Breeding method for inducing watermelon mutation by using EMS

Abstract

The invention belongs to the technical field of mutation breeding, and particularly discloses a breeding method for inducing watermelon mutation by using EMS. The method comprises the following steps: collecting pollen by using a glucose solution, then treating by using an EMS-glucose solution, pollinating female flowers blooming on that day after treatment, uniformly spraying a forchlorfenuron solution with a proper concentration on the pollinated female flower ovary, harvesting and planting M0-generation seeds, obtaining watermelon EMS dominant mutation individuals in the M1 generation, harvesting and planting M1-generation seeds, obtaining watermelon EMS mutation groups with rich variation in the M2 generation, and finally obtaining the watermelon EMS mutation groups with rich variation. And preparing a breeding combination by utilizing the beneficial mutants. According to the invention, various mutants such as stress yellowing, green leaf color, withered leaf margin, male sterility, short vine, plant malformation, leaf malformation, peel and stripe variation, fruit flavor variation and the like can be obtained; the technical bottlenecks of narrow genetic background, shortage of excellent germplasm resources, low germplasm creation efficiency and the like in watermelon functional gene research and variety breeding are effectively solved, and the method can be used for creating excellent breeding new germplasm and cultivating new varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of mutagenesis breeding, relates to watermelon breeding technology, and particularly relates to a breeding method for watermelon inducing mutation by utilizing EMS. Background Art

[0002] Watermelon (Citrullus lanatus), a member of the Cucurbitaceae family, is a globally important horticultural economic crop, accounting for approximately 7% of the world's vegetable production area. my country is the world's largest producer and consumer of watermelon. According to FAO statistics, in 2023, my country's watermelon planting area reached 1.4953 million hectares, and its output reached 63.9604 million tons, accounting for 49.14% and 60.95% of global watermelon production, respectively. Watermelon production holds a significant position among horticultural crops. Originating in Africa, watermelon is an introduced species. my country's watermelon germplasm resources are relatively scarce, and the watermelon industry faces a significant problem of varietal homogeneity. The fundamental reason for this is the narrow genetic background of watermelon, the low frequency of natural variation, the limited availability of effective variants, the lack of in-depth research on functional genes, and the long and inefficient traditional breeding methods. Therefore, the creation of mutant materials through artificially induced mutation is a key approach to conducting functional gene research and creating new germplasm in watermelon.

[0003] Mutagenesis technology is one of the important approaches to germplasm innovation and crop improvement. By generating various mutants through mutagenesis, beneficial traits that are rare in nature or difficult to obtain using conventional methods can be obtained, which can then be applied in breeding. Mutagenesis breeding is a new breeding method with high specificity, short cycle, and significant improvement effect. Mutagenesis breeding includes physical mutagenesis and chemical mutagenesis. Among them, ethyl methanesulfonate (EMS) is one of the most widely used chemical mutagens. EMS mutagenesis has the advantages of convenient operation, high mutation frequency, low chromosomal aberrations and undesirable mutations, and most of the mutations are single-base mutations, which are easy to screen. EMS mutagenesis has been proven to be an effective means of creating crop mutations and is widely used in the breeding of various crops.

[0004] Currently, EMS seed treatment is widely used to create mutants. Utilizable mutants have been successfully created in crops such as rice (CN105695477A), wheat (CN109729972A), soybean (CN117581784A), peanut (CN111670806A), and rapeseed (CN110578015A), providing an important path for the creation of new crop germplasm. Many researchers have invested a lot of manpower, material, and financial resources in important cucurbit crops such as cucumber, wax gourd, melon, and watermelon, using EMS seed treatment to create mutants. For example, CHEN Chen treated cucumber seeds with 1.5% EMS and obtained a series of mutants in the M2 generation with dwarfing, yellow leaves, yellowed fruit skin, and wrinkled leaves (CHEN Chen et al. (2018) An EMS mutant library for cucumber, Journal of Integrative Agriculture, 17(7):1612-1619), Wang Jing used 1% EMS to treat cucumber seeds for 10 hours to induce mutation, constructed a mutant library and found plants with variations in plant type, leaves, flower organs and fruits (Wang Jing et al. (2015). Construction of a mutant library of Changchun spiny cucumber and analysis of some traits. Journal of Nuclear Agricultural Sciences, 29(8):1479-1486); Mi Baobin used 1.2% EMS to treat wax gourd seeds to construct a mutant library (Mi Baobin (2018) Optimization of EMS mutagenesis conditions and mutant screening of wax gourd. Chinese Agricultural Science Bulletin, 34(21):35-41); Hou Yan used 1.0% EMS to treat watermelon seeds and constructed a mutant library with 40 phenotypic variations (Hou Yan et al. (2016). Construction and phenotypic analysis of EMS-induced watermelon mutant library, Journal of Northwest Botany, 12:2411-2 420); Wang Haobo et al. used 0.075% EMS + 5% DMSO to treat watermelon ovaries to obtain induced mutants (CN102715078A); Yin Lijuan used 1.5% EMS to treat watermelon seeds to obtain watermelon mutants with yellowing leaves, connected cotyledons, deformed leaves, incurled leaf margins, juxtaposition of stems, variation in peel stripes, and delayed plant growth and development (Yin Lijuan et al. (2023). Screening and phenotypic analysis of EMS-induced mutants in watermelon. Acta Horticulturae Sinica, 2023, 50(11): 2401-2416); Zhai Yongqi used 2.0% EMS to soak watermelon seeds to obtain mutants with whitening leaves, notches, curling, deformity, yellowing, darkening or lightening of leaf color, changes in leaf shape, and deformed flower organs (Zhai Yongqi et al. (2023). Effects of EMS mutagenesis on watermelon seed germination and agronomic traits. Chinese Vegetables, 2023, (01): 52-59).

[0005] However, the mutants obtained in the above studies mainly focused on the variations in plant, leaf morphology, floral organ morphology and fruit traits obtained by treating seeds with EMS. The treatment concentration was relatively high and the treatment time was relatively long. The obtained variations may be due to abnormal growth and development caused by EMS treatment, and there are few heritable variations. No mutant materials for important traits such as fruit flavor, stress chlorosis, fruit size, peel background color, and fruit shape have been obtained in watermelon. There are few reports on further gene mining using mutants, indicating that the watermelon mutant library obtained by treating seeds with EMS is inefficient.

[0006] Using EMS to treat pollen is a method for inducing mutations in corn. Deng Yun et al. treated watermelon pollen with 0.1% EMS using paraffin oil as a medium, resulting in various mutants, including male sterility, dwarf plants, and tetraploids (CN112640779A). This suggests that using EMS to induce plant pollen to create mutants is feasible. While many researchers have attempted this in other crops, success has so far only been achieved in corn and watermelon. This is partly due to the difficulty of collecting pollen from most crops; and partly because paraffin oil is an oily, viscous medium, pollen separation is difficult after treatment. Furthermore, paraffin oil damages the stigma of many female flowers, resulting in either no fruit set or a very low fruit set rate. Although Deng Yun et al. successfully used EMS-paraffin liquid to treat watermelon pollen, their method not only required manual collection of pollen in advance, which was a cumbersome process, but also made it difficult to separate the pollen from the viscous paraffin oil after EMS treatment. The processing time was long and the efficiency was low, and no variations in fruit flavor, plant stress yellowing, or peel base color were found. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a new and improved method for EMS-induced mutation breeding of watermelon. The present invention uses glucose solution as a medium to collect plant pollen and perform EMS mutagenesis treatment, which can conveniently and effectively create abundant mutant germplasm with economic value.

[0008] The present invention is achieved through the following technical solutions:

[0009] A watermelon breeding method using EMS to induce mutations comprises the following steps:

[0010] (1) Glucose is used to prepare pollen collection solution;

[0011] (2) On the morning of the pollination day, after the male flowers are fully open, collect the male flowers and pick the anthers with pollen;

[0012] (3) placing the male flower anthers picked in step (2) into the glucose pollen collection solution of step (1), shaking thoroughly to allow the pollen to fully scatter from the stigma, and allowing the anthers to stand until the pollen is separated from the anthers, discarding the floating anthers and impurities, and obtaining a pollen precipitation collection mixture;

[0013] (4) adding an appropriate amount of EMS reagent to the pollen collection mixture obtained in step (3), and rapidly and fully shaking for 8-10 minutes to obtain an EMS-glucose pollen mixed treatment solution;

[0014] (5) The EMS-glucose pollen mixed solution obtained in step (4) was allowed to stand for 10-15 minutes until the pollen was obviously precipitated and aggregated. The supernatant was discarded and the pollen precipitate treated with EMS at the bottom was collected;

[0015] (6) Remove the excess liquid from the EMS-treated pollen sediment collected in step (5) and blow dry in a fume hood;

[0016] (7) dipping the pollen treated in step (6) into the female flowers that are open on the day of pollination;

[0017] (8) In the afternoon of the same day, evenly spray the ovaries of the female flowers pollinated in step (7) with a chlorfenapyr solution;

[0018] (9) After the ovary treated in step (8) is enlarged and the fruit is mature, the M0 generation seeds are mixed and harvested;

[0019] (10) sowing the M0 generation seeds harvested in step (9), observing and investigating the phenotype of the M1 generation mutants throughout the growth period, and harvesting the M1 generation seeds from the self-pollinated individual plants;

[0020] (11) sowing the M1 generation seeds harvested in step (10), observing and investigating the phenotypes of the M2 generation mutants throughout the growth period, and obtaining a watermelon EMS-induced mutagenesis population;

[0021] (12) The watermelon EMS mutants obtained in step (11) are hybridized and matched to conduct related gene function research, breeding material creation and breeding combination preparation.

[0022] A further improvement of the present invention is:

[0023] The mass concentration of glucose in the pollen collection solution in step (1) is 8%, the solvent is water, and it is recommended to use distilled water. The pH of the obtained pollen collection solution is neutral or weakly alkaline.

[0024] Furthermore, the shaking time in step (3) is 3-5 minutes; and the standing time is 5-8 minutes.

[0025] Furthermore, the amount of the EMS reagent added in step (4) is 0.5%-2% of the volume of the pollen collection solution.

[0026] Furthermore, the drying time in step (6) is 5-8 minutes.

[0027] Furthermore, the concentration of the forchlorfenuron solution in step (8) is 100-150 ppm.

[0028] Furthermore, the male flowers and female flowers are both from high-generation inbred lines of watermelon.

[0029] Furthermore, the phenotypes of the watermelon EMS mutants obtained in step (11) include but are not limited to plant chlorosis, stress chlorosis, leaf greening, leaf edge scorch, premature senescence of old leaves, leaf deformity, leaf shrinkage, leaf variation, male sterility, petal variation, female flower ovary variation, plant dwarfism, plant deformity, plant type variation, tetraploidy, fruit size variation, fruit shape variation, fruit skin color variation, loss or widening and deepening of fruit skin stripes, fruit bitterness or flavor variation, seed variation, double mutation of leaf chlorosis and fruit skin, and double mutation of leaf chlorosis and plant type.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention uses glucose solution as a medium, which can not only effectively improve turgor pressure, but also supply nutrients to pollen, maintain pollen vitality and promote its germination, and effectively improve the pollination and fruit setting rate. Experiments have shown that the pollination and fruit setting rate of watermelon pollen treated with glucose solution as a medium is close to 95%. In addition, a large amount of watermelon pollen can be quickly collected by separating the watermelon pollen and anthers by sinking and floating in the glucose solution, effectively shortening the processing operation time and enhancing convenience. The manual pollen collection process can be eliminated, overcoming the difficult problem of watermelon pollen collection, and a large amount of watermelon pollen can be obtained simply and efficiently, improving the convenience of operation.

[0032] (2) The present invention utilizes an EMS-glucose reagent to overcome the drawbacks of difficulty in separating pollen from viscous media such as paraffin oil and the damage of paraffin oil to watermelon female flowers, thereby reducing the impact of paraffin oil on watermelon fruit setting. Comparative tests under the same conditions found that the fruit setting rate of watermelon pollen treated with glucose solution as a medium after EMS treatment increased by more than 60% compared with that treated with paraffin oil.

[0033] (3) The method of treating watermelon pollen with EMS provided by the present invention overcomes the disadvantage of EMS damaging watermelon female flowers, reduces the impact of EMS on watermelon fruit setting, and improves the mutagenesis efficiency;

[0034] (4) The method of EMS treatment of watermelon pollen provided by the present invention can obtain a large number of mutants with consistent genetic backgrounds. The method is time-saving, efficient, and can be stably inherited. It can be used to quickly create multiple types of genetic mutations, genetic analysis of important traits, and mutant gene function research. Mutants with beneficial traits can be quickly applied in the field of genetic breeding;

[0035] (5) The method provided by the present invention can be implemented under the condition that watermelon can bloom and fruit normally, and is not affected by season and facility conditions;

[0036] (6) The method provided by the present invention can be used to obtain a large number of mutants with research and application value. The traits of the mutants include but are not limited to plant yellowing, stress yellowing, leaf greening, leaf edge scorch, premature senescence of old leaves, leaf deformity, leaf shrinkage, leaf variation, male sterility, petal variation, female flower ovary variation, plant dwarfism, plant deformity, plant type variation, tetraploidy, fruit size variation, fruit shape variation, fruit skin color variation, loss or widening and deepening of skin stripes, fruit bitterness or flavor variation, seed variation, leaf yellowing and skin double mutation, leaf yellowing and plant type double mutation, etc., which are conducive to large-scale watermelon germplasm innovation and can be used to construct a watermelon mutant library and create breeding materials. The creation cost is low and has good applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is an operational flow chart of the method of the present invention;

[0038] Figure 2 Comparison of the "stress yellowing" mutant and the wild type

[0039] Among them, A is the comparison of the mutant (left) and the wild type (right) at the seedling stage, B is the comparison of the mutant (left) and the wild type (right) plants, C is the comparison of the mutant (left) and the wild type (right) vines, D is the comparison of the mutant (left) and the wild type (right) leaves, E is the comparison of the mutant (top) and the wild type (bottom) fruits;

[0040] Figure 3 Comparison of the "plant yellowing and dwarfing" double phenotypic mutant and the wild type

[0041] Among them, A is the comparison of the mutant (left) and the wild type (right) at the seedling stage, B is the comparison of the mutant (left) and the wild type (right) at the plant stage, C is the comparison of the mutant (left) and the wild type (right) at the leaf stage, and D is the comparison of the mutant (left) and the wild type (right) at the flower stage.

[0042] Figure 4 Comparison of the "low temperature resistant" mutant and the wild type

[0043] Among them, A is the comparison of the mutant (left) and the wild type (right) at the seedling stage, B is the comparison of the mutant (left) and the wild type (right) at the plant stage, C is the comparison of the mutant (left) and the wild type (right) at the leaf stage, and D is the comparison of the mutant (left) and the wild type (right) at the flower stage.

[0044] Figure 5 Comparison of the "mini plant" mutant and the wild type

[0045] Among them, A is the comparison of plants of mutant (left) and wild type (right), B is the comparison of leaves of mutant (left) and wild type (right), and C is the whole plant state of mutant;

[0046] Figure 6 Comparison of the "running and merging" mutant and the wild type

[0047] Among them, A is the creeping state of the mutant (left) and the wild type (right), B is the internode leaf state of the mutant (left) and the wild type (right), and C is the flowering node position and state of the mutant (top) and the wild type (bottom);

[0048] Figure 7 Comparison of the "smaller plant" mutant and the wild type

[0049] Among them, A is the comparison of plants between the mutant (left) and the wild type (right), B is the comparison of internode length between the mutant (left) and the wild type (right), C is the comparison of leaves between the mutant (left) and the wild type (right), and D is the comparison of fruits between the mutant (left) and the wild type (right);

[0050] Figure 8 Comparison of the "leaf scorch" mutant and the wild type

[0051] Among them, A is the comparison of plants of the mutant (middle left) and the wild type (middle right), B is the comparison of leaves of the mutant (left) and the wild type (right), C is the comparison of internodes of the mutant (left) and the wild type (right), and D is the comparison of fruits of the mutant (left) and the wild type (right);

[0052] Figure 9 Comparison of the "plant deformity" mutant and the wild type

[0053] Among them, A is the comparison of mutant (left) and wild type (right) plants, B is the top view of mutant plants in the field, and C is the comparison of leaves of mutant (left) and wild type (right);

[0054] Figure 10 Comparison of fruit mutant and wild type

[0055] Among them, A is the fruit of the oval mutant, and B is the fruit of the wild type;

[0056] Figure 11 Comparison of fruit stripe mutant and wild type

[0057] Among them, A is the fruit of the mutant with chaotic stripes, and B is the fruit of the wild type.

[0058] Figure 12 Genetic analysis and breeding application of fruit skin color mutants

[0059] Among them, A is a comparison of fruits of the darkened skin mutant (left) and the wild type (right); B is the skin color separation of the darkened skin mutant M2 fruit (top is darkened skin color, middle is normal skin color, and bottom is intermediate type); C is the breeding combination (center) of the mutant homozygous material (top) and the high-generation inbred line (bottom);

[0060] Figure 13 Comparison of pollen treated with 8% glucose solution and paraffin oil solution

[0061] Among them, A is a comparison of pollen collected from paraffin solution (left) and 8% glucose solution (right) at the end of shaking; B is a comparison of pollen sedimentation and anther floating separation after pollen was collected from paraffin solution (left) and 8% glucose solution (right) and shaken for 5 minutes; C is a comparison of pollen germination after germination and culture for 2 hours after pollen was treated with paraffin solution (left) and 8% glucose solution (right) for 30 minutes; D is a comparison of female flower stigmas after pollen was treated with paraffin solution (left) and 8% glucose solution (right) for 30 minutes and pollinated; E is a comparison of female flower ovaries after pollen was treated with paraffin solution (left) and 8% glucose solution (right) for 30 minutes and pollinated 2 days later. DETAILED DESCRIPTION

[0062] The present invention is described in detail below with reference to specific embodiments.

[0063] In the following examples, the experimental materials were watermelon inbred lines 1902WMZC38 and 1902WMZC42. The 1902WMZC42 inbred line had a single fruit weighing approximately 2.5 kg, with a dark green peel covered with dark green serrated stripes, red flesh, and a soluble solids content of approximately 11% in the center of the fruit. The 1902WMZC38 inbred line had a single fruit weighing approximately 2 kg, a green peel covered with narrow dark green stripes, bright red flesh, small seeds, and a soluble solids content of approximately 12% in the center of the fruit. EMS (ethyl methanesulfonate) and glucose (analytical grade) were purchased from Sinopharm Group Co., Ltd.

[0064] Example 1: Using EMS to induce mutations in watermelon and its application in breeding

[0065] 1) Preparation of glucose solution

[0066] Using an electronic balance, weigh 16g of analytical grade glucose into a beaker. Add approximately 150mL of distilled water and stir until completely dissolved. Transfer the solution to a 200mL volumetric flask. Rinse the beaker three times with a small amount of distilled water, adding the rinse solution to the volumetric flask. Add distilled water to the mark, cap tightly, and invert to mix thoroughly. Transfer the solution to a clean reagent bottle, label it with the name and date, and store at room temperature or 4°C until needed.

[0067] 2) Preparation of female flowers

[0068] Plant the advanced inbred line 1902WMZC38 and manage it normally until the peak flowering period. Select individual plants with good growth and normal female flower development, and cover the female flowers that are about to open the next day with paper caps made of newspaper to prevent insect pollination; there is no need to cover them in facilities that isolate insects (greenhouses or net houses, etc.).

[0069] 5) Extraction of male flower stamens

[0070] Depending on the weather conditions, collect fully opened fresh male flowers on the morning of the pollination day to ensure sufficient pollen vitality. Use scissors to pick the stamens of the male flowers and place them on absorbent paper. Cut about 30-40 flowers (prepare according to 30 mL of treatment solution in a 50 mL centrifuge tube).

[0071] 6) Pollen collection

[0072] Use a graduated cylinder to measure 30 mL of the glucose solution from step 1 into a clean 50 mL centrifuge tube. Pour the stamens from step 5 into the glucose solution in the centrifuge tube. Tighten the tube cap and shake vigorously up and down for 3-5 minutes. After 5 minutes, the pollen will settle to the bottom. Use tweezers to remove the floating stamens and impurities to collect the pollen sediment mixture.

[0073] 7) Pollen EMS mutagenesis treatment

[0074] Wearing protective gear (lab coat, goggles, mask, double gloves, etc.), in a fume hood, add 300 μL of EMS reagent to the pollen pellet mixture obtained in Step 6 to obtain a 1% EMS pollen solution. Quickly tighten the centrifuge tube (optionally using Parafilm to seal the tube cap) and rapidly vortex to mix thoroughly. Vortex for 8-10 minutes to ensure adequate interaction between the pollen and the EMS.

[0075] 8) Obtaining pollen after EMS treatment

[0076] While wearing protective gear (lab coat, goggles, mask, double gloves, etc.), let the EMS-treated pollen mixture, which was thoroughly stirred in step 7, sit for 10-15 minutes to allow the pollen to settle. Discard the supernatant, retaining only the EMS-treated pollen to obtain the EMS-treated pollen. In a fume hood, blow air through the settled solution for 5-8 minutes to ensure the EMS-treated pollen is as dry as possible.

[0077] 9) Pollination

[0078] Under the premise of taking protective measures (lab coat, goggles, mask, double gloves, etc.), from 9:00 to 11:00 in the morning, use a fine brush to dip the pollen in step 8 and pollinate the female flowers that are open on the same day. Each pollination time should not exceed 1 hour. After 1 hour, use fresh pollen to re-process. It is recommended to work in a group of 2, with 1 person responsible for pollination and the other person responsible for putting on the cap and hanging the tag.

[0079] 10)Spraying treatment

[0080] Under the premise of taking protective measures (lab coat, goggles, mask, double gloves, etc.), evenly spray the ovary of the pollinated female flower with 100-150ppm chlorpyrifos solution between 15:00 and 18:00 in the afternoon of the same day to promote fruit setting.

[0081] The present invention can be implemented under conditions where normal fruit setting is possible, and is not affected by season and facility conditions. In this embodiment, 1902WMZC42 was selected for treatment in the autumn of 2019 and 1902WMZC38 was selected for treatment in the spring of 2020. The pollen treatment pollination and fruit setting rate data statistics are shown in Table 1.

[0082] Table 1 Pollination and fruit setting of watermelon treated with EMS pollen

[0083] Advanced inbred line materials Processing Season Number of pollen treated / flower Number of fruits Fruit setting rate 1902WMZC42 September 2019 40 35 87.5％ 1902WMZC38 April 2020 50 43 86％

[0084] 11) Fruit harvesting

[0085] After pollination, commercial management is adopted throughout the growth period, and the M0 generation seeds are harvested after the fruits mature.

[0086] 12) Phenotypic investigation of M1 generation mutants

[0087] The M0 generation seeds harvested in step 11 were sown, resulting in a total of 3,000 plants, of which 2,616 survived. Throughout the watermelon growth period, the original material 1902WMZC38 was used as a control to observe and investigate the phenotypes of the M1 generation mutants, obtaining watermelon EMS mutants, and statistically analyzing the mutant phenotypes. The mutant traits obtained included bitter fruit flavor, male sterility, dwarf plant development, tetraploidy, premature aging, elongated fruit shape, and peel stripe variation.

[0088] 13) Phenotypic investigation of M2 generation mutants

[0089] The M1 generation seeds harvested in step 12 were randomly selected and sown, with a total of 10,692 plants sown (297 M1 generation lines were randomly selected, and 36 plants were sown in each line), of which 8,938 plants survived. The original material 1902WMZC38 was used as a control during the entire watermelon growth period, and the phenotypes of the M2 generation mutants were observed and investigated. The proportion of each mutant phenotype in the population was counted to obtain a watermelon EMS mutant population with rich variation, and the mutant phenotypes were counted. The statistical results are shown in Table 2, and the mutation percentage is 5.55%. The mutant traits obtained include plant yellowing, stress yellowing, leaf after-greening, leaf edge scorch, premature senescence of old leaves, leaf deformity, leaf shrinkage, leaf variation, male sterility, petal variation, female flower ovary variation, plant dwarfism, plant deformity, plant type variation, tetraploidy, fruit size variation, fruit shape variation, fruit skin color variation, loss or widening and deepening of fruit skin stripes, fruit bitterness or flavor variation, seed variation, double mutation of leaf yellowing and fruit skin, double mutation of leaf yellowing and plant type, etc. The trait comparison diagram of typical mutants is shown in the figure below. Figure 1-12 shown.

[0090] Table 2. Phenotypic statistics of M2 mutations (type, number of strains, percentage)

[0091]

[0092]

[0093] 14) Genetic Analysis of the M2 Generation of Dominant Cold-Tolerant Mutants

[0094] The mutant plants screened in step 12 were self-pollinated and the seeds were harvested separately. The low-temperature resistant plant M1-190 was selected for M2 generation genetic analysis to clearly obtain the genetic stability of the mutant. 36 self-pollinated seeds of the M1-190 mutant plant were randomly selected (M2 generation) and sown in 72-hole trays. The seedling medium was a commercial breeding medium (Huaian Zhongnuo Agricultural Science and Technology Development Co., Ltd.), and seedlings were raised in a glass greenhouse in early spring. Finally, 34 seedlings emerged, and the plant phenotype was observed when 3 leaves and 1 heart began to stretch ( Figure 4 The results of phenotypic and genetic analysis are shown in Table 3. Among the M2 generation plants, 26 plants showed early vine extension and rapid growth at the seedling stage, and 8 plants grew normally. The chi-square test (χ 2 =0.04, df=1, P=0.84), indicating that the trait of rapid vine extension at the seedling stage of watermelon can be stably inherited and conforms to the single dominant gene inheritance model.

[0095] Table 3 Phenotypic segregation ratio and genetic analysis of dominant cold-tolerant mutant M2

[0096]

[0097] 15) Genetic Analysis of the M2 Generation of Dominant Mutant Watermelon with Bitter Fruit Flavor

[0098] The single plant in step 12 was self-pollinated, and the mutant with bitter fruit flesh was screened out, and the seeds were harvested from the single melon. The single plant M1-191 with bitter fruit mutant was selected for M2 generation genetic analysis to clearly obtain the genetic stability of the mutant. 36 self-pollinated seeds of the M1-191 mutant single plant were randomly taken and sown in a 72-hole tray. The seedling medium was a commercial breeding medium (Huaian Zhongnuo Agricultural Science and Technology Development Co., Ltd.), and the temperature of the seedling room was maintained at 25℃ / 15℃ (day / night). Finally, 34 seedlings emerged. After planting, commercial management was carried out throughout the growth period. The hanging vine single vine pruning and single vine single melon cultivation were adopted. The fruit flavor quality was determined after the fruit matured. Finally, 30 fruits were harvested. The results of phenotypic and genetic analysis are shown in Table 4. Among the M2 generation plants, 26 plants had bitter fruits and 4 plants had normal flavor. The chi-square test (χ 2 =2.1778, df=1, P=0.14), indicating that the trait of rapid vine extension at the seedling stage of watermelon can be stably inherited and conforms to the single dominant gene inheritance model.

[0099] Table 4 Phenotypic segregation ratio and genetic analysis of the dominant fruit bitter mutant M2

[0100]

[0101] 16) Genetic Analysis of the M2 Generation of Recessive Root Stress Etiolation Mutants

[0102] The plants that did not show obvious mutations in step 12 were harvested and observed in step 13, and a variety of mutation types were obtained. Among them, the root stress yellowing mutant M1-280 was found. When it grew to 3 leaves in the plug tray, the cotyledons began to turn yellow due to root stress and gradually moved upward to the true leaves. However, after planting, it gradually turned green until the fruit expansion stage. The plant began to gradually enter the senescence stage and began to turn yellow from the base to the whole plant ( Figure 2 ). 36 M1 generation seeds (M2 generation) of M1-280 were randomly selected and sown in 72-hole trays. The seedling medium was a commercial breeding medium (Huaian Zhongnuo Agricultural Science and Technology Development Co., Ltd.). The temperature of the seedling room was maintained at 25℃ / 15℃ (day / night). Finally, 35 seedlings emerged. The phenotype of the strain was observed throughout the growth period. It was found that 25 plants were normal and 10 plants showed stress yellowing ( Figure 2 ), chi-square test (χ 2 =0.24, df=1, P=0.63), indicating that the stress-induced etiolation phenotype conforms to single-gene recessive inheritance, as shown in Table 5.

[0103] Table 5 Phenotype segregation ratio and genetic analysis of M2 representatives of stress-induced etiolation mutants

[0104]

[0105] 17) Genetic Analysis of the M2 Generation of Recessive Dwarf and Etiolation Double Mutants

[0106] The plants that did not show obvious mutations in step 12 were harvested and observed in step 13 to obtain a rich variety of mutation types. 36 M1 generation seeds (M2 generation) of M1-28 were randomly selected and sown in 72-hole trays. The seedling medium was a commercial breeding medium (Huaian Zhongnuo Agricultural Science and Technology Development Co., Ltd.), and the temperature of the seedling room was maintained at 25℃ / 15℃ (day / night). 36 seedlings emerged, of which 9 showed yellowing leaves. After planting, commercial management was adopted throughout the growth period. During the vine extension period, 6 of the 9 yellowing plants also showed shortened internodes, and 2 of the green leaf plants showed shortened internodes, indicating that the M1-28 plant showed a double mutant phenotype of yellowing and plant type. Chi-square test (χ 2 =0, df=1, P=1), as shown in Table 6, indicating that the yellowing mutation phenotype is consistent with single gene recessive inheritance; the chi-square test (χ 2 =0.1482, df=1, P=0.70), as shown in Table 7, indicating that the plant mutation phenotype also conforms to single gene recessive inheritance. This shows that the mutagenesis method of the present invention can produce two non-allelic mutations, enriching the mutation types.

[0107] Table 6 Phenotypic segregation ratio and genetic analysis of the M2 generation of the yellowing mutant phenotype of the "dwarf plant type and yellowing" double mutant

[0108]

[0109] Table 7 Phenotypic segregation ratio and genetic analysis of the M2 generation of the plant type mutation phenotype of the "dwarf plant type and yellow plant type" double mutant

[0110]

[0111] 18) Genetic analysis and breeding applications of a mutant with darker fruit peel color

[0112] The mutant plants with darkened peel base color found in step 12 (good fruit quality, central sugar 12%) were harvested and 36 M1 generation seeds were sown. 36 seedlings emerged and were planted under single vine and single melon hanging vine cultivation. Commercial management was adopted throughout the growth period. 36 fruits were harvested after the fruits matured. It was found that 7 fruits had darkened peel color (dark green), 16 fruits had intermediate color (light dark green), and 7 fruits had normal peel (green), which fully complied with the Mendelian single gene incomplete dominant inheritance ratio of 1:2:1 (Aa×Aa→AA:Aa:aa=1:2:1). The chi-square test (χ 2=0.25, df=2, p=0.88), there was no obvious separation in fruit quality, and the central sugar content was around 12%, which can be directly used in breeding combination configuration. The line with darkened skin color (dark green) was selected and hybridized with the existing high-generation inbred line 2024WMCL42 (flowery skin) to create a new breeding combination. DUS observation was conducted on the prepared new breeding combination, and the fruit phenotype was light dark green skin with stable quality ( Figure 12 ).

[0113] Example 2: Effect of pollen treatment medium

[0114] In this embodiment, watermelon pollen was treated with 8% glucose solution and paraffin oil solution as the medium, respectively. The other operations were roughly the same as those in the relevant steps of Example 1 and will not be repeated here. Statistical analysis of pollen collection efficiency showed that pollen collected with 8% glucose solution as the medium could quickly separate pollen (precipitation) and anthers (floating), while pollen collected with paraffin solution as the medium would quickly precipitate anthers and pollen would diffuse and precipitate in the paraffin solution, resulting in low separation efficiency ( Figure 12 AB), the pollen collection efficiency was greatly improved by using glucose solution as the medium. Further germination culture was conducted on pollen treated with 8% glucose solution and paraffin oil solution as the medium, and it was found that glucose solution could reduce the effect of paraffin solution on pollen germination ( Figure 13 C). 30 watermelon flowers were pollinated with pollen treated with 8% glucose solution and paraffin oil solution respectively. It was found that the paraffin solution would affect the stigma of the female watermelon flower, causing the pollinated stigma to become black and necrotic, affecting the fruit setting rate ( Figure 13 DE), statistics show that the fruit setting rate of pollen treated with paraffin solution was 33.3%, and the fruit setting rate of pollen treated with glucose solution was 96.7% (Table 8).

[0115] Table 8 Comparative analysis of watermelon pollen treated with glucose solution and paraffin solution

[0116] Pollen treatment medium Number of pollinated female flowers / flower Number of successful fruit set Pollination fruit setting rate 8% glucose solution 30 29 96.7％ Paraffin oil stock solution 30 10 33.3％

[0117] Example 3: Effect of EMS treatment solution concentration

[0118] In this example, the volume concentrations of the EMS treatment solution were 0.1%, 0.5%, 1%, and 2%, respectively. Other procedures were largely the same as in Example 1 and are not described here. Statistical analysis of pollen germination activity and pollination fruit set after treatment revealed that pollen treated with 0.1% EMS had a germination activity of 98% after 25 minutes, which falls short of the ideal mutagenic effect. Pollen treated with 0.5% EMS had a germination activity of 82% after 25 minutes, requiring a longer treatment time. Pollen treated with 2% EMS had a germination activity of 34% after 25 minutes, which is not conducive to long-term pollination. Pollen treated with 1% EMS had a germination activity of approximately 60% after 25 minutes, representing an ideal treatment concentration. Even with a pollination time of one hour, the fruit set rate remained above 60%. Based on the ideal pollen collection and processing time, the 1% EMS treatment concentration was optimal.

[0119] In summary, the present invention uses glucose solution as a medium for collecting pollen and inducing mutations using EMS, which not only effectively improves turgor pressure but also supplies pollen nutrients, maintains pollen vitality, and promotes germination, effectively increasing pollination and fruit set rates. Furthermore, the separation of watermelon pollen and stigma, which sink and float in the glucose solution, allows for rapid collection of large quantities of watermelon pollen, effectively shortening processing time and enhancing convenience. The method for EMS mutagenesis of pollen provided by the present invention is also applicable to other crops, such as melons, cucumbers, bitter melons, and peppers, and can produce dominant mutants in the M1 generation and a rich mutant population in the M2 generation.

[0120] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A watermelon breeding method using EMS to induce mutations, characterized in that: The following steps are involved: (1) Using glucose aqueous solution as pollen collection solution; (2) On the morning of the pollination day, after the male flowers are fully open, collect the male flowers and pick the anthers with pollen; (3) placing the male flower anthers picked in step (2) into the pollen collection solution prepared in step (1), shaking thoroughly to allow the pollen to fully scatter from the stigma, and allowing the anthers to stand until the pollen is separated from the anthers, discarding the floating anthers and impurities, and obtaining a pollen precipitation collection mixture; (4) Add an appropriate amount of EMS reagent to the pollen collection mixture obtained in step (3), and shake it quickly and thoroughly for 8-10 minutes to obtain an EMS-glucose pollen mixed treatment solution; (5) The EMS-glucose pollen mixed solution obtained in step (4) was allowed to stand for 10-15 minutes until the pollen was obviously precipitated and aggregated. The supernatant was discarded and the pollen precipitate treated with EMS at the bottom was collected; (6) Remove the excess liquid from the EMS-treated pollen sediment collected in step (5) and blow dry in a fume hood; (7) Dipping the pollen treated in step (6) into the female flowers that are open on the day of pollination; (8) In the afternoon of the same day, evenly spray the ovaries of the female flowers pollinated in step (7) with a solution of chlorfenapyr; (9) After the ovary treated in step (8) is enlarged and the fruit is mature, the M0 generation seeds are mixed and harvested; (10) sowing the M0 generation seeds harvested in step (9), observing and investigating the phenotype of the M1 generation mutants throughout the growth period, and harvesting the M1 generation seeds from the self-pollinated individual plants; (11) sowing the M1 generation seeds harvested in step (10), observing and investigating the phenotypes of the M2 generation mutants throughout the growth period, and obtaining a watermelon EMS-induced mutagenesis population; (12) The watermelon EMS mutants obtained in step (11) are hybridized and matched to conduct related gene function research, breeding material creation and breeding combination preparation.

2. The method for breeding watermelon by inducing mutation using EMS according to claim 1, characterized in that: The mass concentration of glucose in the pollen collection solution in step (1) is 8%.

3. The method for breeding watermelon by inducing mutation using EMS according to claim 1, characterized in that: The shaking time in step (3) is 3-5 minutes; the standing time is 5-8 minutes.

4. The method for breeding watermelon by inducing mutation using EMS according to claim 1, characterized in that: The amount of EMS reagent added in step (4) is 0.5%-2% of the volume of the pollen collection solution.

5. The method for breeding watermelon by inducing mutation using EMS according to claim 1, characterized in that: The drying time in step (6) is 5-8 minutes.

6. The method for breeding watermelon by inducing mutation using EMS according to claim 1, characterized in that: The concentration of the forchlorfenuron solution in step (8) is 100-150 ppm.

7. The method for breeding watermelon by inducing mutation using EMS according to claim 1, characterized in that: The male flowers and female flowers are both from the advanced inbred line of watermelon.

8. The method for breeding watermelon by inducing mutation using EMS according to claim 1, characterized in that: The phenotypes of the watermelon EMS mutants obtained in step (11) include but are not limited to plant yellowing, stress yellowing, leaf greening, leaf edge scorch, premature senescence of old leaves, leaf deformity, leaf shrinkage, leaf variation, male sterility, petal variation, female flower ovary variation, plant dwarfism, plant deformity, plant type variation, tetraploidy, fruit size variation, fruit shape variation, fruit skin color variation, loss or widening and deepening of fruit skin stripes, fruit bitterness or flavor variation, seed variation, double mutation of leaf yellowing and fruit skin, and double mutation of leaf yellowing and plant type.

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