Method for improving cowpea hybridization maturing rate

Abstract

The invention provides a method for improving the cowpea hybridization maturing rate, and belongs to the technical field of cross breeding. The method comprises the following steps: (1) collecting pollen of a male parent within 48 hours after flowering; (2) selecting a variety with strong podding ability as a female parent, and removing stamens 18-72 hours before flowering; and (3) within 0-48 hours of emasculation of the female parent, carrying out artificial pollination by using the pollen collected in the step (1). By adopting the method disclosed by the invention, the cowpea hybridization maturing rate is obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cross-breeding, and particularly relates to a method for improving the cross-fertilization rate of cowpea. Background Art

[0002] Cowpea is an important vegetable, rich in plant protein, dietary fiber, vitamin B group, vitamin C and minerals. Traditional Chinese medicine believes that cowpea is flat in nature and sweet in taste, can strengthen the spleen and tonify the kidney, regulate the middle energizer and replenish qi, and assist in relieving symptoms such as diabetes, indigestion, and constipation. Modern research has confirmed that its phospholipid components can promote insulin secretion and help regulate blood sugar. Cowpea has a long planting history in China. Li Shizhen called it "the top grade among beans". It can be used as both a vegetable and a medicine, and is regarded as the "longevity bean" in some folk customs.

[0003] Cowpea is a strictly self-pollinating crop, and the natural hybridization rate is extremely low under natural conditions (usually less than 1%), resulting in limited genetic diversity and making it difficult to screen excellent traits through natural variation. In addition, traditional varieties are prone to degeneration, and their disease resistance, yield, and commercial quality are difficult to meet the requirements of modern intensive planting. New varieties with characteristics such as high yield, high quality, disease resistance, and stress resistance need to be cultivated. The stamens and pistils of cowpea are tightly wrapped in the keel petal, and the stigma is tender and vulnerable. It is difficult to remove the stamens manually, and improper operation is likely to lead to hybridization failure. Existing literature shows that the cross-fertilization rate of cowpea is about 10%. Therefore, technical improvement is needed to significantly improve the cross-fertilization rate to facilitate the development of cowpea cross-breeding. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for improving the cross-fertilization rate of cowpea. By selecting a variety with strong pod-bearing ability as the female parent and optimizing the pollination measures, the present invention significantly improves the cross-fertilization rate of cowpea.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a method for improving the cross-fertilization rate of cowpea, comprising the following steps:

[0007] (1) Collect the pollen within 48 hours after the male parent blossoms;

[0008] (2) Select a variety with strong pod-bearing ability as the female parent, and remove the stamens 18 - 72 hours before blossoming;

[0009] (3) Conduct artificial pollination with the pollen collected in step (1) within 0 - 48 hours after the female parent is emasculated.

[0010] Preferably, the male parent is any one of Ningjiang No. 3, Loach Bean, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, and Jingyi 506 Cowpea.

[0011] Preferably, the female parent is any one of Ningjiang No. 3, Loach Bean, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, and Jingyi 506 Cowpea.

[0012] Preferably, after removing the stamens in step (2), bagging isolation treatment is further included.

[0013] Preferably, the number of times of artificial pollination is 1 to 3 times.

[0014] Preferably, the interval time between two adjacent artificial pollinations is 3 to 9 h.

[0015] Preferably, after the artificial pollination in step (3), post-hybrid management is further included.

[0016] Preferably, the post-hybrid management includes spraying 15 - 25 mg / L gibberellin or 5 - 15 mg / L naphthaleneacetic acid 12 - 36 h after the artificial pollination.

[0017] Preferably, the spraying amount of the gibberellin or naphthaleneacetic acid is 40 - 60 mL / plant.

[0018] Preferably, the post-hybrid management includes removing non-hybrid flowers and lower old leaves.

[0019] Compared with the prior art, the present invention has the following beneficial effects: By collecting the pollen within 48 h after the male parent blossoms as the pollination male parent, selecting the variety with strong pod-bearing ability as the female parent, removing the stamens 18 - 72 h before blossoming, and conducting artificial pollination within 0 - 48 h after the female parent is emasculated, the hybrid seed-setting rate of cowpea is significantly improved. This method is simple to operate, low in cost, and easy to popularize and use. Specific Embodiments

[0020] The present invention provides a method for improving the hybrid seed-setting rate of cowpea, comprising the following steps:

[0021] (1) Collecting the pollen within 48 h after the male parent blossoms;

[0022] (2) Selecting the variety with strong pod-bearing ability as the female parent and removing the stamens 18 - 72 h before blossoming;

[0023] (3) Conducting artificial pollination with the pollen collected in step (1) within 0 - 48 h after the female parent is emasculated.

[0024] In the present invention, the pollen within 48 h after the male parent blossoms is collected; the male parent is any one of Ningjiang No. 3, Loach Bean, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, and Jingyi 506 Cowpea.

[0025] In the present invention, it is preferred to collect pollen within 48 hours after flowering at 7-9 am on sunny days, and place it in a shaded and dry indoor environment to avoid high temperature and high humidity. Use sterilized forceps to remove the petals, so that all the anthers fall into a plastic round sterile culture dish to collect the pollen. Place the pollen in a desiccator (4°C, relative humidity < 30%), and record information such as pollen parent, collection, and treatment time. The time for collecting pollen after the male parent blooms is preferably within 36 hours, and more preferably within 24 hours.

[0026] In the present invention, a variety with strong pod-bearing ability is selected as the female parent; in the present invention, the female parent is preferably any one of Ningjiang No. 3, Loach Bean, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, and Jingyi 506 Cowpea.

[0027] In the present invention, the stamens are removed 18-72 hours before the female parent blooms; select flower buds with stronger pod-bearing ability at the middle and lower nodes of the main vine. Usually, the inflorescences at the 5-10th nodes of early-maturing varieties and the 8-13th nodes of medium-late-maturing varieties are appropriate. According to the size, color, and morphological characteristics of the flower buds, select flower buds 18-72 hours before blooming to remove the stamens. At this time, the corolla has not unfolded but the stamens have not shed pollen. During the operation, use sterilized forceps to longitudinally cut open the flower bud and remove all 10 stamens, avoiding touching the stigma. After the operation, cover it with a bag for isolation. The time for removing the stamens from the flower buds is preferably 20-60 hours, and more preferably 24-48 hours. When the flower bud length is about 1-2 cm, it enters the later stage, 1-2 days before blooming. When there are purple or pink traces on the flower bud, it is 1-2 days before blooming. The flower bud gradually stretches from being tightened, showing the outline of the petals, 1-2 days before blooming.

[0028] In the present invention, within 0-48 hours after the female parent is emasculated, artificial pollination is carried out with the pollen collected in step (1); select sunny days at 9:00-11:00 am for the pollination. Select pollen collected within 48 hours after blooming, use a small glass pipette with a tip diameter of about 1 mm, dip it into the pollen collection culture dish to make the pollen adhere to the tube wall, and buckle it onto the stigma that has been emasculated in advance. Rotate the pipette to make the stigma fully contact with the pollen on the tube wall. The time for emasculating the female parent is preferably 12-48 hours, and the number of artificial pollinations is 1-3 times; the interval time between two adjacent artificial pollinations is 3-9 hours, preferably 6 hours.

[0029] In the present invention, post-hybrid management is also included. The post-hybrid management includes microclimate control, plant nutrition regulation, growth regulator application, competition source-sink regulation, pest and disease prevention and control, data traceability and improvement. Among them, the microclimate control is as follows: after hybridization, maintain the daily temperature at 25-30°C and the night temperature at 18-22°C, and control the relative humidity at 60%-70%. When high temperature (>35°C) occurs, set up a sunshade net with a shading rate of 30% for cooling treatment to avoid pollen inactivation. The plant nutrition regulation is as follows: after hybridization, increase the application of phosphorus and potassium fertilizers, such as top-dressing 5 g of potassium sulfate per plant, and at the same time reduce the application of nitrogen fertilizers to ensure the good growth of hybrid pods and avoid excessive vegetative growth affecting fruit setting. The growth regulator application is as follows: spray 15-25 mg / L of gibberellin or 5-15 mg / L of naphthaleneacetic acid 12-36 h after artificial pollination. The preferred spraying time is 20-30 h, and more preferably 24 h. The preferred concentration of gibberellin is 18-22 mg / L, and more preferably 20 mg / L. The preferred concentration of naphthaleneacetic acid is 8-12 mg / L, and more preferably 10 mg / L. The spraying amount of gibberellin or naphthaleneacetic acid is 40-60 mL / plant, preferably 45-55 mL / plant, and more preferably 50 mL / plant. To promote ovary swelling and pod setting, and strictly control the concentration to avoid the production of deformed pods. The competition source-sink regulation is as follows: remove non-hybrid flowers and lower old leaves so that nutrients can be concentrated to supply hybrid pods. Retain 1-2 hybrid pods per inflorescence to avoid excessive nutrient dispersion and ensure that hybrid pods obtain sufficient nutrients. The pest and disease prevention and control is as follows: spray a biological pesticide once a week, such as using Bacillus thuringiensis (purchased from Wuhan Kemike Biopharmaceutical Technology Co., Ltd.) to control the bean pod borer and avoid pod drop caused by insect damage. For the prevention of anthracnose, spray difenoconazole (purchased from Shandong Bainong Sida Biotechnology Co., Ltd.) in time after rain. The use concentration and amount of the biological pesticide are both in accordance with the recommended concentration and amount on the pesticide package. The data traceability and improvement is as follows: record information such as the hybridization date, environmental parameters, and operators, establish a correlation model between the fruit setting rate and operation variables, and continuously optimize the hybridization process through data analysis.

[0030] In the present invention, the forceps, culture dishes and pollination utensils used need to be soaked in 75% alcohol to prevent pollen contamination and ensure the accuracy and purity of hybridization operations.

[0031] In the present invention, set up a windbreak or build a plastic greenhouse around the hybridization plot to reduce flower and pod drop caused by mechanical damage and create a stable growth environment for cowpea hybridization.

[0032] In the present invention, the hybridization plot is separated from the ordinary cowpea field by more than 500 m to avoid interference from foreign pollen and ensure the purity of hybrid offspring.

[0033] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Post-hybrid management: (1) Microclimate control: After hybridization, it is necessary to maintain a suitable microclimate environment, with the daily temperature kept at 25 - 30°C, the night temperature at 18 - 22°C, and the relative humidity controlled at 60 - 70%. When high temperatures (>35°C) occur, it will cause pollen inactivation, and a sunshade net (shading rate 30%) needs to be set up for cooling treatment.

[0035] (2) Plant nutrient regulation: To ensure the good growth of hybrid pods, 5 g of potassium sulfate is top-dressed per plant after hybridization, and at the same time, the application of nitrogen fertilizer is reduced to avoid excessive vegetative growth and affect fruit setting.

[0036] (3) Application of growth regulators: 24 hours after pollination, 50 mL of 20 mg / L gibberellin (GA3) or 10 mg / L naphthaleneacetic acid (NAA) is sprayed on the leaves of each plant to promote ovary swelling and pod setting.

[0037] (4) Regulation of competing source-sink: Remove non-hybrid flowers and lower old leaves to concentrate nutrient supply to hybrid pods. Retain 1 - 2 hybrid pods per inflorescence to avoid excessive nutrient dispersion and ensure that hybrid pods obtain sufficient nutrients.

[0038] (5) Prevention and control of pests and diseases: Spray biological pesticides once a week, use Bacillus thuringiensis to control the bean pod borer, and avoid pod drop caused by insect damage. Spray difenoconazole in time after rain to prevent anthracnose.

[0039] (6) Data traceability and improvement: Record information such as hybridization date, environmental parameters, and operators, establish a correlation model between the fruit setting rate and operating variables, and continuously optimize the hybridization process through data analysis.

[0040] The post-hybrid management described in the subsequent embodiments is carried out in the above manner.

[0041] Example 1 Selection of pollen collection time

[0042] At 7 - 9 am on sunny days, anthers in flower buds that are about to open and 48 hours after flowering are collected respectively. Use tweezers to remove the petals so that all anthers fall into a plastic round sterile culture dish to collect pollen. The collected pollen is placed in the shade indoors and stored in a desiccator (4°C, relative humidity <30%) to avoid high temperature and high humidity environment. Record information such as pollen parent, collection, and treatment time. The pollen viability of different varieties of cowpea at different times is measured by the MTT detection method, and the results are shown in Table 1.

[0043] Table 1 Pollen viability of different varieties at different times before and after flowering

[0044]

[0045] As can be seen from Table 1, the pollen collected within 24 hours after flowering has the highest viability. That is, using the pollen collected within 24 hours after flowering as the male parent can maintain a relatively high viability.

[0046] Example 2

[0047] (1) Respectively select Ningjiang No. 3, Loach Bean, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, and Jingyi 506 Cowpea as the male parents, and collect the pollen within 24 hours after the male parents' flowering in the manner of Example 1.

[0048] (2) Respectively select Ningjiang No. 3, Loach Bean, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, and Jingyi 506 Cowpea as the female parents, and select the flower buds with stronger pod-bearing ability at the middle and lower nodes of the main vine. For early-maturing varieties (Ningjiang No. 3, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, Jingyi 506 Cowpea), select the inflorescences at the 5-10th nodes, and for medium-late-maturing varieties (Loach Bean), select the inflorescences at the 8-13th nodes. According to the size, color, and morphological characteristics of the flower buds, select the flower buds 24-48 hours before flowering for emasculation. At this time, the corolla has not unfolded but the stamens have not shed pollen. During the operation, use forceps to longitudinally cut open the flower buds, remove all (10) stamens, avoid touching the stigma, and cover with a bag for isolation after the operation.

[0049] (3) At 9:00-11:00 in the morning on a sunny day, use a small glass pipette with a tip diameter of about 1 mm to dip the pollen in the pollen collection culture dish, so that the pollen adheres to the tube wall, and buckle it onto the stigma that has been emasculated in advance. Rotate the pipette to make the stigma fully contact with the pollen on the tube wall. Use a new pipette for the pollen of different varieties of cowpeas to avoid cross-contamination. Divide the female parents of the 6 varieties into 5 groups according to different emasculation times, namely 0 h, 12 h, 24 h, 36 h, and 48 h after emasculation, and conduct the first cross at 0 h, 12 h, 24 h, 36 h, and 48 h after emasculation. Divide the plants after the first cross into 4 groups on average. Among them, 3 groups are pollinated again at intervals of 3 h, 6 h, and 9 h respectively, and the other 1 group is not pollinated. Conduct post-hybrid management on the hybridized varieties. After the cowpeas are fully mature, count the fruiting situation of the cowpeas. The results are shown in Table 2, where Ningjiang No. 3 × Ideal Glutinous Rice Cowpea (T1), Ideal Glutinous Rice Cowpea × Ningjiang No. 3 (T2), Loach Bean × Spring and Autumn Red Cowpea (T3), Spring and Autumn Red Cowpea × Loach Bean (T4), Golden Collar Crown Cowpea × Jingyi 506 Cowpea (T5), Jingyi 506 Cowpea × Golden Collar Crown Cowpea (T6).

[0050] Fruiting rate (%) = Number of successful hybrids / Total number of pollinations * 100%

[0051] Among them, the number of successful hybrids is the number of small cowpeas, and the total number of pollinations is the number of pollinations.

[0052] Table 2 Hybrid fruiting rates of different varieties at different emasculation times

[0053]

[0054]

[0055] The seed setting rate is an indicator of successful hybridization. If the hybridization is unsuccessful, the flowers of the female parent will naturally fall off 3 to 5 days after hybridization because they have not been successfully pollinated. If the hybridization is successful, the flowers will not fall off, but instead a very thin green cowpea will grow from them. By calculating the number of cowpeas and the number of pollinations, the seed setting rate can be calculated. As can be seen from Table 2, emasculating 24 to 48 hours in advance can increase the hybridization seed setting rate, and after two pollinations, a 6-hour interval between the first pollination and the second pollination can significantly increase the seed setting rate.

[0056] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for improving the hybridization seed setting rate of cowpeas, characterized in that, It includes the following steps: (1) Collect the pollen within 48 hours after the male parent blossoms; (2) Select a variety with strong pod-setting ability as the female parent, and remove the stamens 18 - 72 hours before blossoming; (3) Within 0 - 48 hours after the female parent is emasculated, conduct artificial pollination with the pollen collected in step (1).

2. The method for improving the hybrid seed setting rate of cowpea according to claim 1, characterized in that The male parent is any one of Ningjiang No. 3, Loach Bean, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, and Jingyi 506 Cowpea.

3. The method for improving the hybrid seed setting rate of cowpea according to claim 1, wherein The female parent is any one of Ningjiang No. 3, Loach Bean, Spring and Autumn Red Cowpea, Ideal Glutinous Rice Cowpea, Golden Collar Crown Cowpea, and Jingyi 506 Cowpea.

4. The method for improving the hybridization seed setting rate of cowpea according to claim 1, characterized in that, After removing the stamens in step (2), it also includes bagging and isolation treatment.

5. The method for improving the hybridization seed setting rate of cowpea according to claim 1, characterized in that, The number of times of artificial pollination is 1 - 3 times.

6. The method for improving the hybrid seed setting rate of cowpea according to claim 5, characterized in that, The interval time between two adjacent artificial pollinations is 3 - 9 hours.

7. The method for improving the hybridization seed setting rate of cowpea according to claim 1, characterized in that, After the artificial pollination in step (3), it also includes post-hybrid management.

8. The method for improving the hybridization seed setting rate of cowpea according to claim 7, characterized in that, The post-hybrid management includes spraying 15 - 25 mg / L gibberellin or 5 - 15 mg / L naphthylacetic acid 12 - 36 hours after artificial pollination.

9. The method for improving the hybrid seed setting rate of cowpea according to claim 8, characterized in that, The spraying amount of the gibberellin or naphthylacetic acid is 40 - 60 mL / plant.

10. The method for improving the hybridization seed setting rate of cowpea according to claim 7, characterized in that, The post-hybrid management includes removing non-hybrid flowers and lower old leaves.

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