Avena sativa pollen culture liquid based highland oat hybridization method

Abstract

The application discloses a highland oat hybridization method based on oat pollen culture solution and belongs to the technical field of oat hybridization and breeding. The highland oat hybridization method comprises the following steps: (1) selecting a male parent and a female parent; (2) performing whole ear selection on the female parent; (3) performing emasculation on the female parent; (4) spraying pollen culture solution on the male spike pollen of the male parent; and (5) collecting the pollen of the male parent to perform pollination. The pollen culture solution comprises the following components: sucrose, brassinosteroid, potassium dihydrogen phosphate and calcium nitrate. The highland hybridization method is suitable for oat hybridization in a highland region, can significantly improve the vitality of oat pollen in the highland region, promotes pollen germination, thereby increasing the seed setting rate of hybridized oats and improving the hybridization success rate. Therefore, the hybridization method provides a simple and fast way for the cultivation of excellent oat varieties in the highland region.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oat cross breeding, and particularly relates to a highland oat cross method based on oat pollen culture solution. BACKGROUND

[0002] Oat is an annual herbaceous plant of the family Poaceae and the genus Avena, and likes to grow in a cool summer and a rainy area. Oat is mainly produced in the high-cold area of northwest China, and is an important grain and forage crop in the high-cold area of China. Oat can be divided into green forage, hay and silage, and the stems and leaves are good forage, and the grains are energy feed.

[0003] At present, there is little research on oat cross breeding, and the cross method of oat in the prior art mainly adopts the method of shearing the perianth to remove pollen and shearing the perianth to pollinate. However, due to the low temperature, large diurnal temperature difference and strong wind in the plateau area, and the dry and water shortage in the plateau area, the climate conditions seriously affect the pollen vitality of oat, resulting in a low seed setting rate of cross oat. Therefore, the existing oat cross method is not suitable for the plateau area. Therefore, it is urgent to develop an oat cross method suitable for the plateau area. SUMMARY

[0004] In order to solve the problems in the prior art, the purpose of the present application is to provide a highland oat cross method based on oat pollen culture solution, so as to improve the seed setting rate of cross oat in the plateau area.

[0005] The technical scheme for solving the above technical problems is as follows: a highland oat cross method based on oat pollen culture solution is provided, which comprises the following steps:

[0006] (1) selecting a male parent and a female parent;

[0007] (2) whole ear processing of the female parent;

[0008] (3) dehiscence of the female parent;

[0009] (4) spraying pollen culture solution on the pollen of the male parent;

[0010] (5) collecting the pollen of the male parent for pollination;

[0011] The pollen culture solution comprises the following components in parts by weight: 250-400 parts of sucrose, 0.5-1.0 parts of brassinosteroid, 0.2-0.5 parts of potassium dihydrogen phosphate and 0.01-0.03 parts of calcium nitrate.

[0012] Further, the pollen culture solution comprises the following components in parts by weight: 300 parts of sucrose, 0.5 parts of brassinosteroid, 0.2 parts of potassium dihydrogen phosphate and 0.02 parts of calcium nitrate.

[0013] Further, the paternal parent is at least one of sweet oat, Menglong, Beier, Longyan No.3, Longyan No.4, Qingmu No.3, Mengyan No.6, Longyan No.1 and Bayan No.4.

[0014] Further, the maternal parent is at least one of sweet oat, Menglong, Beier, Longyan No.3, Longyan No.4, Qingmu No.3, Mengyan No.6, Longyan No.1 and Bayan No.4.

[0015] Further, the paternal parent and the maternal parent selected in step (1) are as follows: the maternal parent is selected from individuals with 1 / 2-3 / 4 ear emergence, and the paternal parent is selected from individuals with 3 / 4 ear emergence.

[0016] Further, the whole ear of the maternal parent in step (2) is as follows: the top of the maternal ear has been pollinated, the base is not mature, and the small spike is not straight.

[0017] Further, the emasculation of the maternal parent in step (3) is as follows: the seed coat is pried open with tweezers, the anther and stigma are pinched together, and then the seed coat is restored, the maternal ear is sealed with an isolation bag, and the maternal plant is in a straight state.

[0018] Further, the maternal plant is in a straight state as follows: a fixing device is inserted beside the maternal ear, and the maternal ear is fixed on the fixing device.

[0019] Further, the height of the wooden stick or bamboo stick is 10-50 cm higher than the maternal oat ear.

[0020] Further, the pollen culture solution is sprayed on the paternal pollen in step (4) as follows: the pollen culture solution is sprayed on the paternal pollen one day before pollination, and an isolation bag is put on.

[0021] Further, the paternal pollen is collected for pollination in step (5) as follows: the male ear is cut off, the cut male ear is cut downward into the isolation bag of the maternal ear and resealed, and the isolation bag is bounced with fingers for pollination.

[0022] Further, the pollination time is 5-7 days; and the pollination time is 12:00-14:00 every day.

[0023] The present application has the following beneficial effects:

[0024] (1) Based on the existing oat hybridization method, the present invention sprays oat pollen with pollen culture solution before pollination; the components of the pollen culture solution include sucrose, brassinosteroids, potassium dihydrogen phosphate and calcium nitrate. Among them, sucrose provides a carbon source and energy source 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; brassinosteroids are a plant hormone with a moisturizing effect, which can ensure the humidity conditions during pollination. At the same time, the experimental results of the present invention show that treating oat pollen with pollen culture solution added with brassinosteroids can significantly improve the vitality of oat pollen and promote pollen germination, indicating that brassinosteroids, sucrose, potassium dihydrogen phosphate and calcium nitrate have a significant synergistic effect on pollen germination.

[0025] (2) The plateau oat hybridization method of the present invention is suitable for oat hybridization in plateau areas. It can effectively improve the effects of the low temperature and water shortage environment on the viability of oat pollen, enhance the viability of oat pollen, promote pollen germination, and thus improve the hybridization fruit set rate and success rate. Therefore, the hybridization method provided by the present invention provides a simple and rapid way to cultivate superior oat varieties. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below in conjunction with the examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0027] Examples 1-4 and Comparative Examples 1-3 were all planted in Lhasa.

[0028] Example 1:

[0029] A plateau oat hybridization method based on oat pollen culture solution comprises the following steps:

[0030] (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;

[0031] (2) Whole spike: remove the pollinated spikelets at the top of the female parent spike, the immature spikelets at the base, and the non-erect spikelets;

[0032] (3) Emasculation: Use tweezers to pry 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 spike with an isolation bag, and insert a wooden stick next to the female spike. The height of the wooden stick is 30 cm higher than the female spike. Then fix the female spike on the wooden stick so that the female plant is in an upright position.

[0033] (4) Preparation of pollen culture medium: Weigh 300 parts of sucrose, 0.5 parts of brassinosteroids, 0.2 parts of potassium dihydrogen phosphate, and 0.02 parts of calcium nitrate, dissolve them in 1 L of pure water, and stir evenly to obtain the product;

[0034] (5) Preparation of tassels: After the female plant is emasculated, select a strong plant in the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. The night before pollination, spray the pollen with pollen culture solution using a spray bottle and cover with an isolation bag.

[0035] (6) Pollination: Cut off the male tassel of the male parent and place it with the cut end facing down into the female parent tassel isolation bag and reseal it. Use your fingers to flick the isolation bag at 2:00 PM every day for pollination.

[0036] (7) Repeat steps (5)-(6) and continue pollination for 4 days.

[0037] Example 2:

[0038] A plateau oat hybridization method based on oat pollen culture solution comprises the following steps:

[0039] (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;

[0040] (2) Whole spike: remove the pollinated spikelets at the top of the female parent spike, the immature spikelets at the base, and the non-erect spikelets;

[0041] (3) Emasculation: Use tweezers to pry 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 spike with an isolation bag, and insert a wooden stick next to the female spike. The height of the wooden stick is 30 cm higher than the female spike. Then fix the female spike on the wooden stick so that the female plant is in an upright position.

[0042] (4) Preparation of pollen culture medium: Weigh 250 parts of sucrose, 1.0 part of brassinosteroids, 0.3 parts of potassium dihydrogen phosphate, and 0.01 parts of calcium nitrate, dissolve them in 1 L of pure water, and stir evenly to obtain the following:

[0043] (5) Preparation of tassels: After the female plant is emasculated, select healthy plants in the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. Spray the pollen with pollen culture solution the night before pollination and cover with an isolation bag.

[0044] (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 2:00 pm every day;

[0045] (7) Repeat steps (5)-(6) and continue pollination for 5 days.

[0046] Example 3:

[0047] A plateau oat hybridization method based on oat pollen culture solution comprises the following steps:

[0048] (1) Parent selection: The female parent is selected from individuals drawn from 3 / 4 ears, and the male parent is selected from individuals drawn from 3 / 4 ears; the female parent is Qingmu No. 3, and the male parent is Mengyan No. 6;

[0049] (2) Whole spike: remove the pollinated spikelets at the top of the female parent spike, the immature spikelets at the base, and the non-erect spikelets;

[0050] (3) Emasculation: Use tweezers to pry 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 spike with an isolation bag, and insert a wooden stick next to the female spike. The height of the wooden stick is 30 cm higher than the female spike. Then fix the female spike on the wooden stick so that the female plant is in an upright position.

[0051] (4) Preparation of pollen culture medium: Weigh 400 parts of sucrose, 0.8 parts of brassinosteroids, 0.3 parts of potassium dihydrogen phosphate, and 0.01 parts of calcium nitrate, dissolve them in 1 L of purified water, and stir evenly to obtain the product;

[0052] (5) Preparation of tassels: After the female plant is emasculated, select healthy plants in the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. Spray the pollen with pollen culture solution the night before pollination and cover with an isolation bag.

[0053] (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 2:00 pm every day;

[0054] (7) Repeat steps (5)-(6) and continue pollination for 6 days.

[0055] Example 4:

[0056] A plateau oat hybridization method based on oat pollen culture solution comprises the following steps:

[0057] (1) Parent selection: the female parent is selected from individuals with 3 / 4 ear emergence, and the male parent is selected from individuals with 3 / 4 ear emergence; wherein the female parent is Menglong, and the male parent is Longyan No. 1;

[0058] (2) Ear preparation: remove the small spikes at the top of the female parent ear that have been pollinated, the small spikes at the base that have not developed maturely, and the non-upright spikes;

[0059] (3) Detasseling: use tweezers to pry 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 ear tightly with an isolation bag, insert a wooden stick beside the female parent ear, the height of the wooden stick is 30 cm higher than the female parent ear, then fix the female parent ear on the wooden stick, and make the female parent plant in an upright state;

[0060] (4) Preparation of pollen culture solution: 350 parts of sucrose, 0.6 parts of brassinosteroid, 0.5 parts of potassium dihydrogen phosphate and 0.03 parts of calcium nitrate are weighed into 1 L of pure water, stirred uniformly, and prepared;

[0061] (5) Male ear preparation: after detasseling the female parent, select a healthy plant in the male parent row, cut off 1 / 5 from the top of the small flower and both sides obliquely, spray the pollen culture solution on the pollen the night before pollination, and cover the isolation bag;

[0062] (6) Pollination: cut off the male ear, put the cut male ear into the isolation bag of the female parent ear with the cut end down, and reseal, and use fingers to bounce the isolation bag for pollination every day at 12:00;

[0063] (7) Repeat steps (5) - (6) for 7 days of continuous pollination.

[0064] Comparative Example 1

[0065] A method for crossing oats, comprising the following steps:

[0066] (1) Parent selection: the female parent is selected from individuals with 3 / 4 ear emergence, and the male parent is selected from individuals with 3 / 4 ear emergence; wherein the female parent is Menglong, and the male parent is Longyan No. 1;

[0067] (2) Ear preparation: remove the small spikes at the top of the female parent ear that have been pollinated, the small spikes at the base that have not developed maturely, and the non-upright spikes;

[0068] (3) Detasseling: use tweezers to pry 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 ear tightly with an isolation bag, insert a wooden stick beside the female parent ear, the height of the wooden stick is 30 cm higher than the female parent ear, then fix the female parent ear on the wooden stick, and make the female parent plant in an upright state;

[0069] (4) Preparation of tassels: 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;

[0070] (5) Pollination: Cut off the male tassel of the male parent, place the cut 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 12:00 every day;

[0071] (6) Repeat step (5) and continue pollination for 7 days.

[0072] Comparative Example 2

[0073] A method for hybridizing oats comprises the following steps:

[0074] (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;

[0075] (2) Whole spike: remove the pollinated spikelets at the top of the female parent spike, the immature spikelets at the base, and the non-erect spikelets;

[0076] (3) Emasculation: Use tweezers to pry 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 spike with an isolation bag, and insert a wooden stick next to the female spike. The height of the wooden stick is 30 cm higher than the female spike. Then fix the female spike on the wooden stick so that the female plant is in an upright position.

[0077] (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 them in 1 L of pure water, and stir evenly to obtain the product;

[0078] (5) Preparation of tassels: After the female plant is emasculated, select healthy plants in the male parent row and cut off 1 / 5 of the floret head and both sides diagonally. Spray the pollen with pollen culture solution the night before pollination and cover with an isolation bag.

[0079] (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 2:00 pm every day;

[0080] (7) Repeat steps (5)-(6) and continue pollination for 6 days.

[0081] Comparative Example 3

[0082] A method for hybridizing oats comprises the following steps:

[0083] (1) Parent selection: the female parent is selected from individuals with 3 / 4 ear emergence, and the male parent is selected from individuals with 3 / 4 ear emergence; the female parent is Qingmu No. 3, and the male parent is Mengyan No. 6;

[0084] (2) Whole ear: remove the small spike at the top of the female parent ear that has been pollinated, the small spike at the base that has not developed maturely, and the non-upright spike;

[0085] (3) Detasseling: use tweezers to pry 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 ear tightly with an isolation bag, insert a wooden stick beside the female parent ear, the height of the wooden stick is 30 cm higher than the female parent ear, then fix the female parent ear on the wooden stick, and make the female parent plant in an upright state;

[0086] (4) Preparation of pollen culture solution: 400 parts of sucrose, 0.3 parts of potassium dihydrogen phosphate and 0.01 parts of calcium nitrate are weighed into 1L of pure water, stirred uniformly, and prepared;

[0087] (5) Male ear preparation: after the female parent detasseling is completed, select healthy plants in the male parent row, cut off 1 / 5 from the small flower head and both sides obliquely, spray pollen culture solution on the pollen the night before pollination, and cover the isolation bag;

[0088] (6) Pollination: cut off the male parent tassel, put the cut male parent tassel into the female parent ear isolation bag with the cut end down, and reseal, and use fingers to bounce the isolation bag for pollination at 14:00 every day;

[0089] (7) Repeat steps (5) to (6) for 6 days of continuous pollination.

[0090] Test example

[0091] (1) Effect of pollen culture solution on pollen viability: the male parent tassel was collected back to the laboratory, the anther was taken out with tweezers and placed on white paper, then the stamen was taken out from the tassel to obtain the anther; the anther was cut into pieces to obtain pollen, then the pollen was placed in the pollen culture solution of Examples 1-4 and Comparative Examples 2-3 and water for culture, then microscopic examination was performed under a microscope and the pollen germination rate was counted, and the results are shown in Table 1.

[0092] As shown in 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 a carbon source and energy to support the respiration and metabolic activities of the pollen; potassium dihydrogen phosphate and calcium nitrate provide necessary mineral elements to support the growth and development of the pollen, maintain the osmotic pressure and pH value of the culture solution, and create a suitable growth environment for the pollen. After the pollen was treated with the pollen culture solution of Comparative Example 2, its pollen vitality was higher than that of Comparative Example 3 and water. This shows that treating oat pollen with pollen culture solution containing boric acid can increase the fruit set rate of oats. This may be because boric acid participates in the synthesis of pectin substances, which is beneficial to the formation of pollen tube walls, and synergistically acts with sucrose to promote sugar absorption and metabolism, thereby promoting pollen germination. After the pollen was treated with the pollen culture solution of the application, its pollen vitality 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 vitality 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.

[0093] Table 1 Effects of pollen culture solutions of Examples 1-4 and Comparative Examples 2-3 on pollen viability

[0094]

[0095] (2) After pollination was completed, the fruit set rates of Examples 1-4 and Comparative Examples 1-3 were calculated, and the results are shown in Table 2. As shown in Table 2, the fruit set rate of oats using the hybridization method of Comparative Example 2 was higher than that of Comparative Examples 1 and 3, and the fruit set rate of oats using the hybridization method of Examples 1-4 was significantly higher than that of Comparative Example 2. This indicates that under the same conditions, the use of pollen culture solution containing brassinosteroids can significantly increase the fruit set rate of oats. This may be because, on the one hand, brassinosteroids have a certain moisturizing effect, thereby ensuring the humidity conditions required for pollen during pollination; on the other hand, the above test results confirm that brassinosteroids do have the effect of increasing pollen vitality and promoting pollen germination.

[0096] Table 2 Fruit setting rate of Examples 1-4 and Comparative Examples 1-3

[0097]

[0098] 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A plateau oat hybridization method based on oat pollen culture fluid, characterized in that: The following steps are involved: (1) Select the male and female parent; (2) Trim the ears of the female parent; (3) Emasculation of the female parent; (4) Spray pollen culture solution on the male parent's tassel pollen; (5) Collect 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 plateau oat hybridization method based on oat pollen culture fluid 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 plateau oat hybridization method based on oat pollen culture fluid 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 plateau oat hybridization method based on oat pollen culture fluid 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 plateau oat hybridization method based on oat pollen culture fluid according to claim 1, characterized in that: The emasculation of the female plant in step (3) is as follows: using tweezers to pry open the seed coat, removing the anthers 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 method for highland oat hybridization based on oat pollen culture fluid according to claim 5, characterized in that: The method of placing the female plant in an upright state comprises inserting a fixing device next to the female ear and fixing the female ear on the fixing device.

7. The plateau oat hybridization method based on oat pollen culture fluid 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 method for highland oat hybridization based on oat pollen culture fluid 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 method for highland oat hybridization based on oat pollen culture fluid 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 it, and flicking the isolation bag with a finger for pollination.

10. The method for highland oat hybridization based on oat pollen culture fluid 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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