keeping solution is 6.

0.

3. The method according to claim 1, characterized in that In the step (4), silica gel desiccant and activated carbon powder in a mass ratio of 3:1 are placed in the aluminum foil composite film bag.

4. The method according to claim 1, characterized in that: The storage in step (4) is to be kept away from light throughout the process.

5. The use of Camellia chrysantha pollen stored according to the method of any one of claims 1 to 4, characterized in that: The camellia chrysantha pollen is used for artificial pollination.

6. A method for artificial pollination using Camellia chrysantha pollen stored by the method according to claims 1-5, characterized in that: The method comprises the following steps: 1) Pollen resuscitation and activation: Using a gradient temperature recovery method, first place the stored pollen in an environment of 3-5℃ for 2 hours, and then transfer it to an environment of 22-28℃ for slow release for 1 hour; 2) Use of pollen: After the revived and activated pollen is tested and its germination rate meets the requirements, it can be used for artificial pollination.

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