A soybean heavy ion beam radiation mutation breeding method and its application

Abstract

The present invention provides a soybean heavy ion beam radiation mutagenesis method, which belongs to the field of crop breeding technology. The soybean mutagenesis breeding method of the present invention comprises the following steps: (1) 12 C 6+ (1) irradiating soybean seeds with a radiation source of 40-60 Gy and a radiation time of 100-140 seconds; (2) germinating and culturing the irradiated soybean seeds using a culture medium; the culture medium is supplemented with 10-50 mg / L of vitamin C and 10-30 g / L of mannitol; and (3) transplanting the irradiated soybean seeds into a seedling culture medium after emergence for seedling cultivation, and spraying a water-soluble fertilizer containing 0.1%-0.3% seaweed extract and 0.5%-2% chitin during the seedling cultivation period. The present invention improves the mutant seed germination rate and seedling survival rate by adjusting the radiation dose and post-irradiation management method, promotes the stable expression of the mutant trait, accelerates the breeding process, and improves breeding efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop breeding, and in particular relates to a soybean heavy ion beam radiation mutation breeding method and application thereof. Background Art

[0002] As an important food and cash crop, soybeans play a pivotal role in global agricultural production and the food industry. With population growth, evolving consumption patterns, and the need for sustainable agricultural development, higher demands are being placed on soybean variety improvement, such as increasing yield, enhancing stress resistance, and improving quality (such as protein and oil content and composition).

[0003] Mutation breeding is an important approach for creating new soybean germplasm resources and cultivating new varieties. The principle is to induce genetic mutations in soybeans through physical, chemical, or biological factors, thereby producing individuals with new traits. These individuals are then screened and cultivated to obtain superior varieties. Heavy ion beam mutagenesis is an emerging mutagenesis technique that has garnered widespread attention in plant breeding in recent years. However, research on its application in soybean breeding is relatively limited, and a mature, systematic technical framework has yet to be established.

[0004] Different soybean varieties have different sensitivities to heavy ion beam radiation. If the radiation dose is too high, it will seriously affect seed vitality and seedling growth; if the dose is too low, it may not be possible to obtain mutants of sufficient quantity and quality, and the expected breeding effect cannot be achieved. In addition, the post-irradiation processing technology is not perfect. During the seed germination and seedling cultivation stages after radiation treatment, there is no suitable supporting technology to improve the seed germination rate and seedling survival rate, and promote the stable expression of mutant traits. Existing soybeans are germinated and raised directly in the original environment after radiation mutagenesis, which cannot meet the special nutritional needs and physiological state of soybean seeds and seedlings after radiation, resulting in slow seed germination, weak seedling growth, and increased risk of mutant loss.

[0005] Therefore, it is necessary to provide a more efficient, accurate and reliable mutation breeding technology for soybean breeding to meet the demand of agricultural production for new high-yield, high-quality and stress-resistant soybean varieties. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a soybean heavy ion beam radiation mutagenesis breeding method, which improves the mutant seed germination rate and seedling survival rate by adjusting the radiation dose and post-irradiation management method, and promotes the stable expression of mutant traits.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] A soybean heavy ion beam radiation mutation breeding method, characterized by comprising the following steps:

[0009] (1) 12 C 6+ Soybean seeds were irradiated using a radiation source with a radiation dose of 40-60 Gy and a radiation time of 100-140 s.

[0010] (2) germinating and culturing the irradiated soybean seeds using a culture medium; the culture medium is supplemented with 10-50 mg / L of vitamin C and 10-30 g / L of mannitol;

[0011] (3) After the irradiated soybean seeds emerge, they are transplanted into a seedling medium for seedling cultivation. During the seedling cultivation period, water-soluble fertilizer containing 0.1%-0.3% seaweed extract and 0.5%-2% chitosan is sprayed.

[0012] Preferably, before the radiation treatment, the soybean seeds are disinfected and exposed to the sun; the disinfection treatment is: first soaking in 75% alcohol for 30-60 seconds, then rinsing with sterile water 2-3 times, then soaking in 0.1% mercuric chloride solution for 5-10 minutes, and finally rinsing with sterile water 4-5 times; the exposure treatment is: placing the disinfected seeds on moist sterile filter paper and pre-germinating and culturing in a dark, 25±2°C environment for 24-30 hours.

[0013] Preferably, the culture medium is MS medium.

[0014] Preferably, during the germination culture stage, the temperature is 25-28° C., the humidity is 60-70%, and the light intensity is 2000-3000 Lux.

[0015] Preferably, the seedling culture medium is a mixture of vermiculite, perlite and peat soil in a volume ratio of 1-2:1-2:1-2.

[0016] Preferably, the water-soluble fertilizer further comprises 15-25% N, 10-20% P2O5 and 15-25% K2O.

[0017] More preferably, the water-soluble fertilizer is sprayed once every 5-7 days until the leaves are evenly moistened and no water drips.

[0018] Preferably, during the seedling raising stage, the temperature is 20-25° C., the humidity is 60-70%, and the light intensity is 3000-5000 Lux.

[0019] Preferably, the method also includes screening and identifying mutants.

[0020] The present invention also provides application of the breeding method in soybean mutation breeding.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Improving the germination rate of mutant seeds: By optimizing the radiation dose and radiation time, we can ensure a certain mutagenic intensity while avoiding excessive loss of seed viability, so that a certain proportion of soybean seeds survive and produce more mutations with potential breeding value, which is conducive to subsequent germination. At the same time, adding vitamin C and mannitol to the culture medium lays a good foundation for the subsequent growth of seedlings, thereby effectively improving the germination rate of mutant seeds.

[0023] (2) Improve the survival rate of seedlings: Spraying water-soluble fertilizer containing seaweed extract and chitin during the seedling cultivation period. The synergistic effect of the two, combined with a reasonable proportion of macroelements, can meet the growth needs of seedlings, reduce seedling deaths caused by environmental stress and pests and diseases, and significantly improve the survival rate of seedlings.

[0024] (3) Promote the stable expression of mutant traits: From seed disinfection and whitening treatment before radiation treatment to germination culture and seedling management after radiation, a series of measures provide soybean seeds and seedlings with a suitable growth environment and nutritional conditions, so that they grow well, contribute to the stable expression of mutant genes, reduce the instability or loss of mutant traits caused by environmental factors, facilitate the screening and identification of mutants, accelerate the breeding process, and improve breeding efficiency. DETAILED DESCRIPTION

[0025] The present invention provides a soybean heavy ion beam radiation mutation breeding method, comprising the following steps:

[0026] (1) 12 C 6+ Soybean seeds were irradiated using a 40-60 Gy radiation source and a 100-140 s radiation time.

[0027] Carbon ions have a high linear energy transfer (LET) and relative biological effect (RBE), causing more complex and diverse damage in the soybean seed genome, thereby inducing a rich variety of genetic mutations. Compared to other radiation sources, they can achieve higher mutagenesis efficiencies at lower doses and are highly targeted at specific gene loci, increasing the probability of obtaining beneficial mutations.

[0028] An appropriate radiation dose ensures sufficient mutagenic intensity to effectively mutate soybean seed genes, while preventing excessive radiation doses from completely devitalizing the seeds or producing an excessive number of severely damaged, nonviable mutants. The present invention irradiates soybean seeds with 40-60 Gy for 100-140 seconds, ensuring that a certain proportion of the seeds survive and produce a high number of mutations with potential breeding value. This also prevents excessive inhibition of subsequent germination and growth, facilitating the screening of superior mutants.

[0029] (2) Germination culture is performed on the irradiated soybean seeds using a culture medium; the culture medium is supplemented with 10-50 mg / L of vitamin C and 10-30 g / L of mannitol.

[0030] Vitamin C is an antioxidant. In germination culture media, it can scavenge free radicals generated by radiation, mitigate oxidative damage to biomacromolecules (such as DNA, proteins, and lipids) within seed cells, protect cell integrity and function, reduce the inhibitory effects of radiation on seed germination, and improve germination rates and the initial growth vigor of seedlings. Mannitol, an osmotic regulator, regulates the osmotic pressure within irradiated seed cells, preventing water loss or excessive water absorption due to radiation-induced water imbalance. Mannitol maintains cell turgor, protects cell structure and physiological function, promotes cell elongation and division during seed germination, and promotes root growth and development, laying a good foundation for subsequent seedling growth.

[0031] (3) After the irradiated soybean seeds emerge, they are transplanted into a seedling medium for seedling cultivation. During the seedling cultivation period, water-soluble fertilizer containing 0.1%-0.3% seaweed extract and 0.5%-2% chitosan is sprayed.

[0032] Seaweed extracts are rich in various plant growth regulators, such as auxins, cytokinins, gibberellins, and similar substances, as well as nutrients such as amino acids, polysaccharides, and vitamins. Spraying a water-soluble fertilizer containing an appropriate amount of seaweed extract during the seedling raising period can help these plant growth regulators promote cell division, elongation, and differentiation in the seedlings, regulating their growth and development, making them stronger and more robust, and enhancing their resistance to stresses (such as drought, cold, and disease). Nutrients such as amino acids and polysaccharides directly provide nutrients to the seedlings, supplementing their growth needs. Chitin can induce plant defense responses, enhance plant immunity, and stimulate the production of antimicrobial substances such as phytoalexins and chitinases to resist infection by pathogens. Chitin also promotes root growth, increases root surface area, and improves the root system's ability to absorb nutrients and water. This facilitates the growth and development of seedlings during the raising period, reduces seedling mortality due to environmental stress and pests and diseases, and increases the success rate of mutant screening.

[0033] In the present invention, it is preferred that the soybean seeds be disinfected and exposed to the sun before irradiation. A further preferred disinfection treatment is: first soak in 75% alcohol for 30-60 seconds, then rinse with sterile water 2-3 times, then soak in 0.1% mercuric chloride solution for 5-10 minutes, and finally rinse with sterile water 4-5 times; more preferably, first soak in 75% alcohol for 45 seconds, then rinse with sterile water 3 times, then soak in 0.1% mercuric chloride solution for 7 minutes, and finally rinse with sterile water 5 times. Disinfection creates a sterile environment for germination and growth of seeds after irradiation treatment, reduces experimental errors and seedling death caused by microbial infection, and improves the success rate of breeding. A further preferred exposure treatment is: placing the disinfected seeds on moistened sterile filter paper and pre-germinating and culturing them in a dark, 25±2°C environment for 24-30 hours; more preferably, culturing for 26 hours. The present invention exposes the seeds first and then performs radiation treatment. On the one hand, at this time, the seed cells are more sensitive to radiation, which can improve the mutagenic effect; on the other hand, the seeds after exposing the seeds can adapt to the environment more quickly in the subsequent germination and cultivation process, thereby improving the germination rate and seedling survival rate and shortening the breeding cycle.

[0034] In the present invention, preferably in step (1), the radiation dose is 50 Gy and the radiation time is 120 s.

[0035] In the present invention, preferably in step (2), the culture medium is supplemented with 10-50 mg / L vitamin C and 10-30 g / L mannitol; more preferably, 30 mg / L vitamin C and 20 g / L mannitol are added; more preferably, the culture medium is MS medium.

[0036] In the present invention, in step (2), the temperature during the germination culture stage is preferably 25-28°C, more preferably 26°C; the humidity is preferably 60-70%, more preferably 65%; the light intensity is preferably 2000-3000 Lux, more preferably 2500 Lux, and the more preferred photoperiod is 12-14 h light / 10-12 h dark.

[0037] In the present invention, preferably in step (3), the seedling matrix is ​​formed by mixing vermiculite, perlite and peat soil in a volume ratio of 1-2:1-2:1-2, more preferably 1:1:2. Vermiculite has good water retention and air permeability, can store a large amount of water and slowly release, and its porous structure is conducive to air circulation, providing sufficient oxygen for the seedling root system. Perlite is light in texture and has a large porosity, which can further increase the air permeability of the matrix, prevent the matrix from accumulating water and causing the root system to rot due to lack of oxygen, and promote the respiration and growth of the root system. Peat soil is rich in humus and has rich nutrients (such as nitrogen, phosphorus, potassium and other macroelements and various trace elements), can provide a continuous nutrient supply for seedling growth, and its water retention is also good, which can adjust the moisture condition of the matrix. The three are mixed in a specific proportion, combining their advantages to form a seedling matrix environment that retains water and fertilizer and has good ventilation, which is conducive to the growth and development of the seedling roots after radiation treatment, allowing the roots to fully stretch in the matrix, absorb water and nutrients, promote the healthy growth of the above-ground parts of the seedlings, improve the survival rate and growth quality of the seedlings, and provide a good plant foundation for the subsequent screening and cultivation of mutants.

[0038] In the present invention, preferably in step (3), the water-soluble fertilizer contains 0.2% seaweed extract and 1% chitosan. More preferably, the water-soluble fertilizer also contains 15-25% N, 10-20% P2O5, and 15-25% K2O, and more preferably 20% N, 15% P2O5, and 20% K2O. Preferably, the water-soluble fertilizer is sprayed once every 5-7 days until the leaves are evenly moistened and no water drips. A reasonable proportion of N, P2O5, and K2O can meet the soybean seedlings' demand for macronutrients during the seedling stage, promote balanced growth of the seedlings, improve the quality and stress resistance of the seedlings, and facilitate the subsequent screening and identification of mutants.

[0039] In the present invention, in step (3), the temperature in the seedling raising stage is preferably 20-25°C, more preferably 23°C; the humidity is preferably 60%-70%, more preferably 65%; the light intensity is preferably 3000-5000 Lux, more preferably 4000 Lux, and the more preferred photoperiod is 10-14 h light / 10-14 h dark.

[0040] In the present invention, after the seedling stage is preferably completed, (4) screening and identification of mutants is also included. It is further preferred that during the soybean growth process, phenotypic observations are carried out starting from the seedling stage, and traits such as plant height, leaf shape, and color are regularly recorded; during the flowering period, characteristics such as flowering time, flower color, and flower shape are observed; during the maturity period, agronomic traits such as plant height, number of pods, seed size, shape, and color are focused on. Comparison is made with non-irradiated control plants to screen out suspected mutants with obvious phenotypic differences. For suspected mutants, their leaf tissues are collected, genomic DNA is extracted, and analysis is performed using SSR molecular marker technology to detect whether their DNA levels have changed, and further determine whether they are true mutants.

[0041] The present invention also provides the application of the above breeding method in soybean mutation breeding to accelerate the soybean breeding process and improve the soybean breeding efficiency.

[0042] 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.

[0043] Example 1

[0044] (1) Radiation mutagenesis treatment

[0045] Select mature and plump seeds of the soybean variety "Zhonghuang 13" and first perform disinfection and whitening treatment before radiation treatment. Soak the seeds in 75% alcohol for 30 seconds, then quickly rinse with sterile water 3 times, then transfer to 0.1% mercuric chloride solution and soak for 5 minutes, and finally rinse thoroughly with sterile water 5 times. Place the disinfected seeds on moistened sterile filter paper and pre-germinate and culture in a dark environment at 25℃±2℃ for 24 hours until the seeds turn white. 12 C 6+ The radiation source was set to 40 Gy, the radiation time was set to 140 s, and the soybean seeds after whitening were irradiated.

[0046] (2) Germination culture

[0047] Use MS medium supplemented with 10 mg / L vitamin C and 10 g / L mannitol. Irradiated seeds were inoculated onto the medium and placed in a light incubator set at 25°C, 60% humidity, 2000 Lux, and a photoperiod of 12 hours light / 12 hours dark. During the germination process, closely monitor seed germination and promptly remove any contaminated seeds.

[0048] (3) Seedling management

[0049] After irradiation, soybean seeds are transplanted into a seedling medium. The seedling medium is a mixture of vermiculite, perlite, and peat in a 1:1:1 volume ratio and is sterilized at high temperatures before use. During the seedling period, a water-soluble fertilizer containing 0.1% seaweed extract and 0.5% chitosan is sprayed. The water-soluble fertilizer also contains 15% N, 10% P2O5, and 15% K2O. Spraying is done every five days, with the amount of spraying being based on evenly moistened leaves without dripping. During the seedling stage, the temperature is controlled at 20°C, the humidity is maintained at 60%, the light intensity is 3000 Lux, and the photoperiod is 10 hours of light / 14 hours of darkness.

[0050] (4) Mutant screening and identification

[0051] During soybean growth, phenotypic observations are conducted starting from the seedling stage. Plant height, leaf shape, color, and other traits are regularly recorded. During the flowering period, characteristics such as flowering time, flower color, and flower shape are observed; during the maturity period, agronomic traits such as plant height, number of pods, seed size, shape, and color are focused on. Comparisons are made with unirradiated "Zhonghuang 13" control plants to screen for suspected mutants with significant phenotypic differences. For suspected mutants, leaf tissue is collected, genomic DNA is extracted, and analysis is performed using SSR molecular marker technology to detect changes in DNA levels and further determine whether they are true mutants.

[0052] Example 2

[0053] (1) Radiation mutagenesis treatment

[0054] Select high-quality soybean seeds of the variety "Heihe 43" and perform disinfection and whitening treatment. Soak in 75% alcohol for 60 seconds, rinse twice with sterile water, soak in 0.1% mercuric chloride solution for 10 minutes, and then rinse four times with sterile water. The disinfected seeds are pre-germinated and cultured on moist sterile filter paper in a dark environment at 25℃±2℃ for 30 hours until they turn white. 12 C 6+ The seeds were irradiated with a radiation dose of 50 Gy and a radiation time of 120 s.

[0055] (2) Germination culture

[0056] Use MS medium supplemented with 30 mg / L vitamin C and 20 g / L mannitol. Place the irradiated seeds on the medium and incubate at 26°C, 65% humidity, and 2500 Lux of light intensity with a photoperiod of 14 hours light / 10 hours dark. Maintain a clean culture environment to prevent microbial contamination.

[0057] (3) Seedling management

[0058] After emergence, seedlings were transplanted into a pre-sterilized seedling medium consisting of a 2:1:2 volume ratio of vermiculite, perlite, and peat moss. During the seedling period, a water-soluble fertilizer containing 0.2% seaweed extract and 1% chitosan (20% N, 15% P₂O₅, and 20% K₂O) was sprayed. Application was made every six days, with the amount of spray applied sufficient to ensure evenly moist leaves without dripping. The seedling temperature was maintained at 23°C, humidity at 65%, and light intensity at 4000 Lux, with a photoperiod of 12 hours light / 12 hours dark.

[0059] (4) Mutant screening and identification

[0060] Same as Example 1.

[0061] Example 3

[0062] (1) Radiation mutagenesis treatment

[0063] The soybean variety "Jiyu 47" seeds were used as materials and were first sterilized. The seeds were soaked in 75% alcohol for 45 seconds, rinsed with sterile water three times, soaked in 0.1% mercuric chloride solution for 8 minutes, and finally rinsed with sterile water five times. Then, in a dark environment at 25℃±2℃, the seeds were placed on moist sterile filter paper for pre-germination and culture for 26 hours until they turned white. 12 C 6+ The soybean seeds were irradiated with a radiation source, a radiation dose of 60 Gy, and an irradiation time of 100 s.

[0064] (2) Germination culture

[0065] Use MS medium supplemented with 50 mg / L vitamin C and 30 g / L mannitol. Place the irradiated seeds on the medium and incubate at a temperature of 28°C, 70% humidity, 3000 Lux of light, and a photoperiod of 12 hours light / 12 hours dark. Carefully adjust the incubator's environmental parameters to ensure proper germination.

[0066] (3) Seedling management

[0067] After emergence, transplant the seedlings into a sterilized seedling medium consisting of a 1:2:1 volume ratio of vermiculite, perlite, and peat moss. During seedling cultivation, spray with a water-soluble fertilizer containing 0.3% seaweed extract and 2% chitosan, along with 25% nitrogen, 20% phosphorus oxide, and 25% potassium oxide. Apply spray every seven days, ensuring that the leaves are evenly moistened and free of dripping. During the seedling cultivation phase, maintain a temperature of 25°C, a humidity of 70%, and a light intensity of 5,000 lux, with a photoperiod of 14 hours light / 10 hours dark.

[0068] (4) Mutant screening and identification

[0069] Same as Example 1.

[0070] Example 4

[0071] Screening of mutagenic doses of heavy ion beam radiation

[0072] (1) Test materials and preparation

[0073] Plump seeds of the soybean variety "Zhonghuang 13" were surface disinfected by soaking them in 75% alcohol for 30 seconds, rinsing them three times with sterile water, soaking them in 0.1% mercuric chloride solution for 7 minutes, and finally rinsing them five times with sterile water. The disinfected seeds were placed on moistened sterile filter paper and pre-germinated in a dark environment at 25°C for 26 hours until they turned white.

[0074] (2) Radiation dose setting and processing

[0075] by 12 C 6+ As the radiation source, seven different radiation dose gradients were set: 0 Gy (as a control, no radiation treatment), 20 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy, and 70 Gy, with irradiation for 2 minutes. Each dose treatment was repeated four times, with 50 whitened seeds in each replicate. The whitened seeds were evenly placed in a special radiation sample box and irradiated using a professional heavy ion accelerator according to the set dose.

[0076] (3) Radiation post-processing and data acquisition

[0077] Irradiated seeds were sown in seedling trays containing a mixture of vermiculite and peat (1:1 by volume). The medium was kept moist, the temperature was controlled at 25°C, the light intensity was 3000 Lux, and the photoperiod was 12 hours light / 12 hours dark. Starting from the third day after sowing, the number of germinated seeds in each treatment group was counted daily until the tenth day. Germination rate was calculated as (number of germinated seeds / number of test seeds) × 100%.

[0078] (4) Calculation of median lethality rate and analysis of results

[0079] The median lethality rate (LD50) was calculated based on the germination rate data of the different radiation dose treatment groups. 50 ) was calculated using linear regression, with radiation dose as the independent variable (x) and the probability unit of germination rate as the dependent variable (y). The regression equation y = a + bx was established. Setting y = 5 (corresponding to the median lethality), the value of x was solved for to obtain the median lethal dose.

[0080] (5) Test results

[0081] A regression equation was established: y = 8.53 - 0.07x. When y = 5, the median lethal dose (LD) was calculated to be 50.43 Gy. As shown in Table 1, the germination rate in the 50 Gy group was 52.0 ± 1.6%, close to the LD. Although seed germination and seedling growth were somewhat inhibited at this dose, a large number of distinct and diverse mutant traits emerged, providing a rich resource for subsequent screening of superior mutants. Therefore, 50 Gy can be used as a reference dose for soybean heavy ion beam radiation-induced mutation breeding.

[0082] Table 1 Screening of mutagenic doses of heavy ion beam radiation

[0083]

[0084]

[0085] Example 5

[0086] Effects of culture medium on germination rate of irradiated soybean seeds

[0087] (1) Test materials and preparation

[0088] The seeds of soybean variety “Zhonghuang 13” were selected and sterilized (soaked in 75% alcohol for 45 seconds, rinsed with sterile water three times, soaked in 0.1% mercuric chloride solution for 7 minutes, and rinsed with sterile water five times) and dew-whitened (incubated on moist sterile filter paper in a dark, 25°C environment for 26 hours). 12 C 6+ The white seeds were irradiated with the radiation source, the radiation dose was 50 Gy, and the radiation time was 120 s.

[0089] (2) Culture medium setting and processing

[0090] Prepare 4 different culture media:

[0091] Medium A: MS basic medium (without any special ingredients added).

[0092] Medium B: MS medium supplemented with 30 mg / L vitamin C.

[0093] Medium C: MS medium supplemented with 20 g / L mannitol.

[0094] Medium D: MS medium supplemented with 30 mg / L vitamin C and 20 g / L mannitol. Three replicates were set up for each medium, and each replicate was inoculated with 30 irradiated seeds.

[0095] (3) Germination culture and data collection

[0096] The irradiated seeds were inoculated onto different culture media and cultured in an incubator at 25°C, 65% humidity, 3000 Lux illumination, and a photoperiod of 12 h light / 12 h dark. Starting from the third day after sowing, the number of seeds germinating on each culture medium was counted daily until the tenth day, and the germination rate was calculated. The germination potential and germination index were also recorded during the germination process, using the same calculation method as in Example 4.

[0097] (4) Analysis of test results

[0098] As shown in Table 2, compared with the MS minimal medium (control), the culture medium with the addition of 30 mg / L of vitamin C increased the germination rate to 75.0 ± 2.5%, and the germination potential and germination index also increased, indicating that vitamin C can promote the germination of soybean seeds after radiation to a certain extent and alleviate the inhibitory effect of radiation on seed germination. The culture medium with the addition of 20 g / L of mannitol had a germination rate of 72.0 ± 2.8%, indicating that mannitol also has a positive effect on seed germination. And the preferred culture medium with the addition of 30 mg / L of vitamin C and 20 g / L of mannitol simultaneously significantly increased the germination rate to 85.0 ± 2.0%, and the germination potential and germination index also reached a higher level, indicating that the combination of vitamin C and mannitol has a synergistic promoting effect on the germination of soybean seeds after radiation and can more effectively improve the germination ability of seeds.

[0099] Table 2 Effect of culture medium on germination rate of irradiated soybean seeds

[0100] culture medium Germination rate (%) Germination potential (%) Germination index A 68.0±3.0 55.0±3.5 10.0±1.0 B 75.0±2.5 60.0±3.0 11.5±0.8 C 72.0±2.8 58.0±3.2 11.0±0.9 D 85.0±2.0 70.0±2.5 13.5±0.5

[0101] Example 6

[0102] Effects of water-soluble fertilizer on the survival rate of irradiated soybean seeds and seedlings

[0103] (1) Test materials and preparation

[0104] The soybean variety "Zhonghuang 13" seeds were used as materials, and after disinfection (immersion in 75% alcohol for 45 seconds, washing with sterile water three times, soaking in 0.1% mercuric chloride solution for 8 minutes, and washing with sterile water five times) and dew-whitening (incubation on moist sterile filter paper in a dark, 25℃ environment for 26 hours), the seeds were used as materials. 12 C 6+ White seeds were irradiated using a radiation source at a dose of 50 Gy for 120 seconds. The irradiated seeds were germinated on MS medium (supplemented with 30 mg / L vitamin C and 20 g / L mannitol) and transplanted into a seedling medium (a mixture of vermiculite, perlite, and peat in a 1:1:2 volume ratio) after emergence.

[0105] (2) Setting and processing of water-soluble fertilizer

[0106] Prepare 4 different water-soluble fertilizers:

[0107] Water-soluble fertilizer 1: does not contain seaweed extract and chitin, and only contains N 20%, P2O5 15%, and K2O 20% (basic macro-element water-soluble fertilizer).

[0108] Water-soluble fertilizer 2: contains 0.2% seaweed extract, no chitin, and contains 20% N, 15% P2O5, and 20% K2O.

[0109] Water-soluble fertilizer 3: contains 1% chitosan, no seaweed extract, and contains N 20%, P2O5 15%, and K2O 20%.

[0110] Water-soluble fertilizer 4: Contains 0.2% seaweed extract and 1% chitin, as well as 20% nitrogen, 15% phosphorus oxide, and 20% potassium oxide. Three replicates were set up for each water-soluble fertilizer treatment, with 20 seedlings transplanted into each replicate. During the seedling raising period, spray the water-soluble fertilizer every six days, using a spraying rate sufficient to ensure evenly moist leaves without dripping.

[0111] (3) Data collection and statistical analysis

[0112] During the seedling raising process, the growth of the seedlings was regularly observed, and the number of dead seedlings was recorded until the end of the seedling raising period (30 days). The seedling survival rate was then calculated as (number of surviving seedlings / number of transplanted seedlings) × 100%. At the end of the seedling raising period, growth indicators such as plant height, stem diameter, and leaf number were measured in each treatment group to assess the effect of water-soluble fertilizer on seedling growth.

[0113] (4) Analysis of test results

[0114] As shown in Table 3, compared to the control water-soluble fertilizer without seaweed extract and chitin, the addition of either 0.2% seaweed extract or 1% chitin alone increased seedling survival rates by 80.0±2.5% and 78.0±2.8%, respectively, indicating that seaweed extract and chitin each have a certain promoting effect on seedling growth and survival. Furthermore, the seedling survival rate of the preferred water-soluble fertilizer treatment containing both 0.2% seaweed extract and 1% chitin significantly increased to 92.0±2.0%, indicating that the combination of the two has a synergistic effect, significantly improving the survival rate of irradiated soybean seedlings.

[0115] Table 3 Effects of water-soluble fertilizer on the survival rate of irradiated soybean seeds and seedlings

[0116] water-soluble fertilizer 1 2 3 4 Survival rate% 70.0±3.0 80.0±2.5 78.0±2.8 92.0±2.0

[0117] 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 soybean heavy ion beam radiation mutation breeding method, characterized in that: The following steps are involved: (1) 12 C 6+ Soybean seeds were irradiated using a radiation source with a radiation dose of 40-60 Gy and a radiation time of 100-140 s. (2) germinating and culturing the irradiated soybean seeds using a culture medium; the culture medium is supplemented with 10-50 mg / L of vitamin C and 10-30 g / L of mannitol; (3) After the irradiated soybean seeds germinate, they are transplanted into the seedling medium for seedling cultivation. During the seedling cultivation period, water-soluble fertilizer containing 0.1%-0.3% seaweed extract and 0.5%-2% chitosan is sprayed.

2. The breeding method according to claim 1, characterized in that Before the radiation treatment, the soybean seeds are disinfected and exposed to the sun; the disinfection treatment is as follows: first soaking in 75% alcohol for 30-60 seconds, then rinsing with sterile water 2-3 times, then soaking in 0.1% mercuric chloride solution for 5-10 minutes, and finally rinsing with sterile water 4-5 times; the exposure treatment is as follows: placing the disinfected seeds on moist sterile filter paper and pre-germinating and culturing in a dark, 25±2°C environment for 24-30 hours.

3. The breeding method according to claim 1, characterized in that The culture medium is MS culture medium.

4. The breeding method according to claim 1, characterized in that During the germination and cultivation stage, the temperature is 25-28°C, the humidity is 60%-70%, and the light intensity is 2000-3000Lux.

5. The breeding method according to claim 1, characterized in that The seedling culture medium is formed by mixing vermiculite, perlite and peat soil in a volume ratio of 1-2:1-2:1-2.

6. The breeding method according to claim 1, characterized in that The water-soluble fertilizer further comprises 15-25% N, 10-20% P2O5 and 15-25% K2O.

7. The breeding method according to claim 1 or 6, characterized in that The water-soluble fertilizer is sprayed once every 5-7 days until the leaves are evenly moistened and no water drips.

8. The breeding method according to claim 1, characterized in that During the seedling stage, the temperature is 20-25℃, the humidity is 60%-70%, and the light intensity is 3000-5000Lux.

9. The breeding method according to claim 1, characterized in that It also includes the screening and identification of mutants.

10. Use of the breeding method according to any one of claims 1 to 9 in soybean mutation breeding.

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