Camellia oleifera pollen culture solution and application method thereof in artificial pollination of camellia oleifera

Abstract

The invention provides a camellia oleifera pollen culture solution and an application method of the camellia oleifera pollen culture solution in artificial pollination of camellia oleifera. The camellia oleifera pollen culture solution is prepared from 100g / L of sucrose, 0.005 g / L of boric acid, 0.01 g / L-0. 1g / L of monopotassium phosphate, 0.01 g / L-0. 1g / L of compound sodium nitrophenolate, 0.001 g / L-0. 1g / L of sodium nitrophenolate, 0.001 g / L-0. 1g / L of 4-dichlorphenoxyacetic acid and 0.005 mmol / L-0. 1mol / L of spermidine. According to the method, the pollen can be stably adsorbed on stigmas of female flowers, so that the pollination effect is guaranteed, meanwhile, the germination rate of the camellia oleifera pollen can be remarkably increased, the pollination fruit setting rate of camellia oleifera is increased, and the use amount of the pollen can be reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a camellia oil pollen culture solution and an application method thereof in artificial pollination of camellia oil. Background Art

[0002] Camellia oleifera refers to the general term for oil plants of the genus Camellia in the family Theaceae. China is the origin and main production area of ​​camellia oleifera. Camellia oleifera is a woody edible oil crop that China focuses on developing, with a current cultivated area of ​​more than 70 million mu in China. Camellia oleifera is one of the four major woody oil-bearing tree species in the world. Camellia oleifera seed oil (tea oil) is one of the world's most recognized edible oils of the best quality and is listed as a key healthy edible oil for promotion by the Food and Agriculture Organization of the United Nations. Camellia oleifera seed oil and its by-products are also important pharmaceutical and chemical raw materials, and are widely used in medicine, agriculture and daily chemical industries. Camellia oleifera is a veritable "hard-core crop" with the unique advantages and development potential of benefiting from planting once for many years and not competing with farmers for land or people for food. Developing the camellia oleifera industry is my country's edible oil safety strategy. However, the low fruit setting rate and low yield of camellia oleifera significantly affect the sustainable and healthy development of the camellia oleifera industry, which is a bottleneck problem of the camellia oleifera industry at this stage.

[0003] The main reason for the low fruit yield of Camellia oleifera is poor cross-pollination and poor cross-fertilization caused by unreasonable variety matching, which ultimately leads to the phenomenon of "flowers but no fruits" or "one fruit for a thousand flowers". Camellia oleifera is a strictly insect-pollinated flower, and insufficient insect pollination during the flowering period of Camellia oleifera will lead to poor cross-pollination.

[0004] Therefore, it is necessary to reasonably configure pollination trees or carry out artificial assisted pollination in production. The configuration of pollination trees mainly utilizes the pollination ability of insects for natural pollination. Although it saves labor and effort, it is easily affected by the weather and causes poor pollination and fertilization. Although the artificial spot pollination method in the artificial assisted pollination method has strong selectivity, high accuracy, and small pollen consumption, it has a short operating time, a long time, a lot of labor, and many restrictions. Artificial assisted liquid pollination is a new pollination technology with the characteristics of fast pollination speed and labor saving. It replaces the pollination method of insects (oil tea bees, large tongue bees, etc.) to a certain extent, and can increase the moisture and nutrients of the stigma, and prolong the stigma pollination and fertilization time.

[0005] In the prior art, Chinese patent CN109206249A discloses a method for preparing and using pollen nutrient solution for oil-tea camellia pollination, which can reduce the aging speed of pistils. The raw materials are: 15-20g of oil-tea camellia pollen, 19-22g of potassium sulfate, 31-37g of diammonium phosphate, 120-140g of sucrose, 60-70g of water-soluble chitosan and 32-35g of borax. The method is used for spraying auxiliary pollination, which is not affected by strong winds, low temperatures and other weather conditions. However, the total concentration of the drugs used in the method is high, and the cost is high. Moreover, the fruit setting rate of the oil-tea camellia after spraying is only increased by 1-2 times.

[0006] Therefore, there is an urgent need to develop a camellia oleifera pollen culture solution and apply it to artificial pollination of camellia oleifera to further increase the fruit setting rate of camellia oleifera and achieve high yield of camellia oleifera, while reducing costs compared to existing technologies, which has important application value. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a camellia oil pollen culture solution and an application method thereof in artificial pollination of camellia oil. The technical scheme is as follows:

[0008] The first aspect of the present invention provides a camellia oleifera pollen culture solution, which includes 100g / L sucrose, 0.005g / L boric acid, 0.01g / L-0.1g / L potassium dihydrogen phosphate, 0.01g / L-0.1g / L sodium nitrophenolate, 0.001-0.01g / L 2,4-dichlorophenoxyacetic acid and 0.005mmol / L-0.1mmol / L spermidine.

[0009] The second aspect of the present invention provides a method for preparing a camellia oleifera pollen culture solution, the specific steps of which are as follows:

[0010] Take 1000g sucrose, 0.05g boric acid, 0.1-1g potassium dihydrogen phosphate, 0.1-1g sodium nitrophenolate, 50-1000ul spermidine mother solution, 0.01-0.1g 2,4-dichlorophenoxyacetic acid, add clean water to dissolve all solid substances, and dilute to 10L to obtain Camellia oleifera pollen culture solution;

[0011] The concentration of the spermidine mother solution is 1 mol / L, and the preparation method thereof comprises: taking 2.55 g of spermidine powder, adding clean water to completely dissolve the spermidine powder, and fixing the volume to 10 ml;

[0012] The 0.01-0.1 g of 2,4-dichlorophenoxyacetic acid is first completely dissolved in 1-5 ml of anhydrous ethanol and then used to prepare the camellia oleifera pollen culture solution.

[0013] The third aspect of the present invention provides the use of a camellia oleifera pollen culture solution prepared by the above method in camellia oleifera pollination.

[0014] The fourth aspect of the present invention provides a method for pollinating oil tea, the method comprising: the specific operations are as follows:

[0015] Add fresh camellia oleifera pollen and the camellia oleifera pollen culture solution in a mass ratio of 1:100-500 into an electric sprayer with a stirring function, and quickly stir for 2 minutes to evenly distribute the pollen in the camellia oleifera pollen culture solution, and spray while stirring. The camellia oleifera pollen culture solution with the camellia oleifera pollen added should be used up within 1 hour;

[0016] The fresh camellia oleifera pollen is the camellia oleifera pollen collected by bees within 2 hours;

[0017] Spray the camellia pollen culture solution between 9:00 and 14:00 on a sunny day, aim the nozzle at the flowers, and apply 6-10L / mu in the field. Do not rain within 10 hours after spraying.

[0018] Furthermore, when the electric sprayer is in use, the vertical distance between the spray head and the flowers is 80-100 cm.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention aims to solve the problem of poor cross-pollination of Camellia oleifera and invents a formula for liquid culture solution of Camellia oleifera pollen and its application process. By artificial liquid pollination, a good cross-pollination effect is effectively achieved, the fruit setting rate is improved, and the problem of low yield caused by poor cross-pollination of Camellia oleifera is solved. The invention has good application prospects.

[0021] (2) The present invention can not only improve the pollen germination rate, but also realize pollen germination in a short time. The pollen has germinated before the style ages and dies, thereby improving the speed and quality of pollen germination.

[0022] (3) The total concentration of the drug used in the present invention is low, which reduces costs. The fruit setting rate of the oil tea after spraying is increased by 3-4 times compared with the traditional pollination method, which is far superior to the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Collecting pollen for bees in the field.

[0024] Figure 2The figures are the pollen germination in CK and pollen culture solution. Note: A in treatment 1 is the pollen germination in CK, and B in treatment 1 is the pollen germination in pollen culture solution; A in treatment 2 is the pollen germination in CK, and B in treatment 2 is the pollen germination in pollen culture solution; A in treatment 3 is the pollen germination in CK, and B in treatment 3 is the pollen germination in pollen culture solution; A in treatment 4 is the pollen germination in CK, and B in treatment 4 is the pollen germination in pollen culture solution; A in treatment 5 is the pollen germination in CK, and B in treatment 5 is the pollen germination in pollen culture solution.

[0025] Figure 3 It is used for artificial spraying of pollen culture solution in the field.

[0026] Figure 4 The figures are the pollen germination in CK and culture medium. Note: A in treatment 1 is the pollen germination in CK, and B in treatment 1 is the pollen germination in pollen culture medium; A in treatment 2 is the pollen germination in CK, and B in treatment 2 is the pollen germination in pollen culture medium; A in treatment 3 is the pollen germination in CK, and B in treatment 3 is the pollen germination in pollen culture medium; A in treatment 4 is the pollen germination in CK, and B in treatment 4 is the pollen germination in pollen culture medium; A in treatment 5 is the pollen germination in CK, and B in treatment 5 is the pollen germination in pollen culture medium.

[0027] Figure 5 This is the fruit set after CK and powder spraying treatment. Note: Figure 5 The A in it is CK processing, Figure 5 B in the figure is powder spraying treatment. DETAILED DESCRIPTION

[0028] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0029] Example 1 A method for pollinating oil tea

[0030] (1) Pollen collection:

[0031] The collection of oil-tea pollen is carried out on sunny days. Italian bees or Chinese bees are released in the oil-tea forest, and pollen removal and collection devices are placed on the beehives to collect the pollen collected by the bees (see Figure 1). Collect once every hour, put the collected pollen (in small pieces) into a sealed bag immediately, and add small silica gel (less than 2mm in diameter) at a weight ratio of 1:5. After sealing, rub the pollen immediately to crush the block pollen and make it fully contact with the silica gel to accelerate dehydration. Put it in dry ice and bring it back to the laboratory and store it at -20℃. Within 1 year of storage, the pollen can maintain a germination rate of about 50%.

[0032] (2) Preparation of Camellia oleifera pollen culture medium

[0033] Since the required concentration of spermidine is relatively low and the weighing error is relatively large, a mother solution is prepared first. The preparation method is as follows: 2.55 g of spermidine powder is taken, and clean water is added to completely dissolve the spermidine powder, and the volume is fixed to 10 ml to obtain a 1 mol / L spermidine mother solution.

[0034] The oil-tea camellia pollen culture solution of the present invention is prepared by taking 1000g of sucrose, 0.05g of boric acid, 0.1g of potassium dihydrogen phosphate, 0.1g of sodium nitrophenolate, 50ul of spermidine mother solution, and 0.01g of 2,4-dichlorophenoxyacetic acid (the 2,4-dichlorophenoxyacetic acid is added after being completely dissolved in 1ml of anhydrous ethanol), adding clear water to the above mixture to dissolve all solid substances, and fixing the volume to 10L to obtain the oil-tea camellia pollen culture solution.

[0035] Prepare CK solution: add 1000 g sucrose and 0.05 g boric acid into clean water to completely dissolve the solid matter, adjust the volume to 10 L, and obtain solution CK.

[0036] (3) Pollen germination rate detection

[0037] Fresh camellia oleifera pollen and the camellia oleifera pollen culture solution prepared in step (2) are mixed at a mass ratio of 1:100-500, and fresh camellia oleifera pollen and the CK solution prepared in step (2) are mixed at a mass ratio of 1:100-500, and 1 mL is taken from each of the two mixed solutions for microscopic observation. The fresh camellia oleifera pollen is the camellia oleifera pollen collected by bees within 2 hours.

[0038] 200ul of Camellia oleifera pollen culture solution with pollen added was dropped onto a single concave slide, and the germination of pollen was observed under a 4×4 microscope, and the number of pollen grains was counted. Three parallel slides were made for each sample, and 5 fields of view were taken for each slide. Fields of view with no less than 50 pollen grains and uniform distribution were selected for statistics. The pollen tube length exceeding the pollen grain diameter was used as the germination standard for counting, and the calculation formula for the results was: Pollen germination rate = (number of pollen grains germinated in the field of view / total number of pollen grains in the field of view) * 100%.

[0039] The pollen germination rates of CK and the identification solution treated with the agent of the present invention were different. Figure 2 and Table 1.

[0040] (4) Fruit setting rate detection

[0041] Add fresh Camellia oleifera pollen and the Camellia oleifera pollen culture solution in a mass ratio of 1:100-500 into an electric sprayer with a stirring function, and stir quickly for 2 minutes to evenly distribute the pollen in the Camellia oleifera pollen culture solution, and spray while stirring (see Figure 3 ), the camellia pollen culture solution with camellia pollen added was used up within 1 hour, the vertical distance between the electric sprayer nozzle and the flower was 80-100 cm, and the field application rate was 6-10 L / mu. The fruit setting rate was counted one month after spraying, and the results are shown in Table 1.

[0042] Table 1 Pollen germination rate and fruit setting rate after treatment

[0043]

[0044] Note: The fruit setting rate is calculated based on a total of 100 flowers.

[0045] Example 2 A method for pollinating oil tea

[0046] (1) Pollen collection:

[0047] The collection of oil-tea pollen is carried out on sunny days. Italian bees or Chinese bees are released in the oil-tea forest, and pollen removal and collection devices are placed on the beehives to collect the pollen collected by the bees (see Figure 1 ). Collect once every hour, put the collected pollen (in small pieces) into a sealed bag immediately, and add small silica gel (less than 2mm in diameter) at a weight ratio of 1:5. After sealing, rub the pollen immediately to crush the block pollen and make it fully contact with the silica gel to accelerate dehydration. Put it in dry ice and bring it back to the laboratory and store it at -20℃. Within 1 year of storage, the pollen can maintain a germination rate of about 50%.

[0048] (2) Preparation of Camellia oleifera pollen culture medium

[0049] Since the required concentration of spermidine is relatively low and the weighing error is relatively large, a mother solution is prepared first. The preparation method is as follows: 2.55 g of spermidine powder is taken, and clean water is added to completely dissolve the spermidine powder, and the volume is fixed to 10 ml to obtain a 1 mol / L spermidine mother solution.

[0050] The oil-tea camellia pollen culture solution of the present invention is prepared by taking 1000g of sucrose, 0.05g of boric acid, 1g of potassium dihydrogen phosphate, 1g of sodium nitrophenolate, 1000ul of spermidine mother solution, and 0.1g of 2,4-dichlorophenoxyacetic acid (the 2,4-dichlorophenoxyacetic acid is added after being completely dissolved in 5ml of anhydrous ethanol), adding clear water to the above mixture to dissolve all solid substances, and fixing the volume to 10L to obtain the oil-tea camellia pollen culture solution.

[0051] Prepare CK solution: add 1000 g sucrose and 0.05 g boric acid into clean water to completely dissolve the solid matter, adjust the volume to 10 L, and obtain solution CK.

[0052] (3) Pollen germination rate detection

[0053] Fresh camellia oleifera pollen and the camellia oleifera pollen culture solution prepared in step (2) are mixed at a mass ratio of 1:100-500, and fresh camellia oleifera pollen and the CK solution prepared in step (2) are mixed at a mass ratio of 1:100-500, and 1 mL is taken from each of the two mixed solutions for microscopic observation. The fresh camellia oleifera pollen is the camellia oleifera pollen collected by bees within 2 hours.

[0054] 200ul of Camellia oleifera pollen culture solution with pollen added was dropped onto a single concave slide, and the germination of pollen was observed under a 4×4 microscope, and the number of pollen grains was counted. Three parallel slides were made for each sample, and 5 fields of view were taken for each slide. Fields of view with no less than 50 pollen grains and uniform distribution were selected for statistics. The pollen tube length exceeding the pollen grain diameter was used as the germination standard for counting, and the calculation formula for the results was: Pollen germination rate = (number of pollen grains germinated in the field of view / total number of pollen grains in the field of view) * 100%.

[0055] The pollen germination rates of CK and the identification solution treated with the agent of the present invention are different. Figure 4 and Table 2.

[0056] (4) Fruit setting rate detection

[0057] Add fresh Camellia oleifera pollen and the Camellia oleifera pollen culture solution in a mass ratio of 1:100-500 into an electric sprayer with a stirring function, and stir quickly for 2 minutes to evenly distribute the pollen in the Camellia oleifera pollen culture solution, and spray while stirring (see Figure 3 ), the oil camellia pollen culture solution with oil camellia pollen added was used up within 1 hour, the vertical distance between the electric sprayer nozzle and the flower was 80-100 cm, and the field application rate was 6-10 L / mu. The fruit setting rate was counted one month after spraying, and the results are shown in Table 2 and Figure 5 .

[0058] Table 2 Pollen germination rate and fruit setting rate after treatment

[0059]

[0060] Note: The fruit setting rate is calculated based on a total of 100 flowers.

[0061] Based on Examples 1 and 2, the present invention can improve the pollen germination rate and achieve pollen germination in a short time, and the pollen has germinated before the style ages and dies, thereby improving the speed and quality of pollen germination. The total concentration of the drugs used is low, which reduces costs, and the fruit setting rate of the oil tea after spraying is increased by 3-4 times compared with the traditional pollination method, which is far superior to the existing technology.

[0062] Obviously, the above 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, ordinary technicians in this field can make other different forms of changes or modifications without making creative work, which should fall within the scope of invention protection.

Claims

1. A Camellia oleifera pollen culture solution, characterized in that: The oil-tea camellia pollen culture solution comprises 100 g / L sucrose, 0.005 g / L boric acid, 0.01 g / L-0.1 g / L potassium dihydrogen phosphate, 0.01 g / L-0.1 g / L sodium nitrophenolate, 0.001-0.01 g / L 2,4-dichlorophenoxyacetic acid and 0.005 mmol / L-0.1 mmol / L spermidine.

2. A method for preparing a camellia oleifera pollen culture solution, characterized in that: The specific steps are as follows: Take 1000g sucrose, 0.05g boric acid, 0.1-1g potassium dihydrogen phosphate, 0.1-1g sodium nitrophenolate, 50-1000ul spermidine mother solution, 0.01-0.1g 2,4-dichlorophenoxyacetic acid, add clean water to dissolve all solid substances, and dilute to 10L to obtain Camellia oleifera pollen culture solution; The concentration of the spermidine mother solution is 1 mol / L, and the preparation method thereof comprises: taking 2.55 g of spermidine powder, adding clean water to completely dissolve the spermidine powder, and fixing the volume to 10 ml; The 0.01-0.1 g of 2,4-dichlorophenoxyacetic acid is first completely dissolved in 1-5 ml of anhydrous ethanol and then used to prepare the camellia oleifera pollen culture solution.

3. Use of a camellia oleifera pollen culture solution prepared by the method of claim 2 in camellia oleifera pollination.

4. A method for pollinating oil-tea camellia, characterized in that: The method comprises the following specific operations: Add fresh camellia oleifera pollen and the camellia oleifera pollen culture solution of claim 1 in a mass ratio of 1:100-500 into an electric sprayer with a stirring function, and quickly stir for 2 minutes to evenly distribute the pollen in the camellia oleifera pollen culture solution, and spray while stirring. The camellia oleifera pollen culture solution with the camellia oleifera pollen added should be used up within 1 hour; The fresh camellia oleifera pollen is the camellia oleifera pollen collected by bees within 2 hours; Spray the camellia pollen culture solution between 9:00 and 14:00 on a sunny day, aim the nozzle at the flowers, and apply 6-10L / mu in the field. Do not rain within 10 hours after spraying.

5. A method for pollinating oil-tea camellia according to claim 4, characterized in that: When the electric sprayer is in use, the vertical distance between the spray head and the flowers is 80-100 cm.

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