Method for improving EMS mutagenesis effect of oat

Abstract

The invention belongs to the technical field of crop mutation breeding, and particularly relates to a method for improving the EMS mutagenesis effect of oat. The method comprises the following steps: firstly, treating seeds by using concentrated sulfuric acid to remove hulls, adjusting the water content of the seeds to a suitable range, then screening an optimal ethylmethane sulfonate (EMS) mutagenesis condition, and finally, performing exogenous treatment by using gibberellin to promote germination of the mutagenized seeds and growth of seedlings. Planting the M0 generation in the generation adding room and harvesting the M1 generation seeds. On the basis, an Mgeneration mutant group is constructed, and the agronomic traits of the Mgeneration mutant group are investigated in a milk-ripe stage to evaluate the mutagenesis effect. By integrating seed hull peeling treatment, seed water content adjustment pretreatment, EMS mutagenesis treatment conditions and exogenous gibberellin composite treatment conditions, a set of EMS mutagenesis method capable of remarkably improving the survival rate of mutants and the richness of variation types is established, and reliable technical support is provided for germplasm innovation and variety breeding of the feeding oat.

Description

Technical Field

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

[0002] oat( Avena sativa Oat (L.) is an annual herbaceous plant belonging to the genus *Oat* in the family Poaceae. It is generally divided into two main categories: hulled oats and naked oats. Hulled oats, also known as forage oats, have advantages such as cold resistance, drought resistance, tolerance to poor soil and salinity, high yield, high nutritional value, and good palatability. Forage cultivated oats are allohexaploids, with a highly complex genetic background, posing significant challenges to genetic improvement. This results in long cycles and high difficulty in trait improvement through traditional hybridization breeding, while modern biotechnology is limited by genotype dependence.

[0003] Chemical mutagenesis breeding is an effective way to create new germplasm. Ethyl methanesulfonate (EMS), as one of the most widely used chemical mutagens, has played an important role in crop genetic improvement. Current research on EMS treatment of crops such as wheat, quinoa, and soybean mainly focuses on investigating the effects of mutagen concentration and treatment time on seed germination and seedling growth, and using this to screen for median lethal doses. However, these existing methods suffer from low mutagenesis efficiency when applied to forage oats (hulled oats). Summary of the Invention

[0004] To address the low mutagenesis efficiency of existing methods when applied to feed oats (hulled oats), this invention provides a stable composite mutagenesis method. This invention offers a complete technical method for effectively improving the EMS mutagenesis sensitivity and mutagenesis effect of hulled oats.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for enhancing the EMS mutagenic effect of oats, comprising the following steps: Oat seeds were soaked in concentrated sulfuric acid to remove the husks, thus obtaining dehulled oat seeds.

[0006] The hulled oat seeds are soaked in water until the moisture content reaches 50% to 51%, thus obtaining soaked oat seeds.

[0007] The soaked oat seeds were subjected to ethyl methanesulfonate (EMS) mutagenesis treatment to obtain mutagenized oat seeds.

[0008] The mutated oat seeds were soaked in exogenous gibberellin solution and germinated to obtain germinating seedlings.

[0009] The germinated seedlings were cultured, and after harvesting, M1 generation seeds were obtained. The M1 generation seeds were then sown and planted to obtain the M2 generation mutant population.

[0010] This invention provides a method for improving the EMS mutagenesis effect of oats. The method first involves soaking oat seeds in concentrated sulfuric acid to remove the lemma, effectively overcoming the problem of mutagen penetration due to the lemma barrier. The lemma-removed oat seeds are then soaked in water to achieve a moisture content of 50%–51%, resulting in soaked oat seeds. This soaking process optimizes the seed's physiological state by adjusting the moisture content, promoting efficient entry of the mutagen into the seed and its reach to the embryo cells. Next, EMS mutagenesis conditions are screened to determine the optimal half-lethal dose and treatment time, increasing the mutagenic effect. The soaked oat seeds are then subjected to EMS mutagenesis treatment to obtain mutagenic oat seeds. Finally, the mutagenic oat seeds are germinated, and the germinated seeds are soaked in exogenous gibberellin solution. The germinated seedlings are cultured, and individual plants are harvested to obtain M1 generation seeds. These seeds are then planted in the field using a row-planting method to obtain the M2 generation mutagenesis population. This invention effectively improves the problems of weak seedlings and low survival rates caused by post-mutation damage through the aforementioned multi-step composite mutagenesis method. This invention overcomes EMS penetration barriers by using concentrated sulfuric acid to remove the husk, precisely controls the seed moisture content to 50%–51% to ensure optimal sensitivity, and repairs mutagenesis damage through exogenous gibberellin post-treatment. These three elements synergistically construct a precise pretreatment system of "barrier breaking – physiological synergy – damage repair." This method systematically overcomes key limiting factors such as penetration barriers, inconsistent physiological states, and mutagenesis damage, achieving optimization of the entire oat EMS mutagenesis process and significantly improving mutagenesis efficiency.

[0011] The aforementioned core technologies can overcome the shortcomings of existing technologies and methods that generally neglect the influence of the husks covering the feed oat seeds on the penetration of EMS solution, as well as the germination state of the seeds during mutagenesis and their sensitivity to mutagen. These technologies fail to optimize the mutagenesis efficiency through precise pretreatment, thereby improving the EMS mutagenesis effect of husk oats.

[0012] Furthermore, the EMS mutagenesis process is as follows: The soaked oat seeds were soaked in 1% ethyl methanesulfonate (EMS) for 15-17 hours.

[0013] Furthermore, the soaked oat seeds are soaked in a 1% (w / w) ethyl methanesulfonate (EMS) solution under light-protected conditions.

[0014] Furthermore, the soaked oat seeds are soaked in a 1% (w / w) ethyl methanesulfonate (EMS) solution at a temperature of 18°C ​​to 22°C.

[0015] The 1% EMS solution is prepared by taking EMS as solute and PBS with pH 7.0 as solvent.

[0016] The PBS has a concentration of 1x and a pH of 7.

[0017] Preferably, the time for the oat seeds after mutagenesis to be soaked by the exogenous gibberellin solution is 7d-10d.

[0018] Further, the concentration of the exogenous gibberellin solution is 49mg / L-51mg / L.

[0019] Further, the process for treating the oat seeds after mutagenesis by using the exogenous gibberellin solution is as follows: The oat seeds after mutagenesis are placed in seed germination bags, and the exogenous gibberellin solution with a concentration of 49mg / L-51mg / L is used for exogenous application treatment.

[0020] Further, after mutagenesis treatment, detoxification treatment is performed on the oat seeds after mutagenesis. The process of the detoxification treatment is as follows: The oat seeds after mutagenesis are drained, 4.8%-5.2% sodium thiosulfate solution is added, and the mixture is shaken for 28min-32min, and then rinsed.

[0021] Further, the soaking treatment conditions are as follows: The unhulled oat seeds are soaked in distilled water at 3.5℃-4.5℃ for 3.5h-4.5h.

[0022] Further, the time for the oat seeds to be treated by concentrated sulfuric acid is 5.5min-6.5min.

[0023] Further, the concentrated sulfuric acid is a 97%-99% sulfuric acid solution.

[0024] Further, the conditions for the germinated seedlings to be cultured are as follows: 1980lx-2020lx, 24℃-26℃ / 19℃-21℃, 15h-17h / 7h-9h photoperiod, cycle 3.5 months-4.5 months.

[0025] Further, the oat seeds include 'Zhongtian No.4' unhulled oat seeds.

[0026] The oat seeds are mature and full, and are used as mutagenesis platform materials.

[0027] Further, the exogenous gibberellin treated oat seeds (M0 generation) are sowed in a generation room and single plant seed is collected to obtain M1 generation seeds.

[0028] The M1 generation single plant seeds are planted into M2 generation population according to the plant row method.

[0029] The change of the agronomic traits of the M2 generation mutagenesis population at the milk stage is investigated, the mutagenesis effect is evaluated according to the change range of the agronomic trait indexes of the EMS treatment group and the non-mutagenesis control group, and the mutagenesis efficiency is evaluated according to the change range of the agronomic trait indexes of the non-mutagenesis control group. The M2 generation is planted according to the plant row method, 16 seeds are planted for each plant, the plant spacing is 40 cm, the row spacing is 40 cm, the plant height, the tiller number, the stem diameter, the main ear length, the flag leaf length and width, the fresh weight (biomass) are respectively measured for single plant at the milk stage, and the mutagenesis effect is evaluated.

[0030] The threshold value for evaluating the mutagenesis effect according to the change range of the agronomic trait indexes of the non-mutagenesis control group is that the positive mutation rate is greater than 130% of the average value of the control, and the negative mutation rate is less than 70% of the average value of the control, the number of positive and negative mutations of each trait is used to evaluate the mutagenesis efficiency, and the mutagenesis efficiency is the percentage of the number of mutation single plants of a certain trait to the initial number of M0 generation seeds of each treatment.

[0031] Compared with the prior art, the present application has the following beneficial effects: 1. The application provides a method for improving the EMS mutagenesis effect of oats. The method provided by the application first uses concentrated sulfuric acid to soak and treat oat seeds to remove the hulls, effectively overcoming the problem of difficulty of penetration of the mutagen into the seed coat due to the barrier of the hulls. The oat seeds with the hulls removed are obtained; then, the oat seeds with the hulls removed are placed in water for soaking treatment, so that the water content of the seeds reaches 50-51%, and the soaked oat seeds are obtained. The seed physiological state is optimized by adjusting the seed water content, promoting the efficient entry of the mutagen into the seed and reaching the embryo cells; the optimal semi-lethal dose and treatment time are selected by screening the EMS mutagenesis conditions, and the mutation effect is improved. The soaked oat seeds are subjected to EMS mutagenesis treatment, and the mutagenized oat seeds are obtained. Finally, the mutagenized oat seeds are germinated, and the germinated oat seeds are soaked with exogenous gibberellin solution. The seedlings are cultured, and the M1 generation seeds are obtained after single plant seed collection. After planting in the field according to the plant row method, the M2 generation mutagenized population is obtained. Through the above multi-step composite mutagenesis method, the problem of weak seedlings and low survival rate caused by damage after mutagenesis is effectively improved, and the problem of low mutagenesis efficiency of the existing methods when applied to forage oats (hulled oats) is solved. The present application solves the problem of low mutagenesis efficiency of the existing methods when applied to forage oats (hulled oats) by removing the hulls with concentrated sulfuric acid to solve the EMS penetration barrier, accurately controlling the water content of 50-51% to make the seeds in the best sensitive state, and repairing the mutagenic damage by exogenous gibberellin post-treatment, which cooperates to build a precise pretreatment system of "breakthrough barrier-physiological synergy-damage repair". The method systematically overcomes the key limiting factors such as penetration barrier, different physiological states and mutagenic damage, realizes the optimization of the whole process of oat EMS mutagenesis, and significantly improves the mutagenesis efficiency.

[0032] The above core technology can overcome the defects in the prior art that the existing methods generally ignore the influence of the hulls wrapped outside the forage oat seeds on the penetration of the EMS solution, the germination state of the seeds during mutagenesis and the sensitivity of the seeds to the mutagen, and fail to optimize the mutagenesis efficiency through precise pretreatment, thereby improving the EMS mutagenesis effect of hulled oats.

[0033] 2. The application aims to solve the following three key problems by establishing a technical system integrating seed pretreatment, EMS mutagenesis treatment and post-mutagenesis treatment: (1) determining the semi-lethal conditions of EMS mutagenesis of forage oats, and selecting the appropriate EMS concentration and treatment time; (2) on this basis, removing the hull barrier and regulating the water content of the seeds to make the oat seeds in the most sensitive physiological state to EMS mutagenesis; (3) optimizing the germination conditions of the seeds treated by EMS, and using exogenous gibberellin (GA3) treatment to effectively alleviate the mutagenic damage and improve the germination rate and seedling survival rate.

[0034] By integrating pretreatment for rapid removal of husks and adjustment of seed moisture content, EMS mutagenesis treatment conditions, and exogenous GA3 compound treatment conditions, a set of EMS mutagenesis methods that can significantly improve the survival rate and variety richness of mutants was established, providing reliable technical support for germplasm innovation and variety breeding of feed oats.

[0035] 3. This invention systematically optimizes the EMS mutagenesis technology system for feed oats, achieving significant beneficial effects. First, treatment with concentrated sulfuric acid rapidly and effectively removes the oat husk, significantly promoting the penetration of the EMS mutagen into the embryo and fundamentally improving the efficiency of the mutagenesis treatment. Second, through systematic research on the water absorption changes of seeds, the critical period of sensitivity of oat seeds to EMS mutagenesis was clarified, providing a theoretical basis for determining the timing of mutagenesis. Finally, the innovative use of exogenous gibberellin (GA3) treatment after EMS mutagenesis effectively alleviates the physiological damage caused by mutagenesis, promotes the germination and seedling establishment of mutant seeds, and improves the survival rate of the M1 generation population.

[0036] This invention integrates pretreatment (removing husks and adjusting seed moisture content), EMS mutagenesis, and post-treatment (exogenous GA3 treatment) into a comprehensive technology, establishing a highly efficient, stable, and applicable EMS mutagenesis system for feed oats. This approach not only significantly improves mutagenesis efficiency and the richness of variant types but also strongly supports the subsequent creation and screening of superior germplasm. Furthermore, this method provides valuable technical reference and inspiration for EMS mutagenesis breeding of other gramineous crops (such as wheat, rice, and barley) using seeds as material. Attached Figure Description

[0037] Figure 1 The figure shows the water absorption rate change curve of 'Zhongtian No. 4' over 30 hours in this invention. In the figure, the first stage is the rapid water absorption stage, i.e., the seed swelling stage; the second stage is the stable water absorption stage, i.e., the seed germination stage; and the third stage is the oscillating water absorption stage, i.e., the seed germination stage (when the seed begins to "show white").

[0038] Figure 2 The effect of different EMS concentrations and times on oat germination in this invention is illustrated below: A represents the effect of different EMS concentrations and durations on the germination potential of 'Zhongtian No. 4'. B represents the effect of different EMS concentrations and times on the germination rate of 'Zhongtian No. 4'. C represents the effect of different EMS concentrations and times on the relative germination potential of 'Zhongtian No. 4'. D represents the effect of different EMS concentrations and times on the relative germination rate of 'Zhongtian No. 4'. The lowercase letters in the figure indicate significant differences in concentrations at the same treatment time. P <0.05.

[0039] Figure 3 The effects of different EMS concentrations and durations on oat seedling growth in this invention are illustrated below: A represents the effect of different EMS concentrations and durations on seedling growth of 'Zhongtian No. 4'. B represents the effect of different EMS concentrations and durations on root length in 'Zhongtian No. 4'. The lowercase letters in the figure indicate significant differences in concentrations at the same treatment time. P <0.05.

[0040] Figure 4 The effect of different concentrations of gibberellin on oat germination in this invention is illustrated in the following figures: A represents the effect of different concentrations of gibberellin on germination potential of 'Zhongtian No. 4'. B represents the effect of different concentrations of gibberellin on the germination rate of 'Zhongtian No. 4'. C represents the effect of different concentrations of gibberellin on seedling growth in 'Zhongtian No. 4'. D represents the effect of different concentrations of gibberellin on root length in 'Zhongtian No. 4'. The lowercase letters in the figure indicate significant differences in concentrations at the same treatment time. P <0.05.

[0041] Figure 5 The effect of different seed soaking times on EMS mutagenesis in this invention is illustrated in the following figures: A represents the effect of different soaking times on the germination potential of 'Zhongtian No. 4' induced by EMS mutagenesis. B represents the effect of different soaking times on the germination rate of 'Zhongtian No. 4' induced by EMS mutagenesis. C represents the effect of different soaking times on the growth of 'Zhongtian No. 4' EMS-mutated seedlings; D represents the effect of different soaking times on the root length induced by EMS mutagenesis of 'Zhongtian No. 4'. The lowercase letters in the figure indicate significant differences in concentrations at the same treatment time. P <0.05.

[0042] Figure 6 The effect of exogenous gibberellin treatment on EMS mutagenesis in this invention is illustrated in the following: A represents the effect of exogenous gibberellin treatment on the germination potential of 'Zhongtian No. 4' induced by EMS mutagenesis; B represents the effect of exogenous gibberellin treatment on the germination rate of 'Zhongtian No. 4' induced by EMS mutagenesis. C represents the effect of exogenous gibberellin treatment on the growth of 'Zhongtian 4' EMS-mutated seedlings; D represents the effect of exogenous gibberellin treatment on the root length of 'Zhongtian No. 4' induced by EMS mutagenesis; The lowercase letters in the figure indicate significant differences in concentrations at the same treatment time. P <0.05.

[0043] Figure 7 The images show the treatment of 'Zhongtian No. 4' seeds with concentrated sulfuric acid to remove the lemma and bark, and multiple samples in each image are parallel samples.

[0044] Figure 8 The images show the root seedling phenotypes of 'Zhongtian No. 4' at different EMS concentrations and times in this invention, where multiple samples in each image are parallel samples.

[0045] Figure 9 The images show the effect of different soaking times on EMS mutagenesis in the growth of root seedlings, with multiple samples in each image being parallel samples.

[0046] Figure 10 The images show the effect of gibberellin on EMS mutagenesis in the growth of root seedlings, where multiple samples in each image are parallel samples. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0048] Example 1: Mutagenic effects of EMS mutagenesis breeding on the germination and seedling stages of 'Zhongtian No. 4' hulled oat seeds. Using hulled oat seeds ('Zhongtian No. 4' hulled oat seeds) as the mutagenesis chassis material, a combination of pretreatment with adjusted seed moisture content and EMS mutagenesis was employed. Following this, GA3 was used to post-treat the hulled oat seeds that had undergone EMS semi-lethal treatment. Germination and seedling characteristics were statistically analyzed during the seedling stage to explore the suitable pretreatment conditions for moisture content, EMS mutagenesis treatment conditions, and exogenous gibberellin (GA3) post-treatment conditions for 'Zhongtian No. 4' hulled oat seeds. Specific experimental methods and results are as follows:

[0049] I. Experimental Methods 1. Oat seed husk removal treatment Select uniformly sized and plump oat seeds ('Zhongtian No. 4' hulled oat seeds) and soak them in 98% concentrated sulfuric acid (mass percentage) for 6 minutes. Rinse them with running water for 15 minutes. The tightness of the hulled oat seeds is significantly reduced, and some hulled oat seeds fall off automatically. Wash off the residual concentrated sulfuric acid on the seed surface to avoid toxic effects on seed germination. Use absorbent paper to dry the residual moisture on the seed surface. After drying in an oven at 37℃ for 3 hours, manually peel off the remaining loosened hulled oat seeds. The hulled oat seeds are used for subsequent mutagenesis treatment.

[0050] Among them, the oat seeds ('Zhongtian No. 4' hulled oat seeds) were provided by the Lanzhou Institute of Animal Husbandry and Veterinary Medicine of the Chinese Academy of Agricultural Sciences. They are a new variety of feed oats bred through space breeding technology.

[0051] 2. Pretreatment to adjust seed moisture content Initial seed moisture content determination: The moisture content of oat seeds ('Zhongtian No. 4' hulled oat seeds) was determined according to the seed testing procedures of the International Seed Testing Association (ISTA). After removing the sample box and lid from the desiccator, it was placed in a 130℃ oven for 30 minutes, and then cooled in the desiccator for 30 minutes. The measured mass was recorded as M1 (accurate to 0.001g). 4.5g of oat seeds ('Zhongtian No. 4' hulled oat seeds) were weighed and placed in the sample box, and the measured mass was recorded as M2 (accurate to 0.001g). After opening the sample box and lid, it was placed in the oven. When the temperature reached 130℃, the timer started for 2 hours. After the specified time, it was placed in the desiccator to cool for 30 minutes, and the measured mass was recorded as M3. Three replicates were set up, and the initial moisture content of the oat seeds was calculated according to the following formula:

[0052] Initial moisture content = (M2-M3) / (M2-M1)×100%; In the above formula, the unit of initial water content is %.

[0053] To determine the change in moisture content of oat seeds over 30 hours: 4.5 g ± 0.1 g of mature, plump oat seeds ('Zhongtian No. 4' hulled oat seeds) were weighed and placed into 50 mL numbered centrifuge tubes, and the initial mass was recorded. The experiment was conducted in triplicate, with each replicate containing 4.5 g ± 0.1 g of oat seeds. The centrifuge tubes were placed in a 4°C refrigerator. Seeds were removed every 3 hours, their surface moisture was blotted dry with filter paper, and they were quickly weighed. The time and mass of each weighing were recorded, and the change in water absorption rate for each group of seeds was calculated. Ten consecutive weighings were performed within 30 hours to ensure data accuracy.

[0054] 3. EMS mutagenesis of 'Zhongtian No. 4' hulled oat seeds (1) Reagent preparation Preparation of Phosphate Buffered Sodium (PBS): Dilute 100 mL of 10×PBS buffer to make 1×PBS, adjust the pH of the buffer to neutral, and add distilled water to a final volume of 1 L. The 10×PBS buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd., and its pH is 7.

[0055] Preparation of EMS solutions (mass percentage / mass percentage concentration): Four EMS concentration gradients were set up for the experiment: 0%, 0.5%, 0.75%, and 1%. 0 g, 0.25 g, 0.375 g, and 0.50 g of EMS mutagen were added to 50 mL of PBS buffer, respectively, to prepare EMS solutions with concentrations of 0%, 0.5%, 0.75%, and 1%. The EMS mutagen was provided by Beijing Solarbio Technology Co., Ltd. EMS solution is ethyl methanesulfonate solution.

[0056] Preparation of 5% sodium thiosulfate (Na2S2O3) solution (mass percentage): Add 50g of anhydrous thiosulfate to 1L of pure water and stir until fully dissolved to terminate the mutagenic reaction and alleviate toxicity.

[0057] (2) EMS mutagenesis treatment of 'Zhongtian No. 4' hulled oat seeds EMS mutagenesis treatment of hulled oat seeds ('Zhongtian No. 4' hulled oat seeds): Plump and intact hulled oat seeds were selected and soaked in distilled water at 4°C for 4 hours. The distilled water was drained, and the seeds were washed with absorbent paper to remove residual moisture before being added to EMS solution. Four EMS solution concentrations (0%, 0.50%, 0.75%, and 1.00%) and three time gradients (8 hours, 12 hours, and 16 hours) were established, forming a total of 12 different treatments. The 0% EMS solution concentration served as the control. The treatment temperature was 20°C ± 2°C. Each treatment required 50 seeds, and the experiment was repeated three times. Add 50 mL of EMS solution of different concentrations to the conical flask, seal the seeds of each group, wrap them with tin foil (to prevent EMS from being decomposed by light), and place them in a temperature-controlled shaker at 150 r / min for different times (8 h, 12 h and 16 h) for light-protected shaking mutagenesis. All the above operations were carried out under light-protected conditions.

[0058] Detoxification process: After mutagenesis, the mutagenized oat seeds need to be detoxified. The method is as follows: Drain the mutagenized seeds, add 50 mL of a prepared 5% sodium thiosulfate solution, and shake on a shaker for 30 minutes to completely terminate the reaction and achieve detoxification. Afterward, pour the detoxified seeds into mesh bags and rinse with running water for 2 hours to obtain the mutagenized seeds.

[0059] Germination experiment: Seed germination bags were moistened with distilled water. Mutagenized oat seeds were placed in the germination bags with the ventral groove facing down. Each replicate consisted of 50 seeds, and each mutation treatment was repeated three times. The seed germination bags were placed under a photoperiod of 2000 lx, 25℃ / 20℃, and 16h / 8h. The bags were checked regularly to ensure they remained moist. Germination was defined as the radicle being the same length as the seed and the plumule being approximately half the length of the seed. Germination results for each treatment were recorded.

[0060] (3) Germination index determination During the cultivation process, the daily germination of oat seeds was observed, and the initial germination time was accurately recorded. After 7 days of cultivation, the germination potential of the oat seeds was calculated. After 10 days of cultivation, the germination rate of the oat seeds was calculated, and the relative and absolute germination rates were also calculated to determine the median lethal dose. After the experiment, for each treatment, 10 oat seedlings were randomly selected from each of the three replicates and photographed. The root length and seedling length of the oat seedlings were then measured using ImageJ. The calculation formulas for the relevant indicators of oat seed germination are as follows:

[0061] Germination rate = (Number of seeds germinated on day 10 / Total number of seeds) × 100%; In the above formula, the germination rate is expressed in % %. Relative germination rate = (germination rate of treated seeds / germination rate of control seeds) × 100%; In the above formula, the unit of relative germination rate is %; the germination rate of treated seeds refers to the number of seeds germinated on day 10 after mutagenesis with EMS solutions of mass percentage concentrations of 0.5%, 0.75%, and 1%; the germination rate of control seeds refers to the number of seeds germinated on day 10 after treatment with 0% EMS solution. Germination potential = (Number of seeds germinated on day 7 / Total number of seeds) × 100%; In the above formula, the unit of germination potential is %; Relative germination potential = Germination potential of treated seeds / Germination potential of control seeds × 100%; In the above formula, the unit of relative germination potential is %; the germination potential of treated seeds refers to the number of seeds germinated on day 7 after mutation with EMS solutions of mass percentage concentrations of 0.5%, 0.75%, and 1%; the germination potential of control seeds refers to the number of seeds germinated on day 7 after treatment with 0% EMS solution of mass percentage concentration.

[0062] 4. Post-treatment of exogenous gibberellin (GA3) Uniform, plump, hulled oat seeds ('Zhongtian No. 4' hulled oat seeds) were selected. Four gibberellin (GA3) concentration gradients were set up: 0 mg / L (control), 50 mg / L, 100 mg / L, and 150 mg / L. Each treatment contained 50 seeds, and each treatment was replicated three times. The specific steps were as follows: Seeds were placed in seed germination bags, and gibberellin solution of the corresponding concentration was added until the bags were completely soaked. The bags were then labeled, weighed, and the initial mass was recorded. The germination bags were then incubated at 2000 lx, 25℃ / 20℃, and a photoperiod of 16h / 8h. During incubation, the germination bags were weighed daily, and the water lost due to evaporation was replenished with gibberellin working solution of the corresponding concentration to restore the initial mass. Seed germination was observed and recorded daily. The germination experimental method and measurement indicators were the same as above.

[0063] II. Experimental Results 1. The effect of soaking time on the moisture content of oat seeds Water is essential for seed germination. Studying the water absorption process of oat seeds during germination and identifying key germination moments provides a basis for determining suitable pretreatment conditions for EMS mutagenesis. To screen for appropriate pre-EMS mutagenesis water content in oat seeds, the water absorption rate of oat seeds at 4℃ was measured. The results are shown below. Figure 1 .

[0064] Depend on Figure 1 It was found that oat seeds rapidly absorbed water within the first 1.5 hours, reaching a water absorption rate of 37.39%, four times that of dry seeds, indicating a rapid water absorption phase. From 1.5 to 19 hours, the water absorption rate of oat seeds slowed down, with the water content increasing from 37.39% to 67.19%, indicating a slower water absorption phase. After 19 hours, the water content of the seeds gradually stabilized. Therefore, these three stages can be considered as the seed imbibition stage, the seed germination stage, and the seed sprouting stage (beginning to "show white"), respectively. Based on the phased changes in water absorption rate, six representative soaking times were selected: 0 hours (9.2%), 0.5 hours (30.8%), 2 hours (49.1%), 4 hours (50.4%), 8 hours (56.2%), and 19 hours (67.19%) for pre-treatment of oat seeds. EMS mutagenesis was then performed to screen suitable pre-treatment conditions.

[0065] Therefore, 0h, 0.5h, 2h, 4h, 8h and 19h were selected as suitable pretreatment times for soaking oat seeds.

[0066] 2. Effects of EMS mutagenesis on oat seed germination Germination is an important indicator of mutagenesis efficiency. EMS mutagenesis treatment of hulled oat seeds ('Zhongtian No. 4' hulled oat seeds) at different concentrations and for different durations was performed, and the results are shown in [Figure 1].Figure 2 .

[0067] Depend on Figure 2 It was found that under the control condition (0% EMS), the germination rate of oat seeds was 97%. With the increase of EMS treatment concentration and time, except for the germination rate of different treatment combinations of 0.5% EMS which did not change significantly, the germination potential and germination rate of oat seeds decreased significantly. When the EMS treatment condition was 1% for 16 hours, the germination rate of seeds was 57%, and the relative germination rate was 0.58, which is close to the semi-lethal treatment condition.

[0068] With increasing EMS concentration and treatment time, the root length and seedling length of oat seedlings decreased significantly, and the inhibitory effect was more pronounced with higher concentrations. Under 1% EMS treatment for 16 hours, the seedling length and root length were half that of the control group, indicating that this EMS treatment had a dwarfing effect on oat seedlings. Figure 3 ).

[0069] Therefore, the optimal EMS treatment conditions for 'Zhongtian No. 4' hulled oats are 1% EMS solution for 16 hours.

[0070] 3. Effects of different concentrations of gibberellin on oat seed germination Seeds treated with EMS mutagenesis often have low germination rates and weak seedlings. To improve the germination rate and seedling growth of oats after EMS mutagenesis, different concentrations of exogenous GA3 were used for treatment. Figure 4 It was found that GA3 at concentrations of 50 mg / L to 150 mg / L significantly improved the germination potential of oat seeds, but had no significant effect on the germination rate. This is because the germination rate of oat seeds under the control conditions was 97%, which is close to 100%. Gibberellin at concentrations of 50 mg / L and 100 mg / L significantly promoted the growth of young roots and seedlings. Based on the germination and seedling growth characteristics, and considering the economic dosage, 50 mg / L exogenous gibberellin (GA3) treatment was selected as the most effective treatment for promoting oat seed germination and seedling growth.

[0071] Therefore, treatment with 50 mg / L exogenous GA3 was selected as the optimal post-treatment condition for EMS mutagenesis seeds.

[0072] Example 2: Effect of different soaking times of 'Zhongtian No. 4' hulled oat seeds on EMS mutagenesis I. Experimental Methods 1. Mutagenic effects of different soaking time pretreatments and EMS mutagenesis combinations on the germination period of 'Zhongtian No. 4' hulled oat seeds Based on the moisture content change curve of oat seeds ('Zhongtian No. 4' hulled oat seeds) over 30 hours, germination experiments were conducted using five time points representing the moisture content changes of hulled oat seeds, combined with the selected optimal EMS mutagenesis conditions. The germination experiment methods and measurement indicators were the same as above.

[0073] 2. Mutagenic effect of EMS combined with exogenous gibberellin post-treatment on the germination period of 'Zhongtian No. 4' oats. Uniform, plump, hulled oat seeds were selected and subjected to mutagenesis treatment under optimal EMS conditions. The mutated seeds were then placed in seed germination bags and treated with 50 mg / L gibberellin exogenously. A blank control (CK) was prepared using untreated mutated seeds for germination experiments. The germination experiment methods and measurement indicators were the same as above.

[0074] II. Experimental Results 1. Effects of different soaking times and EMS mutagenesis combinations on oat seed germination Depend on Figure 5 It was found that soaking for 0 hours (dry seeds) and 19 hours (when seeds showed signs of sprouting) resulted in significantly lower germination rates and germination potential compared to other soaking times (0.5 hours, 2 hours, 4 hours, and 8 hours). This indicates that seeds that were not soaked and those soaked for excessively long periods were more sensitive to EMS treatment. In the former case, the cellular metabolic activity of dry seeds was dormant, making them unable to cope with the strong physiological toxicity. In the latter case, prolonged soaking led to active cellular metabolism, resulting in excessive absorption of EMS and irreversible DNA damage, thus severely inhibiting germination. Within the soaking time range of 0.5 hours to 8 hours, the germination potential, germination rate, and root length all increased with prolonged soaking time, indicating that the seeds continuously absorbed water and their physiological functions were activated. At this point, the penetration of EMS was sufficient to induce mutagenesis, but its physiological toxicity had not yet reached the critical point of severely inhibiting germination. Therefore, the seeds germinated relatively normally, exhibiting good early indicators. Based on the germination and seedling growth status, soaking for 8 hours was a suitable pretreatment condition, resulting in the highest germination potential and germination rate, both exceeding 70%, and the highest seedling root length and seedling length, at 8.86 cm and 13.05 cm, respectively. However, to balance the mutation efficiency and plant survival rate of the mutagenesis treatment, 8-hour soaking may lead to excessive DNA damage due to excessive EMS penetration. The negative effects of late-stage seedling death and sterility were not apparent in the early germination stage, but severely restricted mutant survival. Therefore, a 4-hour soaking treatment with moderate germination indicators but potentially higher seedling survival rates is more conducive to obtaining a large-scale, heritable effective mutant library. Contrary to previous literature reports, dry seeds were most sensitive to oat EMS mutagenesis. This may be due to the large water potential gradient between the inside and outside of the seed, allowing the EMS solution to penetrate rapidly. After 8 hours of soaking, the seeds completed the rapid water absorption phase and reached a water saturation state, significantly reducing the biological damage effects of the chemical mutagen.

[0075] 2. Effects of EMS treatment combined with exogenous gibberellin (GA3) post-treatment on oat seed germination Based on a comprehensive analysis of germination potential, germination rate, root length, and seedling length, exogenous gibberellin treatment significantly improved the germination potential and germination rate of EMS-mutated seeds and promoted seedling growth. Figure 7 Following EMS treatment with exogenous gibberellin, the germination rate increased by 8.5%, and seedling and root lengths were also significantly improved. Therefore, 50 mg / L gibberellin is the optimal treatment condition after EMS mutagenesis.

[0076] In summary, the optimal post-treatment technique for EMS mutagenesis in feed oats is as follows: rapid dehulling with concentrated sulfuric acid, followed by rinsing and drying, then soaking in 1% EMS for 16 hours, and finally treatment with 50 mg / L exogenous gibberellin. This significantly improves the sensitivity of oats to EMS mutagenesis, the germination rate after mutagenesis, and the growth of seedlings. The 1% EMS for 16 hours refers to treatment with a 1% (by mass) EMS solution for 16 hours.

[0077] Example 3: Field Mutagenesis Effects of EMS Treatment Combination in Generation M2 I. Experimental Methods 1. Construction of the M1 generation population Uniform and plump hulled oat seeds ('Zhongtian No. 4' hulled oat seeds) were selected. Based on the germination experiment of 'Zhongtian No. 4' with different treatment combinations, the experimental design included 5 moisture content treatments (soaking for 0h, 0.5h, 2h, 4h and 8h), the optimal EMS mutagenesis treatment (1% EMS for 16h), and the exogenous gibberellin post-treatment (50mg / L GA3). Except for the 0h treatment, which involved 358 seeds due to low germination rate, all other treatment combinations involved 208 seeds. Oat seeds soaked for 4 hours and then subjected to EMS mutagenesis without gibberellin treatment (1% EMS for 16 hours) served as the control (A). Simultaneously, untreated seeds were planted as a reference control (CK). Seeds were evenly placed in petri dishes lined with double-layered filter paper and germinated under a photoperiod of 2000 lx, 25℃ / 20℃, and 16h / 8h. After sprouting, seedlings were transferred to flowerpots and continued to grow in a generation chamber under the same conditions. Because EMS is toxic to oat seeds, the vigor of the first-generation mutant plants was relatively weak. During the growth period, timely watering and weekly application of Hoagland's nutrient solution were ensured to provide sufficient water and nutrients. After self-pollination and maturation, individual M1 plants were harvested, threshed, and stored. The 1% EMS 16h means treatment with a 1% EMS solution for 16 hours.

[0078] 2. Construction of the M2 generation group The experimental site is located in Dongcun Village, Lushar Town, Huangzhong District, Qinghai Province (101°59′E, 36°42′N), at an altitude of 2630m. The average annual temperature is 4.6℃, and the annual precipitation ranges from 500mm to 650mm. The previous crop was buckwheat. The total nitrogen content of the topsoil in the experimental site was 1.56 g·kg⁻¹. -1 The total phosphorus content is 2.24 g·kg. -1 The ammonium nitrogen content is 0.79 mg·kg⁻¹. -1 The nitrate nitrogen content is 2.24 mg·kg⁻¹. -1 The organic matter content is 17.90 mg·kg. -1 M2 seeds were sown using the row-planting method, selecting plump and uniform M1 individual seeds, with 16 seeds planted per row. Sowing was done in hills with a row spacing of 40cm, a plant spacing of 40cm, and a sowing depth of 4cm. One row of non-mutated material was planted every 10 rows as a control. Urea (total N≥46.0%) was applied at 75 kg·hm² before sowing. -2 And diammonium phosphate (total nutrients ≥64.0%, of which N≥18.0%, P2O5≥46.0%) 150kg·hm -2 It is used as base fertilizer. Urea and diammonium phosphate were purchased from Qinghai Lvqingxin Agricultural and Animal Husbandry Technology Co., Ltd.

[0079] 3. Measurement Indicators and Methods To compare the EMS mutagenesis effects of different treatment combinations, the variation of M2 generation plants was observed and recorded during the milk stage, and the variation types and rates were statistically analyzed. The methods for observing variant plants are as follows:

[0080] Plant height: Measure the vertical distance from the ground to the tip of the stem using a measuring tape. The unit for plant height is cm.

[0081] Total tillers: The total number of tillers at a height of 10cm from the ground. The unit for total tillers is "each".

[0082] Effective tillers: The number of tillers that produce ears and grains. The unit for effective tillers is individual tillers.

[0083] Leaf length: The length from the base of the second leaf counting down from the top of the main stem of the plant to the tip of the leaf blade. The unit for leaf length is cm.

[0084] Leaf width: The width of the widest part of the second leaf counting down from the top of the main stem of the plant. The unit for leaf width is cm.

[0085] Leaf area: Calculated using the following formula: Leaf area = leaf length × leaf width × R; In the above formula, the unit of leaf area is cm². 2 R is the oat leaf area correction factor, 0.8317.

[0086] Main ear length: The length on the main stem of the plant from the neck node of the ear to the tip of the ear. The unit for main ear length is cm.

[0087] Stem diameter (mm): The diameter of the second internode above ground on the main stem is measured using vernier calipers. The unit for stem diameter is mm.

[0088] II. Experimental Results 1. Evaluation of field phenotypic traits EMS chemical mutagenesis is random; the mutagen interacts with the genome in unpredictable ways, resulting in uncertainty in the site of occurrence, mutation type, and phenotypic effect. Using positive mutation rate (greater than 130% of the control mean) and negative mutation rate (less than 70% of the control mean) to analyze the mutagenized population is an effective method to assess mutagenesis efficiency and reveal significant phenotypic variations. This avoids the high variability generated by random mutations masking valuable extreme phenotypes. Furthermore, comparison with group A (without GA3 post-treatment) accurately assesses the combined effect of different treatments (BF, i.e., different soaking times + EMS + GA3). Results are shown in Table 1 and... Figure 7 .

[0089] Table 1. Mutagenic effects of different treatment combinations on field phenotypes at milk stage. Note: CK refers to untreated seeds, serving as a reference control; A refers to seeds soaked for 4 hours and then subjected to EMS mutagenesis but without gibberellin treatment, serving as a treatment control for evaluating the gibberellin effect; B refers to 0h + EMS + GA3; C refers to 0.5h + EMS + GA3; D refers to 2h + EMS + GA3; E refers to 4h + EMS + GA3; F refers to 8h + EMS + GA3. "—" indicates that positive and negative mutation rates are not calculated for CK.

[0090] Table 1 shows that a higher coefficient of variation (CV%) indicates greater variation in that trait within the population and richer genetic diversity. Compared to group A, most treatments exhibited richer genetic diversity or better positive mutation potential in key traits. The traits with higher variability were effective tiller number, total tiller number, and fresh weight. The CV% of effective tiller number in group E was as high as 60.48%, indicating extremely rich variation. The positive mutation rate of effective tiller number in group B was as high as 29.41%, indicating that nearly one-third of the plants had more effective tillers than the control, and the positive mutation rates in both groups were significantly higher than those in group A. However, the negative mutagenesis rate was also high in all treatment groups, indicating that while mutagenesis produced beneficial mutations, it also produced a large number of unfavorable mutations. The variation trend of total tiller number was similar to that of effective tiller number. The CV% of total tiller number in group E was the highest at 36.21%, producing rich extreme phenotypes, while the positive mutation rate of total tiller number in group B was relatively high at 13.24%. The CV% values ​​of fresh weight in groups D, E, and F were higher than the control, with group E showing the highest value, indicating that EMS significantly increased the variation in fresh weight. Groups C, D, and F showed high negative variation rates (>22%), indicating that a large number of plants suffered biomass loss. However, group A showed symmetrical positive and negative variation (both 13.64%), and group E also retained 9.52% of positive mutants; these are key targets for screening high-yielding mutants. Plant height and ear length are relatively stable traits, with positive variation rates of 0 for both, and their average values ​​did not change much, resulting in relatively low CV% values. This indicates that plant height and main ear length have high genetic stability, making it difficult to generate significantly increased beneficial mutants through EMS mutagenesis. The average leaf area of ​​each treatment was suppressed, but groups B and E produced 8.82% and 4.11% of positive mutants, respectively, indicating the existence of individual plants with larger leaves and the potential to improve photosynthetic capacity. The average stem diameter did not change much, but the positive mutation rate of group F (5.41%) was relatively high, and its average value was the highest (10.46), indicating that this treatment may have screened out a few lodging-resistant mutants with particularly thick stems.

[0091] Systematic comparison with group A showed that exogenous GA3 treatment effectively alleviated EMS damage. Overall, group E produced the most diverse populations with extreme phenotypes, while group B yielded a high proportion of positive mutants in terms of effective tillering and fresh weight, with the smallest overall inhibitory effect. Although mutagenesis reduced the average fresh weight of the population, groups E and B still showed the greatest potential in creating high-yielding positive mutants. In subsequent generations, the focus will be on individual selection of plants with excellent fresh weight performance in group E, combined with screening based on multiple traits such as tiller number, in order to obtain new forage oat germplasm with significantly improved yield traits.

[0092] 2. Analysis of mutagenesis efficiency Mutagenized oat seeds ('Zhongtian No. 4' hulled oat seeds) for each treatment were sown. Individual plants of the M1 generation were harvested for seed production. A systematic agronomic trait survey was conducted on the M2 generation lines to evaluate the efficiency of heritable mutations induced by different EMS synergistic treatments. The results are shown in Table 2.

[0093] Table 2. Field mutation efficiency of comprehensive treatment Table 2 shows that all treatments successfully generated mutants, but there were significant differences in their ability to induce directional variation in different traits. Compared with group A, the GA3 post-treatment (groups A-E) significantly increased the mutation frequency and type richness, resulting in a greater variety of mutation types in the tillering trait, which directly determines yield. Treatment B showed an absolute advantage in the trait of "increased effective tillering," with a mutagenesis efficiency as high as 5.59%, significantly higher than other treatments, indicating that this treatment is the optimal condition for creating high-tillering, high-yielding mutants. Fresh weight, as a direct reflection of biomass, is crucial for mutant screening. The results showed that treatments B and E had the highest mutagenesis efficiency in the trait of "increased fresh weight," at 1.68% and 1.92%, respectively, while treatment E had a relatively moderate efficiency in the trait of "decreased fresh weight," indicating that these two treatments have the greatest potential in creating high-biomass genotypes.

[0094] In summary, all treatments induced a wide range of phenotypic variations, with the E treatment (4-hour soaking) demonstrating a unique comprehensive advantage in creating yield-related mutants. This treatment not only produced the highest proportion of "fresh weight increase" mutants but also induced abundant extreme variations in tiller number. This indicates that the E treatment is the optimal condition for creating a mutant library with yield breakthrough potential, and its progeny populations are more likely to screen for high-yielding new germplasm with synergistic improvements in biomass and tillering.

[0095] As can be seen from the above, the mutagenic conditions for 'Zhongtian No. 4' hulled oat seeds are as follows: seeds with hulls removed by concentrated sulfuric acid are soaked for 4 hours until the moisture content reaches 50%, treated with 1.00% EMS for 16 hours, and then treated with exogenous 50 mg / L gibberellic acid.

[0096] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0097] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A method of increasing the EMS mutagenic effect in oats, characterized in that, The method comprises the following steps: The oat seeds are soaked with concentrated sulfuric acid to remove the hulls, and the oat seeds without hulls are obtained; The oat seeds without hulls are soaked in water to make the water content of the seeds reach 50-51%, and the soaked oat seeds are obtained; The soaked oat seeds are treated with EMS mutagenesis, and the mutagenized oat seeds are obtained; The mutagenized oat seeds are soaked with an exogenous gibberellin solution, and germination is performed to obtain the germinated seedlings; The germinated seedlings are cultured, and the M1 generation seeds are obtained after seed collection; the M1 generation seeds are sowed and planted to obtain the M2 generation mutagenized population.

2. The method of claim 1, wherein, The EMS mutagenesis treatment process is as follows: The soaked oat seeds are soaked in a 1% ethyl methanesulfonate solution for 15-17 hours.

3. The method of claim 2, wherein, The temperature of the soaked oat seeds soaked in the 1% ethyl methanesulfonate solution is 18-22℃.

4. The method of claim 1, wherein, The mutagenized oat seeds are soaked with the exogenous gibberellin solution for 7-10 days.

5. The method of claim 4, wherein, The concentration of the exogenous gibberellin solution is 49-51 mg / L.

6. The method of claim 1, wherein, The soaking treatment conditions are as follows: The oat seeds without hulls are soaked in water at 3.5-4.5℃ for 3.5-4.5 hours.

7. The method of claim 1, wherein, The oat seeds are treated with concentrated sulfuric acid for 5.5-6.5 minutes.

8. The method of claim 7, wherein, The concentrated sulfuric acid is a 97-99% sulfuric acid solution.

9. The method of claim 1, wherein, The germinated seedlings are cultured under the following conditions: 1980-2020 lux, 24-26℃ / 19-21℃, 15-17 hours / 7-9 hours light period, and the cycle is 3.5-4.5 months.

10. The method of claim 1, wherein, The oat seeds include 'Zhongtian No. 4' oat seeds with hulls.

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