A method for creating stress-resistant mutants of common bean using chemical mutagenesis and its application

Abstract

This invention discloses a method and its application for creating stress-resistant mutants of common bean using chemical mutagenesis. The main steps include: determining the screening concentration of hydroxyproline (Hyp), determining the median lethal dose of ethyl methanesulfonate (EMS), and subculturing the stress-resistant mutants. After 3-5 subcultures, the stress-resistant mutants screened by this invention ultimately yielded 31 cotyledonary nodes of common bean resistant to hydroxyproline (Hyp), with a mutation rate of 8.16%. This invention can induce mutagenesis and directionally screen for stress-resistant common bean mutants, providing material support for stress-resistant breeding of common bean.

Description

Technical Field

[0001] This invention belongs to the field of mutation breeding technology, specifically relating to the creation of stress-resistant mutants using chemical mutagenesis. The explants of the cotyledonary nodes of the "Shuangfeng No. 1" common bean variety are treated with the chemical mutagen EMS to induce gene mutations, thereby producing stress-resistant variations. By screening for stress-resistant mutants using hydroxyproline (Hyp), germplasm resources with excellent stress resistance can be obtained, providing material support for breeding new common bean varieties with wide adaptability and strong resistance. In agricultural production, plants often face various adverse conditions, such as drought, salinity, and low temperature. By screening and improving stress-resistant mutants, the adaptability of plants to various adverse conditions can be enhanced, thereby ensuring normal plant growth and development, and improving crop yield and quality. Background Technology

[0002] Mutation breeding is a method that uses chemical and physical means to induce genetic variations in organisms. The core of this method lies in using carefully selected mutagens to induce genetic variations in organisms, and then selecting new varieties with superior characteristics according to breeding objectives (Ji Xiaoyun et al., 2007).

[0003] Chemical mutagens can be applied selectively at different developmental stages of plants to achieve specific breeding goals (Agrawal L et al. 2021). These mutagens can act directly on plant DNA, generating new variants by altering gene sequences (Li Xueping et al. 2019). These variants are of great value to genetics and breeding research, helping us to explore plant trait improvement and genetic mechanisms in greater depth.

[0004] Although chemical mutagenesis has a high mutation rate and frequency, significantly improving breeding efficiency, it can also lead to non-specific mutations. These non-specific mutations may adversely affect plant development and growth, thus increasing breeding uncertainty and risk (Mullins E et al. 2021). To overcome these challenges, we can combine plant tissue culture techniques with chemical mutagenesis. Based on somatic clonal variation, chemical mutagenesis can further increase the probability of variation, thus providing more options for breeding. This combined approach not only helps us to control the mutagenesis process more precisely, but also improves breeding efficiency and success rate (Luo Yuanhua et al. 2022; Wu Zhengjing et al. 2024; Wang Zengzhen et al. 2009).

[0005] Chemical mutagens are generally classified into four categories (Zhang Ruicheng et al. 2017; Peng Bo et al. 2007; Xu Ming et al. 2011; Kishor H et al. 2017; Wang Xiaohua et al. 2010; Han Tingting et al. 2023): (1) alkylating agents; (2) base analogs; (3) acridines and antibiotics; (4) azides. When using chemical mutagens, it is necessary to carefully control the dosage and pay attention to selecting appropriate treatment times and methods. Ethyl methanesulfonate (EMS) is a commonly used alkyl compound with good mutagenic effects in plant mutation breeding. This invention uses EMS as a mutagen for stress-resistant mutagenesis.

[0006] Hydroxyproline (Hyp), an amino acid, plays an important physiological role in plants. In the selection of stress-resistant mutants, hydroxyproline can act as a selection pressure, increasing the stability and elasticity of the cell wall and thus enhancing the plant's ability to adapt to adverse conditions. Summary of the Invention

[0007] This invention overcomes the shortcomings of conventional hybridization breeding techniques and provides a method for obtaining germplasm resources with excellent stress resistance.

[0008] The purpose of this invention is to induce stress-resistant mutants in common beans and successfully screen for them in a targeted manner, providing a material basis for stress-resistant breeding of common beans. To achieve the above, this invention discloses the following technical contents:

[0009] (1) Determination of the screening concentration of hydroxyproline (Hyp)

[0010] Using 5-7 day old cotyledonary nodes of common bean seedlings as material, those with similar growth stages were selected and inoculated into regeneration medium (MS + 2.0 mg / L 6-BA) supplemented with different concentrations of Hyp (1, 1.5, 2, 2.5, 3, 3.5 mmol / L). Adventitious shoot regeneration medium without added hydroxyproline (Hyp) served as the blank control (CK). Ten bottles were inoculated for each treatment, with four cotyledonary nodes inoculated per bottle, and the treatment was repeated three times. After 14 days, the mortality rate of cotyledonary nodes was recorded. The surviving cotyledonary nodes were inoculated onto regeneration medium, and the regeneration rate of cotyledonary nodes on the regeneration medium was recorded after 14 days.

[0011] (2) Determination of the median lethal dose of EMS

[0012] Cotyledonary segments with similar growth status were placed in 100mL Erlenmeyer flasks. Six different volume-to-volume (V / V) EMS solutions were prepared using sterile phosphate buffer as the solvent: 0% (control, no EMS), 0.05%, 0.1%, 0.2%, 0.4%, and 0.6%. 50mL of the prepared EMS solution was added to each Erlenmeyer flask, with four flasks per treatment, each containing 15 cotyledonary segments, and the treatment was repeated three times. The segments were soaked in the dark for 2 hours, then removed and placed on petri dishes containing moistened sterile filter paper. They were then left to stand in the dark for another 12 hours. After 12 hours, the segments were rinsed 3-5 times with sterile water for 3 minutes each time, then transferred to MS medium for recovery culture for 3 days, followed by transfer to regeneration medium (MS + 2mg / L 6-BA). The mortality and regeneration rates of the cotyledonary segments were recorded after 20 days. (Note: EMS refers to ethyl methanesulfonate, a commonly used chemical mutagen).

[0013] (3) EMS mutagenesis and screening of cotyledonary nodes

[0014] The optimal mutagenic dose was used to mutate the cotyledonary nodes of common bean. The mutated cotyledonary nodes were transferred to MS medium without any selection pressure or plant growth regulators for 3 days of recovery culture. After 3 days, the recovered cotyledonary nodes were transferred to regeneration medium supplemented with the optimal selection dose of hydroxyproline (Hyp) for selection. After 20 days of culture, the survival rate and mutation rate of the cotyledonary nodes were counted.

[0015] This invention further discloses the application of the chemical mutagenesis method for creating stress-resistant mutants in obtaining stress-resistant mutants of common bean. Experimental results show that EMS can effectively induce stress-resistant mutations in the cotyledonary nodes of common bean. Adding hydroxyproline to the stress-resistant selection medium allows for targeted screening of stress-resistant mutants. The screened stress-resistant mutants, after subculturing, can grow normally, flower, and bear fruit, thus yielding mutant plants and seeds.

[0016] This invention primarily investigates whether mutants generated from EMS chemical mutagenesis of "Shuangfeng No. 1" bean explants can be regenerated through selection pressure. The key focus is on determining the selection pressure and the optimal mutagenesis dose (the optimal mutagenesis dose refers to the half-lethal dose of the chemical mutagen EMS). The challenge of this invention lies in the screening method for directional selection pressure (directional selection pressure refers to the critical concentration at which explants can survive and regenerate normally in a culture medium without selection agents, but cannot survive normally when selection agents are added to the culture medium; adding this concentration of chemical agent can directionally screen for stress-resistant mutants) and the optimal dose of EMS for mutagenesis of "Shuangfeng No. 1" bean.

[0017] The innovation of this invention lies in using chemical mutagenesis to obtain a highly resistant bean mutant through targeted screening.

[0018] The positive effects of the EMS mutagen induced in common bean explants to obtain stress-resistant mutants disclosed in this invention compared with the prior art are that chemical mutagenesis has a higher mutation ratio and frequency, which can significantly improve breeding efficiency.

[0019] The present invention is described in more detail below:

[0020] Experimental methods and results:

[0021] Using bean cotyledonary nodes as mutagenesis material, EMS at an appropriate concentration was used for mutagenesis treatment. Surviving and well-growing bean cotyledonary nodes were screened for hydroxyproline (Hyp), successfully obtaining hydroxyproline-tolerant mutant plants. The results showed that treating bean cotyledonary nodes with 0.17% EMS solution for 2 h achieved a half-lethal efficiency, with a non-regeneration rate approaching 50%. The hydroxyproline (Hyp) screening pressure was 3.5 mmol / L. After inoculation, the healthy bean cotyledonary nodes were inoculated onto MS medium supplemented with 3.5 mmol / L Hyp and 2 mg / L 6-BA for further culture. After 3-5 subcultures, 31 hydroxyproline-tolerant bean cotyledonary nodes were finally obtained, with a mutation rate of 8.16%.

[0022] Experimental conclusion: Explants treated with EMS can produce variants with strong stress resistance. Hydroxyproline (Hyp) medium can be used to selectively screen for stress-resistant mutants, thereby obtaining stress-resistant mutant plants and seeds. Attached Figure Description

[0023] Figure 1 The growth of EMS-mutated explants on selection medium containing hydroxyproline (Hyp) is shown. A represents bean cotyledonary nodes that were not inoculated onto hydroxyproline selection medium after EMS mutagenesis. B, C, D, and E represent bean cotyledonary nodes that showed browning after treatment with the optimal EMS mutagenesis dose and inoculation onto hydroxyproline (Hyp) selection medium (MS + 2 mg / L 6-BA + 3.5 mmol / L Hyp). F, G, and H represent hydroxyproline-resistant mutants that grew normally on hydroxyproline (Hyp) selection medium after treatment with the optimal EMS mutagenesis dose.

[0024] Figure 2 The growth of hydroxyproline (Hyp) resistant mutant plants selected through targeted screening after transplanting is shown; A and B are M1 generation mutant plants, and C and D are the growth status two weeks after transplanting.

[0025] Figure 3The images show a comparison of leaves and pods from hydroxyproline (Hyp) resistant mutant plants and normal plants. A represents bean leaves without EMS mutagenesis treatment; BH represents deformed leaves (including lobed leaves, B, C, D), inverted heart-shaped leaves (E), and curled leaves (F, G, H)) exhibited by regenerated mutant plants after EMS treatment at the optimal dose. I represents pods from regenerated bean seedlings without EMS mutagenesis treatment; J represents pods from regenerated mutant plants after EMS treatment at the optimal dose, inoculated onto hydroxyproline (Hyp) selection medium (MS + 2 mg / L 6-BA + 3.5 mmol / L Hyp). Detailed Implementation

[0026] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention are also within the scope of protection of this invention. All raw materials and reagents used in this invention are commercially available.

[0027] Example 1

[0028] Test methods

[0029] 1. Determination of the screening concentration of hydroxyproline (Hyp)

[0030] Using 5-7 day old cotyledonary nodes of common bean seedlings as material, those with similar growth stages were selected and inoculated into regeneration medium (MS + 2.0 mg / L 6-BA) supplemented with different concentrations of Hyp (1, 1.5, 2, 2.5, 3, 3.5 mmol / L). Adventitious shoot regeneration medium without added hydroxyproline (Hyp) served as the blank control (CK). Ten bottles were inoculated for each treatment, with four cotyledonary nodes inoculated per bottle, and the treatment was repeated three times. After 14 days, the mortality rate of cotyledonary nodes was recorded. The surviving cotyledonary nodes were inoculated onto regeneration medium, and the regeneration rate of cotyledonary nodes on the regeneration medium was recorded after 14 days.

[0031] Determination of LD2

[0032] 2.1 Preparation of EMS solution

[0033] Weigh 1.36 g KH2PO4, add 0.1 mol / L NaOH solution and dilute with distilled water to 200 mL, adjust the pH to 7.0, sterilize at 121 ℃ and 0.1 MPa for 20 min, and set aside for later use.

[0034] 2.2 Determination of EMS median lethal dose

[0035] Cotyledonary nodes with similar growth status were placed in 100mL Erlenmeyer flasks. Six different volume-to-volume (V / V) EMS solutions were prepared using sterile phosphate buffer as the solvent: 0% (control, no EMS), 0.05%, 0.1%, 0.2%, 0.4%, and 0.6%. 50mL of the prepared EMS solution was added to each Erlenmeyer flask, with four flasks per treatment, each containing 15 cotyledonary nodes, and the treatment was repeated three times. The flasks were soaked in the dark for 2 hours, then the cotyledonary nodes were removed and placed on petri dishes containing moistened sterile filter paper. They were then incubated in the dark for another 12 hours. After 12 hours, the cotyledonary nodes were rinsed 3-5 times with sterile water for 3 minutes each time, then transferred to MS medium for recovery culture for 3 days, followed by transfer to regeneration medium (MS + 2 mg / L 6-BA). The mortality rate and regeneration rate of the cotyledonary nodes were recorded after 20 days.

[0036] 3. EMS mutagenesis and screening of cotyledonary segments

[0037] Mutagenesis was induced in bean cotyledonary nodes using the optimal mutagenesis dose. The mutated cotyledonary nodes were then transferred to MS medium without any selection pressure or plant growth regulators for 3 days of recovery culture. After 3 days, the recovered cotyledonary nodes were transferred to regeneration medium supplemented with the optimal selection doses of polyethylene glycol (PEG-8000) and hydroxyproline (Hyp) for selection. After 20 days of culture, the survival rate and mutation rate of the cotyledonary nodes were calculated.

[0038] 4. Obtaining stress-resistant mutant lines

[0039] The obtained stress-resistant mutant plants (M1 generation) were further propagated to obtain stress-resistant mutant lines. These were then transplanted and domesticated to obtain seeds.

[0040] 5. Data Analysis

[0041] Mortality rate (%) = (Number of dead explants / Total number of inoculated explants) × 100%

[0042] Regeneration rate (%) = (Number of explants that regenerated normally / Total number of inoculated explants) × 100%

[0043] Non-regeneration rate (%) = (Number of explants that cannot regenerate normally / Total number of inoculated explants) × 100%

[0044] Mutation rate (%) = (number of surviving explants / number of explants inoculated) × 100%.

[0045] Survival rate (%) = (Surviving seedlings / Total number of transplanted seedlings) × 100%

[0046] Example 2

[0047] 1. Effect of hydroxyproline (Hyp) on cotyledon node regeneration in common bean

[0048] Table 1. Mortality and non-regeneration rate of cotyledonary nodes of "Shuangfeng No. 1" under different concentrations of hydroxyproline (Hyp) screening pressure.

[0049]

[0050] Note: CK is cotyledon regeneration medium without added Hyp.

[0051] Unmutated bean cotyledonary nodes were placed on selection media supplemented with different concentrations of hydroxyproline (Hyp) for 14 days, and then transferred to regeneration media. Some cotyledonary nodes lost their regeneration ability. Table 1 shows that on the regeneration medium without HYP, the cotyledonary nodes grew well, with a mortality rate of 0% and a non-regeneration rate of 3.32 ± 0.58%. HYP inhibited the growth of adventitious buds. On media supplemented with different concentrations of hydroxyproline (HYP), the mortality rate and non-regeneration rate of bean cotyledonary nodes increased significantly with increasing HYP concentration, indicating a stronger inhibitory effect on adventitious bud regeneration. This suggests that HYP has a strong toxic effect on bean cotyledonary nodes. When the concentration of hydroxyproline was 2 mmol / L, the mortality rate of bean cotyledonary nodes was 45.26 ± 0.74%, and the non-regeneration rate was 63.16 ± 0.91%, indicating severe inhibition of adventitious bud growth. When the hydroxyproline concentration was 3.5 mmol / L, the mortality rate of the cotyledonary nodes of common bean was 98.95 ± 0.67%, and the non-regeneration rate was 100.00 ± 0.00%. Therefore, 3.5 mmol / L is the critical concentration for screening stress-resistant mutants of the cotyledonary nodes of common bean.

[0052] 2. Determination of the median lethal concentration for cotyledon node regeneration in common bean using EMS

[0053] Table 2. Regeneration capacity of cotyledon nodes of "Shuangfeng No. 1" green bean under EMS treatment with different volume concentrations.

[0054]

[0055] Note: ck indicates no EMS treatment, only phosphate buffer is added.

[0056] Cotyledonary nodes soaked in EMS solutions of different concentrations for 2 h were transferred to MS medium for recovery culture for 3 days. During the recovery process, significant differences in cotyledonary node mortality were observed; the explants gradually turned white and died. After 3 days of recovery culture, the cotyledonary nodes were transferred to regeneration medium (MS + 2 mg / L 6-BA). After 20 days, significant differences in cotyledonary node mortality and regeneration rates were observed in each treatment. Table 2 shows that with increasing EMS solution concentration, the inhibitory effect of EMS on the growth of bean cotyledonary nodes increased, and the mortality and non-regeneration rates of bean cotyledonary nodes showed a strong correlation. The EMS mutagen exhibited a significant toxic effect on bean cotyledonary nodes. Without EMS, phosphate buffer had no significant effect on the growth and regeneration of bean cotyledonary nodes; after 3 days of recovery, the cotyledonary nodes could regenerate normally on the regeneration medium. After mutagenesis treatment with different concentrations of EMS solution, the mortality rate of cotyledonary nodes in common bean was significantly increased compared to the control (CK) treatment with phosphate buffer, and the non-regeneration rate of cotyledonary nodes also increased significantly. With increasing EMS mutagen concentration, the mortality rate also increased, and the inhibitory effect on cotyledonary node regeneration strengthened, exhibiting a clear dose-response effect. At an EMS concentration of 0.6%, the mortality rate of the mutagenized cotyledonary nodes was 98.77 ± 0.39%, and the non-regeneration rate was 100%. The inhibitory effect of EMS on the regeneration of common bean cotyledonary nodes reached its peak; that is, when the EMS concentration reached 0.6%, the cotyledonary nodes did not regenerate; when the EMS concentration was 0.8%, the mortality rate and non-regeneration rate of the cotyledonary nodes were both 100%. Therefore, 0.8% EMS solution is the lethal dose for common bean cotyledonary nodes.

[0057] The components of EMS are as follows: ethyl methanesulfonate

[0058] MS formulation reference: Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures[J]. Physiologia Plantarum, 1962,15(3): 473-497.

[0059] To ensure a high mutagenicity rate in bean cotyledonary nodes while minimizing the toxicity of EMS solution to cells and reducing mortality, the optimal mutagenicity condition was determined by the median lethal dose (LD50) of EMS. Adjusting the EMS concentration and treatment time further optimized the mutagenicity conditions, improving mutant screening efficiency and mutagenicity. Table 2 shows that the cotyledonary node mortality rate was 34.48 ± 0.61% when the EMS concentration was 0.1%, and 66.67 ± 0.47% when the EMS volume concentration was 0.2%. Therefore, 0.1%–0.2% represents the LD50 range for EMS mutagen in bean cotyledonary nodes.

[0060] By fitting a logistic curve equation to EMS concentration and its corresponding mortality rate, the following equation is obtained:

[0061]

[0062] Correlation coefficient EMS concentration and mortality rate after conversion The equations show a strong linear correlation, and the median lethal concentration (LD50) of EMS was calculated. 50 =0.17%.

[0063] e represents the natural logarithm, with a value of approximately 2.718.

[0064] x represents the concentration of the EMS solution.

[0065] Effects of 3-hydroxyproline (Hyp) on cotyledonary node regeneration in bean induced by EMS mutagenesis

[0066] Table 3. Growth status of cotyledonary segments after EMS mutagenesis on hydroxyproline (Hyp) selection medium.

[0067]

[0068] Note: CK represents the growth status of cotyledonary segments without EMS mutagenesis on hydroxyproline selection medium.

[0069] See Figure 1 Fresh cotyledonary nodes of common beans were treated with the optimal dose of EMS mutagenesis and then inoculated onto hydroxyproline (Hyp) selection medium (MS + 2 mg / L 6-BA + 3.5 mmol / L Hyp). Observations showed that after EMS mutagenesis followed by inoculation onto the hydroxyproline selection medium, most cotyledonary nodes exhibited browning. In the early stages of browning, adventitious buds could regenerate from the cotyledonary nodes. Most of these newly grown adventitious buds, influenced by EMS and hydroxyproline (Hyp), browned from the base and failed to grow normally. Figure 1BE). A few cotyledonary nodes showed stable growth, a few cotyledons fell off, and there was no browning or death; a very small number showed vigorous growth. Figure 1 (FH); Unmutated cotyledonary segments rapidly browned on hydroxyproline (Hyp) selection medium, and some unbrowned cotyledonary segments could not regenerate. This indicates that EMS mutagenesis treatment had a significant impact on the growth and development of bean cotyledonary segments, producing hydroxyproline-resistant mutants. After EMS mutagenesis treatment, the resistance of bean cotyledonary segments was improved, while untreated cotyledonary segments could not grow and regenerate normally under the same culture conditions.

[0070] Table 3 leads to the following conclusions: Under CK treatment, cotyledonary nodes could not survive on hydroxyproline (Hyp) selection medium. However, a small number of cotyledonary nodes treated with EMS mutagenesis were able to survive on hydroxyproline selection medium. This indicates that EMS mutagenesis can improve the survival rate of cotyledonary nodes on hydroxyproline (Hyp) selection medium, thereby generating hydroxyproline (Hyp) resistant mutants with a mutation rate of 8.61%.

[0071] 4. Obtaining hydroxyproline (Hyp) resistant mutant lines

[0072] Table 4. Growth of regenerated adventitious buds from hydroxyproline-tolerant (Hyp) mutants.

[0073]

[0074] Cotyledonary nodes treated with EMS mutagenesis were inoculated onto hydroxyproline selection medium. The 31 surviving cotyledonary nodes were then inoculated onto MS medium supplemented with 2 mg / L 6-BA for 7-14 days. 23 cotyledonary nodes successfully survived and regenerated, with a regeneration rate of 74.19%. The adventitious bud differentiation efficiency of the regenerated hydroxyproline-resistant cotyledonary nodes was lower than that of untreated cotyledonary nodes. On average, each hydroxyproline-resistant cotyledonary node could induce 2 adventitious buds, exhibiting good growth. This yielded the "Shuangfeng No. 1" hydroxyproline-resistant (Hyp) mutant of common bean. The hydroxyproline-resistant (Hyp) mutant plants were inoculated onto 1 / 2 MS medium supplemented with 1 mg / L IBA and cultured for 14 days to obtain the M1 generation of the hydroxyproline-resistant mutant line. Figure 2 The hydroxyproline-resistant mutant plant M1, induced by EMS in vitro mutagenesis, exhibited significant morphological differences, including leaf deformities, plant clustering, and pod curling. Figure 3 The M1 generation showed lower cotyledon node regeneration ability than the CK, and the number of regenerated adventitious buds was also lower than that of the CK treatment. The M1 mutant plant showed strong rooting ability and fast rooting speed.

[0075] After the detailed description of the preferred embodiments, those skilled in the art will clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above-mentioned claims. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of this invention fall within the scope of this invention. Furthermore, this invention is not limited to the exemplary embodiments described in the specification.

Claims

1. A method for creating stress-resistant mutants of common bean using chemical mutagenesis, characterized in that... Follow these steps: (1) Determination of the screening concentration of hydroxyproline: Using cotyledonary nodes of common bean seedlings aged 5-7 days as material, those with similar growth status were selected and inoculated into regeneration medium MS + 2.0 mg / L 6-BA with different concentrations of hydroxyproline. The hydroxyproline concentration gradients were 1, 1.5, 2, 2.5, 3, and 3.5 mmol / L. The adventitious shoot regeneration medium without hydroxyproline was used as the blank control CK group. Each treatment was inoculated into 10 bottles, with 4 cotyledonary nodes inoculated into each bottle, and the results were repeated 3 times. After 14 days, the mortality rate of cotyledonary nodes was counted. The surviving cotyledonary nodes were inoculated onto regeneration medium, and the regeneration rate of cotyledonary nodes on the regeneration medium was counted after 14 days. (2) Determination of the median lethal dose of EMS: Cotyledonary nodes with similar growth status were placed in 100mL Erlenmeyer flasks. Six different volume concentrations (V / V) of EMS solution were prepared using sterile phosphate buffer as the solvent. 50mL of the prepared EMS solution was added to each flask, with four flasks per treatment. Each flask contained 15 cotyledonary nodes, and the treatment was repeated three times. The flasks were soaked in the dark for 2 hours. Afterward, the cotyledonary nodes were removed and placed on petri dishes containing moistened sterile filter paper. They were then left to stand in the dark for another 12 hours. After standing for 12 hours, the cotyledonary nodes were rinsed 3-5 times with sterile water for 3 minutes each time. They were then transferred to MS medium for recovery culture for 3 days, followed by transfer to regeneration medium MS + 2mg / L 6-BA. The mortality and regeneration rates of the cotyledonary nodes were recorded after 20 days. The six different volume concentrations (V / V) of EMS solution were: 0%, 0.05%, 0.1%, 0.2%, 0.4%, and 0.6%, with 0% representing the control (CK) without added EMS. (3) EMS mutagenesis of cotyledonary nodes and screening of stress-resistant mutants: The cotyledonary nodes of common bean were mutagenized using the optimal mutagenic dose. The mutagenized cotyledonary nodes were then transferred to MS medium without any selection pressure or plant growth regulators for 3 days of recovery culture. After 3 days, the recovered cotyledonary nodes were transferred to regeneration medium supplemented with the optimal selection dose of hydroxyproline for selection. After 20 days of culture, the survival rate and mutation rate of the cotyledonary nodes were calculated. The optimal selection dose of hydroxyproline was 3.5 mmol / L. (4) Creation of stress-resistant mutants of cotyledon nodes in common bean: Common bean cotyledonary nodes were treated with 0.17% EMS solution for 2 hours to achieve a half-lethal efficiency and a non-regeneration rate of 50%. The hydroxyproline selection pressure was 3.5 mmol / L. The mutagenic common bean cotyledonary nodes in good condition were inoculated into MS medium with 3.5 mmol / L hydroxyproline and 2 mg / L 6-BA and cultured for a further 3-5 subcultures to finally obtain hydroxyproline-tolerant common bean cotyledonary nodes.

2. The application of the method for creating stress-resistant mutants of common bean using chemical mutagenesis as described in claim 1 in obtaining stress-resistant common bean mutants.

Images