Method for creating pumpkin allo-tetraploid and application thereof

Abstract

This invention discloses a method for creating allotetraploid pumpkins and its applications, belonging to the field of plant breeding. The invention first provides a method for creating allotetraploid pumpkins, including treating F1 seedlings of interspecific hybrids of pumpkins with a chromosome doubling agent containing amphotericin B2 as an active ingredient, screening for plants with the doubling effect, self-pollinating and collecting seeds, and then identifying chromosome ploidy. The allotetraploid pumpkins obtained using this method exhibit excellent overall traits, with twice the chromosome number of other pumpkins. Furthermore, their self-pollination fruit setting rate, seed quantity per pumpkin, and self-pollination seed germination rate all meet the standards of normal diploid fertile pumpkins, thus fixing interspecific heterosis. They can be used as superior pumpkin varieties for planting or as rootstocks for grafting cucurbit crops, and can also be used as intermediate breeding material to obtain new pumpkins with richer genetics.

Description

Technical Field

[0001] This invention relates to the field of plant breeding, specifically to a method for creating allotetraploid pumpkins and its application. Background Technology

[0002] The genus Cucurbita contains many species that are widely cultivated and consumed, among which the important species is zucchini (Cucurbita spp.) Cucurbita pepo Indian squash ( Cucurbita maxima ) and Chinese pumpkin ( Cucurbita moschata The edible parts are the young fruit (especially zucchini and squash) or the mature fruit (especially squash and squash). All of these species have 40 chromosomes in their somatic cells.

[0003] Interspecific heterosis is a common biological phenomenon, and it is no exception in the Cucurbita genus. For example, interspecific hybrids of Cucurbita indicum (as the female parent) and Cucurbita chinensis (as the male parent) have long been used as rootstocks for cucurbit crops such as watermelon, enabling watermelons to be grown under biotic and abiotic stress conditions. Additionally, there is the interspecific hybrid of Cucurbita indicum and Cucurbita chinensis (…). Cucurbita maxima × Cucurbita moschata Hybrids of these species can also be planted with a pollinating variety to obtain edible fruit, and their adaptability is far better than intraspecific hybrids or fixed varieties. Studies have shown that interspecific hybrids can be obtained from Indian squash and Chinese squash under suitable pairing conditions, and the advantages are very obvious. However, these interspecific hybrids are basically infertile; almost all of the male flowers of these hybrids are sterile. Their female flowers can set fruit and produce a small number of seeds when pollinated with fertile pollen. If this distant hybrid is to be used to produce fruit, it must be planted adjacent to a fertile squash variety (Indian squash or Chinese squash) to provide fertile pollen, that is, it must be paired with a pollinating variety; zucchini ( Cucurbita pepo ) and Indian squash ( Cucurbita maxima ) and Chinese pumpkin ( Cucurbita moschata Hybridization is extremely difficult, and even if successful, the resulting interspecific hybrids are generally fertile. Therefore, the key to utilizing the heterosis of pumpkin interspecific hybrids is to ensure that the hybrids acquire fertility in order to fix and perpetuate this advantage.

[0004] To overcome the lack of pollen in the offspring of interspecific hybrids of pumpkins, some researchers treated the shoot tips of F1 seedlings with two leaves and one heart in a 0.18 mol / L trifluralin solution. After flowering, 70%-80% of the male flowers in the untreated control group had normal and viable pollen, while the male flowers in the untreated control group had no pollen (stigmas but no pollen). However, this method only yielded 15 seeds, far below the normal fertile pumpkin yield of 80-150 seeds per fruit. Furthermore, this patent did not conduct a systematic analysis of the chromosome ploidy of the induced doubling material or the genetic stability of the seed offspring. This indicates that the method only enables the offspring of interspecific hybrids to self-pollinate and produce seeds, but it cannot demonstrate that the method restores the fertility of the hybrids, nor can it prove that the method can be directly applied to interspecific hybridization breeding.

[0005] CN 107950386 A discloses a method for creating interspecific hybrid recombinant inbred lines of pumpkin. The examples show that both the interspecific hybrid combination F1 and the interspecific inbred line BC1F1 are self-sterile. However, BC2F1, obtained after two backcrosses with *Pumpkin spp.* as the male parent, is self-fertile, thus yielding fertile BC2F6 seeds. However, phenotypic segregation still occurred in the BC2F6 inbred line. Whether the BC2F6 seed material can form stable inbred lines through further self-pollination requires further investigation; the fixation and continuation of interspecific heterosis has not yet been achieved.

[0006] In conclusion, it is still necessary to explore methods for fixing and perpetuating interspecific hybrid vigor in pumpkin breeding, and to apply these methods to pumpkin breeding and planting production. Summary of the Invention

[0007] The purpose of this invention is to solve the industry problem of pollen abortion and the inability to fix and continue hybrid vigor in the F1 generation of hybrids between Indian and Chinese pumpkins. It develops a method for fixing and continuing interspecific hybrid vigor in pumpkin breeding, and obtains new germplasm with stable fertility that meets the fertility standards of normal fertile pumpkins.

[0008] To solve the above-mentioned technical problems, the present invention first provides a method for creating pumpkin allotetraploids, characterized by comprising the following steps: S1. After treating the F1 seedlings of interspecific hybrid pumpkins with a chromosome doubling agent containing ammonia-sulfamethoxazole, plants with a doubling effect were screened for self-pollination and seed retention. S2. C2 generation seeds were planted to obtain C2 generation plants. Chromosome ploidy of C2 generation plants was determined to confirm that the number of chromosomes in their somatic cells was twice the number of chromosomes in the interspecific hybrid F1. C2 generation is an allotetraploid germplasm material. in, The doubling effect includes increased pollen grain size and normal pollen fertility; The plants exhibiting the doubling effect are designated as C1 generation plants, and the number of seeds produced by self-pollination of a single melon is comparable to the number of seeds produced by a single melon in an intraspecific diploid F1 plant. The seeds obtained from self-pollination of the C1 generation plants are designated as C2 generation seeds. The germination rate of C2 generation seeds is comparable to that of intraspecific diploid F1 seeds.

[0009] In the above method, the number of seeds per melon in a single melon that is equivalent to the number of seeds in a diploid F1 plant is at least 80 seeds per melon.

[0010] In the above method, the female parent of the interspecific hybrid is the Indian pumpkin (Cucurbita chinensis). Cucurbita maxima The father is the Chinese pumpkin ( Cucurbita moschata ).

[0011] In the above method, the doubling agent is a solution of ammoniasulfonamide and water, and the concentration of ammoniasulfonamide in the doubling agent is 35-70 μmol / L.

[0012] In the above method, the chromosome doubling treatment is applied to interspecific hybrid seedlings that emerge on the day of emergence or the day after emergence.

[0013] In the above method, the chromosome doubling treatment is performed between 5 and 9 a.m. or between 1 and 3 p.m.

[0014] In the above method, the chromosome doubling treatment involves adding the doubling agent between the two cotyledons, allowing the doubling solution to cover the tip of the bud.

[0015] The above method also includes step S3: S3. The obtained allotetraploids are selected by inbreeding pedigrees to obtain allotetraploid inbred lines.

[0016] This invention also protects the application of the above method in pumpkin breeding, wherein the application is to use the allotetraploid inbred line as a parent to configure a hybrid combination to obtain an allopolyploid hybrid, wherein the allopolyploid hybrid includes an allotetraploid hybrid.

[0017] This invention also protects the fertile pumpkin allotetraploid plants created by the above method ( Cucurbita maxchata The self-pollinated offspring of the plant contain ≥80 seeds per melon and have a seed germination rate ≥85%.

[0018] The allotetraploid pumpkins are C1 generation pumpkins obtained by chromosome doubling of interspecific hybrids of Indian and Chinese pumpkins with ammonia-sulfuron, as well as C2 generation pumpkins and higher generations obtained by self-pollination of C1 generation pumpkins.

[0019] In one embodiment of the present invention, the allotetraploid pumpkin ( Cucurbita maxchataThe inbred lines 25SQ43-4X, 25SQ45-4X, WZ103-4X or WZ105-4X, and their C1 generation before their traits stabilize, are derived from the interspecific hybrids 25SQ43, 25SQ45, WZ103 or WZ105 by doubling.

[0020] The present invention also provides the allotetraploid pumpkin ( Cucurbita maxchata The application of this in breeding is to use the plant as a parent to configure hybrid combinations in order to obtain allopolyploid hybrids.

[0021] The allopolyploid hybrids include allotetraploid hybrids.

[0022] This invention also provides the allotetraploid pumpkin ( Cucurbita maxchata It is used as rootstock for grafting cucurbit crops.

[0023] The method of this invention uses Indian pumpkin as the female parent and Chinese pumpkin as the male parent to obtain the F1 generation of interspecific hybridization. Chromosome doubling is achieved by precisely treating the emerging seedlings with 35-70 μmol / L ammonia-sulfamethoxazole. After agronomic trait screening, flow cytometry ploidy identification, and multiple generations of self-pollination, a new allotetraploid pumpkin with 80 chromosomes is created. Cucurbita maxchata This invention has led to the development of several stable lines, including 25SQ43-4X. The new germplasm exhibits normal fertility, with self-pollination fruit set rate and seed quantity per fruit meeting the standards for fertile pumpkins. It demonstrates excellent agronomic traits, a well-developed root system, and strong resistance to adverse conditions. It can be directly used as a cultivar or rootstock for cucurbits, and can also be used as a parent to cultivate tetraploid hybrids and allotriploids. This invention utilizes an environmentally friendly doubling agent, achieving high doubling efficiency. It also provides a feasible scheme for distant hybridization breeding of other cucurbits, broadening the genetic base of pumpkins and possessing significant breeding application value.

[0024] The beneficial effects of this invention are: (1) This invention hypothesizes and verifies that Indian pumpkin ( Cucurbita maxima ) × Chinese Pumpkin ( Cucurbita moschata Interspecific hybrids undergo chromosome doubling, ensuring that each species' genome contains chromosomes capable of homologous pairing. This not only restores fertility but also creates a new allotetraploid pumpkin germplasm with 80 chromosomes in a single somatic cell. Cucurbita maxchata This will help to solidify interspecific hybrid vigor.

[0025] (2) The chromosome doubling of the present invention uses a herbicide, oryzalin, instead of the traditional colchicine, which is safe for humans and the environment and has a higher doubling efficiency.

[0026] (3) Using the method of this invention, a new germplasm with excellent comprehensive traits and normal fertility was created—an allotetraploid pumpkin (Cucurbita maxchata ) ， Its chromosome number is twice that of other pumpkins, and the number of seeds per pumpkin meets the standard for a normal fertile pumpkin (at least 80 seeds per pumpkin).

[0027] (4) Tetraploid material Allopolyploid pumpkin can be used to obtain new pumpkin and rootstock materials that carry richer genetic material.

[0028] (5) The strong interspecific hybrid vigor can be fixed by using the allopolyploid pumpkin created by the present invention.

[0029] (6) The method of the present invention can be used to artificially synthesize new germplasm of cucurbits. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the creation of new interspecific allotetraploid germplasm of pumpkin in an embodiment of the present invention.

[0031] Figure 2 This is a schematic diagram of chromosome doubling treatment in an embodiment of the present invention. In the diagram, A represents seedlings treated with chromosome doubling agent; B represents the germination status of seedling growth points 5 days after treatment; C1 on the left represents seedlings treated with chromosome doubling agent, and F1 on the right represents seedlings not treated with chromosome doubling agent. The early germination and growth of C1 seedlings lags behind that of F1 seedlings, but in the later stages, the growth of C1 seedlings surpasses that of F1 seedlings, showing a significant gene dosage effect.

[0032] Figure 3 This is a diagram showing the differences between diploid and tetraploid plants in an embodiment of the present invention. The first row of the diagram, from left to right, shows young leaves, old leaves, and normal diploid pollen grains of the diploid plant; the second row of the diagram, from left to right, shows young leaves, old leaves, and tetraploid pollen grains of the tetraploid plant.

[0033] Figure 4 The anthers and pollen of male flowers of diploid (F1 generation plant) WZ105 and tetraploid (C1 generation plant) WZ105-4X are shown in the embodiments of the present invention.

[0034] Figure 5 The image shows a dissected fruit (left) and seeds (right) of the tetraploid C1 generation plant WZ105-4X in an embodiment of the present invention.

[0035] Figure 6 The results of potassium iodide activity detection in pollen starch of diploid (F1 generation plants) and tetraploid (C2 generation plants) in the embodiments of the present invention are shown.

[0036] Figure 7 These are flow cytometry diagrams of diploid (F1 generation plants) and tetraploid (C2 generation plants) in an embodiment of the present invention. In the diagram, A is the flow cytometry diagram of the diploid control, and B is the flow cytometry diagram of the tetraploid.

[0037] Figure 8 This is a pedigree chart of 25SQ43-4X in this embodiment of the invention. Since the F1 generation is infertile through self-pollination, the F1 fruits are fruits obtained from pollination with PSB03 pollen, and are not self-pollinated fruits.

[0038] Figure 9 This is a pedigree chart of 25SQ45-4X in an embodiment of the present invention. Since the F1 generation is infertile through self-pollination, the F1 fruits are fruits obtained from pollination with PSB01 pollen, and are not self-pollinated fruits. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] Oryzalin is a herbicide that disrupts the polymerization of microtubules during cell division. Branded under Surflan*-DowElanco, its chemical composition is dinitroaniline, with a molecular weight of 346.3 and CAS number 19044-88-3. It can be purchased from agricultural supply stores or from chemical reagent suppliers in chemically pure form.

[0042] The original breeding material of this invention is not limited by genotype; any Indian squash ( Cucurbita maxima ) and Chinese pumpkin ( Cucurbita moschata Inbred lines, inbred lines, or F1 can all be used to create interspecific hybrids and double their chromosomes to obtain fertile allotetraploid pumpkins.

[0043] The pumpkin materials used in the following examples are as follows: ①Indian pumpkin ( Cucurbita maxima ) Inbred lines: Strain PSB01 is a self-pollinated line of red-skinned chestnut pumpkin, and strain PSB03 is a self-pollinated line of green-skinned chestnut pumpkin. Both were obtained by the applicant from Indian pumpkins ( Cucurbita maxima Hokkaido taro squash and Chinese squash ( Cucurbita moschata Indian squash (a type of squash) was bred from a recombinant population of the American long-stemmed butter squash (Waltham Butternut). Cucurbita maxima) Inbred lines. Among them, Hokkaido Taro Pumpkin is a commercial variety bred by Takii Seed Company of Japan. It is named for its smooth and powdery texture similar to taro. It is a hybrid variety and is numbered 14-33. Waltham Butternut is a commercial variety that originated in Massachusetts, USA. It is internationally recognized as the "standard butternut squash".

[0044] ②Chinese pumpkin ( Cucurbita moschata ) Inbred lines: The PSN01 strain was obtained by the applicant from two Chinese pumpkins ( Cucurbita moschata The Chinese pumpkin (Cucurbita chinensis) bred using conventional breeding methods through line selection from the hybrid offspring of Turkish millstone squash and Hawaii Milk Fragrance No. 1. Cucurbita moschata (Segregated lines). Among them, the Turkish millstone squash is a local Turkish variety, purchased by the applicant in July 2023 from the old city market farmers' market in Seihan district, Adana metropolitan area, Adana province, Turkey (Sar yakupMahallesi, 23002 / 23004 Sokak, 01020 Seyhan / Adana, Türkiye); Xiawei Milk Fragrance No. 1 is a commercial variety purchased from Wuwei Li'an Seed Co., Ltd.

[0045] ③ Pumpkin interspecific hybrid F1 varieties: Weizhan 103 (code: WZ103) and Weizhan 105 (code: WZ105) are commercial rootstock squash varieties, purchased from Wuwei Li'an Seed Co., Ltd., and are a cross between Indian and Chinese squash varieties. Cucurbita maxima × Cucurbita moschata A new hybrid rootstock variety, commonly used as a grafting rootstock for watermelon and melon. The F1 generation of Weizhan 103 (WZ103) and Weizhan 105 (WZ105) cannot self-pollinate and produce fruit.

[0046] For breeding purposes and for ease of obtaining materials, the applicant used the aforementioned materials to demonstrate the present invention. However, those skilled in the art may also use other Indian pumpkins (…). Cucurbita maxima Inbred lines and Chinese pumpkin ( Cucurbita moschata The technical solution of this invention can be reproduced by using inbred lines to obtain interspecific hybrid F1 materials or by using other commercial interspecific hybrid F1 materials.

[0047] Example 1: Obtaining the F1 generation from interspecific hybridization According to the breeding objectives, interspecific hybridization F1 generation was prepared, and the specific hybridization combination information is shown in Table 1. Among them, the F1 generation seeds of Weizhen 103 and Weizhen 105 were directly purchased commercially, while 25SQ45 and 25SQ43 were obtained by interspecific hybridization using Indian pumpkin materials (PSB01, PSB03) as the female parent and Chinese pumpkin material (PSN01) as the male parent.

[0048] Table 1 Information on interspecific hybridization combinations

[0049] The specific steps for interspecific hybridization of combinations PSB01×PSN01 and PSB03×PSN01 are shown below: 1. Parental breeding and flowering period control: Healthy, disease-free, and stable diploid parent plants were selected and planted in a standardized experimental field. Conventional cultivation and management measures were adopted to regulate water, fertilizer, and light to ensure that the flowering period of the two parents was synchronized, laying the foundation for distant hybridization and pollination.

[0050] 2. Distant hybridization pollination: Fresh pollen from the male parent was collected and applied to the stigma of the female parent using a manual pollination method. After pollination, the flowers were bagged for isolation, and the hybridization combination and pollination date were labeled. Fruit set was observed regularly, and unpollinated female flowers were removed promptly to reduce nutrient consumption. After the hybrid fruits matured, the seeds were harvested manually, impurities and shriveled seeds were removed, and the seeds were dried and stored for later use.

[0051] In this embodiment, after artificial pollination, all hybrid fruits (PSB01×PSN01 and PSB03×PSN01) set fruit normally. Approximately 45 days after pollination, the hybrid fruits were fully mature. Upon harvesting and dissection, the internal structure of the fruits from each combination was normal, and the placenta was well-developed. The seed yield per melon was 85-120 seeds, meeting the seed quantity requirements for subsequent experiments in the F1 generation. The seeds were cleaned to remove mucus and chaff, dried in a ventilated environment at 25℃, and then bagged and stored in a refrigerator at 4℃ for later use. The interspecific hybrid F1 obtained by combining PSB01×PSN01 is numbered 25SQ45 (this material is disclosed at https: / / www.pku-iaas.edu.cn / about_93 / , and the public can obtain it from the applicant to replicate the experiment of this application), and the interspecific hybrid F1 obtained by combining PSB03×PSN01 is numbered 25SQ43 (this material is disclosed at https: / / www.pku-iaas.edu.cn / about_94 / , and the public can obtain it from the applicant to replicate the experiment of this application).

[0052] 3. Germination rate of F1 seeds among different species Germination rates of F1 generation seeds from interspecific hybridization of 25SQ45 and 25SQ43 obtained by distant hybridization, as well as F1 generation seeds from interspecific hybridization of purchased Weizhen 103 and Weizhen 105, were tested. The germination rates of all four seeds were above 90%, which provides a physiological basis for chromosome doubling treatment.

[0053] Example 2: Creation of new interspecific allotetraploid germplasm of pumpkin The pedigree chart for the creation of new interspecific allotetraploid germplasm of pumpkin in this application is shown below. Figure 1 The creation method includes the following steps: 1. Chromosome duplication in F1 plants of interspecific hybrids of pumpkin: The compound oryzalin inhibits tubulin polymerization and disrupts spindle formation during cell division, preventing replicated chromosomes from separating to opposite poles. At appropriate concentrations and treatment times, it can lead to chromosome doubling, resulting in polyploid cells. The concentration of the chromosome-doubling agent is related to the cell division activity of the F1 generation seedling shoot tips; the more active and intense the cell division, the lower the concentration required. The level of cell division activity is related to the temperature and humidity conditions of the seedlings. In most cases, concentrations of 35-70 μmol / L can effectively double the chromosomes in dicotyledonous plants. Preliminary experiments can be conducted based on the concentration of the herbicide oryzalin and the seedling growth environment to determine the optimal treatment timing and concentration.

[0054] Chromosome doubling typically involves tissue culture, requiring explant selection, dedifferentiation into callus tissue, redifferentiation into shoots and roots, and finally, the growth of robust plants—a process that takes at least 3-4 months and is cumbersome and complex. Now, however, chromosome doubling can be induced directly in the F1 generation of interspecific hybrids without the need for a complex tissue culture system. The compound used to induce chromosome doubling is the herbicide azoxystrobin, making the process highly efficient, simple, and faster.

[0055] 1.1 Chromosome doubling treatment In this embodiment, the F1 generation seeds from interspecific hybridization were pre-germinated and seedlings were treated when the cotyledons extended on the day of seedling emergence. The chromosome doubling agent was applied to seedlings on the day of emergence or the day after emergence (the later the treatment time, the more original growth and differentiation processes the plant underwent, and the greater the probability of obtaining chimeras; the earlier the treatment time, the fewer chimeras were obtained after doubling). The specific treatment is as follows: Between 5-9 AM or 1-3 PM (during these times, seedlings are actively dividing; and the environment is suitable, so the added doubling solution will not evaporate too quickly due to high temperature and low humidity, especially before 7 AM for the best effect), use a dropper to precisely add the herbicide Oryzalin (chromosome doubling agent diluted with distilled water to the target concentration) at a concentration of 35-70 μmol / L (in this example, 25SQ43, 25SQ45, Weizhen 103, and Weizhen 105 are all 35 μmol / L) between the two cotyledons of F1 generation seedlings (diploid), allowing the doubling solution to cover the tip of the young shoot, inducing chromosome doubling (see...). Figure 2 (A). F1 generation seedlings (diploid) that were not sprayed with ammoniasulfuron-methyl were set up as a control.

[0056] Proceed with routine management and cultivation. See photos of seedlings 5 ​​days after treatment. Figure 2In Figure B, the left side shows the seedlings after doubling treatment (the C1 seedlings after doubling treatment were initially smaller and then larger compared to the control F1 seedlings), and the right side shows the seedlings without doubling treatment.

[0057] 1.2 Observation of morphology and fertility of C1 generation plants: The C1 generation plants after doubling treatment exhibited a significant doubling effect. Taking WZ105 and its doubled plants as examples, the other three diploid F1 generation plants showed similar characteristics after doubling: such as... Figure 3 As shown, the young and mature leaves of diploid pumpkin plants are smaller than those of tetraploid pumpkins. Tetraploid pumpkins have a higher density of serrations along the leaf margins and are generally darker in color. The most obvious difference is in the size of the pollen grains. Figure 4 As shown, the male anthers of the doubled C1 generation plants were well-developed and had abundant pollen, while the male anthers of the undoubled F1 generation plants were small and had no pollen.

[0058] During the flowering period, select C1 generation plants that exhibit significantly enhanced traits compared to diploid plants (such as significantly larger leaves, higher density of leaf margin serrations, darker leaf color under normal conditions, normal pollen fertility, and significantly larger pollen grains), and remove the remaining plants. Continue to cultivate the selected C1 generation plants, self-pollinate them, and harvest the seeds after the fruits mature. After drying, store them for later use and designate them as C2 generation seeds.

[0059] In this embodiment, the C1 generation plants of WZ105 (numbered: WZ105-4X) after chromosome doubling exhibited vigorous growth, normal development of male and female flowers, and a self-pollination fruit set rate exceeding 75% (45 / 58), indicating that chromosome doubling successfully restored the fertility of interspecific hybrids. Forty-five days after pollination, the fruits matured. Post-harvest fruit dissection revealed that the C1 generation WZ105-4X plants produced seeds normally, with an average yield of 90 seeds per fruit. Figure 5 The seeds produced meet the standard for a normal fertile pumpkin (at least 80 seeds per pumpkin). The seeds are plump, with a thousand-seed weight of approximately 250 g. The harvested C2 generation seeds are dried and stored at 4°C for subsequent generations of planting and selection.

[0060] The C1 generation plants of 25SQ43, 25SQ45 and Weizhen 103 developed male and female flowers normally, self-pollinated and set fruit normally, and each fruit had more than 80 seeds.

[0061] 2. Pluripotency identification of C2 generation plants: C2 generation seeds were sown and cultured to obtain C2 generation plants (allogeneic tetraploids). Approximately 100 plants from each of the four interspecific F1 hybrid C2 populations were planted for ploidy identification.

[0062] The following highlights the results of a systematic survey of agronomic traits from the C2 generation (105 plants) of the interspecific hybrid F1 rootstock 105: C2 generation plants did not exhibit phenotypic segregation, and their main traits were similar to those of the corresponding C1 generation plants. However, compared to diploid F1 plants, allotetraploid plants were larger, with thicker stems, larger leaves, darker green color, and thicker texture. Flowers were significantly larger, with abundant and viable pollen. Figure 6 ).

[0063] The relative content of nuclear DNA in C2 generation leaf cells was further detected by flow cytometry to determine whether its DNA content was twice that of the diploid control, thus confirming that it was a true allotetraploid.

[0064] Ploidy detection was performed on leaf samples. First, mature leaves were collected, with untreated diploid F1 plants serving as a control. Ploidy analysis was performed using a flow cytometer (Partec PA, Ploidy Analylzer, Germany). 0.5 cm² leaf tissue was taken and chopped with a sharp blade in a 50 × 12 mm plastic culture dish containing 500 µL of nucleus separation buffer. The standard peak was set at approximately channel 50 of the relative fluorescence intensity, and recalibrated for each measurement.

[0065] Flow cytometry results as follows Figure 7 As shown, compared with the diploid control, the DNA content of the C2 generation plants was twice that of the diploid F1 plants, indicating that they were tetraploid plants. Observation of pollen size and fertility after flowering revealed that the pollen size of the C2 generation WZ105 plants was the same as that of the C1 generation plants, and both were fertile.

[0066] The same ploidy identification was performed on C2 generation plants derived from 25SQ43, 25SQ45 and Weizhen 103. The results showed that the C2 generation plants from the three sources were all tetraploid and their pollen was fertile.

[0067] This indicates that the allotetraploid germplasm created in this invention has successfully overcome the problems of sterility and inability to fix vigor in the F1 generation of distant hybridization, forming stable inbred lines. These tetraploid materials, including C1 generation plants obtained by chromosome doubling, and C2 generation and above plants with stable traits propagated after self-pollination, all belong to new species and are called allotetraploid pumpkins ( Cucurbita maxchata ).

[0068] 3. Further self-pollination and phenotypic selection of allotetraploid pumpkins Tetraploid plants that have undergone C1 generation trait screening and C2 generation ploidy identification are continuously self-pollinated and further screened for ploidy stability and trait consistency until robust, stable, and high-quality allotetraploid materials are obtained.

[0069] In this embodiment, after chromosome doubling in the C1 generation plants, no phenotypic segregation occurred during self-pollination. After one generation of self-pollination, stable inbred lines with stable ploidy, excellent traits, and normal fertility were obtained, thus yielding new allotetraploid germplasms 25SQ43-4X, 25SQ45-4X, WZ103-4X, and WZ105-4X. The correspondence between each new allotetraploid germplasm and the F1 generation of the derived diploid is shown in Table 2. The pedigree charts for 25SQ43-4X and 25SQ45-4X are shown in Table 2. Figure 8 , Figure 9 .

[0070] Table 2. Selected new interspecific allotetraploid germplasm

[0071] Example 3: Observation of agronomic traits and fertility of interspecific allotetraploid new germplasm of pumpkin Taking the new interspecific allotetraploid germplasm WZ105-4X of pumpkin obtained in Example 2 as an example, the agronomic traits and fertility characteristics of interspecific allotetraploid pumpkins are explained.

[0072] WZ105-4X plants exhibit vigorous growth with a full growth period of 90 days. Agronomically, they reach a height of 300 cm, stem diameter of 2 cm, and a leaf shape index of 1. Economically, each plant produces one fruit with a weight of 2000 g, a flesh thickness of 3.5 cm, an average seed yield of 90 seeds per fruit, and a thousand-seed weight of 260 g. Compared to the diploid WZ105, WZ105-4X has a more developed root system, stronger photosynthetic capacity, and significantly greater plant vigor. It exhibits wider adaptability to adverse field environments and a higher tolerance for cultivation management errors. Its flowers are fully developed, overcoming some issues of sterility and low fruit set in the F1 generation of distant hybrids, making propagation and seed production highly feasible.

[0073] Three consecutive generations (C3-C5) of field propagation and ploidy tracking identification of WZ105-4X were conducted. The results showed that the allotetraploid germplasm had good propagation stability. After multiple generations of propagation, the ploidy of the plants did not segregate, and the chromosome set remained stable. The agronomic traits were consistent across generations with no significant segregation, and the heritability of the traits was high. The seed germination rate was stable at over 85%, and the seedling rate reached over 99%. During field propagation, the plants grew uniformly without deformed or weak plants, meeting the stability requirements of germplasm propagation in breeding and laying a solid foundation for subsequent variety selection and application.

[0074] The seed quantity and germination of all four allotetraploid germplasms obtained in Example 1 were statistically analyzed over multiple generations. The results are shown in Tables 3 and 4. All allotetraploid germplasms showed stable and consistent characteristics, with more than 80 seeds per melon and a germination rate of over 85%.

[0075] Table 3. Average number of seeds per melon in new interspecific allotetraploid germplasm.

[0076] Table 4 Seed germination of new interspecific allotetraploid germplasm

[0077] The successful acquisition of the above four new allotetraploid germplasm fully combines the heterosis of distant hybridization with the chromosome doubling advantage of polyploidization, and has extremely high application value and research significance in crop breeding, specifically reflected in: By integrating the superior genes of both parents through distant hybridization, the genetic barriers within the species are broken down, allowing WZ105 to combine the excellent traits of both parents, such as high disease resistance and high yield. The gene recombination brought about by distant hybridization gives the new germplasm super-parental traits that the parents do not possess, such as higher photosynthetic efficiency and wider adaptability, which greatly broadens the genetic base of crops and provides rich genetic resources for breeding.

[0078] The polyploidization advantage brought about by chromosome doubling is the core support for the superior traits of WZ105-4X: on the one hand, after doubling, the plant's vegetative organs (roots, stems, leaves) and reproductive organs (flowers, fruits, grains) are significantly enlarged, directly increasing biomass and yield; on the other hand, the gene dosage effect of polyploids increases the expression of genes related to stress resistance and disease resistance, significantly enhancing the plant's stress resistance and disease resistance, and making it more adaptable; in addition, the chromosome set stability of allotetraploids enables them to overcome the problem of sterility in the F1 generation of diploids from distant hybridization, achieving reproductive stability and providing possibilities for germplasm propagation, seed production, and subsequent breeding applications.

[0079] Example 4: Further hybridization between new allotetraploid germplasm fully utilized the dual advantages of distant hybridization and polyploidization to cultivate excellent super hybrids. 1. Source of material for allotetraploid new germplasm: The sources of WZ103-4X and WZ105-4X are described in Example 2.

[0080] 2. The hybridization process of WZ105-4X and WZ103-4X Using WZ105-4X as the female parent and WZ103-4X as the male parent, an inter-tetraploid hybridization experiment was conducted. The specific process is as follows: (1) The breeding of hybrid parents is synchronized with the flowering period: WZ105-4X (as the female parent) and WZ103-4X (as the male parent) plants with strong growth, stable traits and in the early stage of full bloom are selected and planted in the same experimental field with unified water and fertilizer management.

[0081] (2) Artificial pollination: During the peak flowering period of both parents, at 9-11 am every day (the time when pollen viability is strongest and stigma pollination is optimal), collect fresh and mature pollen from the male parent WZ103-4X and quickly apply it to the stigma of the female parent WZ105-4X. After pollination, label the hybrid combination (WZ105-4X×WZ103-4X), pollination date, and parent information.

[0082] (3) Hybrid fruit cultivation and seed harvesting: After pollination, observe the fruit setting status regularly, remove unfruited buds and deformed fruits in a timely manner to ensure concentrated nutrient supply. After the hybrid fruits are fully mature, harvest them manually, remove the peel and impurities, take out the seeds, dry them and store them for subsequent seedling cultivation and trait investigation.

[0083] Statistics show that the cross pollination and fruit setting rate of this combination (WZ105-4X×WZ103-4X) reached 87.5% (35 / 40). This indicates that interspecific allotetraploids with restored fertility can be used as parental materials for further breeding, and by configuring cross combinations, hybrid fruits can be produced.

[0084] Further analysis of the seeds from the hybrid fruits of this combination (WZ105-4X×WZ103-4X) revealed an average of 108 seeds per melon, with a germination rate of 87%. This indicates that the hybrid combination further configured between interspecific allotetraploids exhibits normal seed reproduction, overcoming the fertility barriers of distant hybridization.

[0085] (4) Hybrid cultivation: Plump and healthy hybrid seeds were selected, and seedlings were cultivated using the plug tray method. The temperature of the seedling environment was controlled at 25-28℃ and the humidity at 60%-70%. When the seedlings grew to one true leaf, they were transplanted to a standardized experimental field for conventional cultivation and management. The results showed that the hybrid plants between interspecific allotetraploids grew and developed normally and could be used for subsequent trait identification.

[0086] This embodiment confirms that interspecific allotetraploids can be normally crossbred to produce normally fertile F1 generation interspecific allotetraploid hybrids. Hybrid vigor is a self-evident genetic principle; hybridization between interspecific allotetraploids combines gene dosage effects with hybrid vigor.

[0087] Furthermore, the allotetraploid germplasm obtained by the method of this invention can be used for other breeding work, such as hybridizing the allotetraploid germplasm with ordinary diploid pumpkin to obtain allotriploids.

[0088] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, contents, and conditions without departing from its spirit and scope or without conducting unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for creating pumpkin allotetraploids, characterized in that, Includes the following steps: S1. After treating the F1 seedlings of interspecific hybrid pumpkins with a chromosome doubling agent containing ammonia-sulfamethoxazole, plants with a doubling effect were screened for self-pollination and seed retention. S2. C2 generation seeds were planted to obtain C2 generation plants. Chromosome ploidy of C2 generation plants was determined to confirm that the number of chromosomes in their somatic cells was twice the number of chromosomes in the interspecific hybrid F1. C2 generation is an allotetraploid germplasm material. in, The doubling effect includes increased pollen grain size and normal pollen fertility; The plants exhibiting the doubling effect are designated as C1 generation plants, and the number of seeds produced by self-pollination of a single melon is comparable to the number of seeds produced by a single melon in an intraspecific diploid F1 plant. The seeds obtained from self-pollination of the C1 generation plants are designated as C2 generation seeds. The germination rate of C2 generation seeds is comparable to that of intraspecific diploid F1 seeds.

2. The method according to claim 1, characterized in that, The doubling effect also includes morphological changes such as darker leaf color, thicker stems, and larger flowers.

3. The method according to claim 1, characterized in that, The interspecific hybrid F1 plant of pumpkin is based on pumpkin (Cucurbita indica). Cucurbita maxima ) as the parent plant, Chinese pumpkin ( Cucurbita moschata () was obtained through interspecific hybridization with the male parent.

4. The method according to claim 1, characterized in that, The doubling agent is a solution made by mixing amiloride and water, and the concentration of amiloride in the doubling agent is 35-70 μmol / L.

5. The method according to claim 4, characterized in that, The chromosome doubling treatment is applied to interspecific hybrid seedlings that emerge on the day of or the day after emergence.

6. The method according to claim 4, characterized in that, The chromosome doubling treatment was performed between 5 and 9 a.m. or between 1 and 3 p.m.

7. The method according to any one of claims 1-6, characterized in that, It also includes step S3: S3. The obtained allotetraploids are selected by inbreeding pedigrees to obtain allotetraploid inbred lines.

8. The application of the method according to any one of claims 1-7 in pumpkin breeding, characterized in that, The application involves using the allotetraploid inbred line as a parent to configure hybridization combinations to obtain allopolyploid hybrids, wherein the allopolyploid hybrids include allotetraploid hybrids.

9. A fertile allotetraploid pumpkin plant created by the method according to any one of claims 1-7, characterized in that, The self-pollinated offspring of the plant contain ≥80 seeds per melon, and the seed germination rate is ≥85%.

10. The application of the allotetraploid inbred line plant described in claim 9 in pumpkin breeding, characterized in that, The application involves using the plant as a parent to create hybrid combinations in order to obtain allopolyploid hybrids.

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