Soybean heavy ion beam radiation mutation breeding method and application thereof

Abstract

The invention provides a soybean heavy ion beam radiation mutagenesis method, and belongs to the technical field of crop breeding. The soybean mutation breeding method comprises the following steps: (1) performing radiation treatment on soybean seeds by taking 12C < 6 + > as a radiation source with the radiation dose of 40-60Gy and the radiation time of 100-140 seconds; (2) carrying out germination culture on the soybean seeds subjected to radiation treatment by utilizing a culture medium; 10-50 mg / L of vitamin C and 10-30 g / L of mannitol are added into the culture medium; and (3) after seedling emergence of the soybean seeds after radiation treatment, transplanting the soybean seeds into a seedling culture medium for seedling culture, and spraying a water-soluble fertilizer containing 0.1-0.3% of seaweed extract and 0.5-2% of chitin during seedling culture. By adjusting the radiation dosage and the radiation post-management mode, the germination rate of mutant seeds and the survival rate of seedlings are increased, stable expression of mutant characters is promoted, the breeding process is accelerated, and the breeding efficiency is improved.

Description

Technical Field

[0001] The 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 crop and cash crop, soybeans play a pivotal role in global agricultural production and the food industry. With the growth of population, the upgrading of consumption structure and the demand for sustainable agricultural development, higher requirements are placed on the improvement of soybean varieties, such as increasing yield, enhancing stress resistance, and improving quality (such as protein and oil content and composition).

[0003] Mutation breeding is one of the important ways to create new soybean germplasm resources and cultivate new varieties. Its principle is to induce gene mutations in soybeans through physical, chemical or biological factors, thereby producing individuals with new traits, and then obtaining excellent varieties through screening and cultivation. Heavy ion beam radiation mutagenesis is an emerging mutagenesis technology that has received widespread attention in the field of plant breeding in recent years. However, there are relatively few studies on the application of heavy ion beam radiation mutagenesis technology in soybean breeding, and a mature and systematic technical system has not yet been formed.

[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 directly germinated and raised 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 and weak seedling growth, which increases the risk of mutant loss.

[0005] Therefore, it is necessary to provide a more efficient, accurate and reliable mutagenesis 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 object 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 and promotes the stable expression of mutant traits by adjusting the radiation dose and post-irradiation management method.

[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 in that it comprises the following steps:

[0009] (1) 12 C 6+ The radiation source is 40-60 Gy, the radiation time is 100-140 s, and the soybean seeds are irradiated;

[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 soak in 75% alcohol for 30-60s, then rinse with sterile water 2-3 times, then soak in 0.1% mercuric chloride solution for 5-10min, and finally rinse with sterile water 4-5 times; the exposed to the sun treatment is: place the disinfected seeds on moist sterile filter paper, and pre-germinate and culture in a dark, 25±2℃ environment for 24-30h.

[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 also includes N15-25%, P 2 O 5 10-20% and K 2 O 15-25%.

[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 further comprises 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 mutagenesis intensity while avoiding excessive loss of seed vitality, 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: During the seedling raising period, water-soluble fertilizer containing seaweed extract and chitosan is sprayed. 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: A series of measures, from seed disinfection and whitening treatment before radiation treatment to germination culture and seedling management after radiation, 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+ The soybean seeds were irradiated with a radiation source, a radiation dose of 40-60 Gy, and a radiation time of 100-140 s.

[0027] Carbon ions have a higher linear energy transfer density (LET) and relative biological effect (RBE), which can cause more complex and diverse damage in the genome of soybean seeds, thereby inducing abundant gene mutations. Compared with other radiation sources, it can achieve higher mutagenesis efficiency at a lower dose, and has a certain targeting effect on mutagenesis of specific gene sites, increasing the probability of obtaining beneficial mutations.

[0028] The appropriate radiation dose can ensure sufficient mutagenic intensity to effectively mutate the genes of soybean seeds, but will not cause the seeds to completely lose vitality or produce too many severely damaged mutants that cannot survive due to excessive radiation dose. The present invention irradiates soybean seeds with 40-60Gy for 100-140s, so that a certain proportion of soybean seeds can survive and produce more mutations with potential breeding value, while the subsequent germination and growth process will not be excessively inhibited, which is conducive to screening out excellent mutants.

[0029] (2) Using a culture medium to germinate and culture the irradiated soybean seeds; 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 the germination medium, it can remove free radicals generated by radiation, reduce the oxidative damage of free radicals to biomacromolecules (such as DNA, protein, lipid, etc.) in seed cells, protect the integrity and function of cells, reduce the inhibitory effect of radiation on seed germination, and improve the germination rate and the initial growth vitality of seedlings. Mannitol, as an osmotic regulating substance, can regulate the osmotic pressure of cells in the intracellular environment of irradiated seed cells, prevent cells from losing water or absorbing too much water due to water imbalance caused by radiation, maintain cell turgor, protect cell structure and physiological function, promote cell elongation and division during seed germination, and is beneficial to the growth and development of the root system, laying a good foundation for the subsequent growth of seedlings.

[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 a variety of plant growth regulators, such as auxins, cytokinins, gibberellins and other analogs, as well as amino acids, polysaccharides, vitamins and other nutrients. During the seedling period, water-soluble fertilizers containing an appropriate amount of seaweed extracts are sprayed. These plant growth regulators can promote the division, elongation and differentiation of seedling cells, regulate the growth and development process of seedlings, make seedlings grow stronger, and enhance the stress resistance of seedlings (such as drought resistance, cold resistance, disease resistance, etc.). Nutrients such as amino acids and polysaccharides can directly provide nutrients for seedlings to supplement their growth needs. Chitin can induce plants to produce defense reactions, enhance plant immunity, stimulate plants to produce antibacterial substances such as phytoalexins and chitinases, and resist infection by pathogens. At the same time, chitin can also promote root growth, increase root surface area, and improve the root system's ability to absorb nutrients and water, which is beneficial to the growth and development of seedlings during the seedling stage, reduce seedling deaths caused by environmental stress and pests and diseases, and improve the success rate of mutant screening.

[0033] In the present invention, preferably, the soybean seeds are disinfected and exposed to the white before radiation treatment. Further preferably, the disinfection treatment is: first soak in 75% alcohol for 30-60s, then rinse with sterile water 2-3 times, then soak in 0.1% mercuric chloride solution for 5-10min, and finally rinse with sterile water 4-5 times; more preferably, first soak in 75% alcohol for 45s, then rinse with sterile water 3 times, then soak in 0.1% mercuric chloride solution for 7min, and finally rinse with sterile water 5 times. Disinfection creates a sterile environment for germination and growth of seeds after radiation treatment, reduces experimental errors and seedling deaths caused by microbial infection, and improves the success rate of breeding. Further preferably, the exposure treatment is: placing the disinfected seeds on moist sterile filter paper, pre-germination culture in a dark, 25±2℃ environment for 24-30h; more preferably, culture for 26h. The present invention first exposes the seeds and then performs radiation treatment. On the one hand, at this time, the seed cells are more sensitive to radiation, which can improve the mutagenesis effect; on the other hand, the seeds after the exposure 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, in step (2), preferably, 10-50 mg / L vitamin C and 10-30 g / L mannitol are added to the culture medium; more preferably, 30 mg / L vitamin C and 20 g / L mannitol are added; more preferably, the culture medium is MS culture medium.

[0036] In the present invention, in step (2), the temperature of 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, in the preferred step (3), the seedling medium is vermiculite, perlite and peat soil mixed 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 large in 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, has abundant nutrients (such as nitrogen, phosphorus, potassium and other macroelements and various trace elements), can provide continuous nutrient supply for seedling growth, and its water retention is also good, and the moisture condition of the matrix can be adjusted. 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 beneficial 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, in step (3), the water-soluble fertilizer preferably contains 0.2% seaweed extract and 1% chitosan, and more preferably the water-soluble fertilizer further comprises N15-25%, P 2 O 5 10-20% and K 2 O 15-25%, more preferably N20%, P 2 O 5 15% and K 2 O20%. It is preferred to spray water-soluble fertilizer once every 5-7 days until the leaves are evenly moistened and no water drips. A reasonable proportion of N, P 2 O 5 and K 2 O can meet the needs of soybean seedlings for a large number of elements during the seedling stage, promote the balanced growth of seedlings, improve the quality and stress resistance of 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 3000-5000Lux, more preferably 4000Lux, and the more preferred photoperiod is 10-14h light / 10-14h dark.

[0040] In the present invention, after the seedling raising stage is preferably completed, (4) screening and identifying mutants is also included. It is further preferred that during the growth of soybeans, phenotypic observations are carried out 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 mature period, agronomic traits such as plant height, number of pods, seed size, shape, and color are focused on. Comparison is made with control plants that have not been irradiated to screen out suspected mutants with obvious phenotypic differences. For suspected mutants, their leaf tissues are collected, genomic DNA is extracted, and SSR molecular marker technology is used for analysis to detect whether their DNA levels have changed, and further determine whether they are true mutants.

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

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] (1) Radiation mutagenesis

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

[0046] (2) Germination culture

[0047] MS culture medium was used, and vitamin C 10 mg / L and mannitol 10 g / L were added thereto. The irradiated seeds were inoculated on the culture medium and placed in a light incubator. The culture temperature was set to 25°C, the humidity was maintained at 60%, the light intensity was 2000 Lux, and the light cycle was 12h light / 12h dark. During the germination culture process, the seed germination was closely observed and the contaminated seeds were removed in time.

[0048] (3) Seedling management

[0049] When the irradiated soybean seeds germinate, they are transplanted into the seedling medium. The seedling medium is made of vermiculite, perlite and peat soil in a volume ratio of 1:1:1 and is sterilized at high temperature before use. During the seedling period, water-soluble fertilizer containing 0.1% seaweed extract and 0.5% chitosan is sprayed. The water-soluble fertilizer also contains 15% N, 2% P and 1% NH4OH. 2 O 5 10% and K 2 O 15%. Spray once every 5 days, and the spraying amount is based on the leaves being evenly moistened and no water dripping. During the seedling stage, the temperature is controlled at 20℃, the humidity is maintained at 60%, the light intensity is 3000Lux, and the photoperiod is 10h light / 14h dark.

[0050] (4) Screening and identification of mutants

[0051] During the growth of soybeans, phenotypic observations are carried out from the seedling stage. Plant height, leaf shape, color and other traits are recorded regularly. During the flowering period, the characteristics of flowering time, flower color, and flower shape are observed; during the maturity period, the focus is on agronomic traits such as plant height, number of pods, seed size, shape, and color. Compare with the non-irradiated "Zhonghuang 13" control plants to screen out suspected mutants with obvious phenotypic differences. For suspected mutants, their leaf tissues are collected, genomic DNA is extracted, and SSR molecular marker technology is used for analysis to detect whether their DNA levels have changed, and further determine whether they are true mutants.

[0052] Example 2

[0053] (1) Radiation mutagenesis

[0054] Select high-quality seeds of soybean variety "Heihe 43" for 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 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 the radiation source, the radiation dose of 50 Gy and the radiation time of 120 s.

[0055] (2) Germination culture

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

[0057] (3) Seedling management

[0058] After germination, the seedlings were transplanted into a seedling medium that was a mixture of vermiculite, perlite and peat in a volume ratio of 2:1:2. The medium was sterilized in advance. During the seedling period, water-soluble fertilizer containing 0.2% seaweed extract and 1% chitosan was sprayed. The water-soluble fertilizer also contained 20% N, P 2 O 5 15%, K 2 O 20%. Spray once every 6 days, and the spraying amount is based on the leaves being evenly moistened and no water dripping. The seedling temperature is maintained at 23℃, humidity is 65%, light intensity is 4000Lux, and the photoperiod is 12h light / 12h dark.

[0059] (4) Screening and identification of mutants

[0060] Same as Example 1.

[0061] Example 3

[0062] (1) Radiation mutagenesis

[0063] The seeds of soybean variety "Jiyu 47" were used as materials and sterilized first. The seeds were soaked in 75% alcohol for 45 seconds, rinsed with sterile water 3 times, soaked in 0.1% mercuric chloride solution for 8 minutes, and finally rinsed with sterile water 5 times. Then, in a dark, 25℃±2℃ environment, 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 a radiation time of 100 s.

[0064] (2) Germination culture

[0065] Use MS medium, add 50mg / L vitamin C and 30g / L mannitol. Place the irradiated seeds on the medium, and the culture environment temperature is 28℃, humidity is 70%, light intensity is 3000Lux, and the light cycle is 12h light / 12h dark. During this period, pay attention to adjusting the environmental parameters in the incubator to ensure normal germination of the seeds.

[0066] (3) Seedling management

[0067] After germination, the seedlings were transplanted into a sterilized seedling medium made of vermiculite, perlite and peat soil in a volume ratio of 1:2:1. When raising seedlings, water-soluble fertilizer containing 0.3% seaweed extract and 2% chitosan was sprayed. The water-soluble fertilizer also contained 25% N, P 2 O 5 20%, K 2 O 25%. Spray once every 7 days, with the leaves evenly moistened and no water dripping as the standard. During the seedling stage, the temperature was controlled at 25℃, the humidity was 70%, the light intensity was 5000Lux, and the photoperiod was 14h light / 10h dark.

[0068] (4) Screening and identification of mutants

[0069] Same as Example 1.

[0070] Example 4

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

[0072] (1) Experimental materials and preparation

[0073] The full seeds of soybean variety "Zhonghuang 13" were selected and surface disinfected. They were first soaked in 75% alcohol for 30 seconds, then rinsed with sterile water for 3 times, then soaked in 0.1% mercuric chloride solution for 7 minutes, and finally rinsed with sterile water for 5 times. The disinfected seeds were placed on moist sterile filter paper and pre-germinated and cultured in a dark, 25°C environment for 26 hours until they turned white.

[0074] (2) Radiation dose setting and processing

[0075] by 12 C 6+ As the radiation source, 7 different radiation dose gradients were set: 0Gy (as a control, no radiation treatment), 20Gy, 30Gy, 40Gy, 50Gy, 60Gy, 70Gy, irradiation for 2min. Four replicates were set for each dose treatment, and 50 seeds after whitening were used in each replicate. The whitening seeds were evenly placed in a special radiation sample box, and the seeds were irradiated according to the set dose using a professional heavy ion accelerator.

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

[0077] The irradiated seeds were sown in seedling trays containing a mixed matrix of vermiculite and peat soil (volume ratio 1:1), the matrix was kept moist, the temperature was controlled at 25°C, the light intensity was 3000 Lux, and the photoperiod was 12h light / 12h dark. Starting from the third day after sowing, the number of germinated seeds in each treatment group was counted every day until the 10th day, and the germination rate was calculated. Germination rate = (number of germinated seeds / number of test seeds) × 100%.

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

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

[0080] (5) Test results

[0081] The regression equation y = 8.53-0.07x was established. When y = 5, the median lethal dose was 50.43 Gy. As shown in Table 1, the germination rate of the 50 Gy dose group was 52.0 ± 1.6%, close to the median lethal dose. At this dose, although seed germination and seedling growth were inhibited to a certain extent, more obvious and diverse mutant traits appeared, providing a rich resource for the subsequent screening of excellent mutants. Therefore, 50 Gy can be used as a reference for the appropriate dose for soybean heavy ion beam radiation mutagenesis 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) Experimental materials and preparation

[0088] The seeds of soybean variety "Zhonghuang 13" were selected and disinfected (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 debonded (cultured on moist sterile filter paper in a dark, 25°C environment for 26 hours). 12 C 6+ The 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 for each medium, and 30 irradiated seeds were inoculated in each replicate.

[0095] (3) Germination culture and data collection

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

[0097] (4) Analysis of test results

[0098] As shown in Table 2, compared with the MS basic medium (control), the medium with 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 germination rate of the medium with 20 g / L of mannitol was 72.0 ± 2.8%, indicating that mannitol also has a positive effect on seed germination. And the preferred medium with 30 mg / L of vitamin C and 20 g / L of mannitol added at the same time, the germination rate significantly increased 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) Experimental materials and preparation

[0104] The seeds of soybean variety "Zhonghuang 13" were used as materials. After disinfection (immersion in 75% alcohol for 45 seconds, washing with sterile water for 3 times, soaking in 0.1% mercuric chloride solution for 8 minutes, and washing with sterile water for 5 times) and de-whitening (incubation on moist sterile filter paper in a dark environment at 25℃ for 26 hours), the seeds were sterilized and then washed with water for 10 minutes. 12 C 6+ The seeds were irradiated with a radiation dose of 50 Gy and a radiation time of 120 s. The irradiated seeds were germinated and cultured on MS medium (added with 30 mg / L vitamin C and 20 g / L mannitol), and after germination, they were transplanted into seedling medium (vermiculite, perlite and peat soil mixed in a volume ratio of 1:1:2).

[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, only contains N 20%, P 2 O 5 15%, K 2 O 20% (basic macro-element water-soluble fertilizer).

[0108] Water-soluble fertilizer 2: Contains 0.2% seaweed extract, no chitin, and contains 20% N, 20% P 2 O 5 15%, K 2 O 20%.

[0109] Water-soluble fertilizer 3: contains chitosan 1%, no seaweed extract, and contains N 20%, P 2 O 5 15%, K 2 O20%.

[0110] Water-soluble fertilizer 4: contains 0.2% seaweed extract and 1% chitosan, as well as 20% N, 20% P 2 O 5 15%, K 2 O20%. Each water-soluble fertilizer treatment was set up with 3 replicates, and 20 seedlings were transplanted in each replicate. During the seedling raising period, water-soluble fertilizer was sprayed once every 6 days, and the spraying amount was based on the leaves being evenly moistened and no water dripping.

[0111] (3) Data collection and statistical analysis

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

[0113] (4) Analysis of test results

[0114] As shown in Table 3, compared with the control water-soluble fertilizer without seaweed extract and chitosan, adding seaweed extract 0.2% or chitosan 1% alone can improve the survival rate of seedlings, reaching 80.0±2.5% and 78.0±2.8%, respectively, indicating that seaweed extract and chitosan each have a certain promoting effect on the growth and survival of seedlings. The survival rate of seedlings in the preferred water-soluble fertilizer treatment group containing both seaweed extract 0.2% and chitosan 1% was significantly increased to 92.0±2.0%, indicating that the combination of the two has a synergistic effect and can greatly improve the survival rate of soybean seedlings after radiation.

[0115] Table 3 Effect of water-soluble fertilizer on the survival rate of irradiated soybean seed 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 principle of the present invention. These improvements and modifications should also be regarded as 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+ The radiation source is 40-60 Gy, the radiation time is 100-140 s, and the soybean seeds are irradiated; (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 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.

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 for 2-3 times, then soaking in 0.1% mercuric chloride solution for 5-10 minutes, and finally rinsing with sterile water for 4-5 times; the exposed to the sun treatment is as follows: placing the disinfected seeds on moist sterile filter paper, and pre-germinating and culturing them in a dark environment at 25±2°C 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 culture stage, the temperature is 25-28° C., the humidity is 60%-70%, and the light intensity is 2000-3000 Lux.

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 also includes N15-25%, P2O510-20% and K2O 15-25%.

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 raising stage, the temperature is 20-25° C., the humidity is 60%-70%, and the light intensity is 3000-5000 Lux.

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

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

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