A method for hybrid seed production using strong female lines of balsam pear

Abstract

This invention belongs to the field of plant breeding technology and discloses a method for hybrid seed production using a strong female line of bitter gourd. The method includes: (1) selecting a strong female line, which is defined as having ≤5 male flowers on the main vine, a female flower node rate ≥90%, and a strong female plant rate ≥95%; (2) selecting a superior male inbred line; (3) propagating and preserving the strong female line through artificially assisted self-pollination; (4) isolating the strong female line as the female parent and the male inbred line as the male parent, utilizing the characteristic of the female parent having very few male flowers, and controlling pollination by removing emasculation during the pollination period, so that the female parent mainly receives pollen from the male parent, effectively preventing the production of false hybrid seeds through self-pollination, and harvesting hybrid seeds. This invention makes full use of the characteristic of the strong female line having very few male flowers, reducing the pollination workload during the flowering period in the seed production process by more than 60%, reducing the seed production cost by about 50%, and achieving a hybrid seed purity of ≥98%. Moreover, the parental propagation does not require chemical male induction, making the operation simple and suitable for large-scale commercial hybrid seed production of bitter gourd.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of plant breeding technology, and particularly relates to a method for hybrid seed production using a strong female line of bitter gourd. Background Technology

[0002] Bitter melon (Momordica charantia L.) is an annual climbing herbaceous plant belonging to the genus Momordica in the Cucurbitaceae family. It is an important vegetable crop widely cultivated in southern my country, possessing both edible and medicinal value. The annual planting area of ​​bitter melon in China reaches more than 3 million mu (approximately 200,000 hectares). With the deepening understanding of the nutritional and health benefits of bitter melon, its market demand is increasing, placing higher demands on the innovation of bitter melon breeding materials and the selection of new varieties.

[0003] Bitter gourd is generally a monoecious, separate-flowering plant in its natural state. Traditional hybrid seed production requires cumbersome processes such as artificially bagging (tying) female flowers for isolation, artificial pollination, and pollination marking. In particular, artificial bagging (tying) is not only labor-intensive but also difficult to scale up for seed production. Using strong female lines (including all-female lines and strongly female lines) as the female parent for seed production can significantly simplify the seed production process, save labor, and reduce production costs, and has become an important development direction for utilizing the heterosis of bitter gourd.

[0004] In production practice, plants with ≤5 male flowers and ≥90% female flower node rate within 50 nodes of the main vine are usually defined as strong female plants, and lines with ≥95% strong female plant rate are defined as strong female lines. However, the existing strong female line breeding technology still has the following shortcomings: (1) It is difficult to coordinate the screening of strong female traits and excellent economic traits. Simply pursuing female enhancement can easily lead to an increase in the rate of deformed fruits and a decline in vitality; (2) A complete technical solution covering strong female line breeding, standardized screening to hybrid seed production has not yet been formed; (3) When strong female lines are used as female parents for seed production, their core advantage of having very few male flowers is not fully utilized, and there is still room for optimization of pollination control methods.

[0005] While existing patents (such as a breeding method for a white-type strong female hybrid bitter gourd, application number 201310422227) involve the breeding of strong female lines, their focus is on the selection and breeding of strong female lines themselves, and they do not systematically solve the technical problem of how to maximize the use of the extremely low number of male flowers in the large-scale seed production process.

[0006] Therefore, developing a hybrid breeding method for bitter gourd with strong female traits that has clear screening criteria, excellent economic traits, simple propagation and preservation, and high hybrid seed production efficiency has important theoretical significance and industrial application value.

[0007] Based on the above analysis, the urgent technical problems that need to be solved in the existing technology are:

[0008] Existing breeding techniques for strong female lines of bitter gourd have drawbacks, such as difficulty in co-screening female indicators and economic traits, and low efficiency due to failure to fully utilize the characteristics of strong female lines during hybrid seed production. Summary of the Invention

[0009] To address the problems existing in the prior art, this invention provides a method for hybrid seed production using a strong female line of bitter gourd.

[0010] This invention is implemented as follows: a method for hybrid seed production using a strong female line of bitter gourd includes:

[0011] (1) Selection and breeding of strong female parent lines:

[0012] Using bitter gourd germplasm resources carrying strong female traits as maternal material and bitter gourd inbred lines with excellent comprehensive traits as recurrent paternal parents, the process involves continuous self-pollination and purification after hybridization. Female indicators are systematically screened across generations, and single-plant directional selection is conducted in conjunction with target superior economic traits until a strong female line with stable female characteristics and excellent comprehensive traits is developed. The strong female line is defined as an inbred line with ≤5 male flowers within 50 nodes of the main vine, a female flower node rate ≥90%, and a strong female plant rate ≥95% within the strong female line.

[0013] (2) Selection and breeding of paternal inbred lines:

[0014] Using bitter gourd germplasm resources with various target superior traits as materials, stable male inbred lines were bred through continuous self-pollination and purification, combined with field identification and screening of target traits.

[0015] (3) Reproduction and preservation of strong female lines:

[0016] The seeds of the strong female line obtained in step (1) are directly sown for propagation. The strong female line plants differentiate female flowers and limited male flowers on their own. During the male flower opening period, pollen is collected to artificially assist self-pollination of the female flowers of the same plant or the same line, so as to achieve self-pollination and seed saving.

[0017] (4) Preparation of hybrids:

[0018] The strong female line obtained in step (1) is used as the female parent, and the male inbred line obtained in step (2) is used as the male parent. They are planted in isolation according to the planting ratio of the female and male parents. The female and male parents are planted separately, and the female plants are subjected to controlled pollination treatment. The controlled pollination treatment includes: under the isolated planting conditions, the female flowers of the female plants are not bagged or tied, and the female plants are subjected to emasculation treatment during the pollination period. That is, during the artificial hybridization pollination period, all male flowers and male flower buds on the female plants are continuously removed until the pollination ends, so that the female plants mainly receive pollen from the male parents. The hybrid seeds are harvested from the female plants, which yields the bitter gourd hybrid.

[0019] Furthermore, in step (1), the single-plant female flower node rate of the strong female line is ≥90%.

[0020] Furthermore, the target superior economic traits in step (1) are selected from one or more of the following: early maturity, high yield, disease resistance, commercial fruit shape, fruit color, and quality.

[0021] Furthermore, the disease resistance refers to resistance to powdery mildew, wilt, and viral diseases.

[0022] Furthermore, the number of generations of continuous self-pollination purification in steps (1) and (2) is 5 to 7.

[0023] Furthermore, in step (4), the planting ratio of the maternal parent to the paternal parent is 10 to 15:1.

[0024] Furthermore, the time for emasculation during the pollination period in step (4) is 15 to 18 days.

[0025] Furthermore, in step (4), the spatial isolation distance for isolated planting shall not be less than 1000 meters, or a greenhouse shall be used and covered with insect-proof netting (30-40 mesh) for isolation.

[0026] Furthermore, in step (4), to ensure that the flowering periods coincide, the male parent is sown 7 to 10 days earlier than the female parent.

[0027] Furthermore, in step (4), one day before the male flowers of the male parent open, the male flowers that will open the next day are collected and prepared for use. After the male flowers are collected, they are tied into a bunch of 30 to 50 flowers with the flower stems facing down and placed in a basin with a water layer of about 3 cm deep and placed indoors. If the temperature is below 25°C, a 100-watt incandescent lamp is hung about 1 meter above the basin for heating, or the male flowers are placed indoors at about 30°C.

[0028] In step (4), artificial hybridization pollination is carried out from 5:00 to 9:00 a.m. on the day the female flower opens. Each male flower of the male parent is used to pollinate 3 to 5 female flowers. After pollination, the female flower is tied with a string at the flower stalk as a pollination mark.

[0029] In step (4), mature fruits with pollination marks on the mother plant are harvested 23 to 26 days after pollination, and the seeds obtained are the F1 hybrid seeds.

[0030] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0031] This invention provides clear screening criteria for strong female traits, facilitating industrial application. It specifies quantitative indicators such as ≤5 male flowers on the main vine, ≥90% female flower node rate per plant, and ≥95% strong female plant rate, thus resolving the problem of inconsistent standards.

[0032] This invention enables the simultaneous breeding of strong female traits and excellent economic traits. It avoids the problems of increased deformed fruit rate and decreased viability caused by simply pursuing enhanced femaleness.

[0033] This invention simplifies breeding and preservation, eliminating the need for chemical male-inducing treatment. It also saves the cumbersome process of chemical male-inducing required for all-female lines.

[0034] This invention significantly reduces seed production costs. Due to the use of a strong female-line seed production scheme, the workload of pollination during the flowering period is reduced by more than 60% compared to traditional methods, eliminating the need for bagging or tying flowers. Calculations show that labor costs can be saved by approximately 3,000 to 5,000 yuan per acre of seed production field.

[0035] This invention produces hybrids with high purity and strong heterosis. The purity of the hybrids can reach over 98%. The F1 generation hybrids inherit the strong female characteristics of the maternal parent, exhibiting numerous female flowers and early maturity, which is conducive to achieving high yields and can meet the growing demand for high-level, high-quality cultivation in modern facilities.

[0036] This invention is simple to operate and has broad prospects for industrial application. The process is clear, requires no chemical reagents to induce male reproduction, and is suitable for large-scale commercial production.

[0037] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: Using the method of this invention for bitter gourd hybrid seed production, the labor cost per mu of seed production field can be directly saved by 3,000 to 5,000 yuan. Based on an annual promotion of seed production area of ​​10,000 mu, the annual cost savings can be 30 million to 50 million yuan. At the same time, the purity of the hybrid seeds is ≥98%, the seed quality is high, and the market competitiveness is strong. The hybrid seeds produced have the characteristics of early maturity, many female flowers, and high yield. After promotion and planting, the yield of bitter gourd per mu can be increased by more than 50%, significantly increasing farmers' income. It has extremely high commercial promotion value and market prospects.

[0038] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally: This invention is the first to integrate clearly defined quantitative indicators of strong female lines (≤5 male flowers within 50 nodes of the main vine, female flower node rate ≥90%, strong female plant rate ≥95%) with the controlled pollination technology system of emasculation during the pollination period, forming a complete technical solution from parent selection, propagation and preservation to hybrid seed production. It solves the technical gaps in existing technologies, such as inconsistent screening standards for strong female traits and failure to fully utilize the characteristic of very few male flowers in strong female lines in the seed production process. In particular, it has reached the leading level both domestically and internationally in achieving efficient seed preservation and low-cost seed production without chemical emasculation.

[0039] (3) The technical solution of this invention solves a long-standing technical problem that people have long desired to solve but have never been able to achieve: For a long time, the field of bitter gourd hybrid seed production has been eager to solve the technical problem of how to significantly reduce or eliminate the arduous process of manual bagging / flower binding while ensuring the purity of hybrid seeds. Existing technologies either rely on all-female lines but require chemical induction of males (increasing costs and operations), or use strong female lines but still follow the traditional bagging / flower binding thinking. This invention, by deeply exploring the essential characteristic of strong female lines having very few male flowers, creatively proposes a emasculation scheme during the pollination period. Under the condition of eliminating bagging / flower binding, by efficiently removing a very small number of male flowers from the female parent, the proportion of false hybrid seeds is successfully controlled to below 2% (seed purity ≥98%), while reducing pollination labor by more than 60%, thus solving this long-standing technical bottleneck that has plagued the seed industry.

[0040] (4) The technical solution of this invention overcomes technical prejudice: In the field of bitter gourd hybrid seed production technology, there has long been a technical prejudice that to ensure seed purity, the female flowers of the maternal parent must be bagged / tethered for isolation. Researchers generally believe that without bagging / tethering for isolation, it is impossible to effectively prevent self-pollination and foreign pollen contamination. This invention utilizes the biological characteristic of strong female lines having very few male flowers, combined with continuous emasculation during the concentrated pollination period, to prove that even without bagging, by managing a very small number of male flowers of the maternal parent, seed purity can be achieved or even exceeded by the traditional bagging method (98% vs 95%), completely breaking the industry technical prejudice that seed production must be bagged / tethered for isolation, and providing a new seed production technology idea for cucurbitaceous crop hybrid seed production. Attached Figure Description

[0041] Figure 1 This is a flowchart of a method for hybrid seed production using a strong female line of bitter gourd provided in an embodiment of the present invention.

[0042] Figure 2 This is a flowchart illustrating the technical route for the selection and hybridization of strong female lines of bitter gourd provided in this embodiment of the invention.

[0043] Figure 3 This is a comparison chart of seed production efficiency and seed production purity provided in the embodiments of the present invention.

[0044] Figure 4 This invention provides the effect of different degrees of emasculation on the purity and false hybrid ratio of hybrids. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0046] like Figure 1As shown in the figure, an embodiment of the present invention provides a method for hybrid seed production using a strong female line of bitter gourd, comprising the following steps:

[0047] S101, selection and breeding of strongly female parent lines:

[0048] Using bitter gourd germplasm resources carrying strong female traits as maternal material and bitter gourd inbred lines with excellent comprehensive traits as recurrent paternal parents, the process involves continuous self-pollination and purification after hybridization. Female indicators are systematically screened across generations, and single-plant directional selection is conducted in conjunction with target superior economic traits until a strong female line with stable female characteristics and excellent comprehensive traits is developed. The strong female line is defined as an inbred line with ≤5 male flowers within 50 nodes of the main vine, a female flower node rate ≥90%, and a strong female plant rate ≥95%.

[0049] S102, breeding of paternal inbred lines:

[0050] Using bitter gourd germplasm resources with various target superior traits as materials, stable male inbred lines were bred through continuous self-pollination and purification, combined with field identification and screening of target traits.

[0051] S103, propagation and preservation of the strongly female line:

[0052] The seeds of the strong female line obtained in step S101 are directly sown for propagation. The strong female line plants differentiate female flowers and limited male flowers on their own. During the male flower opening period, pollen is collected to artificially assist self-pollination of female flowers of the same plant or the same line to achieve self-pollination and seed saving.

[0053] S104, preparation of hybrid varieties:

[0054] The strong female line obtained in step S101 is used as the female parent, and the male inbred line obtained in step S102 is used as the male parent. They are planted in isolation according to the planting ratio of the female and male parents, and the female and male parents are planted separately. The female plants are subjected to controlled pollination treatment. The controlled pollination treatment includes: under the isolated planting conditions, the female flowers of the female plants are not bagged or tied, and the female plants are subjected to emasculation treatment during the pollination period. That is, during the artificial hybridization pollination period, all male flowers and male flower buds on the female plants are continuously removed until the pollination is completed, so that the female plants receive pollen from the male parents. The hybrid seeds are harvested from the female plants, which yields the bitter gourd hybrid.

[0055] In step S101 of this embodiment of the invention, the single-plant female flower node rate of the strongly female line is ≥90%.

[0056] The target superior economic traits mentioned in step S101 of the present invention are selected from one or more of the following: early maturity, high yield, disease resistance, marketable fruit shape, fruit color, and quality.

[0057] The disease resistance provided by the embodiments of the present invention is resistance to powdery mildew, resistance to wilt, and resistance to viral diseases.

[0058] In the embodiments of the present invention, the number of generations of continuous self-pollination purification in steps S101 and S102 is 5 to 7.

[0059] In step S104 of this embodiment of the invention, the planting ratio of the maternal parent to the paternal parent is 10 to 15:1.

[0060] The time for emasculation during the pollination period in step S104 of this embodiment of the invention is 15 to 18 days.

[0061] In step S104 of this embodiment of the invention, the spatial isolation distance for isolated planting is not less than 1000 meters, or a greenhouse is used and covered with insect-proof netting (30-40 mesh) for isolation.

[0062] In step S104 of this embodiment of the invention, in order to ensure that the flowering periods coincide, the male parent is sown 7 to 10 days earlier than the female parent.

[0063] In step S104 of this embodiment of the invention, one day before the male flowers of the male parent open, the male flowers that will open the next day are collected and prepared for use. After being collected, the male flowers are tied into a bunch of 30 to 50 flowers with the flower stems facing down and placed in a basin containing a water layer of about 3 cm deep and placed indoors. If the temperature is below 25°C, a 100-watt incandescent lamp is hung about 1 meter above the basin for heating, or the male flowers are placed indoors at about 30°C.

[0064] In step S104, artificial hybridization pollination is carried out from 5:00 to 9:00 a.m. on the day the female flower opens. Each male flower of the male parent pollinates 3 to 5 female flowers. After pollination, the female flower is marked with a string tied to the pedicel.

[0065] In step S104, mature fruits bearing pollination marks on the mother plant are harvested 23–26 days after pollination, and the resulting seeds are F1 hybrid seeds.

[0066] Specific implementation of the present invention:

[0067] like Figure 2 As shown, the technical route of this invention is as follows: Strong female germplasm is hybridized with superior inbred lines, and after multiple generations of directional screening through self-pollination (screening criteria: ≤5 male flowers within 50 nodes of the main vine, female flower node rate ≥90%, strong female plant rate ≥95%), a strong female line is obtained. This strong female line is used for propagation and seed preservation through natural self-pollination; on the other hand, it is used as the female parent for hybrid seed production in isolated planting, and controlled pollination is achieved by emasculating the male parent inbred line through artificial pollination, ultimately obtaining F1 hybrid seeds.

[0068] Example 1: Breeding of a strong female line of bitter gourd

[0069] (1) Parental materials: The maternal material is Xiafeng bitter gourd germplasm material carrying strong female traits. The recurrent parent is Chizhu bitter gourd inbred line with excellent comprehensive traits (large top shape, early maturity, high yield, and resistance to powdery mildew).

[0070] (2) Hybridization and self-pollination purification: In spring, the two were hybridized to obtain F1, and in autumn, F1 was planted and self-pollinated to obtain F2. The F2 population was sown, and the female indicators of each individual plant were investigated during the flowering period. Individual plants with high female flower node rate, few male flowers (≤5 male flowers within 50 nodes of the main vine), and excellent agronomic traits were selected for self-pollination and seed saving. The above screening was repeated for the F3 and F4 generations.

[0071] (3) Stable system: After seven generations of self-pollination and directional selection, a strong female line, 90-01A, with stable female characteristics and excellent and consistent comprehensive traits was obtained. According to the identification, the number of male flowers within 50 nodes of the main vine of this line is about 2 to 3 on average, the rate of strong female plants is over 95%, the rate of female flower nodes is 95%, and the strong female genetic stability is stable.

[0072] (4) Comprehensive evaluation of economic traits: This strong female line has large top-shaped fruit, oily green color, early maturity, and resistance to powdery mildew, with excellent comprehensive traits.

[0073] Example 2: Breeding and Preservation of Strong Female Lines

[0074] Seeds of the strong female line 90-01A are sown in spring and planted in isolation at a distance of at least 1000 meters. The planting density is approximately 1500 plants per acre. After flowering, remove any weeds and inferior plants with excessive male flowers. The strong female line can naturally differentiate into female flowers and a small number of male flowers. On the day the male flowers open, collect pollen from them and artificially pollinate them on the stigma of female flowers on the same plant or on the stigma of female flowers from other strong female lines. After the fruit matures, harvest the pollinated melons to obtain seeds for propagation. Compared with all-female lines that rely on chemically induced male flowers for propagation, the method of this invention does not require any chemical reagents, is simple to operate, and has a lower cost.

[0075] Example 3: Preparation of Hybrids

[0076] (1) Selection of parents: The mother is the strong female line 90-01A, and the father is the excellent inbred line 94-03B (large top shape, bright green and glossy fruit color, disease resistant).

[0077] (2) Isolation zone setup: Fields that have not been planted with cucurbit crops within the past 3 years should be selected, preferably loam soil where rice was previously grown, as seed production fields. The spatial isolation distance around the seed production area should be more than 1000 meters. The female and male lines are planted separately, with a planting ratio of 12:1. Approximately 1500 female lines and 2000 male lines are planted per mu. The male lines are sown 7 days earlier than the female lines.

[0078] (3) Elimination of Impurities and Inferior Plants: The elimination of impurities and inferior plants should be carried out throughout the entire seed production process to ensure the purity of the F1 hybrid seeds. During the seedling stage, heteromorphic plants and diseased or weak plants in the female and male parents should be removed in a timely manner. Before artificial hybridization and pollination, the strong female lines of the female parent should be thoroughly examined, and suspected heteromorphic plants and inferior plants with too many male flowers on the main vine should be removed, leaving only strong female plants. The male parent needs to be closely observed, and all suspected heteromorphic plants should be completely removed from the budding stage to improve the purity of the parent lines.

[0079] (4) Plant adjustment and emasculation: After retaining the main stem and two lateral stems of the female parent, all other lateral stems should be removed promptly to facilitate emasculation and pollination. Lateral stems of the male parent are generally not removed. Training and removal of lateral stems should be carried out on sunny afternoons. Before formal pollination, all open female and male flowers and identifiable male flower buds on the female parent plant should be removed; thereafter, at least one inspection should be conducted each afternoon and morning before artificial pollination to remove any missed male flowers and buds of the female parent. Timely and thorough removal of male flowers from the female parent to prevent the production of false hybrid seeds through self-pollination is one of the key steps in ensuring seed purity.

[0080] (5) Controlled pollination treatment (preferred scheme of the present invention): The plants are inspected before the female parent flowers for pollination. Since the strong female line has very few male flowers, this embodiment adopts a emasculation scheme during the pollination period. That is, before formal pollination, a small number of male flower buds appearing on the female parent plant are inspected and removed, and during the pollination period, continuous inspections are carried out to remove newly grown male flower buds in a timely manner until the pollination is completed. Field trials show that the workload of emasculation pollination under this scheme is about 15 hectares / acre, which can reduce the workload of pollination during the flowering period by more than 60% compared with conventional parental bagging or flower tying isolation pollination. Under this scheme, the proportion of false hybrid seeds produced by self-pollination of the female parent plant after artificial pollination is less than 2%, and the seed purity can reach more than 98%.

[0081] (6) Pollination and Harvesting: Artificial pollination is used. After flowering, from 5:00 AM to 9:00 AM, gently rub the stamens of the male flowers of the male parent that were in bloom the previous evening onto the stigma of the female flowers of the female parent that were in bloom that day, so that the pollen is evenly distributed on the stigma. Mark the pedicel of the pollinated female flower with a string. Usually, one male flower can pollinate 3 to 5 female flowers. Each hybrid plant pollinates 6 to 10 flowers, selects 4 to 6 seed fruits, and removes the remaining unpolluted female flowers and poorly developed young fruits that have been pollinated as soon as possible. After pollination, stop emasculating and prune the main and lateral vines. About 25 days after pollination, harvest the mature fruits with pollination marks on the female parent plant. After seed extraction and drying, the bitter gourd hybrid variety Zaofeng No. 3 is obtained. The seed yield is about 25 to 40 kg / mu.

[0082] (7) Control Example (Traditional Manual Bagging or Flower Binding): As a control, the same maternal parent was used, but the maternal parent was not emasculated. However, all female flowers on the maternal parent plant were bagged or tied with cotton yarn one day before opening. The next day, after artificial hybridization and pollination, the pollinated female flowers were bagged or tied to mark the pollination. The results showed that the purity of the hybrid was about 95%, but the labor required for bagging or tying the flowers was more than twice that of the emasculation method during the pollination period of this invention.

[0083] Example 4: Comparison of Seed Production Effects

[0084] The method of this invention is compared with the traditional conventional inbred line (female flower node rate of about 15%) artificial bagging or flower binding seed production method:

[0085]

[0086] Example 5: Application Effects of Hybrids

[0087] The bitter gourd hybrid variety Zaofeng 3, bred using the method of this invention (approved by the Guangdong Provincial Crop Variety Approval Committee in 2001 (Approval No.: Yue Shen Cai 200109), and selected by the Shantou Baisha Vegetable Seed Research Institute), has been tested in production trials in Guangdong and Hainan. Results show that the first female flower node of this hybrid is 3-5 nodes lower than the control male parent 94-03B, and the first harvest is 7-10 days earlier; the yield is 2500-4000 kg / mu, an increase of 50-100% compared to the control male parent 94-03B; the fruit has good marketability; and it shows resistance to powdery mildew and wilt in the field.

[0088] Example 6: Effect of different degrees of emasculation on the purity of hybrids

[0089] To further verify the technical effectiveness of the emasculation scheme during pollination in this invention, the following comparative treatment was set up (the maternal parent was the same strong female line 90-01A, the paternal parent was the same 94-03B, and the isolation conditions were the same):

[0090]

[0091] The results showed that the pollination-period emasculation method, with a emasculation workload of only 40% of that of complete emasculation, still achieved a hybrid purity of 98.3%, with a false hybrid rate of only 1.7%, fully meeting the national seed quality standard (false hybrid rate ≤ 5%). While the complete emasculation method could achieve a hybrid purity of 98.5%, the workload increased significantly, resulting in a low cost-effectiveness ratio. The emasculation-free method further reduced labor input, but the false hybrid rate rose to 18.8%, failing to meet the national seed quality standard (false hybrid rate ≤ 5%). Therefore, the pollination-period emasculation method represents the optimal balance point in terms of cost-effectiveness. All pollination-period emasculation methods claimed in this invention are covered within the above-mentioned schemes, with pollination-period emasculation being the most preferred option.

[0092] Comparison of Example 7 with other existing patented technologies

[0093] The method of this invention is compared with a breeding method for a white, strongly female hybrid bitter gourd (application number 201310422227) in the prior art:

[0094]

[0095] Therefore, it can be seen that the present invention is significantly superior to the prior art in terms of the clarity of the screening criteria for strong female lines and the simplification of the seed production process.

[0096] Evidence related to the technical effects obtained by the embodiments of the present invention.

[0097] Figure 3 Comparison of seed production efficiency and seed purity;

[0098] Figure 4 The effects of different degrees of emasculation on the purity and false hybrid ratio of hybrids;

[0099] I. Evidence of Improved Seed Production Efficiency

[0100] 1. Percentage reduction in pollination workload during flowering period

[0101] The present invention, in Example 3, Part (5), clearly states that the emasculation method during the pollination period can reduce the workload of pollination by more than 60% compared with the conventional parental bagging or flower binding.

[0102] 2. The extent of reduction in seed production labor costs

[0103] Example 4 provides quantitative comparison data:

[0104]

[0105] 3. Amount saved in labor costs per acre

[0106] Article (4) of the beneficial effects of the invention states that each mu of seed production field can save about 3,000 to 5,000 yuan in labor costs.

[0107] II. Evidence for Improved Purity of Hybrids

[0108] 1. Seed purity indicators

[0109] Example 3, paragraph (5) states that the proportion of false hybrid seeds is less than 2%, and the seed purity can reach more than 98%.

[0110] According to the comparison example (traditional bagging / flower binding): the purity of the hybrid is about 95%.

[0111] 2. Summary of Purity Comparison

[0112] Example 4 table: The purity of the conventional method is above 95%, while the purity of the method of the present invention is above 98%, which is an increase of about 3 percentage points.

[0113] III. Optimized Evidence for the Relationship Between Eunuchization Degree and Purity

[0114]

[0115] Example 6 Key Optimization Data:

[0116] Conclusion: The emasculation method during pollination achieves 98.3% purity with only 40% of the workload, and the false hybrid rate is only 1.7%, which fully meets the national standard (false hybrid rate ≤ 5%). It represents the optimal balance point for cost-effectiveness.

[0117] IV. Evidence of the Field Application Effects of Hybrids

[0118] Example 5 Field Trial Data:

[0119] Early maturity: The first female flower node is 3-5 nodes lower than the control male parent 94-03B, and the first harvest is 7-10 days earlier.

[0120] High yield: 2500-4000 kg / mu, an increase of 50-100% compared to the control parent 94-03B.

[0121] Marketability: The fruit has good marketability.

[0122] Disease resistance: In the field, it shows resistance to powdery mildew and wilt.

[0123] V. Evidence of Breeding and Preservation Efficiency

[0124] Example 2 describes:

[0125] Strongly female lines can naturally differentiate into female flowers and a small number of male flowers, achieving self-pollination and seed production without the need for chemical induction of males.

[0126] Compared to all-female lines that rely on chemical induction of male flowers for reproduction, this method requires no chemical reagents, is simple to operate, and is less expensive.

[0127] VI. Evidence comparing with existing patented technologies

[0128] Example 7 Comparison Results:

[0129]

[0130] In the description of this invention, unless otherwise stated, "multiple" means two or more; the terms "upper," "lower," "left," "right," "inner," "outer," "front end," "rear end," "head," and "tail," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0131] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for hybridization seed production using a strong female line of bitter gourd, characterized in that, The method includes the following steps: (1) Selection and breeding of strong female parent lines: Using bitter gourd germplasm resources carrying strong female traits as maternal material and bitter gourd inbred lines with excellent comprehensive traits as recurrent paternal parents, the process involves continuous self-pollination and purification after hybridization. Female indicators are systematically screened across generations, and single-plant directional selection is conducted in conjunction with target superior economic traits until a strong female line with stable female characteristics and excellent comprehensive traits is developed. The strong female line is defined as an inbred line with ≤5 male flowers within 50 nodes of the main vine, a female flower node rate ≥90%, and a strong female plant rate ≥95% within the strong female line. (2) Selection and breeding of paternal inbred lines: Using bitter gourd germplasm resources with various target superior traits as materials, stable male inbred lines were bred through continuous self-pollination and purification, combined with field identification and screening of target traits. (3) Reproduction and preservation of strong female lines: The seeds of the strong female line obtained in step (1) are directly sown for propagation. The strong female line plants differentiate female flowers and limited male flowers on their own. During the male flower opening period, pollen is collected to artificially assist self-pollination of the female flowers of the same plant or the same line, so as to achieve self-pollination and seed saving. (4) Preparation of hybrids: The strong female line obtained in step (1) is used as the female parent, and the male inbred line obtained in step (2) is used as the male parent. They are planted in isolation according to the planting ratio of the female and male parents. The female and male parents are planted separately, and the female plants are subjected to controlled pollination treatment. The controlled pollination treatment includes: under the isolated planting conditions, the female flowers of the female plants are not bagged or tied, and the female plants are subjected to emasculation treatment during the pollination period. That is, during the artificial hybridization pollination period, all male flowers and male flower buds on the female plants are continuously removed until the pollination ends, so that the female plants mainly receive pollen from the male parents. The hybrid seeds are harvested from the female plants, which yields the bitter gourd hybrid.

2. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 1, characterized in that, The female flower node rate of the strong female line in step (1) is ≥90%.

3. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 1, characterized in that, The target superior economic traits mentioned in step (1) are selected from one or more of the following: early maturity, high yield, disease resistance, commercial fruit shape, fruit color, and quality.

4. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 3, characterized in that, The disease resistance refers to resistance to powdery mildew, wilt, and / or viral diseases.

5. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 1, characterized in that, The number of generations for continuous self-pollination purification in steps (1) and (2) is 5 to 7.

6. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 1, characterized in that, The planting ratio of the maternal parent to the paternal parent in step (4) is 10 to 15:

1.

7. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 1, characterized in that, The time for emasculation during the pollination period in step (4) is 15 to 18 days.

8. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 1, characterized in that, In step (4), the spatial isolation distance for isolated planting shall not be less than 1,000 meters, or a greenhouse shall be used and covered with insect-proof netting for isolation.

9. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 1, characterized in that, In step (4), to ensure that the flowering periods coincide, the male parent is sown 7 to 10 days earlier than the female parent.

10. The method for hybridization and seed production using a strong female line of bitter gourd as described in claim 1, characterized in that, In step (4), one day before the male flowers of the male parent open, the male flowers that will open the next day are picked and stored for later use. After picking, the male flowers are tied into a bunch of 30 to 50 flowers with the flower stems facing down and placed in a basin with a water layer of about 3 cm deep and placed indoors. If the temperature is below 25°C, a 100-watt incandescent lamp is hung about 1 meter above the basin for heating, or the male flowers are placed indoors at about 30°C. In step (4), artificial hybridization pollination is carried out from 5:00 to 9:00 a.m. on the day the female flower opens. Each male flower of the male parent is used to pollinate 3 to 5 female flowers. After pollination, the female flower is tied with a string at the flower stalk as a pollination mark. In step (4), mature fruits with pollination marks on the mother plant are harvested 23 to 26 days after pollination, and the seeds obtained are the F1 hybrid seeds.

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