A method for improving the ems mutagenic effect of barley

Abstract

The present application belongs to the technical field of barley genetic breeding, and provides a method for improving the EMS mutagenesis effect of barley, comprising the following steps: detoxifying the barley seeds after EMS mutagenesis by using sodium thiosulfate to obtain the detoxified barley seeds; soaking the detoxified barley seeds by using cell division factor and gibberellin; and performing seed germination after the soaking is completed. The method of the present application is synergistically coordinated in multiple steps, significantly improves the mutation frequency and mutation type richness of EMS mutagenesis, reduces the damage of mutagenesis to the viability of barley seeds, improves the acquisition rate of effective mutants, is simple and easy to operate, and has low cost, and can be widely applied to barley genetic breeding and mutant library construction, and provides efficient technical support for barley mutant library construction and excellent variety breeding.

Description

Technical Field

[0001] This invention belongs to the field of barley genetic breeding technology, specifically relating to a method for improving the EMS mutagenesis effect in barley. Background Technology

[0002] Ethyl methanesulfonate (EMS), a commonly used chemical mutagen, boasts advantages such as high mutagenicity, diverse mutation types, and low chromosomal aberration rate, making it widely applied in the creation of mutants and genetic breeding of crops like barley. Its core mechanism of action involves alkylation of DNA bases, initiating base transitions (primarily G–A or C–T transitions) to generate heritable gene mutations, thereby obtaining mutants with superior traits. This provides abundant germplasm resources for barley functional genomics research and genetic breeding.

[0003] Currently, existing barley EMS mutagenesis technology only optimizes the concentration and time of the mutagenesis stage, completely lacking a standardized post-mutation treatment system, which is the core problem of poor mutagenesis results. Without post-mutation detoxification treatment, residual EMS continues to poison the seeds. Simultaneously, the lack of seedling growth control measures leads to the death of many materials containing mutation sites due to growth inhibition, resulting in an extremely low survival rate of effective mutants and a mutation frequency generally below 5%, failing to meet the needs of large-scale mutant screening. Due to the lack of post-treatment methods, it is difficult to balance mutagenesis efficiency and seed vigor; simply adjusting mutagenesis parameters cannot fundamentally improve the problem of low mutagenesis yield.

[0004] To address the shortcomings of existing technologies, there is an urgent need for a barley EMS mutagenesis method that is systematic, easy to operate, and has a significant mutagenesis effect. Through multi-step synergistic optimization, this method can improve the mutation frequency while protecting seed vigor, thus solving the problems of low mutagenesis efficiency and significant seed damage in existing methods. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the EMS mutagenesis effect in barley. The method described in this invention increases the mutation frequency and mutation type richness of EMS mutagenesis, while reducing the damage to barley seed vigor caused by mutagenesis, providing efficient technical support for the construction of barley mutant libraries and the breeding of superior varieties.

[0006] This invention provides a method for enhancing the EMS mutagenesis effect in barley, comprising the following steps: Barley seeds induced by EMS were detoxified using sodium thiosulfate to obtain detoxified barley seeds. The detoxified barley seeds were soaked in cytokinin and gibberellin, and then the seeds were germinated after soaking.

[0007] As a preferred embodiment, the sodium thiosulfate has a mass concentration of 0.5% to 1.5%, the detoxification treatment time is 0.5 to 1.0 h, and the detoxification treatment temperature is 24 to 26 °C.

[0008] As a preferred embodiment, the concentration of the cytokinin is 0.1~1.0 mg / L; the concentration of the gibberellin is 45~55 mg / L.

[0009] As a preferred embodiment, the soaking time is 24-36 hours and the soaking temperature is 24-26°C.

[0010] As a preferred embodiment, the method for preparing EMS-mutated barley seeds includes the following steps: mixing barley seeds that have sprouted and EMS mutagenesis solution, and then inducing mutagenesis culture to obtain EMS-mutated barley seeds.

[0011] As a preferred embodiment, the mass concentration of the EMS mutagenesis solution is 0.4%~0.5%, and the volume-to-mass ratio of the EMS mutagenesis solution to the seed is 4~6 mL:1 g.

[0012] As a preferred embodiment, the mutagenesis culture time is 8-18 hours, and the mutagenesis culture temperature is 24-26℃.

[0013] As a preferred embodiment, the method for preparing barley seeds after they have sprouted includes the following steps: soaking the barley seeds to achieve a seed moisture content of 35% to 40%, and then culturing them in the dark after the soaking process. The temperature for soaking the barley seeds is 24~26℃.

[0014] As a preferred embodiment, the seed germination temperature is 24~26℃, the seed germination light intensity is 2000~3000lx, and the seed germination light cycle is 16h light / 8h darkness.

[0015] As a preferred option, the barley seed variety includes "Ganpi 8".

[0016] Beneficial Effects: This invention provides a method for improving the EMS mutagenesis effect in barley, comprising the following steps: detoxifying EMS-mutated barley seeds with sodium thiosulfate to obtain detoxified barley seeds; soaking the detoxified barley seeds in cytokinin and gibberellin, followed by seed germination after soaking. The method of this invention utilizes multiple synergistic steps to significantly increase the mutation frequency and mutation type richness of EMS mutagenesis, reduce the damage to barley seed viability caused by mutagenesis, and improve the yield of effective mutants. It is simple to operate, low in cost, and can be widely applied to barley genetic breeding and mutant library construction, providing efficient technical support for the construction of barley mutant libraries and the selection of superior varieties.

[0017] Compared with the prior art, the present invention also has the following beneficial effects: 1. Significantly enhanced mutagenic effect: Through multi-step synergistic optimization, the seed whitening treatment activates embryonic cell metabolism, enhancing the seed's sensitivity to EMS mutagenesis. Combined with the synergistic regulation of cytokinins and gibberellins and thorough detoxification with sodium thiosulfate, the mutation frequency of barley EMS mutagenesis is increased to 8%~12%, as in the example where the mutation frequency reaches 10.2%, far exceeding the less than 5% of the comparative mutagenesis method (only 3.9% in the comparative example), representing an increase of 6.3 percentage points in mutation frequency. At the same time, the mutation types are more diverse, covering various mutation types such as leaf color, plant height, tiller number, ear size, and growth period. The proportion of various mutant lines in the example is balanced, effectively solving the problems of low mutagenesis efficiency and single mutation type in conventional methods.

[0018] 2. Simple operation and low cost: All processing steps use conventional equipment and reagents, without the need for professional and complicated instruments. The steps of seed selection, cleaning, soaking, and mutagenesis are simple to operate and easy to master. Reagents such as 1% sodium thiosulfate and gibberellin are inexpensive and readily available, and can be promoted and applied on a large scale. They are suitable for the actual needs of barley breeding, especially for grassroots breeding units and large-scale breeding scenarios.

[0019] 3. Minimal damage to seed viability: Immersion in 1% sodium thiosulfate completely inactivates residual EMS, preventing its continued damage to seeds; combined with hormone treatment, it alleviates the damage to embryo cells caused by mutation, maintaining a seed germination rate of over 85% after mutation (reaching 88.67% in the example), significantly better than the comparative mutation method (germination rate is usually below 70%, 68.33% in the comparative example). At the same time, the seedling survival rate is increased to over 80% (reaching 84.9% in the example), an increase of more than 25 percentage points compared to the comparative method (59.25%), effectively reducing mutation costs and improving the economics of mutation.

[0020] 4. High versatility and wide range of applications: This method is applicable to barley seeds. When using this method for EMS mutagenesis, parameters such as EMS concentration and soaking time can be fine-tuned according to the characteristics of different varieties. It has strong adaptability and can be widely used in the fields of barley mutant library construction, screening of superior traits, and functional gene research, providing efficient technical support for barley genetic breeding. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1Images of mutant barley obtained by the mutagenesis method of this invention are shown below; where A is barley with white striped leaves obtained by mutagenesis; B is barley with albino seedlings obtained by mutagenesis; C is barley with multiple stem nodes obtained by mutagenesis; D is barley with yellow mosaic leaves obtained by mutagenesis; E is barley with mosaic leaves obtained by mutagenesis; F is the control spike of "Ganpi 8"; G is barley with early-maturing straight spikes obtained by mutagenesis; H is barley with awns obtained by mutagenesis; and I is barley with dense spikes obtained by mutagenesis. Figure 2 This is an image of the mature spikelet of mutant barley obtained by the mutagenesis method of this invention. Detailed Implementation

[0023] This invention provides a method for enhancing the EMS mutagenesis effect in barley, comprising the following steps: Barley seeds induced by EMS were detoxified using sodium thiosulfate to obtain detoxified barley seeds. The detoxified barley seeds were soaked in cytokinin and gibberellin, and then the seeds were germinated after soaking.

[0024] This invention first detoxifies EMS-mutated barley seeds with sodium thiosulfate to obtain detoxified barley seeds.

[0025] The sodium thiosulfate of this invention has a mass concentration of 0.5% to 1.5%, the detoxification treatment time is 0.5 to 1.0 h, and the detoxification treatment temperature is 24 to 26 °C. As one specific embodiment, the mass concentration of the sodium thiosulfate can be any value from 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%, or an intermediate value of any two of these values. As one specific embodiment, the detoxification treatment time can be any value from 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, and 1.0 h, or an intermediate value of any two of these values. As one specific embodiment, the detoxification treatment temperature can be any value from 24 °C, 25 °C, and 26 °C, or an intermediate value of any two of these values. Experimental examples of this invention show that soaking in sodium thiosulfate is significantly more effective than simple rinsing. In the rinsing group, the average germination potential at 3 days was 48.3%, and the average germination rate at 5 days was 50.7%, both the lowest among all groups. In the soaking group, the germination potential (78.0%) and germination rate (88.67%) of seeds soaked for 1 hour both reached their peak values. Soaking for 0.5 hours was insufficient for detoxification, resulting in a significant decrease in germination indicators. While extending the soaking to 1.5 hours ensured a relatively high final germination rate (84.0%), it delayed early germination, reducing the germination potential to 72.7%. Therefore, soaking in 1% sodium thiosulfate for 1 hour after mutagenesis is the optimal detoxification treatment for this system.

[0026] The method for preparing barley seeds after EMS mutagenesis according to the present invention includes the following steps: mixing barley seeds after they have sprouted and EMS mutagenesis solution, and then mutagenesis culture to obtain barley seeds after EMS mutagenesis.

[0027] The method for preparing barley seeds after sprouting according to the present invention includes the following steps: soaking the barley seeds to achieve a seed moisture content of 35% to 40%; after soaking, culturing the seeds in the dark until more than 70% sprout; the soaking temperature is 24 to 26°C. As a specific embodiment, after soaking, the seed moisture content can reach any value among 35%, 36%, 37%, 38%, 39%, and 40%, or any midpoint between any two of these values. As a specific embodiment, the soaking temperature can be any value among 24°C, 25°C, and 26°C, or any midpoint between any two of these values. The embodiments of the present invention show that under the treatment conditions of soaking seeds for 8 hours to achieve a water absorption of 35.8% and soaking seeds for 9 hours to achieve a water absorption of 40%, the germination rate of the seeds reaches its maximum value of 98.67% and 98% after 5 days, respectively. In the present invention, seed sprouting can activate seed metabolism and enhance mutagenicity.

[0028] Before the barley seeds undergo the whitening process, the method includes seed selection and washing. In a specific embodiment of the invention, the seed selection method includes: selecting plump, undamaged, disease-free, and uniformly sized barley seeds; removing shriveled, broken, insect-damaged seeds and impurities to ensure seed purity ≥98% and a thousand-grain weight variation coefficient ≤5%. The selected seeds are then placed in a cool, ventilated place to dry for 2-3 hours, maintaining a seed moisture content below 12%. This avoids uneven mutagenesis caused by differences in seed moisture content, laying a uniform physiological foundation for subsequent mutagenesis treatment and enhancing the seeds' sensitivity to EMS mutagenesis. As a specific implementation, the drying time for the selected seeds in a cool, ventilated place can be any value from 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, and 3h, or any two values ​​in between. The seed selection method of the present invention lays the foundation for mutagenesis and improves the uniformity of mutagenesis. In a specific embodiment of the present invention, the seed cleaning method includes: placing the selected barley seeds in a clean container and rinsing them 3-4 times with distilled water, each rinse lasting 1-2 minutes, to thoroughly remove dust, impurities, and residual seed coat debris from the seed surface; then soaking them in sterile water for 10-15 minutes to further clean the seed surface, draining the surface water before use. As one specific embodiment, the number of times the seeds are rinsed with distilled water can be 3 or 4 times. As one specific embodiment, the rinsing time for each rinse can be any value from 1 minute, 1.1 minutes, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, 1.6 minutes, 1.7 minutes, 1.8 minutes, 1.9 minutes, and 2 minutes, or an intermediate value of any two of these values. As one specific embodiment, the subsequent soaking time in sterile water can be any value from 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, and 15 minutes, or an intermediate value of any two of these values. The seed cleaning step described in this invention can prevent impurities on the seed surface from reacting with EMS, reduce the interference of impurities on the mutagenesis effect, and at the same time ensure that the seed surface is clean, so that EMS can contact the seed surface evenly and improve the uniformity of mutagenesis.

[0029] The EMS mutagenesis solution of this invention has a mass concentration of 0.4%~0.5%, a volume-to-mass ratio of EMS mutagenesis solution to seeds of 4~6 mL:1 g, a mutagenesis culture time of 8~18 h, and a mutagenesis culture temperature of 24~26 °C. As a specific embodiment, the mass concentration of the EMS mutagenesis solution can be any value from 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, and 0.5%, or an intermediate value of any two of these values. As a specific embodiment, the volume-to-mass ratio of the EMS mutagenesis solution to seeds can be 4 mL:1 g, 5 mL:1 g, or 6 mL:1 g. As a specific embodiment, the mutagenesis treatment time can be any value from 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, and 18 h, or an intermediate value of any two of these values. In one specific implementation, the temperature for the mutagenesis culture can be any value among 24℃, 25℃, and 26℃, or an intermediate value between any two of these values. The EMS mutagenesis treatment step described in this invention is the core mutagenesis step, through which the mutagenesis intensity can be controlled.

[0030] After obtaining the detoxified barley seeds, the detoxified barley seeds were soaked in cytokinin and gibberellin, and then the seeds were germinated after soaking.

[0031] The concentration of the cytokinin described in this invention is 0.1~1.0 mg / L; the concentration of the gibberellin is 45~55 mg / L; the soaking time is 24~36 h; and the soaking temperature is 24~26 °C. As a specific embodiment, the concentration of the cytokinin can be any value selected from 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, and 1.0 mg / L, or an intermediate value between any two of these values. As a specific embodiment, the soaking time can be any value selected from 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, and 36 h, or an intermediate value between any two of these values. As one specific implementation method, the soaking temperature can be any value among 24℃, 25℃, and 26℃, or an intermediate value between any two of these values. Experimental examples of this invention show that during the 30min-12h gibberellin treatment process, hormone absorption is insufficient, dormancy-breaking effect is poor, and germination is uneven. 24h gibberellin treatment can effectively break dormancy, significantly improving germination potential and germination rate. Seven days after gibberellin treatment, seeds are prone to oxygen deficiency and seed rot, radicle deformities, and increased rot rate. The CTK and GA combination group (T1) achieved the best results in all indicators: germination potential 78.00%, germination rate 88.67%, seedling height 11.43 cm, taproot length 10.50 cm, single-plant leaf fresh weight 0.11g, and single-plant root fresh weight 0.093g. The two hormones exhibit a significant synergistic effect, simultaneously repairing seed vigor damage caused by mutagenesis and promoting balanced growth of above-ground stems and leaves and underground roots, making it the optimal germination-promoting hormone combination under this mutagenesis system.

[0032] The seed germination temperature of the present invention is 24-26℃, the light intensity for seed germination is 2000-3000 lx, and the photoperiod for seed germination is 16 hours of light / 8 hours of darkness. As one specific embodiment, the seed germination temperature can be any value among 24℃, 25℃, and 26℃, or an intermediate value between any two of these values. As one specific embodiment, the light intensity for seed germination can be any value among 2000 lx, 2100 lx, 2200 lx, 2300 lx, 2400 lx, 2500 lx, 2600 lx, 2700 lx, 2800 lx, 2900 lx, and 3000 lx, or an intermediate value between any two of these values.

[0033] This invention involves all treatments performed at 24-26℃. In one specific embodiment, the barley seed variety includes "Ganpi 8". Experimental examples of this invention show that at 20-25℃, seed germination potential, germination rate, leaf length, and root length all significantly increase with increasing treatment temperature. A low temperature of 20℃ severely inhibits seed germination and seedling development, with a germination potential of only 32.70% and a germination rate of only 39.30%. A moderate temperature of 23℃ can slightly improve germination, but the optimization effect is limited. Under a treatment temperature of 25℃ throughout the entire process, the seed germination potential is 78.00%, the germination rate is 88.67%, the leaf length is 11.43cm, and the root length is 10.50cm. All four indicators are optimal for each group, significantly alleviating the damage to seeds caused by mutation, simultaneously improving germination uniformity, total seedling emergence, and cultivating strong seedlings. This is the preferred treatment temperature for the mutation process of this invention.

[0034] To further illustrate the present invention, the following detailed description of a method for improving the EMS mutagenesis effect in barley, in conjunction with embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0035] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0036] Example 1: A method for enhancing the EMS mutagenesis effect in barley This embodiment uses the malting barley variety "Ganpi No. 8" as the experimental material. This variety is of high quality, disease-resistant, and lodging-resistant. It was bred by the Gansu Academy of Agricultural Sciences and is the main cultivated variety in the local area. The specific implementation steps are as follows: Step 1: Seed Selection "Ganpi No. 8" barley seeds were selected, and shriveled, broken, insect-eaten, and impurity grains were manually removed. Plump, undamaged, and uniformly sized seeds were selected. The seed purity was tested to be 99.2%, and the coefficient of variation for thousand-grain weight was 3.8%. The selected seeds were then placed in a cool and ventilated place to dry for 2.5 hours to keep the seed moisture content below 12%.

[0037] Step 2: Seed washing Place the selected seeds in a clean beaker and rinse them four times with distilled water for 1.5 minutes each time to thoroughly remove dust and impurities from the seed surface. Then soak them in sterile water for 12 minutes and drain off the surface water for later use.

[0038] Step 3: Seed sprouting treatment Place the cleaned seeds in a sterile petri dish, add an appropriate amount of sterile water (3 cm above the seeds), and soak at 25°C in the dark for 8 hours, stirring once every 2 hours. After soaking, pour off the excess water, place a layer of moist sterile filter paper at the bottom of the petri dish, and continue to cultivate at 25±1°C in the dark until more than 70% of the seeds show signs of sprouting.

[0039] Step 4: EMS mutagenesis treatment Prepare an EMS mutagenesis solution with a mass concentration of 0.5% (using distilled water as the solvent), adjust the pH value to 7.1 with NaOH, and place the seeds that have sprouted into the EMS mutagenesis solution. The volume ratio of the mutagenesis solution to the seed mass is 5 mL: 1 g. Place the solution on a shaker at 90 r / min and shake for 16 h. During this period, shake the beaker once every 2 h to ensure that the seeds and the mutagenesis solution are in full contact.

[0040] Step 5: Sodium thiosulfate detoxification treatment After the mutagenesis was completed, the mutagenesis solution was recovered, and a 1% sodium thiosulfate solution was immediately poured into the seeds. The seeds were then soaked at 25±1℃ for 1 hour, with stirring once during the process.

[0041] Step 6: Synergistic treatment with cytokinins and gibberellins After detoxification, the seeds were removed and rinsed quickly with sterile water 6 times, 2 minutes each time. Then they were placed in a solution of 0.1 mg / L cytokinin (CK) + 50 mg / L gibberellin (GA) and soaked at 25±1℃ for 24 hours, with gentle stirring 3 times during the process.

[0042] Step 7: Subsequent cultivation After treatment, the seeds were removed, rinsed four times with sterile water, drained, placed in a petri dish lined with filter paper, and 6 ml of sterile water was added. The dish was then cultured at 25±1℃, with a light intensity of 3000 lx and a photoperiod of 16 h light / 8 h dark.

[0043] Comparative Example 1 A barley EMS mutagenesis method similar to Example 1 is described, except that after the EMS mutagenesis treatment in step 4, the seeds are rinsed 3 times with 1% sodium thiosulfate and then quickly rinsed 6 times with sterile water for 2 minutes each time to ensure that no treatment liquid remains on the seed surface. Subsequently, the seeds are placed in a gibberellin (GA) solution with a mass concentration of 50 mg / L and soaked at 25±1℃ for 24 hours, during which time they are gently stirred 2 to 3 times.

[0044] Experimental Example 1 In Example 1 and Comparative Example 1, germination potential was recorded on day 3 of subsequent culture, germination rate was recorded on day 5, and germination index, shoot length, root length, shoot fresh weight, root fresh weight, total number of surviving individuals, total number of mutants, mutation frequency, and mutant survival rate were statistically analyzed on day 7. Except for the total number of treated seeds, all other data in this experiment are averages from three replicates. The calculation formulas for the relevant indicators are as follows: Germination rate (%) = (Total number of germinated seeds on day 5 of culture / Total number of seeds) × 100%; Germination index = ∑Gt / Dt.

[0045] In the formula: Gt is the number of germinations on day t, and Dt is the corresponding number of germination days.

[0046] Bud length (cm) = Average bud length on day 7 of culture; Root length (cm) = Average root length on day 7 of culture; Fresh bud weight (g / plant) = Average fresh bud weight on day 7 of culture; Fresh root weight (g / plant) = Average fresh root weight on day 7 of culture; Mutation frequency (%) = Total number of mutant plants / Total number of surviving plants after treatment × 100%; Mutant survival rate (%) = Number of surviving mutants / Total number of mutants × 100%.

[0047] The results are shown in Table 1: In Example 1, after transplanting to the seedling substrate and normal cultivation, the seedling survival rate was 84.9%. When the seedlings reached the 4-leaf stage, the average plant height was 6.8 cm, and the average number of tillers was 1.9. During the subsequent growth period, mutant screening was conducted, and a total of 128 mutant lines were screened, with a mutation frequency of 10.2%. Among them, 42 plants (32.8%) had leaf color mutations, including yellowing, whitening, and striped leaves; 35 plants (27.3%) had plant height mutations, including dwarfing (plant height ≤ 4 cm) and tall (plant height ≥ 10 cm); 28 plants (21.9%) had tiller number mutations, including multiple tillers (≥ 3 tillers) and few tillers (0 tillers); and 23 plants (18.0%) had ear and growth period mutations, including ear type variation, and early or late heading (see images of different mutation types). Figure 1 See the image of the induced mature spikelet. Figure 2 Furthermore, the seedling malformation rate was only 3.8%, with the main type of malformation being leaf curling, and no embryo necrosis.

[0048] Comparative Example 1 showed that the seed germination rate was only 68.33%, and the seedling survival rate was 59.25%. When the seedlings reached the 4-leaf stage, the average plant height was 4.2 cm, and the average number of tillers was 1.1, indicating a significantly weaker growth pattern than in Example 1. Only 25 mutant lines were screened, with a mutation frequency of 3.9%, significantly lower than the 10.2% in Example 1. The mutation type was singular, with only leaf color mutations observed; no mutations related to plant height, number of tillers, ear size, or growth period were found. The seedling malformation rate was as high as 18.5%, with 12 seedlings exhibiting embryo necrosis and 8 seedlings exhibiting stem malformation in addition to leaf curling.

[0049] Table 1 Comparison of mutation effects between the present invention and Comparative Example 1

[0050] Note: Except for the total number of seeds treated, all other data in this experiment are the average of three replicates. The test seeds were the variety "Ganpi 8" bred by the Gansu Academy of Agricultural Sciences, which is the main barley variety cultivated in the area. Different lowercase letters in the table represent significant differences.

[0051] Experiment Example 2 This experiment set up 4 groups to verify the effects of different methods of sodium thiosulfate termination treatment on the germination potential and germination rate of mutagenic seeds.

[0052] Steps: Take the seeds from step 4 of Example 1 after EMS mutagenesis treatment and treat them with sodium thiosulfate solutions in different ways: Experimental Group 1: Soaking in 1% sodium thiosulfate solution for 0.5 h was the final treatment. The treated seeds were placed in a container of 1% sodium thiosulfate solution and soaked for 0.5 h at a temperature of 25±1℃, with one stirring during the soaking period to ensure full contact between the solution and the seeds. The seeds were then rinsed quickly 6 times with sterile water for 2 min each time to ensure no residual treatment liquid was removed from the seed surface. Subsequently, the seeds were soaked in a compound solution of 0.1 mg / L cytokinin (CK) + 50 mg / L gibberellin (GA) for 24 h at a temperature of 25±1℃, with three gentle stirrings during the soaking period. After the hormone treatment, the seeds were removed, rinsed 4 times with sterile water, and drained. The seeds were then placed in a petri dish lined with sterile filter paper, and 6 ml of sterile water was added. The dish was cultured at 25±1℃, with a light intensity of 3000 lx and a photoperiod of 16 h light / 8 h dark. Results were recorded on the third and fifth days.

[0053] Experimental Group 2: The treated seeds were placed in a container containing a 1% sodium thiosulfate solution and soaked for 1 hour. The remaining steps were the same as in Experimental Group 1.

[0054] Experimental Group 3: The treated seeds were placed in a container containing a 1% sodium thiosulfate solution and soaked for 1.5 hours. The remaining steps were the same as those in Experimental Group 1.

[0055] Experimental Group 4: The treated seeds were simply rinsed 3 times with 1% sodium thiosulfate, and the remaining steps were the same as in Experimental Group 1.

[0056] This experiment compared the effects of different termination treatments using 1% sodium thiosulfate on the germination of mutagenic seeds. The results (Table 2) showed that soaking in sodium thiosulfate was significantly more effective than rinsing alone. The average germination potential at 3 days (48.3%) and average germination rate at 5 days (50.7%) in the rinsing group were the lowest among all groups. In the soaking group, the germination potential (78.0%) and germination rate (88.67%) of seeds soaked for 1 hour both reached their peak values. Soaking for 0.5 hours was insufficient for detoxification, resulting in a significant decrease in germination indicators. While extending the soaking to 1.5 hours maintained a high germination rate (84.0%), it delayed early germination, reducing the germination potential to 72.7%. In conclusion, soaking in 1% sodium thiosulfate for 1 hour after mutagenesis was the optimal termination treatment for this system.

[0057] Table 2 Termination Test of Sodium Thiosulfate Note: Different lowercase letters in the table represent significant differences.

[0058] Experimental Example 3 This experiment demonstrates the effects of gibberellin (CA) treatment and cytokinin (CTK) treatment alone on germination parameters in barley seeds: Procedure: After the seeds were detoxified with sodium thiosulfate solution in Example 1, they were quickly rinsed 6 times with sterile water for 2 minutes each time to ensure that no treatment liquid remained on the seed surface. Experiments were conducted using the following hormones for different treatment times: T1: Barley seeds were treated with gibberellin (CA) alone and cytokinin (CTK) alone for 30 min.

[0059] The treated seeds were placed in a gibberellin (GA) solution with a mass concentration of 50 mg / L and soaked for 30 min at a soaking temperature of 25 ± 1 °C, with gentle stirring 2 to 3 times during the soaking period, to obtain seeds treated with gibberellin alone.

[0060] The treated seeds were placed in a 0.1 mg / L cytokinin (CK) solution and soaked for 30 min at a temperature of 25 ± 1 °C, with gentle stirring 2 to 3 times during the soaking period, to obtain seeds treated with cytokinin alone.

[0061] After treatment, remove the seeds, rinse them four times with sterile water, drain the surface water, and place them in labeled petri dishes lined with sterile filter paper. Add 6 ml of sterile water and incubate them at 25±1℃, with a light intensity of 3000 lx and a photoperiod of 16 h light / 8 h dark until the 5th day of germination.

[0062] T2: Barley seeds were treated with gibberellin (CA) alone and cytokinin (CTK) alone for 12 h.

[0063] The seeds were soaked in a 50 mg / L gibberellin (GA) solution for 12 hours, and the subsequent steps were the same as for “T1 treatment”.

[0064] The treated seeds were soaked in a 0.1 mg / L cytokinin (CK) solution for 12 hours, and the subsequent steps were the same as for “T1 treatment”.

[0065] T3: Barley seeds were treated with gibberellin (CA) alone and cytokinin (CTK) alone for 24 hours.

[0066] The treated seeds were placed in a gibberellin (GA) solution with a mass concentration of 50 mg / L and soaked for 24 h. Subsequent steps were the same as for “T1 treatment”.

[0067] The treated seeds were placed in a 0.1 mg / L cytokinin (CK) solution and soaked for 24 h. Subsequent steps were the same as for “T1 treatment”.

[0068] T4: Barley seeds were treated with gibberellin (CA) alone and cytokinin (CTK) alone for 7 days.

[0069] The seeds were soaked in a 50 mg / L gibberellin (GA) solution for 7 days, and the subsequent steps were the same as for “T1 treatment”.

[0070] The treated seeds were soaked in a 0.1 mg / L cytokinin (CK) solution for 7 days, and the subsequent steps were the same as for "T1 treatment".

[0071] Germination results analysis (Table 3): During the 30-12h treatment with gibberellin and cytokinin alone, insufficient hormone absorption resulted in poor dormancy breaking and uneven germination. During the 24h treatment with gibberellin and cytokinin alone, dormancy was effectively broken, and the germination rate was significantly improved. Seven days after treatment with gibberellin and cytokinin alone, seeds were prone to oxygen deficiency and seed rot, resulting in deformed radicles and an increased rate of decay.

[0072] Table 3. Effects of gibberellin (CA) and cytokinin (CTK) treatment time alone on seed germination. Note: Different lowercase letters in the table represent significant differences.

[0073] Experiment Example 4 This experimental example sets up four processes: Procedure: After the seeds from Example 1 were mutagenized and detoxified with sodium thiosulfate solution, they were quickly rinsed 6 times with sterile water for 2 minutes each time to ensure that no treatment liquid remained on the seed surface. Then, they were treated with the following different hormones: Blank control: The treated seeds were then placed directly into a petri dish lined with sterile filter paper, 6 ml of sterile water was added, and the dish was cultured at 25±1℃, with a light intensity of 3000 lx and a photoperiod of 16 h light / 8 h dark until the seeds germinated into seedlings.

[0074] T1 (CK+GA compound): Soak the treated seeds in a compound solution of 0.1 mg / L cytokinin (CTK) + 50 mg / L gibberellin (GA) for 24 hours at a temperature of 25±1℃, stirring gently 2-3 times during the soaking period. After hormone treatment, remove the seeds, rinse them 4 times with sterile water, drain the surface water, place the seeds in a petri dish lined with sterile filter paper, add 6 ml of sterile water, and culture at 25±1℃, light intensity of 3000 lx, and a photoperiod of 16 h light / 8 h dark until the seeds germinate into seedlings.

[0075] T2 (GTK alone): Soak the treated seeds in a 0.1 mg / L cytokinin (CK) solution for 24 hours at a temperature of 25 ± 1 °C, stirring gently 2-3 times during the soaking process. After hormone treatment, remove the seeds, rinse them four times with sterile water, drain the surface moisture, and place them in a petri dish lined with sterile filter paper. Add 6 ml of sterile water and incubate at 25 ± 1 °C under a light intensity of 3000 lx and a photoperiod of 16 h light / 8 h dark until the seeds germinate into seedlings.

[0076] T3 (GA alone): Soak the treated seeds in a 50 mg / L gibberellin (GA) solution for 24 hours at a temperature of 25 ± 1 °C, stirring gently 2-3 times during the soaking process. After hormone treatment, remove the seeds, rinse them four times with sterile water, drain the surface moisture, and place them in a petri dish lined with sterile filter paper. Add 6 ml of sterile water and incubate at 25 ± 1 °C under a light intensity of 3000 lx and a photoperiod of 16 h light / 8 h dark until the seeds germinate into seedlings.

[0077] The results showed (Table 4) that all hormone treatments significantly improved various indicators of seed germination and seedling growth in the induced mutation. The CTK and GA combination group (T1) achieved the best results in all indicators, including germination potential of 78.00%, germination rate of 88.67%, shoot length of 11.43 cm, taproot length of 10.50 cm, fresh weight of leaves per plant of 0.11 g, and fresh weight of roots per plant of 0.093 g. The two hormones showed a significant synergistic effect, which could simultaneously repair the seed vigor damage caused by mutation and promote the balanced growth of aboveground stems and leaves and underground roots. This combination of hormones was the optimal germination-promoting hormone combination in this mutation system.

[0078] Table 4 Effects of different hormone treatments on germination indices Note: Different lowercase letters in the table represent significant differences.

[0079] Experimental Example 5 This embodiment is only for screening the optimal seed soaking time and water absorption. After soaking and water absorption, the seeds are directly germinated without EMS mutagenesis treatment. T1: Seeds soaked for 5 hours, absorbing 13.8% of their water; T2: Seeds soaked for 7 hours, water absorption reached 31.4%; T3: Seeds soaked for 8 hours, absorbing 35.8% of their water; T4: Soak seeds for 9 hours until they absorb 40% water. After soaking, drain the water and proceed with germination directly.

[0080] The experimental results (Table 5) show that under the T3 and T4 treatments, the germination rate of the seeds reached its maximum value after 5 days, indicating that the experimental seeds can achieve maximum germination results when they absorb 35.8% of the water after 8 hours of water absorption.

[0081] Table 5. Effects of seed soaking water absorption on seed germination Note: Different lowercase letters in the table represent significant differences.

[0082] Experimental Example 6 The method is carried out according to Example 1, except that the processing temperature is different for each step: T1: All steps are performed at a temperature of 20°C; T2: All steps are performed at a temperature of 23°C; T3: All steps are performed at a temperature of 25°C.

[0083] The results (Table 6) showed that at 20-25℃, seed germination potential, germination rate, leaf length, and root length all significantly increased with increasing treatment temperature. Low temperature at 20℃ severely inhibited seed germination and seedling development, with a germination potential of only 32.70% and a germination rate of only 39.30%. Medium temperature at 23℃ could slightly improve germination, but the optimization effect was limited. Under the 25℃ treatment throughout the entire process, seed germination potential was 78.00%, germination rate was 88.67%, leaf length was 11.43cm, and root length was 10.50cm. All four indicators were the best among the groups, significantly alleviating the damage to seeds caused by mutation, simultaneously improving germination uniformity, total seedling emergence, and cultivating strong seedlings. This is the preferred treatment temperature for the mutation process of this invention.

[0084] Table 6. Effects of mutagenesis temperature on germination parameters Note: Different lowercase letters in the table represent significant differences.

[0085] Therefore, this invention significantly improves the mutation frequency and mutation type richness of EMS mutagenesis through a multi-step synergistic approach involving seed selection, washing, soaking in the bud, EMS mutagenesis, regulation by cytokinins and gibberellins, and detoxification with sodium thiosulfate. It also reduces the damage to barley seed viability caused by mutagenesis and increases the yield of effective mutants. The operation is simple and low-cost, and it can be widely used in barley genetic breeding and mutant library construction.

[0086] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for enhancing the EMS mutagenic effect of barley, characterized in that, Includes the following steps: Barley seeds induced by EMS were detoxified using sodium thiosulfate to obtain detoxified barley seeds. The detoxified barley seeds were soaked in cytokinin and gibberellin, and then the seeds were germinated after soaking.

2. The method according to claim 1, characterized in that, The sodium thiosulfate has a mass concentration of 0.5% to 1.5%, the detoxification treatment takes 0.5 to 1.0 h, and the detoxification treatment temperature is 24 to 26 °C.

3. The method according to claim 1, characterized in that, The concentration of the cytokinin is 0.1~1.0 mg / L; the concentration of the gibberellin is 45~55 mg / L.

4. The method according to claim 1, characterized in that, The soaking time is 24-36 hours, and the soaking temperature is 24-26°C.

5. The method according to claim 1, characterized in that, The method for preparing EMS-mutated barley seeds includes the following steps: mixing barley seeds after they have sprouted with EMS mutagenesis solution, mutagenesis culture, and obtaining EMS-mutated barley seeds.

6. The method according to claim 5, characterized in that, The mass concentration of the EMS mutagenesis solution is 0.4%~0.5%, and the volume-to-mass ratio of the EMS mutagenesis solution to the seed is 4~6 mL:1 g.

7. The method according to claim 5, characterized in that, The mutagenesis culture time is 8-18 hours, and the mutagenesis culture temperature is 24-26℃.

8. The method according to claim 5, characterized in that, The method for preparing barley seeds after they have sprouted includes the following steps: soaking the barley seeds to make the seed moisture content reach 35%~40%; after soaking, culturing them in the dark. The temperature for soaking the barley seeds is 24~26℃.

9. The method according to claim 1, characterized in that, The seed germination temperature is 24~26℃, the light intensity for seed germination is 2000~3000lx, and the photoperiod for seed germination is 16h light / 8h darkness.

10. The method according to any one of claims 1 to 9, characterized in that, The barley seed varieties include "Ganpi 8".

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