Method for preparing watermelon hybrid seeds through bee pollination

Abstract

The invention belongs to the technical field of plant breeding, and particularly discloses a method for preparing watermelon hybrid seeds by bee pollination, which is characterized in that a watermelon dominant sterile female parent line is created based on a watermelon dominant male sterile material, and haploids can be quickly, accurately and efficiently obtained by using a gene editing or immature embryo rescue culture technology. According to the watermelon hybrid seed production method, dominant male sterile double haploids are obtained through doubling, the dominant male sterile double haploids are hybridized with fertile female parents to efficiently obtain batches of completely sterile female parent seeds, and then bee pollination replaces artificial pollination to produce watermelon hybrid seeds, so that the labor cost of watermelon seed production can be reduced by 44%, and the yield of watermelon hybrid seeds is improved. And the purity of the hybrid seeds is improved to 100%. The problem of insufficient labor force can be effectively solved, the production cost of hybrid seeds is reduced, and the method has high application value in large-scale seed production of watermelons.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, and relates to watermelon seed production through bee pollination, and particularly to a method for propagating hybrid watermelon seeds based on watermelon dominant sterile materials combined with bee pollination. Background Technology

[0002] Watermelon is one of the world's five major fruits. my country accounts for approximately 50% of the global watermelon planting area and over 60% of global production, both ranking first in the world. However, watermelon seed production requires a large labor force, mainly because watermelons are cross-pollinated plants, and artificial pollination is necessary to harvest F1 hybrid seeds during seed propagation (hybrid seed production). Currently, most of the frontline workers engaged in watermelon hybrid seed production in my country are between 50 and 70 years old. Coupled with the aging population and declining birth rate in my country, the future use of artificial pollination for watermelon hybrid seed production will face high costs and even a shortage of personnel. Therefore, using insects such as bees for pollination in watermelon hybrid seed production will become a major technological innovation in watermelon hybrid seed production.

[0003] Insect pollination for seed production is widely used in horticultural crops such as cabbage, but watermelon hybrid seed production still relies on traditional artificial pollination, mainly due to the lack of usable male-sterile resources. Watts obtained the first male-sterile material in watermelon through gamma-ray induction, but this material exhibited weak growth, poor disease resistance, and low seed production, thus hindering significant research progress. Subsequently, Xia Xitong, Li Qian, Wang Wei, Tan Suying, Zhang Xian, and others discovered several male-sterile watermelon materials, all controlled by recessive male-sterile genes. To utilize male sterility in watermelon for insect pollination to produce hybrid watermelon seeds, for recessive male-sterile plants as the female parent, 3 / 4 to 1 / 2 of the fertile plants need to be removed, while for dominant male-sterile plants, 1 / 2 of the fertile plants need to be removed completely, leaving no fertile plants. This requires high levels of technology and labor, resulting in high production costs and difficulty in ensuring seed purity. However, if dominant male-sterile double haploids are used for female seed propagation, manual removal is not required, which can save more labor (see Table 1). Therefore, dominant male sterility is more valuable.

[0004] Table 1

[0005] However, while domestic and international teams have discovered several male-sterile watermelon materials, all of which are controlled by recessive male-sterile genes, these genes have not been truly transformed and applied to watermelon hybrid seed production. Furthermore, no reports or applications of dominant male-sterile materials in small-fruited watermelons have been found to date. Our team, in collaboration with the Institute of Modern Agriculture at Peking University, constructed a mutant library based on EMS-treated watermelon pollen and using the watermelon backbone line G42 as a background. We discovered a dominant male-sterile mutant, which, except for fertility, retains all other agronomic traits and can be directly applied to breeding and seed production. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing watermelon hybrid seeds through bee pollination. Without altering the cultivation environment or increasing costs associated with mechanized operations, this invention creatively proposes a watermelon seed production method based on dominant male sterility using bee pollination. This method includes the use of dominant male sterility double haploid creation technology, watermelon completely sterile female parent seed propagation technology, and insect-based watermelon hybrid seed production operations under both facility and open-field conditions. The seed production method provided by this invention can reduce the labor cost of watermelon seed production by 44% and increase the purity of hybrid seeds to 100%.

[0007] This invention is achieved through the following technical solution: A method for creating a dominant male-sterile double haploid material from watermelon, comprising the following steps: (1) Creating a dominant male-sterile maternal line for watermelon: Using dominant male-sterile watermelon material as maternal parent P1, select its male-sterile plant P1 (Aa) and cross it with the maternal line P2 of watermelon hybrid. Then use the maternal line P2 as the recurrent parent for backcrossing. Only the hybrid seeds of the male-sterile plants are harvested in each generation. After 4-5 generations, the dominant male-sterile maternal line P3 of watermelon can be obtained, of which 50% are male-sterile plants and 50% are fertile plants. (2) Creating watermelon dominant male sterile double haploid material: Using the dominant heterozygous male sterile plant P3(Aa) obtained in step (1) as the female parent, hybridize it with the haploid induction line or pollinate it with radiation pollen with different genetic backgrounds and then rescue the embryo to obtain dominant male sterile haploid material P3(A) or P3(a); use the obtained dominant male sterile haploid material to create watermelon dominant male sterile double haploid material P3(AA) or P3(aa) by doubling it with asulfanilamide or colchicine, and then screen the sterile plants by molecular markers to finally obtain P3(AA); (3) Propagation: Using the watermelon double haploid material P3 (AA) obtained in step (2), rapid propagation is carried out through tissue culture or grafting; (4) Propagation of male-sterile watermelon maternal seeds: Using a certain number of male-sterile double haploid plants P3 (AA) obtained in step (3) as maternal parents, crossbreeding them with fertile plants P3 (aa) to produce a large number of heterozygous male-sterile plants P3 (Aa) maternal watermelon seeds. (5) Propagation of hybrid watermelon seeds by bee pollination: Using the heterozygous male sterile line P3 (Aa) as the female parent and the inbred line P4 as the male parent, hybrid watermelon seeds are propagated by combining facility seed production or open field seed production with bee pollination. During the propagation process, pest and disease control and fertilizer and water management are required. After the seeds are harvested, they are disinfected, screened and tested for germination rate. Once qualified, they can be packaged and sold.

[0008] A further improvement to the present invention is as follows: The process of creating dominant sterile haploid materials is as follows: a ClDMP4 gene-edited mutant was obtained through CRISPR / Cas9 technology. The resulting haploid inducible line was used as the male parent, and the dominant heterozygous male sterile plant P3(Aa) obtained in step (1) was used as the female parent for hybridization. Seeds were harvested. The seeds were dehulled to expose the embryos. The embryos were screened in a dark room using a handheld fluorescence detector or a stereofluorescence microscope. The embryos without fluorescence were haploids P3(A) or P3(a), and the diploids that emitted green fluorescence were removed. Subsequently, the haploid plants were doubled using sulfadiazine or colchicine. Then, DNA from some plant tissues was extracted and fertility was identified using dCAPS labeling (MS-F1: 5'-AAAAGCAGCGTCCATGATGTTGTCCTTCTT-3', MS-R1: 5'-TTCCTTCACCACTCAAGATG-3', DdeI digestion). Male sterile double haploid plants P3(AA) were screened.

[0009] Furthermore, the following process can be used to create dominant sterile haploid materials: using the dominant heterozygous male sterile plant P3 (Aa) obtained in step (1) as the female parent and inbred lines with different genetic backgrounds as the male parent, the pollen of the male parent is irradiated with X-rays at a dose of 200-600 Gry to pollinate the female parent. About 10-15 days after pollination, the embryo rescue culture is carried out to create haploid plants.

[0010] Furthermore, the specific operation for rescuing the immature embryo is as follows: The retrieved watermelon fruit is sprayed with 75% ethanol, placed in a stainless steel dish, and the surface of the watermelon is moistened with a small amount of 95% ethanol. The watermelon is then sterilized by burning. The watermelon is cut open with a sterile scalpel, and the immature seeds are removed. About 1 / 3 of the seed's distal embryo is cut off, and the remaining seeds are placed flat on a haploid induction medium. The medium formula is: MS + 20 g / L sucrose + 20 g / L mannitol + 0.01 mg / L IAA. The seeds are cultured on the induction medium for 3-10 weeks. When the seeds swell, the epidermis turns brown, or the cotyledons emerge, the seed coat is removed, and the seeds are transferred to a medium of 1 / 2 MS + 20 g / L sucrose + 20 g / L mannitol for subculture. The subcultured plants are then transferred to a rooting medium of 1 / 2 MS + 20 g / L sucrose + 20 g / L mannitol + 1 mg / L IBA for rooting culture to obtain haploid plants.

[0011] Furthermore, the concentration of sulfamethoxazole in step (2) is 2 mg / L, and the concentration of colchicine is 200 mg / L.

[0012] Furthermore, the fertility identification process of the obtained double haploid plants is as follows: samples of the obtained double haploid plants are taken, and the genome of the samples is extracted using the CTAB method. Molecular marker identification and screening are performed. Samples with the same genotype as the maternal parent are screened for fertility identification using dCAPS markers (MS-F1: 5'-AAAAGCAGCGTCCATGATGTTGTCCTTCTT-3', MS-R1: 5'-TTCCTTCACCACTCAAGATG-3', DdeI restriction enzyme digestion). At the same time, watermelon ploidy is identified by root tip staining or flow cytometry.

[0013] Furthermore, the tissue culture process described in step (3) is as follows: (a) Take terminal buds or lateral shoots (with 1-2 axillary buds) 3-5 cm long from healthy field-grown male-sterile double haploid mother plants, preferably newly sprouted shoots in spring; disinfection process: rinse with running water for 30 minutes, soak in 75% alcohol for 30 seconds, rinse 3 times with sterile water, soak in 0.1% HgCl2 (mercuric chloride) or 2% NaClO (sodium hypochlorite) for 8-12 minutes, rinse 5 times with sterile water, and blot with filter paper. (b) Primary culture induction of shoot clusters: Primary culture medium formula: MS + 6-BA (0.5~1.0 mg / L) + NAA (0.05~0.1 mg / L) + sucrose 30 g / L + agar 6 g / L (pH adjusted to 5.8, autoclaved at 121℃ for 20 minutes). Sterilized explants were inserted into the primary culture medium. Culture conditions: temperature 25±2℃, light intensity 1500~2000 lx, light duration 12 h / day. After 20~30 days of culture, axillary buds germinated to form shoot clusters. (c) Rapid propagation by subculture: Subculture medium formula: MS + 6-BA (0.3~0.5mg / L) + NAA (0.01~0.05mg / L), reduce hormone concentration to prevent malformation; cut the clustered buds into single buds or small bud clusters with 2~3 bud points, transfer to subculture medium, and the propagation cycle is 25~30 days; (d) Rooting culture: Rooting medium formula: 1 / 2 MS + IAA (0.5~1.0 mg / L). Transfer the tissue from the subculture medium to the rooting medium. Before transfer, cut off a part of the old tissue to form a new wound. Insert the new wound into the culture medium, which is conducive to rooting. Transplant after 15~20 days when the root system is 2~3 cm long. (e) Hardening off seedlings in bottles: Open the bottle to allow the rooted seedlings to breathe for 2-3 days at 90% humidity. Then wash the agar off the roots and plant them in a sterilized vermiculite:perlite = 1:1 substrate. Cover with a transparent cover to keep the soil moist and gradually ventilate to reduce humidity. Remove the cover after 2 weeks and maintain a temperature of 22-28℃ and a diffused light environment.

[0014] Further, the grafting process described in step (3) is as follows: When the main vine of the dominant male sterile double haploid plant grows 4-5 true leaves, pinch off the growing point (tender tip) at the very top of the main vine with sterilized scissors or fingers, retaining the strong leaves at the base. When the lateral vines grow to 20-30 cm, the strong lateral vines can be pinched off again to stimulate the growth of tertiary vines (secondary lateral vines) and further increase the number of fruiting branches. When there are enough newly grown lateral branches, the growing point at the top is picked for grafting. Pumpkin is selected as the rootstock for grafting and propagation. The grafting method is used. Before grafting, both the scion and the rootstock are disinfected with fungicide. After grafting, the seedlings are shaded and covered with a moisture-retaining cover for 5 days. The temperature is controlled at around 26℃. Then, the grafted seedlings are transferred to a cool, low-light outdoor place to continue moisturizing for 5 days. During this period, attention should be paid to intermittent ventilation and root moisture retention. After 5 days, the moisture-retaining cover is removed, and the seedlings are transplanted after 2-3 days of recovery. Grafting is carried out continuously throughout the year according to the seed production needs to ensure the propagation base.

[0015] Furthermore, the process of facility seed production in step (5) is as follows: Select a single plastic greenhouse or a multi-span plastic greenhouse with a width of 6-8 m, cover the ventilation openings and doorways with insect-proof netting, and the mesh count of the insect-proof netting should not be less than 40 meshes; select the planting season according to the local climate, ensure that the minimum temperature inside the greenhouse is above 15℃ during the pollination period, and cultivate on the ground or with vines. For ground cultivation, plant 800-2000 plants per mu and prune 2-4 vines; for vine cultivation, plant 1800-3500 plants per mu; the planting ratio of the parents is 1:4-1:8, with the male parent planted between two rows of female parents; plant the male parent first, and plant the heterozygous male sterile (Aa) female parent 7-10 days later. Release bees for pollination at the beginning of the opening of the first female flower of the female parent, and remove the male parent after pollination.

[0016] Furthermore, the process of open-field seed production in step (5) is as follows: the open field should have good isolation conditions, and there should be no watermelon fields within 1000m. The crop rotation should be selected according to the local climate, and the minimum temperature during the pollination period should not be lower than 15℃. Direct sowing or seedling transplanting should be adopted. The number of female plants should be 1000-4500 per mu. The planting ratio of male and female plants should be 1:4-1:8. The male plants should be directly sown or planted between two rows of female plants. The male plants should be sown or planted first, and the hybrid male sterile plants (Aa) should be sown or planted 7-10 days later. Bees should be released to pollinate the first female flower of the female plant at the beginning of its opening. After pollination, the male plants should be removed.

[0017] Furthermore, in the above-mentioned facility-based or open-field seed production process, the number of pollinating bees is 1-1.5 standard hives per acre, with approximately 25,000-30,000 worker bees per hive.

[0018] Furthermore, the pest and disease control and fertilizer and water management process described in step (5) is as follows: pest and disease control is carried out before the bees enter the shed, and harmless pesticides are selected after the bees enter the shed. After the male parent is removed, the beehive is moved out of the shed and routine pest and disease control is carried out; fertilizer and water management: fertilizer is applied with water during the vine extension period, with 0.5 kg to 1 kg of nitrogen fertilizer (N), 0.5 kg to 1 kg of phosphorus fertilizer (P2O5), and 0.5 kg to 1 kg of potassium fertilizer (K2O) per mu. Fruit setting period: Two days after pollination, apply fertilizer with irrigation water, using 1.5-2 kg of nitrogen (N), 1.5-2 kg of phosphorus (P2O5), and 0.5-1 kg of potassium (K2O) per acre; 12 days after pollination, apply fertilizer with irrigation water, using 1.5-2 kg of nitrogen (N), 1.5-2 kg of phosphorus (P2O5), and 0.5-1 kg of potassium (K2O) per acre; irrigate until the soil is moist but not waterlogged, and then stop watering until harvest. If the soil cracks, a small amount of drip irrigation can be used. Strictly control the soil moisture content throughout the growing season.

[0019] Furthermore, the seeds are harvested 35 to 55 days after pollination. After harvesting, the melons can be placed in a cool, dry place to ripen for 5 to 7 days. Miscellaneous fruits and diseased fruits that do not conform to the shape, size, and color of the seeds of the variety should be removed in time, and the seeds should be taken out and washed in time. Furthermore, the disinfection process is as follows: after cleaning the seeds, soak them in a 0.3% sodium hypochlorite solution or a 0.8% calcium hypochlorite solution for 20 minutes, and then rinse them thoroughly with clean water. After disinfection, the seeds are air-dried in a ventilated place, then dried at 35℃~40℃, turning them over every 4~6 hours until the seed moisture content is ≤8%.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: Several male-sterile watermelon materials have been discovered by domestic and international teams, but all are controlled by recessive male-sterile genes and have not been truly applied to watermelon hybrid seed production. To date, there are no reports or applications of dominant male-sterile watermelon materials for hybrid seed production. This invention utilizes gene editing or embryo rescue culture technology to rapidly, accurately, and efficiently obtain haploid induction lines, thereby obtaining dominant male-sterile double haploids. Crossing these dominant male-sterile double haploids with fertile maternal parents can efficiently obtain batches of completely sterile maternal parent seeds, enabling bee pollination to replace artificial pollination in watermelon hybrid seed production. This effectively overcomes labor shortages and reduces hybrid seed production costs. Therefore, the dominant male-sterile maternal line with superior traits used in this study is innovative and of significant importance (Table 2).

[0021] Table 2. Comparison of the advanced nature of this invention with similar technologies at home and abroad. Technical content Status of similar technologies at home and abroad Advanced features of this invention Application of dominant male sterile materials All reported applications of male-sterile materials in hybrid seed production have been recessive male-sterile; no reports have been found of applications of dominant male-sterile watermelon materials. Using dominant male-sterile materials, a series of dominant male-sterile maternal lines were created. Application of male sterile materials in seed production There are no reports of using dominant male-sterile materials in watermelon hybrid production. Gene editing or embryo rescue culture technology can be used to quickly, accurately and efficiently obtain haploid induction lines, obtain dominant male-sterile double haploids, and use dominant male-sterile double haploids to cross with fertile maternal parents to efficiently obtain a large number of completely sterile maternal parent seeds, thereby realizing the production of watermelon hybrids by replacing artificial pollination with bee pollination. Attached Figure Description

[0022] Figure 1 This is a technical roadmap of the present invention; Figure 2 This is a screenshot of the SSR tagging and filtering results from Example 1. Figure 3 This is a diagram showing the fertility results of plants labeled with dCAPS in Example 1; Figure 4 This is a graph showing the results of sequencing identification of plant fertility in Example 1. The arrows indicate T as the sterile plant and C as the fertile plant. Figure 5 The planting pattern is as described in Example 1; Figure 6 Seed yield under the planting mode of Example 1; Figure 7 The image shows the results of seed purity detection using the SSR marker BVWS00208F / R in Example 1. A represents the seed purity of seeds produced under this model, B represents the seed purity of seeds produced through artificial pollination, and the arrow points to the detected female parent. Figure 8 This is the planting pattern for Example 2. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all obtained through commercial purchase.

[0025] In this invention, the dominant male-sterile watermelon line G42MS is used. This line was obtained by mutagenesis using the method described in Chinese Patent CN115385994B and has been registered as a new plant variety (Announcement No.: CNA090372E).

[0026] Example 1: Production of SM5 watermelon hybrids using bee pollination and vine hanging in plastic greenhouses in Huai'an City, Jiangsu Province Step 1: Creation of a dominant sterile maternal line for watermelons Using the dominant sterile line G42MS obtained by mutagenesis as the female parent, the sterile plants of G42MS were selected and crossed with the female parent line G33 of the watermelon hybrid SM5. Then, G33 was used as the recurrent parent for backcrossing. Only the hybrid seeds of the sterile plants were harvested in each generation. After 4-5 generations, the dominant sterile female parent line G33MS of watermelon (50% of which are sterile plants and 50% are fertile plants) could be obtained.

[0027] Step Two: Seed Propagation Technology of Completely Sterile Female Parent for Watermelon Hybrids (1) Creation of watermelon dominant male sterile double haploid material Method 1: Obtaining via CRISPR / Cas9 technology ClDMP4 Gene-edited mutants (haploid inducible lines) were used as the male parent and the dominant heterozygous male-sterile plant G33MS(Aa) as the female parent for hybridization. After obtaining the fruit, haploid selection was performed using fluorescent labeling: the seeds in the fruit were dehulled to expose the embryos. Embryos were screened in a dark room using a handheld fluorescence detector or a stereofluorescence microscope. Those without fluorescence were haploids G33MS(A) or G33MS(a), while diploids emitting green fluorescence were removed. Subsequently, the haploid plants were doubled using sulfadiazine or colchicine. DNA was then extracted from some plant tissues and fertility was determined using dCAPS labeling (MS-F1: 5'-AAAAGCAGCGTCCATGATGTTGTCCTTCTT-3', MS-R1: 5'-TTCCTTCACCACTCAAGATG-3', DdeI digestion). Male-sterile double haploid plants G33MS(AA) were selected.

[0028] Method 2: Using the dominant heterozygous male-sterile plant G33MS(Aa) as the female parent and inbred lines with different genetic backgrounds as the male parent, pollination of the female parent is achieved by irradiating the male parent's pollen with 200-600 Gry of X-rays. Approximately 10-15 days after pollination, embryo rescue culture is performed to create haploid plants. The specific procedures are as follows: The collected watermelon fruits are sprayed with 75% ethanol, placed in a stainless steel dish, and the surface is moistened with a small amount of 95% ethanol. The fruit is then sterilized by incineration. The watermelon is cut open with a sterile scalpel, and the immature seeds are removed. Approximately one-third of the seed's distal embryo is cut off, and the remaining seeds are laid flat on the haploid induction medium. The medium formula is: MS + 20 g / L sucrose + 20 g / L mannitol + 0.01 mg / L IAA. Culture the seeds on induction medium for 3-10 weeks. When the seeds swell, the epidermis turns brown, or the cotyledons emerge, remove the seed coat and transfer them to 1 / 2 MS medium + 20 g / L sucrose + 20 g / L mannitol for subculture. Simultaneously, collect samples into EP tubes and label both the tubes and the flasks. The rooting medium formula is: 1 / 2 MS + 20 g / L sucrose + 20 g / L mannitol + 1 mg / L IBA. Watermelon ploidy is determined using root tip staining or flow cytometry to screen for haploid plants G33MS(A) or G33MS(a). Haploid plants were created by doubling watermelon haploids using asulfuron-methyl (2 mg / L) or colchicine (200 mg / L): The haploid seeds were soaked in sterile water for 24 hours, then placed on moist sterile filter paper to germinate for another 24 hours. Once the embryo began to germinate, the seeds were soaked in asulfuron-methyl (2 mg / L) or colchicine (200 mg / L) solution for 8 hours, followed by rinsing with running water for 8 hours. The treated seeds were then sown in a greenhouse under normal fertilization and irrigation management. Seedlings were observed; those developing normally were considered successfully doubled diploids.

[0029] Fertility identification of the obtained double haploid plants: Samples were taken from the double haploid plants obtained by method two, and the genome of the samples was extracted using the CTAB method. Molecular marker identification and screening were performed using SSR markers (BVWS00208F: 5'-TGCTTCAAAATCTATTCACAATTTGC-3', BVWS00208R: 5'-TTCTTGGTTTCGGGTTTCTTTACA-3'). The results are as follows: Figure 2 As shown, using the SSR marker BVWS00208F / R, 24 single plants with the same genotype as G33MS were screened from 323 single plants (indicated by the arrow).

[0030] Samples with the same genotype as the maternal parent were screened and then subjected to fertility identification using dCAPS markers (MS-F1: 5'-AAAAGCAGCGTCCATGATGTTGTCCTTCTT-3', MS-R1: 5'-TTCCTTCACCACTCAAGATG-3', DdeI digestion). Male-sterile double haploid plants G33MS(AA) were screened. Simultaneously, watermelon ploidy was determined using root tip staining or flow cytometry to identify successfully doubled diploid plants. Results are shown below. Figure 3 The fertility of 24 plants was detected using the dCAPS marker MS-F1 / R1, among which plant number 15 was the sterile genotype. Figure 4 As shown, sequencing further confirmed that plant No. 15 had the sterile genotype T / A, while plant No. 3 had the fertile genotype C / G.

[0031] (2) Rapid propagation technology of watermelon double haploids Method 1: Rapid propagation via tissue culture using obtained watermelon double haploid plants: Take terminal buds or tender shoots (with 1-2 axillary buds) from healthy mother plants in the field, 3-5 cm in length. Prioritize newly sprouted shoots in spring. Disinfection procedure: Rinse with running water for 30 minutes, soak in 75% alcohol for 30 seconds, rinse 3 times with sterile water, soak in 0.1% HgCl2 (mercuric chloride) or 2% NaClO (sodium hypochlorite) for 8-12 minutes, rinse 5 times with sterile water, and blot dry with filter paper.

[0032] Primary culture (induction of shoot clusters): Primary culture medium formula: MS + 6-BA (0.5~1.0 mg / L) + NAA (0.05~0.1 mg / L) + sucrose 30 g / L + agar 6 g / L (pH adjusted to 5.8, autoclaved at 121℃ for 20 minutes). Sterilized explants were inserted into the primary culture medium. Culture conditions: temperature 25±2℃, light intensity 1500~2000 lx, light duration 12 h / day. After 20~30 days of culture, axillary buds germinated to form shoot clusters.

[0033] Subculture proliferation (rapid propagation): Subculture medium formula: MS + 6-BA (0.3~0.5mg / L) + NAA (0.01~0.05mg / L) (reduce hormone concentration to prevent malformation). Cut the clustered buds into single buds or small bud clusters (containing 2~3 bud points), transfer them to subculture medium, and the proliferation cycle is 25~30 days.

[0034] Rooting culture: Rooting medium formula: 1 / 2 MS + IAA (0.5~1.0 mg / L). Transfer the tissue from the subculture medium to the rooting medium. Before transfer, cut off a portion of the aging tissue to create new wounds. Inserting the tissue into the culture medium through these new wounds promotes rooting. Transplant after 15~20 days of culture, when the roots are 2~3 cm long.

[0035] Hardening off seedlings: Hardening off seedlings in bottles: Open the bottle to allow air to circulate for 2-3 days (humidity 90%), then wash the agar off the roots, plant them in a sterilized vermiculite:perlite = 1:1 substrate, cover with a transparent cover to keep them moist, gradually ventilate to reduce humidity, remove the cover after 2 weeks, and maintain a temperature of 22-28℃ and a diffused light environment.

[0036] Method 2: Propagation using grafting techniques on obtained watermelon double haploid plants: When the main vine has 4-5 true leaves, pinch off the very tip (tender shoot) of the main vine with sterilized scissors or your fingers, retaining the strong leaves at the base. When the lateral vines reach 20-30 cm in length, pinch off the tips again to stimulate the growth of secondary lateral vines, further increasing the number of fruiting branches. Once enough new lateral branches have grown, graft the tips of the lateral vines onto pumpkin rootstock for propagation. Use the patch grafting method. Before grafting, both the scion and rootstock should be disinfected with a fungicide. After grafting, cover the grafted seedlings with a moisture-retaining cover for 5 days, maintaining a temperature of around 26℃. Then, move the grafted seedlings to a cool, low-light outdoor location and continue moisturizing for another 5 days, ensuring intermittent ventilation and root moisture retention. After 5 days, remove the moisture-retaining cover and transplant the seedlings after 2-3 days of recovery. Grafting should be carried out continuously throughout the year to ensure a sufficient propagation population, based on seed production needs.

[0037] (3) Propagation of male-sterile watermelon maternal seeds: A certain number of male-sterile double haploid plants G33MS(AA) obtained by rapid propagation were used as maternal parents and crossed with their fertile plants G33MS(aa) to produce a large number of heterozygous male-sterile plants G33(Aa) maternal watermelon seeds.

[0038] Step 3: Producing hybrid watermelon seeds by pollinating and trellising the vines with bees. (1) Requirements for greenhouse facilities: Plastic greenhouses with 8-meter wide insect-proof netting, the mesh count of the insect-proof netting should not be less than 60 meshes.

[0039] (2) Pollinating bee species and quantity: Select Chinese honeybee. Configure 1.5 standard hives per mu (approximately 25,000-30,000 worker bees per hive).

[0040] (3) Bee pollination and crop rotation: For spring cultivation, the male parent G38 is planted on March 20th, and the heterozygous male-sterile plant (Aa) is planted in early April. Bees are placed in 1.5 standard hives per mu (approximately 0.067 hectares) at the beginning of the first female flower opening on the female parent. The male parent is removed 2-3 weeks after bee pollination. The female parent's fruit is harvested 35-50 days after pollination to obtain hybrid seeds.

[0041] (4) Parent planting method based on bee pollination: For vine cultivation, about 900 watermelon seedlings are planted in a plastic greenhouse with a plant spacing of 30cm and an 8m wide insect-proof net, with a parent ratio of about 1:4.

[0042] according to Figure 5Planting model for parent plants: Plastic greenhouse trellis cultivation according to Figure 5 Land preparation and bed making: In the diagram, green represents the bed surface. An 8-meter-wide, 40-meter-long plastic greenhouse with insect-proof netting can accommodate 8 beds. One bed (1 meter wide) is located on the east and west sides, planted with the male parent (yellow icon) and female parent (red icon) respectively, in a 1:4 ratio (one male parent plant followed by four female parent plants). The three middle beds (2 meters wide) are also planted with the same ratio of male parent (yellow icon) and female parent (red icon), with one male parent plant followed by four female parent plants. The wider middle beds only require alternating rows of male parent plants with the female parent plants, saving space and increasing the fruit set rate and seed yield of the female parent. Beehives are placed in the center of the greenhouse to facilitate the radiating of bees for nectar collection and pollination, and to allow for artificial sap supplementation.

[0043] (5) Pest and disease control and water and fertilizer management: Pest and disease control should be carried out before the bees enter the greenhouse. After the bees enter the greenhouse, no more insecticides should be applied. Disease treatment should be carried out according to the routine procedure. After the male parent is removed, the beehives should be moved out of the greenhouse before routine pest and disease control can be carried out. For details, please refer to the "Technical Specification for Hybrid Seed Production of Watermelon in Facility" DB 3208 / T 222—2024. Fertilizer and water management: Apply fertilizer with irrigation during the vine extension period. Apply 0.5 kg of nitrogen fertilizer (N), 0.5 kg of phosphorus fertilizer (P2O5), and 0.5 kg of potassium fertilizer (K2O) per mu. Fruit setting period: Two days after pollination, apply fertilizer with irrigation water, using 1.5 kg of nitrogen (N), 1.5 kg of phosphorus (P2O5), and 0.5 kg of potassium (K2O) per acre; 12 days after pollination, apply fertilizer with irrigation water, using 1.5 kg of nitrogen (N), 1.5 kg of phosphorus (P2O5), and 0.5 kg of potassium (K2O) per acre; water until the soil is moist but not waterlogged, and then stop watering until harvest. If the soil cracks, a small amount of drip irrigation can be used. Strictly control the soil moisture content throughout the growing season.

[0044] Step 4: Seed harvesting and disinfection (1) Harvesting: Seed melons should be harvested 35-50 days after pollination. After harvesting, they can be placed in a cool, dry place to ripen for 5 days. Miscellaneous fruits and diseased fruits that do not conform to the shape, size, and color of the seeds of the variety should be removed in time. Take out the melon seeds, wash them in time, and do not let them ferment.

[0045] (2) Disinfection: After cleaning the seeds, soak them in a 0.3% sodium hypochlorite solution or a 0.8% calcium hypochlorite solution for 20 minutes, then rinse them with clean water. After disinfection, air-dry the seeds in a ventilated place, then dry them at 40℃, turning them over every 4 hours until the seeds are dry (moisture content ≤8%). Then place the seeds in a 70℃ oven to continue drying for 72 hours to avoid the spread of watermelon quarantine disease CGMMV.

[0046] (3) Screening and germination rate test: Use an air separator or color sorter to screen the disinfected seeds and remove immature seeds. Take three parallel samples of 100 seeds from each batch, soak them in warm water for 20 minutes, pat dry the surface water, wrap them in a clean container with a slightly damp towel, and incubate at 30℃. Record the germination rate at 24, 48, and 72 hours. If the germination rate reaches 90% after 72 hours, the seeds can be packaged and sold; otherwise, continue screening.

[0047] Seed production yield and cost data: (1) Seed yield: According to the planting model in this embodiment, the number of seeds harvested is as follows: Figure 6 As shown: The number of seeds harvested in each row varies slightly, 320m 2 A total of 4654.4 g of hybrid seeds were harvested from the greenhouse. Currently (taking Huai'an, Jiangsu as an example), the yield of artificially pollinated seeds in a greenhouse of the same area is about 5000 g.

[0048] (2) Seed purity: Because the female parent has male sterility, theoretically, the hybrid offspring will not have self-pollinated fruits and seeds from the female parent, and the purity of the hybrid seeds obtained should be 100%. To verify the purity of the hybrid seeds, we used SSR molecular markers BVWS00208F: 5'-TGCTTCAAAATCTATTCACAATTTGC-3' and BVWS00208R: 5'-TTCTTGGTTTCGGGTTTCTTTACA-3' to detect the purity of the hybrid seeds. Figure 7 As shown in Figure A, among all 98 hybrids tested, no maternal parent was detected (the tested samples showed consistent bands, all belonging to the hybrid genotype), indicating that the male sterility trait and bee pollination method can indeed guarantee 100% purity of the hybrid seeds. The seed purity of artificially pollinated seeds (Figure B) was 96 / 98 = 97.95%, with the arrow pointing to the detected maternal parent.

[0049] (3) Comparison of input costs: The creation of breeding stock in the early stage of this technology requires a lot of cost, such as gene editing to obtain haploids and double haploids for propagation. However, once a certain number of double haploids are obtained, the cost of obtaining breeding stock will be greatly reduced. In other words, once the breeding stock creation system is established in the early stage, the larger the scale of breeding in the later stage, the lower the cost. The comparison of labor costs in the later stage of bee pollination (taking Huai'an, Jiangsu as an example) is shown in Table 3: Table 3 Comparison of input costs in Example 1 900 plants per greenhouse (8m×40m) Bee pollination based on dominant male sterility Ordinary artificial pollination Seedling transplanting 16.25 yuan / hour * 2 hours = 32.5 yuan 16.25 yuan / hour * 2 hours = 32.5 yuan Hanging vines 16.25 yuan / hour * 8 hours = 125 yuan 16.25 yuan / hour * 8 hours = 125 yuan Pruning and side cuttings 16.25 yuan / hour * 2 hours = 32.5 yuan 16.25 yuan / hour * 8 hours = 125 yuan Male pollination 300 yuan (bee) 16.25 yuan / hour * 40 hours = 625 yuan Harvesting 16.25 yuan / hour * 2 hours = 32.5 yuan 16.25 yuan / hour * 2 hours = 32.5 yuan Total / Yuan 522.5 940

[0050] Step 1: Propagation of Watermelon Hybrid Seeds with Completely Sterile Female Parent Seeds (1) Creation of dominant male-sterile maternal lines for watermelons Using the dominant sterile line MSG42 obtained through mutagenesis as the female parent, sterile plants of MSG42 were selected and crossed with the female parent line G33 of the watermelon hybrid SM5. Then, G33 was used as the recurrent parent for backcrossing. Only the hybrid seeds of sterile plants were harvested in each generation. After 4-5 generations, the dominant sterile female parent line G33MS of watermelon (of which 50% are sterile plants and 50% are fertile plants) could be obtained.

[0051] (2) Creating dominant sterile double haploid materials for small-fruited watermelons Method 1: Obtaining via CRISPR / Cas9 technology ClDMP4 Gene-edited mutants (haploid induction lines) were used as the male parent and a dominant heterozygous male-sterile plant (Aa) as the female parent for hybridization to obtain haploid plants (A). Haploid plants were screened using fluorescent labeling: seeds were dehulled to expose the embryos, and embryos were screened in a dark room using a handheld fluorescence detector or a stereofluorescence microscope. Green fluorescence indicated diploid plants, while no fluorescence indicated haploid plants. For specific methods, please refer to the research paper entitled "Production of double haploid watermelon via maternal haploid induction" published by Xu Yong's team at the Beijing Academy of Agricultural and Forestry Sciences. Subsequently, haploid plants were doubled using sulfadiazine or colchicine. Then, DNA was extracted from some plant tissues and fertility was identified using dCAPS markers (MS-F1: 5'-AAAAGCAGCGTCCATGATGTTGTCCTTCTT-3', MS-R1: 5'-TTCCTTCACCACTCAAGATG-3', DdeI digestion). Male-sterile double haploid plants G33MS(AA) were screened.

[0052] Method 2: Using a dominant heterozygous male-sterile line (Aa) as the female parent and inbred lines with different genetic backgrounds as the male parent, pollination of the female parent is achieved by irradiating the male parent's pollen with 300 Gry of X-rays. After approximately 12 days, embryo rescue culture is performed to create haploid plants. The specific procedures are as follows: The retrieved watermelon fruits are sprayed with 75% ethanol, placed in a stainless steel dish, and the surface is moistened with a small amount of 95% ethanol. The fruit is then sterilized by incineration. The watermelon is cut open with a sterile scalpel, and the immature seeds are removed. Approximately one-third of the seed's distal embryo is cut off, and the remaining seeds are laid flat on a haploid induction medium. The medium formula is: MS + 20 g / L sucrose + 20 g / L mannitol + 0.1 mg / L IBA. Culture the seeds on induction medium for 2-5 weeks. When the seeds swell, the epidermis turns brown, or the cotyledons emerge, remove the seed coat and transfer them to rooting medium. Simultaneously, sample the seeds into EP tubes and label both the EP tubes and the bottle. Rooting medium formulation: 1 / 2 MS + 20 g / L sucrose + 20 g / L mannitol + 1 mg / L IBA. Extract the genome from the samples in the EP tubes using the CTAB method and screen using molecular markers. Samples with the same genotype as the maternal parent are identified for fertility using dCAPS markers (MS-F1: 5'-AAAAGCAGCGTCCATGATGTTGTCCTTCTT-3', MS-R1: 5'-TTCCTTCACCACTCAAGATG-3', DdeI digestion) or root tip staining.

[0053] The above two methods were used to create dominant sterile haploid materials for small-fruited watermelons, and double haploid materials for watermelons were created by doubling watermelon haploids using sulfadiazine (2 mg / L) or colchicine (200 mg / L).

[0054] (3) Rapid propagation of watermelon double haploids Method 1: Rapid propagation via tissue culture using the obtained watermelon double haploid material: Take terminal buds or lateral shoots (with 1-2 axillary buds) from healthy mother plants in the field, 3-5 cm in length. Prioritize newly sprouted shoots in spring (high vigor, low contamination rate). Disinfection procedure: Rinse with running water for 30 minutes, soak in 75% alcohol for 30 seconds, rinse 3 times with sterile water, soak in 0.1% HgCl2 (mercuric chloride) or 2% NaClO (sodium hypochlorite) for 8-12 minutes, rinse 5 times with sterile water, and blot dry with filter paper.

[0055] Primary culture (induction of shoot clusters): Primary culture medium formula: MS + 6-BA (1.0 mg / L) + NAA (0.1 mg / L) + sucrose 30 g / L + agar 6 g / L (pH adjusted to 5.8, autoclaved at 121℃ for 20 minutes). Sterilized explants were inserted into the primary culture medium. Culture conditions: temperature 25 ± 2℃, light intensity 2000 lx, light duration 12 h / day. After 30 days of culture, axillary buds germinated to form shoot clusters.

[0056] Subculture proliferation (rapid propagation): Subculture medium formula: MS + 6-BA (0.5 mg / L) + NAA (0.05 mg / L) (reduce hormone concentration to prevent malformation). Cut the clustered shoots into single shoots or small shoot clusters (containing 3 bud points), transfer them to subculture medium, and the proliferation cycle is 30 days.

[0057] Rooting culture: Rooting medium formula: 1 / 2 MS + IAA (1.0 mg / L). Transfer tissue from the subculture medium to the rooting medium. Before transfer, cut off a portion of the aged tissue to create new wounds. Inserting the new wounds into the culture medium promotes rooting. Transplant after 20 days of culture, when the roots are 3 cm long.

[0058] Hardening off seedlings: Hardening off seedlings in bottles: Open the bottle to allow ventilation for 3 days (humidity 90%), then wash off the agar from the roots, plant in a sterilized vermiculite:perlite = 1:1 substrate, cover with a transparent cover to keep moist, gradually ventilate to reduce humidity, remove the cover after 2 weeks, maintain a temperature of 22-28℃ and a diffused light environment.

[0059] Method 2: Propagation using grafting techniques on obtained watermelon double haploid plants: When the main vine has 5 true leaves, pinch off the very tip (tender shoot) of the main vine with sterilized scissors or your fingers, retaining the strong leaves at the base. When the lateral vines reach 30 cm in length, pinch off the tips again to stimulate the growth of secondary lateral vines, further increasing the number of fruiting branches. Once enough new lateral branches have grown, graft the tips of the lateral vines onto pumpkin rootstock. Use the patch grafting method. Before grafting, both the scion and rootstock should be disinfected with a fungicide. After grafting, cover the grafted seedlings with a moisture-retaining cover for 5 days, maintaining a temperature of around 26℃. Then, move the grafted seedlings to a cool, low-light outdoor location and continue moisturizing for another 5 days, ensuring intermittent ventilation and root moisture retention. After 5 days, remove the moisture-retaining cover and transplant the seedlings after 2-3 days of recovery. Grafting should be carried out continuously throughout the year to ensure a sufficient propagation population, based on seed production needs.

[0060] (4) Propagation of male-sterile watermelon maternal seeds: A certain number of male-sterile double haploid plants G33MS(AA) obtained by rapid propagation were used as maternal parents and hybridized with fertile plants G33MS(aa) to produce a large number of heterozygous male-sterile G33MS(Aa) maternal watermelon seeds.

[0061] Step 2: Producing hybrid watermelon seeds by using bees for pollination and crawling on the ground. (1) Requirements for greenhouse facilities: Plastic greenhouses with 6m wide insect-proof netting, the mesh count of the insect-proof netting should not be less than 60 meshes.

[0062] (2) Pollinating bee species and quantity: Select Chinese honeybee. Configure 1 standard hive per mu (approximately 25,000-30,000 worker bees per hive).

[0063] (3) Bee pollination and crop rotation: For spring cultivation, the male parent G38 is planted on March 20th, and the heterozygous male-sterile plant (Aa) is planted in early April. Bees are placed in 1.5 standard hives per mu (approximately 0.067 hectares) at the beginning of the first female flower opening on the female parent. The male parent is removed 2-3 weeks after bee pollination. The female parent's fruit is harvested 35-50 days after pollination to obtain hybrid seeds.

[0064] (4) Parental planting method based on bee pollination: For ground cultivation, select a plastic greenhouse with a plant spacing of 20cm, a length of 40m, and a width of 6m for insect prevention netting, and plant approximately 400 watermelon seedlings. The ratio of parents is approximately 1:4. Parental plants can be planted according to the pattern shown in the diagram below ( Figure 8 ).

[0065] Plastic greenhouse ground-creeping cultivation according to Figure 8 Prepare the land by making raised beds. In the diagram, green represents the bed surface. A 6-meter-wide, 40-meter-long plastic greenhouse with insect-proof netting can accommodate two beds, one 1 meter wide on the east and one on the west. Plant the male parent (yellow icon) and the female parent (red icon) on each side, with a parent-to-female ratio of 1:4 (one male parent plant followed by four female parent plants). Place the beehives in the center of the greenhouse to facilitate the bees' radial spread for nectar collection and pollination, and to allow for artificial sap supplementation.

[0066] (5) Pest and disease control and water and fertilizer management: Pest and disease control should be carried out before the bees enter the greenhouse. After the bees enter the greenhouse, no more insecticides should be applied. Disease treatment should be carried out according to the routine procedure. After the male parent is removed, the beehives should be moved out of the greenhouse before routine pest and disease control can be carried out. For details, please refer to the "Technical Specification for Hybrid Seed Production of Watermelon in Facility" DB 3208 / T 222—2024. Fertilizer and water management: Apply fertilizer with irrigation during the vine extension period, with 1 kg of nitrogen fertilizer (N), 1 kg of phosphorus fertilizer (P2O5), and 1 kg of potassium fertilizer (K2O) per mu. Fruit setting period: Two days after pollination, apply fertilizer with irrigation water at a rate of 2 kg N, 2 kg P2O5, and 1 kg K2O per acre; 12 days after pollination, apply fertilizer with irrigation water at a rate of 2 kg N, 2 kg P2O5, and 1 kg K2O per acre; water until the soil is moist but not waterlogged, and then stop watering until harvest. If the soil cracks, a small amount of drip irrigation can be applied. Strictly control the soil moisture content throughout the growing season.

[0067] Step 3: Seed harvesting and disinfection (1) Harvesting: Seed melons should be harvested 45 days after pollination. After harvesting, they can be placed in a cool, dry place to ripen for 7 days. Miscellaneous fruits and diseased fruits that do not conform to the shape, size, and color of the seeds of the variety should be removed in time. Take out the melon seeds, wash them in time, and do not let them ferment.

[0068] (2) Disinfection: After cleaning the seeds, soak them in a 0.3% sodium hypochlorite solution or a 0.8% calcium hypochlorite solution for 20 minutes, then rinse them with clean water. After disinfection, air-dry the seeds in a ventilated place, then dry them at 35℃, turning them over every 4 hours until the seeds are dry (moisture content ≤8%). Then place the seeds in a 70℃ oven to continue drying for 72 hours to avoid the spread of watermelon quarantine disease CGMMV.

[0069] (3) Screening and germination rate test: Use an air separator or color sorter to screen the disinfected seeds and remove immature seeds. Take three parallel samples of 100 seeds from each batch, soak them in warm water for 20 minutes, pat dry the surface water, wrap them in a clean container with a slightly damp towel, and incubate at 30℃. Record the germination rate at 24, 48, and 72 hours. If the germination rate reaches 90% after 72 hours, the seeds can be packaged and sold; otherwise, continue screening.

[0070] Seed production yield and cost data: (1) Seed yield: According to the planting pattern of this example, 320m 2 A total of 2932.2 g of hybrid seeds were harvested from the greenhouse. At present (taking Huai'an, Jiangsu as an example), the yield of artificially pollinated seeds in a greenhouse of the same area is 2700 g.

[0071] (2) Seed purity: Since the female parent has male sterility, theoretically the hybrid offspring will not have self-pollinated fruits and seeds of the female parent, and the purity of the hybrid seeds obtained should be 100%. To verify the purity of the hybrid seeds, we used SSR molecular markers BVWS00209F: 5'-TGCTTCAAAATCTATTCACAATTTGC-3' and BVWS00209R: 5'-TTCTTGGTTTCGGGTTTCTTTACA-3' to detect the purity of the hybrid seeds. The final data showed that no female parent was detected in all 96 hybrids tested, indicating that the male sterility trait and bee pollination method can indeed guarantee the purity of the hybrid seeds.

[0072] (3) Comparison of input costs: The creation of breeding stock in the early stage of this technology requires a lot of cost, such as gene editing to obtain haploids and double haploids for propagation. However, once a certain number of double haploids are obtained, the cost of obtaining breeding stock will be greatly reduced. In other words, once the breeding stock creation system is established in the early stage, the larger the scale of breeding in the later stage, the lower the cost. The comparison of labor costs in the later stage of bee pollination (taking Huai'an, Jiangsu as an example) is shown in Table 4: Table 4 Comparison of input costs in Example 2 400 plants per greenhouse (6m x 40m) Bee pollination based on dominant male sterility Ordinary artificial pollination Seedling transplanting 16.25 yuan / hour * 2 hours = 32.5 yuan 16.25 yuan / hour * 2 hours = 32.5 yuan Pruning and side cuttings 16.25 yuan / hour * 8 hours = 130 yuan 16.25 yuan / hour * 8 hours = 130 yuan Male pollination 300 yuan (bee) 16.25 yuan / hour * 32 hours = 520 yuan Harvesting 16.25 yuan / hour * 2 hours = 32.5 yuan 16.25 yuan / hour * 2 hours = 32.5 yuan Total / Yuan 495 715

[0073] Step 1: Propagation of Watermelon Hybrid Seeds with Completely Sterile Female Parent Seeds (1) Creation of dominant male-sterile maternal lines for watermelons Using the dominant sterile line MSG42 obtained through mutagenesis as the female parent, sterile plants of MSG42 were selected and crossed with the female parent line G33 of the watermelon hybrid SM5. Then, G33 was used as the recurrent parent for backcrossing. Only the hybrid seeds of sterile plants were harvested in each generation. After 4-5 generations, the dominant sterile female parent line G33MS of watermelon (of which 50% are sterile plants and 50% are fertile plants) could be obtained.

[0074] (2) Creating dominant sterile double haploid materials for small-fruited watermelons Method 1: Obtaining CRISPR / Cas9 technology ClDMP4 Gene-edited mutants (haploid induction lines) were used as the male parent and a dominant heterozygous male-sterile G33MS (Aa) as the female parent for hybridization to obtain fruits and seeds. Haploid plants were screened using fluorescent labeling: seeds were dehulled to expose the embryos, and embryos were screened in a dark room using a handheld fluorescence detector or a stereofluorescence microscope. Green fluorescence indicated diploid plants, while no fluorescence indicated haploid G33MS (A) or G33MS (a). Specific methods can be found in the research paper titled "Production of double haploid watermelon via maternal haploid induction" published by Xu Yong's team at the Beijing Academy of Agricultural and Forestry Sciences. Subsequently, haploid plants were doubled using sulfadiazine or colchicine. Then, DNA was extracted from some plant tissues and fertility was identified using dCAPS markers (MS-F1: 5'-AAAAGCAGCGTCCATGATGTTGTCCTTCTT-3', MS-R1: 5'-TTCCTTCACCACTCAAGATG-3', DdeI digestion). Male-sterile double haploid plants G33MS(AA) were screened.

[0075] Method 2: Using a dominant heterozygous male-sterile line (Aa) as the female parent and inbred lines with different genetic backgrounds as the male parent, pollination of the female parent is achieved by irradiating the male parent's pollen with 400 Gry of X-rays. After approximately 15 days, embryo rescue culture is performed to create haploid plants. The specific procedures are as follows: The retrieved watermelon fruits are sprayed with 75% ethanol, placed in a stainless steel dish, and the surface is moistened with a small amount of 95% ethanol. The fruit is then sterilized by incineration. The watermelon is cut open with a sterile scalpel, and the immature seeds are removed. Approximately one-third of the seed's distal embryo is cut off, and the remaining seeds are laid flat on a haploid induction medium. The medium formula is: MS + 20 g / L sucrose + 20 g / L mannitol + 0.1 mg / L IBA. Culture the seeds on induction medium for 2-5 weeks. When the seeds swell, the epidermis turns brown, or the cotyledons emerge, remove the seed coat and transfer them to rooting medium. Simultaneously, take samples into EP tubes and label both the EP tubes and the bottle. Rooting medium formulation: 1 / 2 MS + 20 g / L sucrose + 20 g / L mannitol + 1 mg / L IBA. Extract the genome from the samples in the EP tubes using the CTAB method and identify and screen them using molecular markers. Samples with the same genotype as the maternal parent are identified for fertility using dCAPS markers (MS-F: 5'-AAAAGCAGCGTCCATGATGTTGTCCTTCTT-3', MS-R: 5'-TTCCTTCACCACTCAAGATG-3', DdeI digestion) or root tip staining.

[0076] The above two methods were used to create dominant sterile haploid materials for small-fruited watermelons, and double haploid materials for watermelons were created by doubling watermelon haploids using sulfadiazine (2 mg / L) or colchicine (200 mg / L).

[0077] (3) Rapid propagation of watermelon double haploids Method 1: Rapid propagation of watermelon double haploid materials through tissue culture: Take terminal buds or tender shoots (with 1-2 axillary buds) from healthy mother plants in the field, 3-5 cm in length. Prioritize newly sprouted shoots in spring (high vigor, low contamination rate). Disinfection process: Rinse with running water for 30 minutes, soak in 75% alcohol for 30 seconds, rinse 3 times with sterile water, soak in 0.1% HgCl2 (mercuric chloride) or 2% NaClO (sodium hypochlorite) for 8-12 minutes, rinse 5 times with sterile water, and blot dry with filter paper.

[0078] Primary culture (induction of shoot clusters): Primary culture medium formula: MS + 6-BA (0.8 mg / L) + NAA (0.08 mg / L) + sucrose 30 g / L + agar 6 g / L (pH adjusted to 5.8, autoclaved at 121℃ for 20 minutes). Sterilized explants were inserted into the primary culture medium. Culture conditions: temperature 25 ± 2℃, light intensity 1500 lx, light duration 12 h / day. After 20-30 days of culture, axillary buds germinated to form shoot clusters.

[0079] Subculture proliferation (rapid propagation): Subculture medium formula: MS + 6-BA (0.4 mg / L) + NAA (0.04 mg / L) (reduce hormone concentration to prevent malformation). Cut the clustered buds into single buds or small bud clusters (containing 2-3 bud points), transfer them to subculture medium, and the proliferation cycle is 25-30 days.

[0080] Rooting culture: Rooting medium formula: 1 / 2 MS + IAA (0.8 mg / L). Transfer the tissue from the subculture medium to the rooting medium. Before transfer, cut off a portion of the aging tissue to create new wounds. Inserting the new wounds into the culture medium promotes rooting. Transplant after 15-20 days of culture, when the roots are 2-3 cm long.

[0081] Hardening off seedlings: Hardening off seedlings in bottles: Open the bottle to allow air to circulate for 2-3 days (humidity 90%), then wash the agar off the roots, plant them in a sterilized vermiculite:perlite = 1:1 substrate, cover with a transparent cover to keep them moist, gradually ventilate to reduce humidity, remove the cover after 2 weeks, and maintain a temperature of 22-28℃ and a diffused light environment.

[0082] Method 2: Propagation of the obtained watermelon double haploid plants through grafting: When the main vine has grown 4-5 true leaves, pinch off the very tip (tender shoot) of the main vine with sterilized scissors or your fingers, retaining the strong leaves at the base. When the lateral vines grow to 20-30 cm, pinch off the tips of the strong lateral vines again to stimulate the growth of secondary lateral vines, further increasing the number of fruiting branches. When enough new lateral branches have grown, graft the tips of the lateral vines onto pumpkin rootstock for propagation. Use the patch grafting method. Before grafting, both the scion and rootstock should be disinfected with a fungicide. After grafting, cover the grafted seedlings with a moisture-retaining cover for 5 days, maintaining a temperature of around 26℃. Then, move the grafted seedlings to a cool, low-light outdoor location and continue moisturizing for another 5 days, paying attention to intermittent ventilation and root moisture retention. After 5 days, remove the moisture-retaining cover and transplant the seedlings after 2-3 days of recovery. Grafting should be carried out continuously throughout the year according to seed production needs to ensure a sufficient propagation population.

[0083] (4) Propagation of male-sterile watermelon maternal seeds: A certain number of male-sterile double haploid plants G33MS(AA) obtained by rapid propagation were used as maternal parents and hybridized with fertile plants G33MS(aa) to produce a large number of heterozygous male-sterile G33MS(Aa) maternal watermelon seeds.

[0084] Step 2: Using bee pollination to propagate watermelon hybrid seeds in open fields (1) Open field requirements: There must be no watermelon planting area within 1000 meters around the seed production plot.

[0085] (2) Pollinating insect species and quantity: Natural pollination is carried out using field insects. If the number of insects is insufficient, bees can be placed to assist in pollination. Chinese honeybees are selected. Ensure that 100-150 worker bees leave the hive to collect nectar per acre per day.

[0086] (3) Bee pollination and crop rotation: Watermelon is planted in open fields starting in May each year. The male parent G38 is planted first, and a week later, a heterozygous male sterile plant (Aa) is planted as the female parent. Bees are released to pollinate the plants 15-20 days later. After 2-3 weeks of bee pollination, the male parent plants and their fruits are removed according to the fruit set. After the male parent plants are completely removed, the female parent fruits are allowed to mature before the seeds are harvested.

[0087] (4) Parent planting method based on bee pollination: For open field cultivation, set a row spacing of 2m and a plant spacing of 15-20cm. The ratio of parent to parent line should be between 1:8 and 1:20. The male line should be planted in the middle of the two rows of female line (i.e., in the middle of the bed). The placement and arrangement of beehives should be determined according to factors such as bee flight radius, sunlight, and wind direction. Beehives can be placed in the middle of the planting area or on one side of the field, but should be far away from irrigation areas, wells, and other damp areas. Once the location of the beehives is determined, try not to change the location arbitrarily, or change the location of the beehives under the guidance of beekeepers.

[0088] (5) Pest and disease control and water and fertilizer management: Before the bees enter the field, growers need to do a good job in the prevention and control of crop pests and diseases, and choose pesticides or insecticides and fungicides that are pollution-free, have low residue, and are non-toxic or low-toxic to ensure the safety of pollinating bee colonies and ensure the pollination efficiency of bees. After the male parent is removed, the beehives are moved out of the planting area before the conventional pest and disease control for open-field watermelon planting can be carried out. Fertilizer and water management: Follow the conventional water and fertilizer management for open-field watermelon planting.

[0089] Step 3: Seed harvesting and disinfection (1) Harvesting: Seed melons should be harvested 35-45 days after pollination. After harvesting, they can be placed in a cool, dry place to ripen for 5-7 days. Miscellaneous fruits and diseased fruits that do not conform to the shape, size, and color of the seeds of the variety should be removed in time. Take out the melon seeds, wash them in time, and do not let them ferment.

[0090] (2) Disinfection: After cleaning the seeds, soak them in a 0.3% sodium hypochlorite solution or a 0.8% calcium hypochlorite solution for 20 minutes, and then rinse them with clean water. After disinfection, dry the seeds in the sun or air, turning them over every 4-6 hours until the seed moisture content is ≤8%. Then place the seeds in a 40℃ oven to dry for 24 hours, and then adjust the oven temperature to 72℃ for 72 hours. After that, turn off the oven and let the temperature drop to room temperature before storing them.

[0091] (3) Screening and germination rate test: Seeds after dry heat treatment were screened using an air separator or color sorter to remove immature seeds. Three parallel samples of 100 seeds were taken from each batch, soaked in warm water for 20 minutes, dried, wrapped in a slightly damp towel in a clean container, and incubated at 30°C. Germination rates were recorded at 24, 48, and 72 hours. Seeds with a germination rate of 90% after 72 hours were ready for storage; otherwise, screening continued.

[0092] Seed production yield and cost data: (1) Seed yield: According to the planting pattern in this example, a total of 7560g / 667m² of hybrid seeds were harvested. 2 Currently (taking Gansu as an example), the yield of artificially pollinated seed production is 6210g / 667m³. 2 .

[0093] (2) Seed purity: Because the female parent has male sterility, theoretically, the hybrid offspring will not have self-pollinated fruits or seeds from the female parent, and the purity of the hybrid seeds obtained should be 100%. To verify the purity of the hybrid seeds, we used SSR molecular markers BVWS00209F: 5'-TGCTTCAAAATCTATTCACAATTTGC-3' and BVWS00209R: 5'-TTCTTGGTTTCGGGTTTCTTTACA-3' to detect the purity of the hybrid seeds. The results showed that no female parent was detected in all 96 hybrids tested, indicating that the male sterility trait and bee pollination method can indeed guarantee the purity of the hybrid seeds to be 100%.

[0094] (3) Comparison of input costs: The creation of breeding stock in the early stage of this technology requires a lot of cost, such as gene editing to obtain haploids and double haploids for propagation. However, once a certain number of double haploids are obtained, the cost of obtaining breeding stock will be greatly reduced. In other words, once the breeding stock creation system is established in the early stage, the larger the scale of breeding in the later stage, the lower the cost. The comparison of labor costs (taking Gansu as an example) in the later stage of bee pollination is shown in Table 5: Table 5 Comparison of input costs in Example 3 <![CDATA[Open area 667 m 2 > Bee pollination based on dominant male sterility Ordinary artificial pollination Seedling transplanting 12.5 yuan / hour * 16 hours = 200 yuan 12.5 yuan / hour * 16 hours = 200 yuan Pruning and side cuttings 12.5 yuan / hour * 16 hours = 200 yuan 12.5 yuan / hour * 16 hours = 200 yuan Male pollination 300 yuan (bee) 12.5 yuan / hour * 80 hours = 1000 yuan Harvesting 12.5 yuan / hour * 16 hours = 200 yuan 12.5 yuan / hour * 16 hours = 200 yuan Total / Yuan 900 1600 The above description of the embodiments is only for illustrating the technical concept and features of the present invention. Its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. Those skilled in the art can obviously easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the above embodiments should not be used to limit the scope of protection of the present invention. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing watermelon hybrid seeds through bee pollination, characterized in that, Includes the following steps: (1) Creating a dominant male-sterile maternal line for watermelon: Using dominant male-sterile watermelon material as maternal parent P1, select its male-sterile plant P1 (Aa) and cross it with the maternal line P2 of watermelon hybrid. Then use the maternal line P2 as the recurrent parent for backcrossing. Only the hybrid seeds of the male-sterile plants are harvested in each generation. After 4-5 generations, the dominant male-sterile maternal line P3 of watermelon can be obtained, of which 50% are male-sterile plants and 50% are fertile plants. (2) Creating watermelon dominant male sterile double haploid material: Using the dominant heterozygous male sterile plant P3(Aa) obtained in step (1) as the female parent, hybridize it with the haploid induction line or pollinate it with radiation pollen with different genetic backgrounds and then rescue the embryo to obtain dominant male sterile haploid material P3(A) or P3(a); use the obtained dominant male sterile haploid material to create watermelon dominant male sterile double haploid material P3(AA) or P3(aa) by doubling it with asulfanilamide or colchicine, and then screen the sterile plants by molecular markers to finally obtain P3(AA); (3) Propagation: Using the watermelon double haploid material P3 (AA) obtained in step (2), rapid propagation is carried out through tissue culture or grafting; (4) Propagation of male-sterile watermelon maternal seeds: Using a certain number of male-sterile double haploid plants P3 (AA) obtained in step (3) as maternal parents, crossbreeding them with fertile plants P3 (aa) to produce a large number of heterozygous male-sterile plants P3 (Aa) maternal watermelon seeds. (5) Propagation of hybrid watermelon seeds by bee pollination: Using the heterozygous male sterile line P3 (Aa) as the female parent and the inbred line P4 as the male parent, hybrid watermelon seeds are propagated by combining facility seed production or open field seed production with bee pollination. During the propagation process, pest and disease control and fertilizer and water management are required. After the seeds are harvested, they are disinfected, screened and tested for germination rate. Once qualified, they can be packaged and sold.

2. The method for preparing watermelon hybrid seeds by bee pollination according to claim 1, characterized in that: The process of creating dominant sterile haploid materials is as follows: the ClDMP4 gene-edited mutant is obtained through CRISPR / Cas9 technology. The obtained haploid induction line is used as the male parent and the dominant heterozygous male sterile plant P3(Aa) obtained in step (1) is used as the female parent for hybridization to obtain haploid plants and harvest seeds. The seeds are dehulled to expose the embryos. The embryos are screened in a dark room using a handheld fluorescence detector or a stereofluorescence microscope. The embryos without fluorescence are haploids P3(A) or P3(a). The diploids that emit green fluorescence are removed.

3. The method for preparing watermelon hybrid seeds by bee pollination according to claim 1, characterized in that: The process of creating dominant sterile haploid materials is as follows: using the dominant heterozygous male sterile plant P3 (Aa) obtained in step (1) as the female parent and inbred lines with different genetic backgrounds as the male parent, the pollen of the male parent is irradiated with 200-600 Gry of X-rays to pollinate the female parent P3 (Aa). About 10-15 days after pollination, the embryo rescue culture is carried out to create haploid plants P3 (A).

4. The method for preparing watermelon hybrid seeds by bee pollination according to claim 3, characterized in that: The specific procedures for rescuing the immature embryos are as follows: The retrieved watermelon fruit is sprayed with 75% ethanol, placed in a stainless steel dish, and the surface is moistened with a small amount of 95% ethanol. The fruit is then sterilized by burning. The watermelon is cut open with a sterile scalpel, and the immature seeds are removed. Approximately one-third of the seed's distal embryo is cut off, and the remaining seeds are placed flat on a haploid induction medium. The medium formulation is: MS + 20 g / L sucrose + 20 g / L mannitol + 0.01 mg / L IAA. The seeds are cultured on the induction medium for 3-10 weeks. When the seeds swell, the epidermis turns brown, or the cotyledons emerge, the seed coat is removed, and the seeds are transferred to a medium of 1 / 2 MS + 20 g / L sucrose + 20 g / L mannitol for subculture. The subcultured plants are then transferred to a rooting medium of 1 / 2 MS + 20 g / L sucrose + 20 g / L mannitol + 1 mg / L IBA for rooting culture to obtain haploid plants.

5. The method for preparing watermelon hybrid seeds by bee pollination according to claim 1, characterized in that: The concentration of sulfamethoxazole in step (2) is 2 mg / L, and the concentration of colchicine is 200 mg / L.

6. The method for preparing watermelon hybrid seeds by bee pollination according to claim 1, characterized in that: The tissue culture process described in step (3) is as follows: (a) Take the terminal buds or lateral shoots of healthy field mother plants with dominant male sterile double haploids, 3-5 cm in length, and disinfect them; (b) Primary culture to induce shoot clusters: Primary culture medium formula: MS + 6-BA (0.5~1.0mg / L) + NAA (0.05~0.1mg / L) + sucrose 30g / L + agar 6g / L; Insert the sterilized explants from step (a) into the primary culture medium. Culture conditions: temperature 25±2℃, light intensity 1500~2000lx, light 12h / day. After 20~30 days of culture, axillary buds germinate to form shoot clusters. (c) Rapid propagation by subculture: Subculture medium formula: MS + 6-BA (0.3~0.5mg / L) + NAA (0.01~0.05mg / L), reduce hormone concentration to prevent malformation; cut the clustered buds into single buds or small bud clusters with 2~3 bud points, transfer to subculture medium, and the propagation cycle is 25~30 days; (d) Rooting culture: Rooting medium formula: 1 / 2 MS + IAA (0.5~1.0 mg / L). Transfer the tissue from the subculture medium to the rooting medium. Before transfer, cut off a part of the old tissue to form a new wound. Insert the new wound into the culture medium, which is conducive to rooting. Transplant after 15~20 days when the root system is 2~3 cm long. (e) Hardening off seedlings in bottles: Open the bottle to allow the rooted seedlings to breathe for 2-3 days at 90% humidity. Then wash the agar off the roots and plant them in a sterilized vermiculite:perlite = 1:1 substrate. Cover with a transparent cover to keep the soil moist and gradually ventilate to reduce humidity. Remove the cover after 2 weeks and maintain a temperature of 22-28℃ and a diffused light environment.

7. The method for preparing watermelon hybrid seeds by bee pollination according to claim 1, characterized in that: The grafting process described in step (3) is as follows: When the main vine of the dominant male sterile double haploid plant grows 4-5 true leaves, pinch off the growing point at the very top of the main vine with sterilized scissors or fingers, leaving the strong leaves at the base. When the lateral vines grow to 20-30 cm, the strong lateral vines can be pinched off again to stimulate the growth of tertiary vines and further increase the number of fruiting branches. When there are enough new lateral branches, the growing point at the top is removed for grafting. Pumpkin is selected as the rootstock for grafting and propagation. The grafting method is used. Before grafting, both the scion and the rootstock are disinfected with fungicide. After grafting, the seedlings are shaded and covered with a moisture-retaining cover for 5 days, with the temperature controlled at around 26℃. Then, the grafted seedlings are transferred to a cool, low-light outdoor place to continue moisturizing for 5 days, during which time attention is paid to intermittent ventilation and root moisture retention. After 5 days, the moisture-retaining cover is removed, and the seedlings are transplanted after 2-3 days of recovery. Grafting is carried out continuously throughout the year according to the seed production needs to ensure the propagation base.

8. The method for preparing watermelon hybrid seeds by bee pollination according to claim 1, characterized in that: The process of facility seed production in step (5) is as follows: select the planting season according to the local climate, ensure that the minimum temperature in the greenhouse is above 15℃ during the pollination period, and carry out ground cultivation or hanging vine cultivation. For ground cultivation, plant 800-2000 plants per mu and prune 2-4 vines; for hanging vine cultivation, plant 1800-3500 plants per mu; the planting ratio of parents is 1:4-1:8, and the male parent is planted between two rows of female parents; plant the male parent first, and plant the heterozygous male sterile plant (Aa) female parent 7-10 days later. Release bees for pollination at the beginning of the opening of the first female flower of the female parent, and remove the male parent after pollination.

9. The method for preparing watermelon hybrid seeds by bee pollination according to claim 1, characterized in that: The process of open-field seed production in step (5) is as follows: select the crop rotation according to the local climate, ensure that the minimum temperature during the pollination period is not lower than 15℃, adopt direct seeding or seedling transplanting, ensure 1000-4500 seedlings per mu of female parent, the planting ratio of parent parent is 1:4-1:8, the male parent is directly sown or planted in the middle of two rows of female parent; first sow or plant the male parent, and 7-10 later sow or plant the heterozygous male sterile plant (Aa) female parent, release bees for pollination in the early stage of the opening of the first female flower of the female parent, and remove the male parent after pollination.

10. The method for preparing watermelon hybrid seeds by bee pollination according to claim 1, characterized in that: The process of pest and disease control and fertilizer and water management described in step (5) is as follows: Before the bees enter the shed, pest and disease control should be carried out. After the bees enter the shed, pesticides that are harmless to the bees should be selected. After the male parent is removed, the beehives should be moved out of the shed and conventional pest and disease control should be carried out. Fertilizer and water management: During the vine extension period, fertilizer should be applied with water, with 0.5 kg to 1 kg of nitrogen fertilizer, 0.5 kg to 1 kg of phosphorus fertilizer, and 0.5 kg to 1 kg of potassium fertilizer per mu. During the fruit setting period: 2 days after pollination, fertilizer should be applied with water, with 1.5 kg to 2 kg of nitrogen fertilizer, 1.5 kg to 2 kg of phosphorus fertilizer, and 0.5 kg to 1 kg of potassium fertilizer per mu. 12 days after pollination, fertilizer should be applied with water, with 1.5 kg to 2 kg of nitrogen fertilizer, 1.5 kg to 2 kg of phosphorus fertilizer, and 0.5 kg to 1 kg of potassium fertilizer per mu. It is advisable to water until the soil is moist but not waterlogged. Watering should be stopped until harvest. If the soil is dry and cracked, a small amount of drip irrigation can be used. The soil moisture content should be strictly controlled throughout the growing season. And / or, the seeds are harvested 35 to 55 days after pollination. After harvesting, the melons can be placed in a cool, dry place to ripen for 5 to 7 days. Miscellaneous fruits and diseased fruits that do not conform to the shape, size and color of the seeds of the variety should be removed in time, and the seeds should be taken out and washed in time. And / or, the disinfection process is as follows: after cleaning the seeds, soak them in a 0.3% sodium hypochlorite solution or a 0.8% calcium hypochlorite solution for 20 minutes, and then rinse them thoroughly with clean water. After disinfection, air-dry the seeds in a ventilated place, then dry them at 35℃~40℃, turning them over every 4~6 hours until the seed moisture content is ≤8%.

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