Plateau oat hybridization method based on oat pollen culture solution

Abstract

The invention discloses a plateau oat hybridization method based on an oat pollen culture solution, and belongs to the technical field of oat hybridization breeding. The highland oat hybridization method comprises the following steps: (1) selecting a male parent and a female parent; (2) trimming ears of the female parent; (3) emasculating the female parent; (4) spraying a pollen culture solution to male parent tassel pollen; (5) collecting male parent pollen for pollination; wherein the pollen culture solution is prepared from the following components: cane sugar, brassinosterols, monopotassium phosphate and calcium nitrate. The hybridization method for the plateau is suitable for oat hybridization in the plateau area, the activity of oat pollen in the original area can be remarkably improved, pollen germination is promoted, and therefore the maturing rate of the hybrid oat is increased, and the hybridization success rate is increased. Therefore, the hybridization method provided by the invention provides a simple, convenient and rapid way for the cultivation of fine oat varieties in plateau areas.

Description

Technical Field

[0001] The invention relates to the technical field of oat hybrid breeding, and in particular to a plateau oat hybridization method based on oat pollen culture fluid. Background Art

[0002] Oats are an annual herbaceous plant of the Poaceae family. They prefer to grow in areas with cool summers and abundant rainfall. They are mainly produced in the high-altitude cold regions of northwest my country and are important crops used for both food and feed in these areas. Oats can be divided into green fodder, hay, silage, etc. Its stems and leaves are good forage, while its seeds are energy feed.

[0003] At present, there are few studies on oat hybrid breeding. In the prior art, oat hybridization methods mostly adopt the methods of pruning emasculation and pruning pollination. However, due to the low temperature, large temperature difference between day and night, strong wind, and dryness and water shortage in plateau areas, such climatic conditions have a serious impact on the viability of oat pollen, resulting in a low oat hybridization fruit setting rate. It can be seen that the existing oat hybridization method is not suitable for plateau areas. Therefore, it is urgent to develop an oat hybridization method suitable for plateau areas. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a plateau oat hybridization method based on oat pollen culture solution to improve the fruit setting rate of hybrid oats in plateau areas.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a method for highland oat hybridization based on oat pollen culture solution is provided, comprising the following steps: (1) Select the male and female parent; (2) Trimming the ears of the female parent; (3) Emasculating the female parent; (4) Spraying pollen culture solution on the male tassel pollen; (5) Collecting pollen from the male parent for pollination; The pollen culture solution comprises the following components in parts by weight: 250-400 parts of sucrose, 0.5-1.0 parts of brassinosteroids, 0.2-0.5 parts of potassium dihydrogen phosphate and 0.01-0.03 parts of calcium nitrate.

[0006] Further, the pollen culture solution includes the following components in parts by weight: 300 parts of sucrose, 0.5 parts of brassinosteroids, 0.2 parts of potassium dihydrogen phosphate and 0.02 parts of calcium nitrate.

[0007] Further, the male parent is at least one of Sweet Oats, Menglong, Beile, Longyan No. 3, Longyan No. 4, Qingmu No. 3, Mengyan No. 6, Longyan No. 1, and Bayan No. 4; Furthermore, the female parent is at least one of Sweet Oats, Menglong, Beile, Longyan No. 3, Longyan No. 4, Qingmu No. 3, Mengyan No. 6, Longyan No. 1, and Bayan No. 4.

[0008] Furthermore, in step (1), the male parent and the female parent are selected as follows: the female parent is selected from individuals drawn from 1 / 2-3 / 4 of the ears, and the male parent is selected from individuals drawn from 3 / 4 of the ears.

[0009] Furthermore, in step (2), the ear trimming of the mother plant is performed by removing the pollinated spikelets at the top of the ear of the mother plant and the spikelets that are not mature at the base and are not erect.

[0010] Furthermore, in step (3), the female plant is emasculated by: using tweezers to break open the seed coat, removing the anther and stigma together, then restoring the seed coat, sealing the female plant spike with an isolation bag, and then placing the female plant in an upright state; Furthermore, the mother plant is placed in an upright state by inserting a fixing device next to the ear of the mother plant and fixing the ear of the mother plant on the fixing device.

[0011] Furthermore, the height of the wooden stick or bamboo stick should be 10-50 cm higher than the oat ear of the mother plant.

[0012] Furthermore, in step (4), the pollen culture solution is sprayed on the male parent's tassel pollen, and the pollen culture solution is sprayed on the male parent's pollen one day before pollination, and then an isolation bag is placed on the bag.

[0013] Furthermore, in step (5), pollen of the male parent is collected for pollination by cutting off the male spike, placing the cut male spike with the cut end facing downward into the female parent spike isolation bag and resealing it, and flicking the isolation bag with fingers for pollination.

[0014] Furthermore, the pollination days are 5-7 days; the pollination time is 12:00-14:00 every day.

[0015] The present invention has the following beneficial effects: (1) Based on the existing oat hybridization method, the present invention sprays pollen culture solution on oat pollen before pollination; the components of the pollen culture solution include sucrose, brassinosteroid, potassium dihydrogen phosphate and calcium nitrate. Among them, sucrose provides carbon source and energy to support the respiration and metabolic activities of pollen; potassium dihydrogen phosphate and calcium nitrate provide necessary mineral elements to support the growth and development of pollen, maintain the osmotic pressure and pH value of the culture solution, and create a suitable growth environment for pollen; brassinosteroid is a plant hormone with a moisturizing effect, which can ensure the humidity conditions during pollen pollination. At the same time, the test results of the present invention show that the use of pollen culture solution with brassinosteroid added to treat oat pollen can significantly improve the vitality of oat pollen and promote pollen germination, indicating that brassinosteroid, sucrose, potassium dihydrogen phosphate and calcium nitrate have a significant synergistic effect on pollen germination.

[0016] (2) The plateau oat hybridization method of the present invention is suitable for oat hybridization in plateau areas, and can effectively improve the influence of the low temperature and water shortage environment of the plateau on the vigor of oat pollen, and can improve the vigor of oat pollen and promote pollen germination, thereby improving the hybridization fruit setting rate and success rate. Therefore, the hybridization method provided by the present invention provides a simple and fast way for the breeding of superior oat varieties. DETAILED DESCRIPTION

[0017] The principles and features of the present invention are described below in conjunction with the examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0018] Examples 1-4 and Comparative Examples 1-3 were all planted in Lhasa.

[0019] Embodiment 1: A plateau oat hybridization method based on oat pollen culture solution comprises the following steps: (1) Parent selection: The female parent is selected from individuals with 3 / 4 ears, and the male parent is selected from individuals with 3 / 4 ears; the female parent is sweet oats and the male parent is Longyan No. 3; (2) Whole ear: remove the pollinated spikelets at the top of the mother ear, the immature spikelets at the base, and the non-erect spikelets; (3) Emasculation: Use tweezers to break open the seed coat, find 3 anthers and pinch them off, and use tweezers to pinch off the remaining 6 anthers together with 2 stigmas, restore the seed coat, seal the female parent spike with an isolation bag, and insert a wooden stick next to the female parent spike. The height of the wooden stick is 30 cm higher than the female parent spike. Then fix the female parent spike on the wooden stick so that the female parent plant is in an upright state. (4) Preparation of pollen culture solution: Weigh 300 parts of sucrose, 0.5 parts of brassinosteroids, 0.2 parts of potassium dihydrogen phosphate and 0.02 parts of calcium nitrate respectively, dissolve in 1 L of pure water, and stir evenly to obtain; (5) Preparation of male spikes: After the female plant is emasculated, select healthy plants from the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. The night before pollination, use a spray bottle to spray the pollen with pollen culture solution and put it in an isolation bag. (6) Pollination: Cut off the male tassel of the male parent, place the cut male tassel with the cut end facing down into the female parent tassel isolation bag and reseal it. Use your fingers to flick the isolation bag for pollination at 14:00 every day; (7) Repeat steps (5)-(6) and continue pollination for 4 days.

[0020] Embodiment 2: A plateau oat hybridization method based on oat pollen culture solution comprises the following steps: (1) Parent selection: The female parent is selected from individuals drawn from 1 / 2 of the ears, and the male parent is selected from individuals drawn from 3 / 4 of the ears; the female parent is Beile and the male parent is Bayan No. 4; (2) Whole ear: remove the pollinated spikelets at the top of the mother ear, the immature spikelets at the base, and the non-erect spikelets; (3) Emasculation: Use tweezers to break open the seed coat, find 3 anthers and pinch them off, and use tweezers to pinch off the remaining 6 anthers together with 2 stigmas, restore the seed coat, seal the female parent spike with an isolation bag, and insert a wooden stick next to the female parent spike. The height of the wooden stick is 30 cm higher than the female parent spike. Then fix the female parent spike on the wooden stick so that the female parent plant is in an upright state. (4) Preparation of pollen culture solution: Weigh 250 parts of sucrose, 1.0 part of brassinosteroids, 0.3 parts of potassium dihydrogen phosphate and 0.01 parts of calcium nitrate respectively, dissolve in 1 L of pure water, and stir evenly to obtain; (5) Preparation of male spikes: After the female plant is emasculated, select healthy plants from the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. Spray pollen culture solution on the pollen the night before pollination and put it in an isolation bag. (6) Pollination: Cut off the male spike of the male parent, place the cut male spike with the cut end facing down into the female parent spike isolation bag and reseal it. Use your fingers to flick the isolation bag for pollination at 14:00 every day; (7) Repeat steps (5)-(6) and continue pollination for 5 days.

[0021] Embodiment 3: A plateau oat hybridization method based on oat pollen culture solution comprises the following steps: (1) Parent selection: The female parent is selected from individuals with 3 / 4 ears, and the male parent is selected from individuals with 3 / 4 ears; the female parent is Qingmu No. 3, and the male parent is Mengyan No. 6; (2) Whole ear: remove the pollinated spikelets at the top of the mother ear, the immature spikelets at the base, and the non-erect spikelets; (3) Emasculation: Use tweezers to break open the seed coat, find 3 anthers and pinch them off, and use tweezers to pinch off the remaining 6 anthers together with 2 stigmas, restore the seed coat, seal the female parent spike with an isolation bag, and insert a wooden stick next to the female parent spike. The height of the wooden stick is 30 cm higher than the female parent spike. Then fix the female parent spike on the wooden stick so that the female parent plant is in an upright state. (4) Preparation of pollen culture solution: Weigh 400 parts of sucrose, 0.8 parts of brassinosteroids, 0.3 parts of potassium dihydrogen phosphate and 0.01 parts of calcium nitrate respectively, dissolve in 1 L of pure water, and stir evenly to obtain; (5) Preparation of male spikes: After the female plant is emasculated, select healthy plants from the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. Spray pollen culture solution on the pollen the night before pollination and put it in an isolation bag. (6) Pollination: Cut off the male spike of the male parent, place the cut male spike with the cut end facing down into the female parent spike isolation bag and reseal it. Use your fingers to flick the isolation bag for pollination at 14:00 every day; (7) Repeat steps (5)-(6) and continue pollination for 6 days.

[0022] Embodiment 4: A plateau oat hybridization method based on oat pollen culture solution comprises the following steps: (1) Parent selection: The female parent is selected from individuals with 3 / 4 ears, and the male parent is selected from individuals with 3 / 4 ears; the female parent is Menglong and the male parent is Longyan No. 1; (2) Whole ear: remove the pollinated spikelets at the top of the mother ear, the immature spikelets at the base, and the non-erect spikelets; (3) Emasculation: Use tweezers to break open the seed coat, find 3 anthers and pinch them off, and use tweezers to pinch off the remaining 6 anthers together with 2 stigmas, restore the seed coat, seal the female parent spike with an isolation bag, and insert a wooden stick next to the female parent spike. The height of the wooden stick is 30 cm higher than the female parent spike. Then fix the female parent spike on the wooden stick so that the female parent plant is in an upright state. (4) Preparation of pollen culture solution: Weigh 350 parts of sucrose, 0.6 parts of brassinosteroids, 0.5 parts of potassium dihydrogen phosphate and 0.03 parts of calcium nitrate respectively, dissolve in 1 L of pure water, and stir evenly to obtain; (5) Preparation of male spikes: After the female plant is emasculated, select healthy plants from the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. Spray pollen culture solution on the pollen the night before pollination and put it in an isolation bag. (6) Pollination: Cut off the male spike of the male parent, place the cut male spike with the cut end facing down into the female parent spike isolation bag and reseal it. Use your fingers to flick the isolation bag for pollination at 12:00 every day; (7) Repeat steps (5)-(6) and continue pollination for 7 days.

[0023] Comparative Example 1 A method for hybridizing oats comprises the following steps: (1) Parent selection: The female parent is selected from individuals with 3 / 4 ears, and the male parent is selected from individuals with 3 / 4 ears; the female parent is Menglong and the male parent is Longyan No. 1; (2) Whole ear: remove the pollinated spikelets at the top of the mother ear, the immature spikelets at the base, and the non-erect spikelets; (3) Emasculation: Use tweezers to break open the seed coat, find 3 anthers and pinch them off, and use tweezers to pinch off the remaining 6 anthers together with 2 stigmas, restore the seed coat, seal the female parent spike with an isolation bag, and insert a wooden stick next to the female parent spike. The height of the wooden stick is 30 cm higher than the female parent spike. Then fix the female parent spike on the wooden stick so that the female parent plant is in an upright state. (4) Preparation of male spikes: After the female plant is emasculated, select a strong plant in the male plant row and cut off 1 / 5 of the floret head and both sides diagonally; (5) Pollination: Cut off the male spike of the male parent, place the cut male spike with the cut end facing down into the female parent spike isolation bag and reseal it. Use your fingers to flick the isolation bag for pollination at 12:00 every day; (6) Repeat step (5) and continue pollination for 7 days.

[0024] Comparative Example 2 A method for hybridizing oats comprises the following steps: (1) Parent selection: The female parent is selected from individuals with 3 / 4 ears, and the male parent is selected from individuals with 3 / 4 ears; the female parent is sweet oats and the male parent is Longyan No. 3; (2) Whole ear: remove the pollinated spikelets at the top of the mother ear, the immature spikelets at the base, and the non-erect spikelets; (3) Emasculation: Use tweezers to break open the seed coat, find 3 anthers and pinch them off, and use tweezers to pinch off the remaining 6 anthers together with 2 stigmas, restore the seed coat, seal the female parent spike with an isolation bag, and insert a wooden stick next to the female parent spike. The height of the wooden stick is 30 cm higher than the female parent spike. Then fix the female parent spike on the wooden stick so that the female parent plant is in an upright state. (4) Preparation of pollen culture solution: Weigh 400 parts of sucrose, 0.8 parts of boric acid, 0.3 parts of potassium dihydrogen phosphate and 0.01 parts of calcium nitrate respectively, dissolve in 1 L of pure water, and stir evenly to obtain; (5) Preparation of male spikes: After the female plant is emasculated, select healthy plants from the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. Spray pollen culture solution on the pollen the night before pollination and put it in an isolation bag. (6) Pollination: Cut off the male spike of the male parent, place the cut male spike with the cut end facing down into the female parent spike isolation bag and reseal it. Use your fingers to flick the isolation bag for pollination at 14:00 every day; (7) Repeat steps (5)-(6) and continue pollination for 6 days.

[0025] Comparative Example 3 A method for hybridizing oats comprises the following steps: (1) Parent selection: The female parent is selected from individuals with 3 / 4 ears, and the male parent is selected from individuals with 3 / 4 ears; the female parent is Qingmu No. 3, and the male parent is Mengyan No. 6; (2) Whole ear: remove the pollinated spikelets at the top of the mother ear, the immature spikelets at the base, and the non-erect spikelets; (3) Emasculation: Use tweezers to break open the seed coat, find 3 anthers and pinch them off, and use tweezers to pinch off the remaining 6 anthers together with 2 stigmas, restore the seed coat, seal the female parent spike with an isolation bag, and insert a wooden stick next to the female parent spike. The height of the wooden stick is 30 cm higher than the female parent spike. Then fix the female parent spike on the wooden stick so that the female parent plant is in an upright state. (4) Preparation of pollen culture solution: Weigh 400 parts of sucrose, 0.3 parts of potassium dihydrogen phosphate and 0.01 parts of calcium nitrate respectively, dissolve them in 1L of pure water, and stir evenly to obtain; (5) Preparation of male spikes: After the female plant is emasculated, select healthy plants from the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. Spray pollen culture solution on the pollen the night before pollination and put it in an isolation bag. (6) Pollination: Cut off the male spike of the male parent, place the cut male spike with the cut end facing down into the female parent spike isolation bag and reseal it. Use your fingers to flick the isolation bag for pollination at 14:00 every day; (7) Repeat steps (5)-(6) and continue pollination for 6 days.

[0026] Test example (1) Effect of pollen culture solution on pollen viability: The male tassels were collected and brought back to the laboratory. The anthers were taken out with tweezers and placed on white paper. The stamens were then taken out from the tassels to obtain the anthers. The anthers were cut into pieces to obtain pollen. The pollen was then cultured in the pollen culture solutions of Examples 1-4 and Comparative Examples 2-3 and in clean water, respectively. The pollen was then examined under a microscope and the pollen germination rate was counted. The results are shown in Table 1.

[0027] As can be seen from Table 1, after the pollen was treated with the pollen culture solution of Comparative Example 3, its pollen germination rate was improved compared with the water group. This may be because sucrose provides carbon source and energy to support the respiration and metabolic activities of pollen; potassium dihydrogen phosphate and calcium nitrate provide necessary mineral elements to support the growth and development of pollen, maintain the osmotic pressure and pH value of the culture solution, and create a suitable growth environment for pollen. After the pollen was treated with the pollen culture solution of Comparative Example 2, the activity of the pollen was higher than that of Comparative Example 3 and water. It shows that the treatment of oat pollen with pollen culture solution containing boric acid can increase the fruiting rate of oats. This may be because boric acid participates in the synthesis of pectin substances, is conducive to the formation of pollen tube walls, and synergizes with sucrose to promote the absorption and metabolism of sugar, thereby promoting the germination of pollen. After the pollen was treated with the pollen culture solution of the application, its pollen activity was significantly higher than that of Comparative Examples 2-3 and water, indicating that the pollen culture solution of the present application can significantly improve the activity of pollen and promote pollen germination. Therefore, brassinosteroids, sucrose, potassium dihydrogen phosphate, and calcium nitrate used together have a significant synergistic effect on pollen germination.

[0028] Table 1 Effects of pollen culture solutions of Examples 1-4 and Comparative Examples 2-3 on pollen viability

[0029] (2) After pollination, the fruit setting rates of Examples 1-4 and Comparative Examples 1-3 were counted, and the results are shown in Table 2. As shown in Table 2, the fruit setting rate of oats using the hybridization method of Comparative Example 2 is higher than that of Comparative Examples 1 and 3, and the fruit setting rate of oats using the hybridization method of Examples 1-4 is significantly higher than that of Comparative Example 2. This indicates that under the same conditions, the use of pollen culture solution with brassinosteroid added can significantly increase the fruit setting rate of oats. The reason may be that, on the one hand, brassinosteroid has a certain moisturizing effect, thereby ensuring the humidity conditions required by pollen during pollination; on the other hand, the above test results confirm that brassinosteroid does have the effect of improving pollen vitality and promoting pollen germination.

[0030] Table 2 Fruiting rate of Examples 1-4 and Comparative Examples 1-3

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for highland oat hybridization based on oat pollen culture solution, characterized in that: The following steps are involved: (1) Select the male and female parent; (2) Trimming the ears of the female parent; (3) Emasculating the female parent; (4) Spraying pollen culture solution on the male tassel pollen; (5) Collecting pollen from the male parent for pollination; The pollen culture solution comprises the following components in parts by weight: 250-400 parts of sucrose, 0.5-1.0 parts of brassinosteroids, 0.2-0.5 parts of potassium dihydrogen phosphate and 0.01-0.03 parts of calcium nitrate.

2. The highland oat hybridization method based on oat pollen culture solution according to claim 1, characterized in that: The pollen culture solution comprises the following components in parts by weight: 300 parts of sucrose, 0.5 parts of brassinosteroids, 0.2 parts of potassium dihydrogen phosphate and 0.02 parts of calcium nitrate.

3. The highland oat hybridization method based on oat pollen culture solution according to claim 1, characterized in that: The selection of the male parent and the female parent in step (1) is as follows: the female parent is selected from individuals drawn from 1 / 2-3 / 4 ears, and the male parent is selected from individuals drawn from 3 / 4 ears.

4. The highland oat hybridization method based on oat pollen culture solution according to claim 1, characterized in that: The step (2) of trimming the ear of the mother plant is to remove the pollinated spikelets at the top of the ear of the mother plant, the immature spikelets at the base and the non-erect spikelets.

5. The highland oat hybridization method based on oat pollen culture solution according to claim 1, characterized in that: The step (3) of emasculating the female plant is as follows: using tweezers to pry open the seed coat, removing the anther and stigma together, then restoring the seed coat, sealing the female plant spike with an isolation bag, and then placing the female plant in an upright position.

6. The highland oat hybridization method based on oat pollen culture solution according to claim 5, characterized in that: The method of making the female plant stand upright comprises inserting a fixing device beside the female ear and fixing the female ear on the fixing device.

7. The method for highland oat hybridization based on oat pollen culture solution according to claim 6, characterized in that: The height of the fixing device is 10-50 cm higher than the oat ear of the female parent.

8. The highland oat hybridization method based on oat pollen culture solution according to claim 1, characterized in that: The step (4) of spraying the pollen culture solution on the male parent's tassel pollen is as follows: one day before pollination, the pollen culture solution is sprayed on the male parent's pollen and an isolation bag is placed on the bag.

9. The highland oat hybridization method based on oat pollen culture solution according to claim 1, characterized in that: The step (5) of collecting pollen from the male parent for pollination is as follows: cutting off the male spike, placing the cut male spike with the cut end facing downward into the female parent spike isolation bag and resealing the bag, and flicking the isolation bag with fingers for pollination.

10. The highland oat hybridization method based on oat pollen culture solution according to claim 9, characterized in that: The pollination days are 5-7 days; the pollination time is 12:00-14:00 every day.

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