Breeding method of sweet-taste and crisp-meat muskmelons

Abstract

The invention provides a breeding method of sweet-taste and crisp-meat muskmelons. The breeding method comprises the following steps: S1, parent selection; s2, parent pretreatment; s3, hybrid seed collection and initiation treatment; s4, accelerating germination and raising seedlings; s5, seedling transplanting and microorganism-assisted cultivation; s6, F1-generation seeds are harvested; s7, culturing and screening generations F2-F4; s8, high-generation stability screening and homozygous strain breeding; and S9, carrying out a regionalization test. Through multi-generation strict pedigree method screening and intra-strain consistency identification, the genetic homozygosity of the final strain is ensured. The stability and the eurytopic adaptability of the system in different environments are verified through further multi-site regionalization tests. The finally bred variety not only has excellent comprehensive characters, but also can be stably expressed in different regions, and has extremely high popularization value.

Description

Technical Field

[0001] This invention relates to the field of melon and fruit breeding technology, specifically to a method for breeding sweet and crisp melons. Background Technology

[0002] Melon, a globally popular cucurbitaceous fruit, has won widespread popularity for its diverse shapes, captivating aroma, and sweet, refreshing flavor. Whether it's the netted Hami melon, the golden and tempting Yellow River honey melon, or the emerald green, thin-skinned sweet melon, its rich variety caters to different consumer tastes. Beyond its exceptional taste, melon is also a treasure trove of nutritional value. It is rich in vitamins C and A, as well as essential minerals like potassium and calcium. Its high soluble solids content (mainly sugar) is the direct source of its sweetness, while its abundant antioxidants and dietary fiber play a positive role in promoting health and maintaining intestinal function. However, traditional melon breeding methods rely on phenotypic selection, which is time-consuming, inefficient, and fails to meet market demands. Summary of the Invention

[0003] In view of this, the present invention proposes a method for breeding sweet and crisp melons to solve the above problems.

[0004] The technical solution of this invention is implemented as follows:

[0005] A method for breeding sweet and crisp melons includes the following steps:

[0006] S1. Parental selection: M3839, which has a stable crisp meat trait, was selected as the female parent, and M4035A, which has a stable high sugar content trait, was selected as the male parent.

[0007] S2. Parental pretreatment: One week before the flowering period of the female parent M3839 and the male parent M4035A, ultrasonic treatment was performed for 5-7 days; then, one day before the female flowers of the female parent M3839 opened, artificial emasculation and bagging were carried out; the next day, fresh fresh pollen from the male parent M4035A, which had undergone ultrasonic pretreatment, was collected to pollinate the female parent, and the parent was immediately bagged, isolated, and tagged for record-keeping.

[0008] S3. Collection and initiation treatment of hybrid seeds: Harvest mature hybrid fruits, remove seeds, wash and dry them, and select plump and healthy F0 generation seeds; place the seeds in an alternating magnetic field for treatment, and then place the treated seeds in a compound initiation solution consisting of 200-400 mg / L seaweed extract, 80-150 mg / L Eucalyptus leaf extract, and 80-120 mg / L Centella asiatica extract and incubate in the dark.

[0009] S4. Germination and seedling raising: The seeds that have undergone priming treatment are sown in seedling trays for germination to obtain F1 generation seedlings;

[0010] S5. Seedling transplanting and microbial-assisted cultivation: When the F1 generation seedlings have grown to 3-4 true leaves, they are transplanted into the experimental field. Compound probiotics are applied to the roots of the seedlings at the time of transplanting and two weeks after transplanting. Uniform plant spacing and fertilizer and water management are adopted.

[0011] S6 and F1 generation seed harvesting: Among the fruits harvested from the F1 generation, select fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% and harvest the seeds they produce;

[0012] Cultivation and screening of S7, F2-F4 generations: Seeds harvested from the F1 generation were sown to establish an F2 generation segregating population. When the F2 generation seedlings grew to 3-4 true leaves, they were transplanted into the experimental field. Compound probiotics were applied to the roots of the seedlings at the time of transplanting and two weeks after transplanting. Uniform plant spacing and fertilizer and water management were adopted. Fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% were selected, and their seeds were harvested. The F3 and F4 generations were cultivated and screened in the same way to obtain F4 generation seeds. F4 generation seeds were stored individually.

[0013] S8. High-generation stability screening and homozygous line selection: Seeds from the selected F4 generation single plants were sown separately as individual lines to establish F5 generation line groups. 20-30 plants were planted in each line, and the growth vigor, flowering period, fruit shape, reticulation, and flesh color of each plant within each F5 generation line were observed and measured to ensure uniformity. Lines with severe segregation and unstable traits were eliminated. The retained lines were bagged and self-pollinated. Seeds with fruits having a hardness ≥4.0 kg / cm² and a central sugar content ≥16.5% were selected, and individual plants were preserved to obtain F5 generation seeds. F5 generation seeds were sown separately as individual lines of F4 generation seeds to establish F6 generation line groups. In the F6 generation, intra-line uniformity identification and agronomic trait evaluation were continued to screen out two genetically stable homozygous high-generation lines with a hardness of 4.0 kg / cm² and a central sugar content ≥16.5%.

[0014] S9. Regionalized Trials: The selected stable high-generation strains will be tested in different regions using randomized block design. The yield, sweetness, and crispness will be comprehensively evaluated to select one widely adaptable and superior strain.

[0015] Furthermore, in S2, the plants were treated with sound waves at a frequency of 1.0-1.5kHz for 1.5-2.5 hours every morning.

[0016] Furthermore, the alternating magnetic field strength in S3 is 5-15 mT, the treatment time is 25-35 min, and it is incubated in a dark environment at 14-18℃ for 18-24 h.

[0017] Furthermore, the Gynostemma pentaphyllum leaf extract in S3 was prepared by the following method: Pest-free Gynostemma pentaphyllum leaves were collected in autumn, washed, air-dried in a cool place, and pulverized into 40-60 mesh powder to obtain Gynostemma pentaphyllum leaf powder; subsequently, an ethanol reflux extraction method was used, with 60-70 wt% ethanol solution as the solvent, and the Gynostemma pentaphyllum leaf powder was added to the ethanol solution at a material-to-liquid ratio of 1:10-15 (g / mL). The mixture was refluxed at 80-85℃ for 2-3 hours, and the extraction was repeated once. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 50-60℃ to 10-20% of the original volume, thus obtaining the Gynostemma pentaphyllum leaf extract.

[0018] Furthermore, the seaweed extract in S3 is prepared by the following method: Dried *Sargassum fusiforme*, *Laminaria japonica*, or *Sargassum fusiforme* are washed to remove surface salts and impurities, drained, and broken into 1-5 mm pieces to obtain seaweed fragments. Using a water extraction method, the seaweed fragments are added to deionized water at a material-to-liquid ratio of 1:10-15 (g / mL). The mixture is extracted in a water bath at 40-45℃ and 100-200 rpm for 5-7 hours. The extract is first coarsely filtered through gauze and then filtered through a Buchner funnel to obtain the primary seaweed extract. The primary seaweed extract is then concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 45-50℃ to 10-15% of its original volume, yielding the seaweed extract.

[0019] Furthermore, the Centella asiatica extract in S3 was prepared by the following method: the dried whole Centella asiatica herb was pulverized and passed through a 20-40 mesh sieve to obtain Centella asiatica powder; then, an ethanol reflux extraction method was used, with 70-80wt% ethanol solution as solvent, and Centella asiatica powder was added to the ethanol solution at a material-to-liquid ratio of 1:10-15 g / mL, and refluxed at 75-80℃ for 2.5-3.5 h. The extraction was repeated once, and the filtrates were combined. The filtrates were concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 60-65℃ to 8-15% of the original volume to obtain the Centella asiatica extract.

[0020] Furthermore, S4 is germinated in an environment with daytime temperature of 28-30℃, nighttime temperature of 25-28℃, and air humidity of 85-90%.

[0021] Furthermore, the compound probiotics in S5 and S7 are composed of Bacillus jellyii, Bacillus polymyxa, and Trichoderma harzianum in a mass ratio of (1.8-2.2):(1.4-1.6):(0.8-1.0).

[0022] Furthermore, the uniform plant spacing in S5 is 30cm between plants and 100cm between rows. The specific fertilizer and water management is as follows: at the time of transplanting, apply 3000-4000 kg / mu of well-rotted manure, 30-40 kg / mu of balanced compound fertilizer, 15-20 kg / mu of calcium magnesium phosphate fertilizer, and 10-15 kg / mu of potassium sulfate; 7-10 days after transplanting, apply 5-8 kg / mu of high-nitrogen water-soluble fertilizer; during the fruit enlargement period, apply 10-15 kg / mu of high-potassium water-soluble fertilizer; keep the soil moist from transplanting to the seedling establishment period; during the fruit enlargement period, use drip irrigation to supply water; stop watering 7 days before the fruit ripens.

[0023] Furthermore, the uniform plant spacing in S7 is 30cm between plants and 100cm between rows. The specific fertilizer and water management is as follows: at the time of transplanting, apply 2000-2500 kg / mu of well-rotted manure, 25-30 kg / mu of balanced compound fertilizer, and 9-11 kg / mu of potassium sulfate; 7-10 days after transplanting, apply 4-6 kg / mu of balanced water-soluble fertilizer; during the fruit enlargement period, apply 8-12 kg / mu of high-potassium water-soluble fertilizer; keep the soil moist from transplanting to the seedling establishment period; during the fruit enlargement period, use drip irrigation to supply water; stop watering 7 days before the fruit ripens.

[0024] Furthermore, S9 conducts regionalized trials in at least three different regions, with two replicates.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. This invention enhances the vigor of hybrid seeds and the uniformity and robustness of F1 seedlings by pretreating the parents with ultrasound and treating the seeds with a magnetic field and a compound initiator. This provides a more fully expressed and consistent segregating population for subsequent generations, enabling earlier and more accurate identification of superior individual plants that meet the target criteria in the F2-F4 segregating population, thus shortening the number of generations required for phenotypic homozygosity. The continuous application of compound probiotics ensures that plants can fully express their genetic potential in a healthy state in each generation, reducing the interference of environmental variations on phenotypic expression. This makes targeted screening based on these traits more accurate and reliable, reducing the probability of misselection and missed selection, and increasing the success rate of each generation's screening.

[0027] 2. This invention, by selecting a high-sugar parent plant and utilizing probiotics to promote potassium absorption (potassium is a key element in sugar transport), and optimizing fertilization and irrigation (applying high-potassium fertilizer during fruit enlargement), ensures efficient sugar synthesis and full translocation to the fruit, maintaining a stable central sugar content above 16.5%. By selecting a crisp-fleshed parent plant and utilizing probiotics to promote the absorption of calcium and other micronutrients and healthy root development, a solid material foundation is provided for the construction and maintenance of fruit cell walls, resulting in a firm and crisp fruit flesh structure with a flesh firmness reaching or exceeding 4.0 kg / cm². Ultrasonic treatment, magnetic field treatment, and initiator treatment lay the foundation for strong seedlings. Furthermore, the compound probiotics' phosphorus and potassium solubilization and disease inhibition comprehensively optimize the rhizosphere environment, greatly improving fertilizer and water utilization efficiency and ensuring robust plant growth and continuous fruit setting. Detailed Implementation

[0028] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0029] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0031] Example 1

[0032] A method for breeding sweet and crisp melons includes the following steps:

[0033] S1. Parental selection: M3839, which has a stable crisp meat trait, was selected as the female parent, and M4035A, which has a stable high sugar content trait, was selected as the male parent.

[0034] S2. Parental pretreatment: One week before the flowering period of the female parent M3839 and the male parent M4035A, the plants were treated with a 1.0kHz sound wave for 2.5 hours every morning for 7 days. Then, one day before the female flowers of the female parent M3839 opened, the plants were manually emasculated and bagged. The next day, fresh pollen from the male parent M4035A, which had been pretreated with the sound wave, was collected to pollinate the female parent. The plants were then immediately bagged, isolated, and tagged for record-keeping.

[0035] S3. Collection and initiation treatment of hybrid seeds: Harvest mature hybrid fruits, remove seeds, wash and dry them, and select plump and healthy F0 generation seeds; place the seeds in an alternating magnetic field with an intensity of 5mT for 35min, and then place the treated seeds in a compound initiation solution composed of 200mg / L seaweed extract, 80mg / L Eucalyptus leaf extract and 80mg / L Centella asiatica extract and incubate in a dark environment at 14℃ for 24h.

[0036] The extract of Eucalyptus globulus leaf was prepared by the following method: Eucalyptus globulus leaves free from pests were collected in autumn, washed, air-dried in a cool place, and pulverized into 40-mesh powder to obtain Eucalyptus globulus leaf powder. Subsequently, an ethanol reflux extraction method was used, with 60wt% ethanol solution as the solvent. The Eucalyptus globulus leaf powder was added to the ethanol solution at a material-to-liquid ratio of 1:10 (g / mL). The mixture was refluxed at 80℃ for 3 hours. After repeating the extraction once, the filtrates were combined and concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 50℃ to 20% of the original volume, thus obtaining the Eucalyptus globulus leaf extract.

[0037] The seaweed extract was prepared by the following method: Dried *Alternaria solani* was washed to remove surface salt and impurities, drained, and broken into 1mm pieces to obtain seaweed fragments. Using a water extraction method, the seaweed fragments were added to deionized water at a material-to-liquid ratio of 1:10 (g / mL), and extracted in a water bath at 40℃ and 100rpm for 7 hours. The extract was first coarsely filtered through gauze and then filtered through a Buchner funnel to obtain the primary seaweed extract. The primary seaweed extract was then concentrated under reduced pressure using a rotary evaporator at 45℃ in a water bath to 15% of its original volume, yielding the seaweed extract.

[0038] The Centella asiatica extract was prepared by the following method: the dried whole Centella asiatica herb was pulverized and passed through a 20-mesh sieve to obtain Centella asiatica powder; then, the ethanol reflux extraction method was used, with 70wt% ethanol solution as solvent, and Centella asiatica powder was added to the ethanol solution at a material-to-liquid ratio of 1:10 (g / mL). The mixture was refluxed at 75℃ for 3.5h, and the extraction was repeated once. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 60℃ to 15% of the original volume to obtain the Centella asiatica extract.

[0039] S4. Germination and Seedling Raising: Sow the seeds that have completed the priming treatment in a seedling tray and germinate them in an environment of 28℃ during the day, 25℃ at night, and 85% air humidity to obtain F1 generation seedlings.

[0040] S5. Seedling Transplanting and Microbial-Assisted Cultivation: When the F1 generation seedlings reached 3 true leaves, they were transplanted into the experimental field. Compound probiotics were applied to the roots of the seedlings at the time of transplanting and two weeks after transplanting. Uniform plant spacing and fertilizer and water management were adopted. The plant spacing was 30cm and the row spacing was 100cm. The specific fertilizer and water management was as follows: at the time of transplanting, 3000 kg / mu of decomposed manure, 30 kg / mu of balanced compound fertilizer, 15 kg / mu of calcium magnesium phosphate fertilizer, and 10 kg / mu of potassium sulfate were applied. Seven days after transplanting, 5 kg / mu of high-nitrogen water-soluble fertilizer was applied. During the fruit enlargement period, 10 kg / mu of high-potassium water-soluble fertilizer was applied. The soil was kept moist from transplanting to the seedling establishment period. During the fruit enlargement period, drip irrigation was used to supply water. Watering was stopped 7 days before the fruit ripened.

[0041] S6 and F1 generation seed harvesting: Among the fruits harvested from the F1 generation, select fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% and harvest the seeds produced from them.

[0042] Cultivation and selection of S7, F2-F4 generations: Seeds harvested from the F1 generation were sown to establish an F2 generation segregating population. When the F2 seedlings reached 3 true leaves, they were transplanted into the experimental field. Compound probiotics were applied to the roots of the seedlings at transplanting time and two weeks after transplanting. Uniform plant spacing and fertilizer and water management were adopted. The plant spacing was 30cm and the row spacing was 100cm. The specific fertilizer and water management was as follows: 2000 kg / mu of decomposed manure, 25 kg / mu of balanced compound fertilizer, and 9 kg / mu of potassium sulfate were applied at transplanting time; 4 kg / mu of balanced water-soluble fertilizer was applied 7 days after transplanting; 8 kg / mu of high-potassium water-soluble fertilizer was applied during the fruit enlargement period; the soil was kept moist from transplanting to the seedling establishment period; drip irrigation was used to supply water during the fruit enlargement period; watering was stopped 7 days before the fruit ripened. Fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% were selected, and their seeds were harvested. F3 and F4 generations were cultivated and screened in the same way to obtain F4 generation seeds, and F4 generation seeds were preserved as individual plants.

[0043] S8. High-generation stability screening and homozygous line selection: Seeds from the selected F4 generation single plants were sown separately as individual lines to establish F5 generation line groups. Twenty plants were planted in each line, and the growth vigor, flowering period, fruit shape, reticulation, and flesh color of each plant within each F5 generation line were observed and measured to ensure uniformity. Lines with severe segregation and unstable traits were eliminated. The retained lines were bagged and self-pollinated. Seeds with fruits having a hardness ≥4.0 kg / cm² and a central sugar content ≥16.5% were selected and preserved as individual plants to obtain F5 generation seeds. F5 generation seeds were sown separately as individual lines of F4 generation seeds to establish F6 generation line groups. In the F6 generation, intra-line uniformity identification and agronomic trait evaluation were continued to screen out two genetically stable homozygous high-generation lines with a hardness of 4.0 kg / cm² and a central sugar content ≥16.5%.

[0044] S9. Regional Trials: The selected stable high-generation strains will be tested in Lingshui, Ledong, and Dongfang using a randomized block design with two replicates. The yield, sweetness, and crispness will be comprehensively evaluated to select one widely adaptable and superior strain.

[0045] The compound probiotics in S5 and S7 consist of Bacillus jellyii, Bacillus polymyxa, and Trichoderma harzianum in a mass ratio of 1.8:1.4:0.8.

[0046] Example 2

[0047] A method for breeding sweet and crisp melons includes the following steps:

[0048] S1. Parental selection: M3839, which has a stable crisp meat trait, was selected as the female parent, and M4035A, which has a stable high sugar content trait, was selected as the male parent.

[0049] S2. Parental pretreatment: One week before the flowering period of the female parent M3839 and the male parent M4035A, the plants were treated with a 1.2kHz sound wave for 2.0 hours every morning for 6 days. Then, one day before the female flowers of the female parent M3839 opened, the plants were manually emasculated and bagged. The next day, fresh pollen from the male parent M4035A, which had been pretreated with the sound wave, was collected to pollinate the female parent. The plants were then immediately bagged, isolated, and tagged for record-keeping.

[0050] S3. Collection and initiation treatment of hybrid seeds: Harvest mature hybrid fruits, remove seeds, wash and dry them, and select plump and healthy F0 generation seeds; place the seeds in an alternating magnetic field with an intensity of 10mT for 30min, and then place the treated seeds in a composite initiation solution consisting of 300mg / L seaweed extract, 120mg / L Eucalyptus leaf extract and 100mg / L Centella asiatica extract and incubate in a dark environment at 16℃ for 20h.

[0051] The extract of Eucalyptus globulus leaf was prepared by the following method: Eucalyptus globulus leaves free from pests were collected in autumn, washed, air-dried in a cool place, and pulverized into 50-mesh powder to obtain Eucalyptus globulus leaf powder. Subsequently, an ethanol reflux extraction method was used, with 65wt% ethanol solution as the solvent. The Eucalyptus globulus leaf powder was added to the ethanol solution at a material-to-liquid ratio of 1:12 (g / mL), and refluxed at 82℃ for 2.5 hours. The extraction was repeated once, and the filtrates were combined. The filtrates were then concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 55℃ to 15% of the original volume, yielding the Eucalyptus globulus leaf extract.

[0052] The seaweed extract was prepared by the following method: Dried kelp was washed to remove surface salt and impurities, drained, and broken into 3mm pieces to obtain seaweed fragments. Using a water extraction method, the seaweed fragments were added to deionized water at a material-to-liquid ratio of 1:12 (g / mL), and extracted in a water bath at 43℃ and 150rpm for 6 hours. The extract was first coarsely filtered through gauze and then filtered through a Buchner funnel to obtain the initial seaweed extract. The initial seaweed extract was then concentrated under reduced pressure using a rotary evaporator at 48℃ in a water bath to 12% of its original volume, yielding the seaweed extract.

[0053] The Centella asiatica extract was prepared by the following method: the dried whole Centella asiatica herb was pulverized and passed through a 30-mesh sieve to obtain Centella asiatica powder; then, the ethanol reflux extraction method was used, with 75wt% ethanol solution as solvent, and Centella asiatica powder was added to the ethanol solution at a material-to-liquid ratio of 1:12 (g / mL). The mixture was refluxed at 78℃ for 3.0 h, and the extraction was repeated once. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 63℃ to 11% of the original volume to obtain the Centella asiatica extract.

[0054] S4. Germination and Seedling Raising: The seeds that have undergone priming treatment are sown in seedling trays and germinated in an environment of 29°C during the day, 26°C at night, and 88% air humidity to obtain F1 generation seedlings.

[0055] S5. Seedling Transplanting and Microbial-Assisted Cultivation: When the F1 generation seedlings reached 4 true leaves, they were transplanted into the experimental field. Compound probiotics were applied to the roots of the seedlings at the time of transplanting and two weeks after transplanting. Uniform plant spacing and fertilizer and water management were adopted. The plant spacing was 30cm and the row spacing was 100cm. The specific fertilizer and water management was as follows: at the time of transplanting, 3500 kg / mu of decomposed manure, 35 kg / mu of balanced compound fertilizer, 18 kg / mu of calcium magnesium phosphate fertilizer, and 12.5 kg / mu of potassium sulfate were applied. Eight days after transplanting, 6.5 kg / mu of high-nitrogen water-soluble fertilizer was applied as a top dressing. During the fruit enlargement period, 12.5 kg / mu of high-potassium water-soluble fertilizer was applied as a top dressing. The soil was kept moist from transplanting to the seedling establishment period. During the fruit enlargement period, drip irrigation was used to supply water. Watering was stopped 7 days before the fruit ripened.

[0056] S6 and F1 generation seed harvesting: Among the fruits harvested from the F1 generation, select fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% and harvest the seeds produced from them.

[0057] Cultivation and selection of S7, F2-F4 generations: Seeds harvested from the F1 generation were sown to establish an F2 generation segregating population. When the F2 seedlings reached 4 true leaves, they were transplanted into the experimental field. Compound probiotics were applied to the roots of the seedlings at transplanting time and two weeks after transplanting. Uniform plant spacing and fertilizer and water management were adopted. The plant spacing was 30cm and the row spacing was 100cm. The specific fertilizer and water management was as follows: 2200 kg / mu of decomposed manure, 28 kg / mu of balanced compound fertilizer, and 10 kg / mu of potassium sulfate were applied at transplanting time; 5 kg / mu of balanced water-soluble fertilizer was applied 8 days after transplanting; 10 kg / mu of high-potassium water-soluble fertilizer was applied during the fruit enlargement period; the soil was kept moist from transplanting to the seedling establishment period; drip irrigation was used to supply water during the fruit enlargement period; watering was stopped 7 days before the fruit ripened. Fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% were selected, and their seeds were harvested. F3 and F4 generations were cultivated and screened in the same way to obtain F4 generation seeds, and F4 generation seeds were preserved as individual plants.

[0058] S8. High-generation stability screening and homozygous line selection: Seeds from the selected F4 generation single plants were sown separately as individual lines to establish F5 generation line groups. Twenty plants were planted in each line, and the growth vigor, flowering period, fruit shape, reticulation, and flesh color of each plant within each F5 generation line were observed and measured to ensure uniformity. Lines with severe segregation and unstable traits were eliminated. The retained lines were bagged and self-pollinated. Seeds with fruits having a hardness ≥4.0 kg / cm² and a central sugar content ≥16.5% were selected and preserved as individual plants to obtain F5 generation seeds. F5 generation seeds were sown separately as individual lines of F4 generation seeds to establish F6 generation line groups. In the F6 generation, intra-line uniformity identification and agronomic trait evaluation were continued to screen out two genetically stable homozygous high-generation lines with a hardness of 4.0 kg / cm² and a central sugar content ≥16.5%.

[0059] S9. Regional Trials: The selected stable high-generation strains will be tested in Lingshui, Ledong, and Dongfang using a randomized block design with two replicates. The yield, sweetness, and crispness will be comprehensively evaluated to select one widely adaptable and superior strain.

[0060] The compound probiotics in S5 and S7 consist of Bacillus jellyii, Bacillus polymyxa, and Trichoderma harzianum in a mass ratio of 2.0:1.5:0.9.

[0061] Example 3

[0062] A method for breeding sweet and crisp melons includes the following steps:

[0063] S1. Parental selection: M3839, which has a stable crisp meat trait, was selected as the female parent, and M4035A, which has a stable high sugar content trait, was selected as the male parent.

[0064] S2. Parental pretreatment: One week before the flowering period of the female parent M3839 and the male parent M4035A, the plants were treated with a 1.5kHz sound wave for 1.5 hours every morning for 5 days. Then, one day before the female flowers of the female parent M3839 opened, the plants were manually emasculated and bagged. The next day, fresh pollen from the male parent M4035A, which had been pretreated with the sound wave, was collected to pollinate the female parent. The plants were then immediately bagged, isolated, and tagged for record-keeping.

[0065] S3. Collection and initiation treatment of hybrid seeds: Harvest mature hybrid fruits, remove seeds, wash and dry them, and select plump and healthy F0 generation seeds; place the seeds in an alternating magnetic field with an intensity of 15mT for 25min, and then place the treated seeds in a compound initiation solution composed of 400mg / L seaweed extract, 150mg / L Eucalyptus leaf extract and 120mg / L Centella asiatica extract and incubate in a dark environment at 18℃ for 18h.

[0066] The extract of Eucalyptus globulus leaf was prepared by the following method: Eucalyptus globulus leaves free from pests were collected in autumn, washed, air-dried in a cool place, and pulverized into 60-mesh powder to obtain Eucalyptus globulus leaf powder. Subsequently, an ethanol reflux extraction method was used, with 70wt% ethanol solution as the solvent. The Eucalyptus globulus leaf powder was added to the ethanol solution at a material-to-liquid ratio of 1:15 (g / mL). The mixture was refluxed at 85℃ for 2 hours. After repeating the extraction once, the filtrates were combined and concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 60℃ to 10% of the original volume, thus obtaining the Eucalyptus globulus leaf extract.

[0067] The seaweed extract was prepared by the following method: Dried Sargassum seaweed was washed to remove surface salt and impurities, drained, and broken into 5mm pieces to obtain seaweed fragments. Using a water extraction method, the seaweed fragments were added to deionized water at a material-to-liquid ratio of 1:15 (g / mL), and extracted in a water bath at 45℃ and 200rpm for 5 hours. The extract was first coarsely filtered through gauze and then filtered through a Buchner funnel to obtain the initial seaweed extract. The initial seaweed extract was then concentrated under reduced pressure using a rotary evaporator at 0℃ in a water bath to 10% of its original volume, yielding the seaweed extract.

[0068] The Centella asiatica extract was prepared by the following method: the dried whole Centella asiatica herb was pulverized and passed through a 40-mesh sieve to obtain Centella asiatica powder; then, an ethanol reflux extraction method was used, with 80wt% ethanol solution as solvent, and Centella asiatica powder was added to the ethanol solution at a material-to-liquid ratio of 1:15 (g / mL). The mixture was refluxed at 80℃ for 2.5h, and the extraction was repeated once. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 65℃ to 8% of the original volume, thus obtaining the Centella asiatica extract.

[0069] S4. Germination and Seedling Raising: Sow the seeds that have completed the priming treatment in a seedling tray and germinate them in an environment of 30℃ during the day, 28℃ at night, and 90% air humidity to obtain F1 generation seedlings.

[0070] S5. Seedling Transplanting and Microbial-Assisted Cultivation: When the F1 generation seedlings reached 4 true leaves, they were transplanted into the experimental field. Compound probiotics were applied to the roots of the seedlings at the time of transplanting and two weeks after transplanting. Uniform plant spacing and fertilizer and water management were adopted. The plant spacing was 30cm and the row spacing was 100cm. The specific fertilizer and water management was as follows: at the time of transplanting, 4000 kg / mu of decomposed manure, 40 kg / mu of balanced compound fertilizer, 20 kg / mu of calcium magnesium phosphate fertilizer, and 15 kg / mu of potassium sulfate were applied. Ten days after transplanting, 8 kg / mu of high-nitrogen water-soluble fertilizer was applied as a top dressing. During the fruit enlargement period, 15 kg / mu of high-potassium water-soluble fertilizer was applied as a top dressing. The soil was kept moist from transplanting to the seedling establishment period. During the fruit enlargement period, drip irrigation was used to supply water. Watering was stopped 7 days before the fruit ripened.

[0071] S6 and F1 generation seed harvesting: Among the fruits harvested from the F1 generation, select fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% and harvest the seeds they produce;

[0072] Cultivation and selection of S7, F2-F4 generations: Seeds harvested from the F1 generation were sown to establish an F2 generation segregating population. When the F2 seedlings grew to 4 true leaves, they were transplanted into the experimental field. Compound probiotics were applied to the roots of the seedlings at the time of transplanting and two weeks after transplanting. Uniform plant spacing and fertilizer and water management were adopted. The plant spacing was 30cm and the row spacing was 100cm. The specific fertilizer and water management was as follows: 2500 kg / mu of decomposed manure, 30 kg / mu of balanced compound fertilizer, and 11 kg / mu of potassium sulfate were applied at the time of transplanting; 6 kg / mu of balanced water-soluble fertilizer was applied 10 days after transplanting; 12 kg / mu of high-potassium water-soluble fertilizer was applied during the fruit enlargement period; the soil was kept moist from transplanting to the seedling establishment period; drip irrigation was used to supply water during the fruit enlargement period; watering was stopped 7 days before the fruit ripened. Fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% were selected, and their seeds were harvested. F3 and F4 generations were cultivated and screened in the same way to obtain F4 generation seeds, and F4 generation seeds were preserved as individual plants.

[0073] S8. High-generation stability screening and homozygous line selection: Seeds from the selected F4 generation single plants were sown separately as individual lines to establish F5 generation line groups. Twenty plants were planted in each line, and the growth vigor, flowering period, fruit shape, reticulation, and flesh color of each plant within each F5 generation line were observed and measured to ensure uniformity. Lines with severe segregation and unstable traits were eliminated. The retained lines were bagged and self-pollinated. Seeds with fruits having a hardness ≥4.0 kg / cm² and a central sugar content ≥16.5% were selected and preserved as individual plants to obtain F5 generation seeds. F5 generation seeds were sown separately as individual lines of F4 generation seeds to establish F6 generation line groups. In the F6 generation, intra-line uniformity identification and agronomic trait evaluation were continued to screen out two genetically stable homozygous high-generation lines with a hardness of 4.0 kg / cm² and a central sugar content ≥16.5%.

[0074] S9. Regional Trials: The selected stable high-generation strains will be tested in Lingshui, Ledong, and Dongfang using a randomized block design with two replicates. The yield, sweetness, and crispness will be comprehensively evaluated to select one widely adaptable and superior strain.

[0075] The compound probiotics in S5 and S7 consist of Bacillus jellyii, Bacillus polymyxa, and Trichoderma harzianum in a mass ratio of 2.2:1.6:1.0.

[0076] field trials

[0077] The strains obtained from Examples 1-3, M3839, and M4035A were used in simultaneous field trials in Lingshui, Ledong, and Dongfang, selecting fields with uniform soil fertility and convenient irrigation. A randomized block design was adopted, with three replicates. Each experimental plot was 15m² in size. 2 Two rows of 25 melons each were planted, for a total of 50 plants. The plant spacing was 30cm × 100cm. The same cultivation and management measures were adopted, such as fertilization, irrigation, and pest and disease control. From cultivation to fruit harvest, the yield of each experimental plot of the varieties selected in Examples 1-3, M3839, and M4035A was statistically analyzed, and the yield per mu (unit of land area) was calculated and averaged. Yield per mu = (yield per experimental plot / area of ​​experimental plot) × 666.7. From the melons harvested from each experimental plot, five melons of equal size were randomly selected for firmness and central sugar content testing, and the average value was taken. The results are shown in Table 1.

[0078] Table 1

[0079] Example 1 Example 2 Example 3 M3839 M4035A Yield per mu (kg / mu) 2348 2406 2351 2195 2287 Hardness (Kg / cm²) 4.4 4.5 4.3 4.2 3.3 Center sugar content % 16.9 17.2 17.0 14.5 17.0

[0080] As can be seen from Table 1, the new strains bred by the breeding method of the present invention can have high yield, high sugar content, and high hardness (high hardness makes them more brittle).

[0081] In the breeding process of this invention, ultrasonic treatment is used. The micro-mechanical vibrations and cavitation effects generated by ultrasound "micro-massage" the cells of the parent plants, making it easier for them to expel metabolic waste and transport external nutrients and their own synthesized hormones, thereby enhancing the metabolic activity of the parent plant cells. It also enables more efficient transport of photosynthetic products from the leaves to the floral organs (pollen and ovules), promoting the flow and accumulation of nutrients, thus improving pollen viability and promoting better ovule development, resulting in high-quality, plump F0 generation hybrid seeds.

[0082] This invention involves placing seeds in an alternating magnetic field during the breeding process to enhance the activity of enzymes and cell membrane permeability within the seeds, breaking seed dormancy and synchronizing the germination process. This promotes uniform and robust emergence of F1 generation seedlings, laying the foundation for a homogeneous and high-yielding population. Further soaking in a compound initiator, rich in natural plant growth regulators (such as cytokinins, auxins, and gibberellins), seaweed polysaccharides, amino acids, and trace elements, awakens seed metabolism, promotes cell division, and provides comprehensive nutritional and signal support for embryonic development. The *Eucalyptus globulus* leaf extract, rich in eucalyptol, scavenges harmful reactive oxygen species during the critical stage of seed imbibition, a period of high oxygen consumption and free radical generation, protecting fragile cell membranes, proteins, and DNA from oxidative damage. The *Centella asiatica* extract, rich in asiaticoside, promotes wound healing, repairs cell damage, and enhances cell vitality. It can repair minor damage incurred during harvesting and storage and enhance the cells' resilience. Through the synergistic effect of these three factors, metabolism is efficiently activated, damage is effectively repaired, and seeds germinate uniformly, resulting in more vigorous seedlings.

[0083] In the breeding process of this invention, a compound probiotic is irrigated and colonized around the roots of the melon. Among these, *Bacillus mucilaginosus* converts unavailable potassium fixed in the soil into an absorbable form, strongly promoting fruit enlargement and increasing individual fruit weight. *Bacillus polymyxa* releases phosphorus fixed in the soil, meeting the plant's enormous needs for energy metabolism and genetic material synthesis, ensuring vigorous growth and flowering / fruit setting. *Trichoderma harzianum* effectively prevents soil-borne diseases such as wilt, ensuring healthy plant growth.

[0084] Therefore, this method organically combines physical (sound waves, magnetic fields), biological (probiotics, plant extracts), and chemical (precision fertilization and irrigation) methods to create optimized internal (physiological) and external (environmental) conditions for the hybridization and breeding process. This strongly promotes the expression of genetic potential, resulting in the breeding of breakthrough melon varieties with high yield, crisp flesh, and high sugar content. Compared to the traditional method of breeding stable new varieties after about 10 generations, the breeding method of this invention can breed high-yielding and stable new varieties in about 7 generations, greatly shortening the breeding cycle and improving breeding efficiency.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for breeding sweet and crisp melons, characterized in that, Includes the following steps: S1. Parental selection: M3839, which has a stable crisp meat trait, was selected as the female parent, and M4035A, which has a stable high sugar content trait, was selected as the male parent. S2. Parental pretreatment: One week before the flowering period of the female parent M3839 and the male parent M4035A, ultrasonic treatment was performed for 5-7 days; then, one day before the female flowers of the female parent M3839 opened, artificial emasculation and bagging were carried out; the next day, fresh fresh pollen from the male parent M4035A, which had undergone ultrasonic pretreatment, was collected to pollinate the female parent, and the parent was immediately bagged, isolated, and tagged for record-keeping. S3. Collection and initiation treatment of hybrid seeds: Harvest mature hybrid fruits, remove seeds, wash and dry them, and select plump and healthy F0 generation seeds; Seeds were treated in an alternating magnetic field, and then the treated seeds were placed in a compound initiation solution consisting of 200-400 mg / L seaweed extract, 80-150 mg / L Eucalyptus leaf extract, and 80-120 mg / L Centella asiatica extract and incubated in the dark. S4. Germination and seedling raising: The seeds that have undergone priming treatment are sown in seedling trays for germination to obtain F1 generation seedlings; S5. Seedling transplanting and microbial-assisted cultivation: When the F1 generation seedlings have grown to 3-4 true leaves, they are transplanted into the experimental field. Compound probiotics are applied to the roots of the seedlings at the time of transplanting and two weeks after transplanting. Uniform plant spacing and fertilizer and water management are adopted. S6 and F1 generation seed harvesting: Among the fruits harvested from the F1 generation, select fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% and harvest the seeds they produce; Cultivation and screening of S7, F2-F4 generations: Seeds harvested from the F1 generation were sown to establish an F2 generation segregating population. When the F2 generation seedlings grew to 3-4 true leaves, they were transplanted into the experimental field. Compound probiotics were applied to the roots of the seedlings at the time of transplanting and two weeks after transplanting. Uniform plant spacing and fertilizer and water management were adopted. Fruits with a firmness ≥3.5 kg / cm² and a central sugar content ≥16.0% were selected, and their seeds were harvested. F3 and F4 generations were cultivated and screened in the same way to obtain F4 generation seeds, and individual F4 generation seed plants were preserved. S8. High-generation stability screening and homozygous line selection: Seeds from the selected F4 generation single plants were sown separately as individual lines to establish F5 generation line groups. 20-30 plants were planted in each line, and the growth vigor, flowering period, fruit shape, reticulation, and flesh color of each plant within each F5 generation line were observed and measured to ensure uniformity. Lines with severe segregation and unstable traits were eliminated. The retained lines were bagged and self-pollinated. Seeds with fruits having a hardness ≥4.0 kg / cm² and a central sugar content ≥16.5% were selected, and individual plants were preserved to obtain F5 generation seeds. F5 generation seeds were sown separately as F4 generation seeds to establish F6 generation line groups. In the F6 generation, intra-line uniformity identification and agronomic trait evaluation were continued to select two genetically stable homozygous high-generation lines with a hardness ≥4.0 kg / cm² and a central sugar content ≥16.5%. S9. Regionalized Trials: The selected stable high-generation strains will be tested in different regions using randomized block design. The yield, sweetness, and crispness will be comprehensively evaluated to select one superior strain with broad adaptability.

2. The method for breeding sweet and crisp melons as described in claim 1, characterized in that, In S2, the plants are treated with sound waves at a frequency of 1.0-1.5kHz for 1.5-2.5 hours every morning.

3. The method for breeding sweet and crisp melons as described in claim 1, characterized in that, The alternating magnetic field strength in S3 is 5-15 mT, the processing time is 25-35 min, and it is incubated in a dark environment at 14-18℃ for 18-24 h.

4. The method for breeding sweet and crisp melons as described in claim 1, characterized in that, The Gynostemma pentaphyllum leaf extract in S3 is prepared by the following method: Pest-free Gynostemma pentaphyllum leaves are collected in autumn, washed, air-dried in a cool place, and pulverized into 40-60 mesh powder to obtain Gynostemma pentaphyllum leaf powder. Subsequently, an ethanol reflux extraction method is used, with 60-70 wt% ethanol solution as the solvent. The Gynostemma pentaphyllum leaf powder is added to the ethanol solution at a material-to-liquid ratio of 1:10-15 (g / mL). The mixture is refluxed at 80-85℃ for 2-3 hours. After repeating the extraction once, the filtrates are combined and concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 50-60℃ to obtain the Gynostemma pentaphyllum leaf extract.

5. The method for breeding sweet and crisp melons as described in claim 1, characterized in that, Germination is carried out in S4 under an environment of 28-30℃ during the day, 25-28℃ at night, and 85-90% air humidity.

6. The method for breeding sweet and crisp melons as described in claim 1, characterized in that, The compound probiotics in S5 and S7 are composed of Bacillus jellyii, Bacillus polymyxa, and Trichoderma harzianum in a mass ratio of (1.8-2.2):(1.4-1.6):(0.8-1.0).

7. The method for breeding sweet and crisp melons as described in claim 1, characterized in that, The uniform plant spacing in S5 is 30cm between plants and 100cm between rows. The specific fertilizer and water management is as follows: at the time of transplanting, apply 3000-4000 kg / mu of well-rotted manure, 30-40 kg / mu of balanced compound fertilizer, 15-20 kg / mu of calcium magnesium phosphate fertilizer, and 10-15 kg / mu of potassium sulfate; 7-10 days after transplanting, apply 5-8 kg / mu of high-nitrogen water-soluble fertilizer; during the fruit enlargement period, apply 10-15 kg / mu of high-potassium water-soluble fertilizer; keep the soil moist from transplanting to the seedling establishment period; during the fruit enlargement period, use drip irrigation to supply water; stop watering 7 days before the fruit ripens.

8. The method for breeding sweet and crisp melons as described in claim 1, characterized in that, The uniform plant spacing in S7 is 30cm between plants and 100cm between rows. The specific fertilizer and water management is as follows: at the time of transplanting, apply 2000-2500 kg / mu of well-rotted manure, 25-30 kg / mu of balanced compound fertilizer, and 9-11 kg / mu of potassium sulfate; 7-10 days after transplanting, apply 4-6 kg / mu of balanced water-soluble fertilizer; during the fruit enlargement period, apply 8-12 kg / mu of high-potassium water-soluble fertilizer; keep the soil moist from transplanting to the seedling establishment period; during the fruit enlargement period, use drip irrigation to supply water; stop watering 7 days before the fruit ripens.

9. The method for breeding sweet and crisp melons as described in claim 1, characterized in that, In S9, regionalized experiments are conducted in at least three different regions, with two replicates.