Pumpkin haploid breeding method
Through heat shock treatment and specific culture medium cultivation of pumpkin unpollinated ovule, the problem of haplogenic species of pumpkin is solved, the formation rate of callus and embryonic bodies is improved, and the rapid breeding of new pumpkin varieties is achieved.
Patent Information
- Application Number
- CN202510768614.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-10
- Publication Date
- 2025-08-12
AI Technical Summary
In the prior art, it is difficult to succeed in ex vivo culture of pumpkin without pollination ovule, which leads to a lack of haplogenic squash methods, limiting the breeding speed of new pumpkin varieties.
The pumpkin was sliced unpollinated ovule on the day of flowering and was heat shocked, and inoculated in a specific culture medium. After callus induction, bud induction and rooting culture, and finally treated with colchicine to obtain a homozygous parental plant of pumpkin.
The callus formation rate and embryonic body formation rate were improved, and the haplogenic pumpkin species method was successfully created, which significantly accelerated the selection and breeding of new pumpkin varieties.
Smart Images
Figure BDA0005442349270000091 
Figure BDA0005442349270000092 
Figure BDA0005442349270000101
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of pumpkin haploid breeding, in particular to a pumpkin haploid breeding method. Background Art
[0002] Pumpkin (Cucurbita moschata) is a key vegetable crop in the Cucurbitaceae family. It boasts rich nutritional and health benefits, earning it the title of "world vegetable." With rising living standards, consumers are demanding a greater diversity of pumpkin varieties. However, traditional breeding methods require a long time—at least three to four years—to obtain homozygous parents. This significantly limits the speed of breeding new pumpkin hybrids with significant heterosis. Rapidly obtaining high-quality homozygous parents is crucial for breeding new pumpkin varieties, and experiments have demonstrated that haploid breeding is the most effective method for rapidly obtaining pure lines.
[0003] Haploid breeding refers to the use of methods such as androgenic culture (anther culture, pollen culture) and gynogenic culture (unpollinated ovaries or ovules, irradiated pollen culture, etc.) to induce the production of haploid plants, and then through artificial induction and other methods to double their single chromosomes into pairs, becoming viable, homozygous plants that can normally produce fruit, thereby breeding new varieties. Anther and pollen culture techniques are widely used and mature in plants such as the Cruciferae, Poaceae, and Solanaceae, but it is more difficult to successfully obtain haploids in Cucurbitaceae plants. In vitro gynogenic culture technology, which promotes the development of gynogenetics in unfertilized ovules or ovaries of some plants in vitro, has become an important way to obtain haploids in the Cucurbitaceae family.
[0004] Unpollinated ovule in vitro culture is one of the techniques for artificially inducing haploid plants. In recent years, domestic and foreign scholars have done a lot of exploratory work in this area. Successive studies have been conducted on unpollinated ovules or ovaries in vitro culture of plants such as wheat, tobacco, corn, poplar, and sugar beet, and most of them have obtained haploid plants. Compared with anther culture, haploid plants induced by unpollinated ovule in vitro culture have three unique advantages: (1) Anther culture of some plants has been difficult to succeed or the induction rate is too low, while unpollinated ovule in vitro culture can provide a new way for haploid breeding; (2) For male sterile plants that cannot be cultured with anthers to obtain haploids, unpollinated ovule culture can be used for induction; (3) The offspring of unpollinated ovule culture can produce more green seedlings. Cultivating unpollinated ovules or ovaries of local varieties or hybrids can quickly obtain high-purity germplasm resources, which has theoretical research and practical application value in efficiently utilizing germplasm resources and improving breeding results. However, there are few reports on methods for haploid breeding using in vitro culture of unpollinated pumpkin ovules. Both zucchini and pumpkin belong to the Cucurbitaceae family, and while several methods for in vitro culture of unpollinated ovules in zucchini are available in the prior art, these methods are not applicable to pumpkin. Therefore, there is a need to provide a method for haploid breeding of pumpkin to address this gap in haploid breeding. Summary of the Invention
[0005] In view of this, the present invention provides a pumpkin haploid breeding method to solve the problem of difficulty in in vitro culture of unpollinated pumpkin ovules.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The present invention provides a pumpkin haploid breeding method, comprising the following steps:
[0008] S1. Take unpollinated pumpkin ovules from the day of flowering and cut them into 1-2 mm slices.
[0009] S2. Inoculate the ovules into the pretreatment medium and heat shock them at 36-38°C for 2-4 days.
[0010] S3. The heat-shocked ovules were inoculated into callus induction medium for culture; callus was obtained after 48 to 72 hours of culture;
[0011] S4. After 1-2 subcultures, the callus was transferred to a bud induction medium for culture. The bud induction medium was: MS + sucrose 1.5-2.5 wt% + TDZ 0.03-0.05 mg / L + GA3 0.6-0.8 mg / L + anthocyanin 10-20 mg / L + agar 4-6 wt%;
[0012] S5. After culturing in a bud induction medium for 30 to 35 days, embryoids were obtained; the embryoids were transferred to a rooting medium and cultured for 10 to 20 days to obtain haploid plants;
[0013] S6. Treating the root system of the haploid plant with colchicine to obtain a pumpkin homozygous parent plant.
[0014] Preferably, the heat shock treatment and callus culture are performed under dark conditions.
[0015] Preferably, the culturing in steps S4 and S5 includes light culturing and dark culturing, the light culturing time is 16 h / d, the dark culturing time is 8 h / d, and the light intensity is 1500-2000 LX.
[0016] Preferably, the temperature for light culture is 25-27°C, and the temperature for dark culture is 22-24°C.
[0017] Preferably, the pretreatment culture medium is: MS+sucrose 2.5-3.5wt%+2,4-D 0.8-1.2mg / L+NAA 0.4-0.6mg / L+agar 7-9wt%.
[0018] Preferably, the callus induction medium is: MS+sucrose 2.5-3.5wt%+2,4-D 0.8-1.2mg / L+IAA 0.4-0.6mg / L+agar 7-9wt%.
[0019] Preferably, the callus subculture is: MS+sucrose 2.5-3.5wt%+2,4-D 0.4-0.6mg / L+6BA 0.8-1.2mg / L+agar 7-9wt%.
[0020] Preferably, the rooting medium is: 1 / 2MS+CaCl20.2-0.3 mg / L+sucrose 2.5-3.5 wt%+agar 7-9 wt%.
[0021] Preferably, in step S1, the ovule is sliced by cross-section.
[0022] By adopting the above technical solution, the present invention has the following beneficial effects: the present invention uses unpollinated pumpkin ovules on the day of flowering as explants, slices them, heat-shocks them, and then inoculates them in a callus induction medium for culture to obtain callus tissue; the callus tissue is then inoculated into a bud differentiation medium for culture to obtain embryoids; the embryoids are then inoculated into a rooting medium to obtain haploid plants, and after the haploid plants are treated with colchicine, homozygous pumpkin parent plants are obtained. The callus tissue culture medium of the present invention increases the callus formation rate, and the heat shock treatment and bud induction medium of the present invention increase the embryoid formation rate. The present invention successfully creates a pumpkin haploid breeding method, which is of great significance to pumpkin breeding. DETAILED DESCRIPTION
[0023] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0024] The present invention provides a pumpkin haploid breeding method, comprising the following steps:
[0025] S1. Take unpollinated pumpkin ovules from the day of flowering and cut them into 1-2 mm slices.
[0026] S2. Inoculate the ovules into the pretreatment medium and heat shock them at 36-38°C for 2-4 days.
[0027] S3. The heat-shocked ovules were inoculated into callus induction medium for culture; callus was obtained after 48 to 72 hours of culture;
[0028] S4. After 1-2 subcultures, the callus was transferred to a bud induction medium for culture. The bud induction medium was: MS + sucrose 1.5-2.5 wt% + TDZ 0.03-0.05 mg / L + GA3 0.6-0.8 mg / L + anthocyanin 10-20 mg / L + agar 4-6 wt%;
[0029] S5. After culturing in a bud induction medium for 30 to 35 days, embryoids were obtained; the embryoids were transferred to a rooting medium and cultured for 10 to 20 days to obtain haploid plants;
[0030] S6. Treating the root system of the haploid plant with colchicine to obtain a pumpkin homozygous parent plant.
[0031] In the present invention, unpollinated pumpkin ovules are taken on the day of flowering and disinfected with alcohol having a volume fraction of 75%; the skin is removed on a sterile clean bench and sliced into thin slices using a cross-section method, the thickness of the slices being 1 to 2 mm, more preferably 1.5 mm; the ovules are disinfected with sodium hypochlorite having a concentration of 6 wt% and a disinfection time of 10 minutes; and the ovules are then rinsed 3 to 4 times with sterile water until no sodium hypochlorite remains. The ovule slices are inoculated into a pretreatment medium and subjected to a heat shock treatment, the heat shock treatment being carried out in the dark at a temperature of 36 to 38°C, more preferably 36.5 to 37.5°C, and more preferably 37°C; the heat shock treatment lasts for 2 to 4 days, more preferably 3 days. The pretreatment culture medium is: MS + sucrose 2.5-3.5wt% + 2,4-D 0.8-1.2mg / L + NAA 0.4-0.6mg / L + agar 7-9wt%; in the pretreatment culture medium, the concentration of sucrose is preferably 2.8-3.2wt%, more preferably 3wt%; the concentration of 2,4-D is preferably 0.9-1.1mg / L, more preferably 1mg / L; the concentration of NAA is preferably 0.45-0.55mg / L, more preferably 0.5mg / L; the concentration of agar is preferably 7.5-8.5wt%, more preferably 8wt%.
[0032] The present invention inoculates heat-shocked ovule slices into a callus induction medium for culture, obtaining callus after 48 to 72 hours. The callus culture is carried out in the dark. The callus induction medium comprises: MS + 2.5 to 3.5 wt% sucrose + 0.8 to 1.2 mg / L 2,4-D + 0.4 to 0.6 mg / L IAA + 7 to 9 wt% agar. In the callus culture medium, the concentration of sucrose is preferably 2.8 to 3.2 wt%, more preferably 3 wt%; the concentration of 2,4-D is preferably 0.9 to 1.1 mg / L, more preferably 1 mg / L; the concentration of IAA is preferably 0.45 to 0.55 mg / L, more preferably 0.5 mg / L; and the concentration of agar is preferably 7.5 to 8.5 wt%, more preferably 8 wt%.
[0033] After 1-2 subcultures, the callus tissue is transferred to a bud induction medium for culture. The subculture medium for the callus tissue is: MS+2.5-3.5wt% sucrose+0.4-0.6mg / L 2,4-D+0.8-1.2mg / L 6BA+7-9wt% agar. In the subculture medium for the callus tissue, the concentration of sucrose is preferably 2.7-3.3wt%, more preferably 3wt%; the concentration of 2,4-D is preferably 0.45-0.55mg / L, more preferably 0.5mg / L; and the concentration of agar is preferably 7.5-8.5wt%, more preferably 8wt%.
[0034] The bud induction medium comprises: MS, sucrose 1.5-2.5%, TDZ 0.03-0.05 mg / L, GA3 0.6-0.8 mg / L, anthocyanin 10-20 mg / L, and agar 4-6 wt%. In the bud induction medium, the concentration of sucrose is preferably 1.7-2.2 wt%, more preferably 2 wt%. The concentration of TDZ is preferably 0.04 mg / L. The concentration of GA3 is preferably 0.65-0.75 mg / L, more preferably 0.7 mg / L. The concentration of anthocyanin is preferably 13-18 mg / L, more preferably 15 mg / L. The concentration of agar is preferably 4.5-5.5 wt%, more preferably 5 wt%. Under the combined action of TDZ, GA3, and anthocyanin, the formation of embryoids is more favorable.
[0035] After culturing in a bud induction medium for 30 to 35 days, embryoids are obtained; the embryoids are then transferred to a rooting medium and cultured for 10 to 20 days to obtain haploid plants. The rooting medium comprises: 1 / 2 MS + 0.2 to 0.3 mg / L CaCl2 + 2.5 to 3.5 wt% sucrose + 7 to 9 wt% agar; the CaCl2 concentration is preferably 0.22 to 0.28 mg / L, more preferably 0.25 mg / L; the sucrose concentration is preferably 2.7 to 3.2 wt%, more preferably 3 wt%; and the agar concentration is preferably 7.5 to 8.5 wt%, more preferably 8 wt%. The inventors have discovered that adding CaCl2 to the rooting medium promotes rooting of the embryoids.
[0036] In the present invention, bud induction culture and root induction culture include light culture and dark culture; the light culture time is 16h / d, and the dark culture time is 8h / d; the light intensity is 1500-2000LX, preferably 1700-1900LX, more preferably 1800LX; the light culture temperature is 25-27°C, preferably 25.5-26.5°C, more preferably 26°C; the dark culture temperature is 22-24°C, preferably 22.5-23.5°C, more preferably 23°C.
[0037] After obtaining haploid plants, the present invention treats the root systems of the haploid plants with colchicine to obtain pumpkin homozygous parent plants; the concentration of the colchicine is 0.2-0.4wt%, preferably 0.25-0.35wt%, more preferably 3wt%, and the treatment time is 20-30h, preferably 22-27h, more preferably 24h.
[0038] Example 1
[0039] In this embodiment, the pretreatment culture medium is: MS+sucrose 2.5 wt%+2,4-D 0.8 mg / L+NAA 0.4 mg / L+agar 7 wt%.
[0040] The callus induction medium was: MS+sucrose 2.5 wt%+2,4-D 0.8 mg / L+IAA 0.4 mg / L+agar 7 wt%.
[0041] The callus subculture was: MS+sucrose 2.5wt%+2,4-D 0.4mg / L+6BA 0.8 mg / L+agar 7wt%.
[0042] The bud induction medium is: MS+sucrose 1.5wt%+TDZ 0.03mg / L+GA30.6 mg / L+anthocyanidin 10mg / L+agar 4wt%.
[0043] The rooting medium is: 1 / 2MS+CaCl20.2 mg / L+sucrose 2.5wt%+agar 7wt%.
[0044] (1) Unpollinated pumpkin ovules were taken on the day of flowering and disinfected with 75% alcohol; the skin was removed on a sterile clean bench and cut into 1-2 mm thin slices using a cross-section method; the ovules were disinfected with 6 wt% sodium hypochlorite for 10 min; and then rinsed with sterile water for 4 times.
[0045] (2) The sterilized ovule slices were inoculated into the pretreatment medium and heat-shocked at 36°C in the dark for 4 days.
[0046] (3) The heat-shocked ovules were inoculated into callus induction medium and cultured in the dark; callus was obtained after 72 hours of culture.
[0047] (4) After two subcultures, the callus tissue was transferred to bud induction medium for culture. The light culture time was 16 h / d, and the dark culture time was 8 h / d; the light intensity was 1500 LX, the light culture temperature was 27°C, and the dark culture temperature was 24°C.
[0048] (5) After culturing in bud induction medium for 35 days, embryoids were obtained; the embryoids were transferred to rooting medium and cultured for 15 days to obtain haploid plants.
[0049] (6) The roots of the haploid plants were treated with colchicine at a concentration of 0.2 wt % for 30 h to obtain pumpkin homozygous parent plants.
[0050] Example 2
[0051] In this embodiment, the pretreatment culture medium is: MS+sucrose 3wt%+2,4-D 1mg / L+NAA 0.5mg / L+agar 8wt%.
[0052] The callus induction medium was: MS+sucrose 3wt%+2,4-D 1mg / L+IAA 0.5mg / L+agar 8wt%.
[0053] The callus subculture medium was: MS+sucrose 3wt%+2,4-D 0.5mg / L+6BA 1mg / L+agar 8wt%.
[0054] The bud induction medium is: MS+sucrose 2wt%+TDZ 0.04mg / L+GA30.7mg / L+anthocyanidin 15mg / L+agar 5wt%.
[0055] The rooting medium is: 1 / 2MS + CaCl2 0.25 mg / L + sucrose 3% + agar 8%.
[0056] (1) Unpollinated pumpkin ovules were taken on the day of flowering and disinfected with 75% alcohol; the skin was removed on a sterile clean bench and cut into 1-2 mm thin slices using a cross-section method; the ovules were disinfected with 6 wt% sodium hypochlorite for 10 min; and then rinsed with sterile water for 4 times.
[0057] (2) The sterilized ovule slices were inoculated into the pretreatment medium and heat-shocked at 37°C in the dark for 3 days.
[0058] (3) The heat-shocked ovules were inoculated into callus induction medium and cultured in the dark; callus was obtained after 72 hours of culture.
[0059] (4) After two subcultures, the callus tissue was transferred to bud induction medium for culture. The light culture time was 16 h / d, and the dark culture time was 8 h / d; the light intensity was 1800 LX, the light culture temperature was 26°C, and the dark culture temperature was 23°C.
[0060] (5) After culturing in bud induction medium for 35 days, embryoids were obtained; the embryoids were transferred to rooting medium and cultured for 15 days to obtain haploid plants.
[0061] (6) The roots of the haploid plants were treated with colchicine at a concentration of 0.3 wt % for 24 h to obtain pumpkin homozygous parent plants.
[0062] Example 3
[0063] In this embodiment, the pretreatment culture medium is: MS+sucrose 3.5 wt%+2,4-D 1.2 mg / L+NAA 0.6 mg / L+agar wt 9%.
[0064] The callus induction medium was: MS+sucrose 3.5 wt%+2,4-D 1.2 mg / L+IAA 0.6 mg / L+agar 9 wt%.
[0065] The callus subculture medium was: MS+sucrose 3.5wt%+2,4-D 0.6mg / L+6BA 1.2mg / L+agar 9wt%.
[0066] The bud induction medium is: MS+sucrose 2.5wt%+TDZ 0.05mg / L+GA30.8mg / L+anthocyanidin 20mg / L+agar 6wt%.
[0067] The rooting medium is: 1 / 2MS+CaCl20.3 mg / L+sucrose 3.5wt%+agar 9wt%.
[0068] (1) Unpollinated pumpkin ovules were taken on the day of flowering and disinfected with 75% alcohol; the skin was removed on a sterile clean bench and cut into 1-2 mm thin slices using a cross-section method; the ovules were disinfected with 6 wt% sodium hypochlorite for 10 min; and then rinsed with sterile water for 4 times.
[0069] (2) The sterilized ovule slices were inoculated into the pretreatment medium and heat-shocked at 38°C in the dark for 2 days.
[0070] (3) The heat-shocked ovules were inoculated into callus induction medium and cultured in the dark; callus was obtained after 72 hours of culture.
[0071] (4) After two subcultures, the callus tissue was transferred to bud induction medium for culture. The light culture time was 16 h / d, and the dark culture time was 8 h / d; the light intensity was 2000 LX, the light culture temperature was 25°C, and the dark culture temperature was 22°C.
[0072] (5) After culturing in bud induction medium for 35 days, embryoids were obtained; the embryoids were transferred to rooting medium and cultured for 15 days to obtain haploid plants.
[0073] (6) The roots of the haploid plants were treated with colchicine at a concentration of 0.4 wt % for 20 h to obtain pumpkin homozygous parent plants.
[0074] Comparative Example 1
[0075] The difference from Example 2 is that the temperature of the heat shock treatment is 32°C.
[0076] Comparative Example 2
[0077] The difference from Example 2 is that the IAA in the callus induction medium was replaced with NAA of equal concentration.
[0078] Comparative Example 3
[0079] Different from Example 2, the concentration of 2,4-D in the callus induction medium is 0.5 mg / L.
[0080] Comparative Example 4
[0081] The difference from Example 2 is that the concentration of sucrose in the bud induction medium is 3.5 wt %.
[0082] Comparative Example 5
[0083] The difference from Example 2 is that the bud induction medium does not contain GA3.
[0084] Comparative Example 6
[0085] The difference from Example 2 is that the concentration of TDZ in the shoot induction medium is 0.08 mg / L.
[0086] Comparative Example 7
[0087] Different from Example 2, the bud induction medium does not contain anthocyanin.
[0088] Comparative Example 8
[0089] The difference from Example 2 is that the anthocyanin in the bud induction medium is replaced by AgNO3 of equal concentration.
[0090] Comparative Example 9
[0091] The difference from Example 2 is that the concentration of agar in the bud induction medium is 8 wt %.
[0092] Experimental example
[0093] The experiment was conducted from August to December 2024, using the pumpkin variety "Weinuo No. 1 (WF001)." Twelve groups were divided, and pumpkin haploid cultivation was performed according to the methods described in Examples 1-3 and Comparative Examples 1-9. Each group was inoculated with 100 ovule slices, and three replicates were set up for each group. The callus induction rates for Examples 1-3 and Comparative Examples 1-9 were statistically analyzed and shown in Tables 1 and 1.
[0094] Callus induction rate = total number of ovule slices producing callus / total number of inoculated ovule slices × 100%.
[0095] Table 1 Number of pumpkin ovule slices producing callus and callus induction rate in each group
[0096]
[0097] As shown in Table 1, the callus induction rates in Examples 1 to 3 of the present invention were between 35.3% and 37.3%, which were higher than those in Comparative Examples 2 and 3. This indicates that changing the type and concentration of plant hormones was not conducive to the formation of pumpkin callus.
[0098] The calli in Comparative Examples 1 and 5-9 of Examples 1-3 were subcultured twice and then inoculated into bud induction medium for culture. 50 calli were inoculated in each group, and three replicates were set for each group. The embryoid formation rate was counted after 35 days. The results are shown in Table 2.
[0099] Embryoid formation rate = number of embryoids formed / total number of inoculated callus tissues × 100%.
[0100] Table 2 Embryoid formation rate of each group
[0101]
[0102]
[0103] As shown in Table 2, the embryoid formation rates in Examples 1 to 3 of the present invention were between 22.7% and 24.0%, which were higher than those in Comparative Examples 1 and 4 to 9. This indicates that changing the components or contents in the bud induction medium was not conducive to embryoid formation.
[0104] To test the doubling success rate, plantlets obtained after colchicine treatment were transplanted into nutrient soil with a moisture content of 60% (a mixture of peat soil and perlite in a volume ratio of 3:1). After two weeks, newly grown leaves were removed and tested for ploidy using a BD FACSCalibur flow cytometer to distinguish haploids, doubled haploids, and tetraploids. The doubled haploid doubling success rate is the ratio of the number of doubled haploid plants tested to the total number of regenerated plants tested. The doubling success rate of the present invention reached 85%.
[0105] As can be seen from the above examples, the present invention provides a pumpkin haploid breeding method that improves the callus formation rate, embryoid formation rate and doubling success rate.
[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A pumpkin haploid breeding method, characterized in that: The following steps are involved: S1. Take unpollinated pumpkin ovules from the day of flowering and cut them into 1-2 mm slices. S2. Inoculate the ovules into the pretreatment medium and heat shock them at 36-38°C for 2-4 days. S3. The heat-shocked ovules were inoculated into callus induction medium for culture; callus was obtained after 48 to 72 hours of culture; S4. After 1-2 subcultures, the callus was transferred to a bud induction medium for culture. The bud induction medium was: MS + sucrose 1.5-2.5 wt% + TDZ 0.03-0.05 mg / L + GA3 0.6-0.8 mg / L + anthocyanin 10-20 mg / L + agar 4-6 wt%; S5. After culturing in a bud induction medium for 30 to 35 days, embryoids were obtained; the embryoids were transferred to a rooting medium and cultured for 10 to 20 days to obtain haploid plants; S6. Treating the root system of the haploid plant with colchicine to obtain a pumpkin homozygous parent plant.
2. The pumpkin haploid breeding method according to claim 1, wherein Heat shock treatment and callus culture were carried out in the dark.
3. The pumpkin haploid breeding method according to claim 1, wherein The cultivation in steps S4 and S5 includes light cultivation and dark cultivation, the light cultivation time is 16 h / d, and the dark cultivation time is 8 h / d; the light intensity is 1500-2000 LX.
4. The pumpkin haploid breeding method according to claim 3, characterized in that The temperature for light culture is 25-27°C, and the temperature for dark culture is 22-24°C.
5. The pumpkin haploid breeding method according to claim 1, characterized in that: The pretreatment culture medium is: MS+sucrose 2.5-3.5wt%+2,4-D 0.8-1.2mg / L+NAA 0.4-0.6mg / L+agar 7-9wt%.
6. The pumpkin haploid breeding method according to claim 1, characterized in that: The callus induction medium is: MS+sucrose 2.5-3.5wt%+2,4-D 0.8-1.2mg / L+IAA 0.4-0.6mg / L+agar 7-9wt%.
7. The pumpkin haploid breeding method according to claim 1, characterized in that: The callus subculture is as follows: MS+sucrose 2.5-3.5wt%+2,4-D 0.4-0.6mg / L+6BA 0.8-1.2mg / L+agar 7-9wt%.
8. The pumpkin haploid breeding method according to claim 1, characterized in that: The rooting culture medium comprises: 1 / 2MS+CaCl2 0.2-0.3 mg / L+sucrose 2.5-3.5 wt%+agar 7-9 wt%.
9. The pumpkin haploid breeding method according to claim 1, characterized in that: In step S1, the ovule is sliced by transverse section.
10. The pumpkin haploid breeding method according to claim 1, characterized in that: The concentration of colchicine is 0.2-0.4 wt %, and the treatment time is 20-30 hours.