A method for screening drought-resistant rice mutants

Abstract

This invention relates to the fields of agricultural biotechnology and crop breeding technology, specifically a method for screening drought-resistant rice mutants. The method includes the following steps: using a space-induced mutation SP₂ generation population as material, under stress with 22% PEG-6000 solution, initial drought-resistant germplasm is selected based on germination rate; the initial selected germplasm is cultivated to the three-leaf stage, and under stress with 24% PEG-6000 solution, a second screening is conducted based on chlorophyll content ratio; the malondialdehyde (MDA), proline content, and antioxidant enzyme activity of the selected plants are measured to screen drought-resistant mutants with excellent physiological indicators. This invention utilizes a multi-level progressive screening system—primary phenotypic screening, secondary phenotypic screening, and physiological verification—to achieve efficient and accurate identification of drought-resistant mutants from space-induced mutations, significantly reducing the false positive rate and providing fully validated superior germplasm resources for drought-resistant rice breeding.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology and crop breeding technology, specifically a method for screening drought-resistant rice mutants. Background Technology

[0002] Rice (Oryza sativa L.), as one of the most important food crops in China and even globally, is highly dependent on water resources for its growth. With the intensification of global climate change and the increasing frequency and severity of droughts, water scarcity has become a key factor limiting the stability and sustainable development of rice production. Therefore, exploring and utilizing drought-resistant germplasm resources and cultivating new drought-resistant rice varieties is of great strategic significance for ensuring national food security.

[0003] Drought resistance in rice is a complex quantitative trait controlled by multiple genes, and its phenotypic identification is easily affected by environmental factors. As the initial stage of the rice life cycle, the strength of drought resistance during germination directly affects field emergence rate, seedling uniformity, and robustness, forming the cornerstone of later yield formation. Therefore, establishing efficient and accurate drought resistance identification methods during germination is crucial for discovering drought-resistant germplasm. However, existing technologies face two major problems in drought resistance identification during germination: First, the screening stress standards are inconsistent. Polyethylene glycol (PEG-6000) is a commonly used osmotic regulator for simulating drought stress, but the suitable concentration used for screening rice during germination varies greatly in existing studies, ranging from 12.5% ​​to 25%. This difference stems from variations in the genotype of the tested varieties, seed physiological state, and experimental conditions. Too low a stress concentration fails to effectively distinguish drought resistance; too high a concentration severely inhibits germination, leading to the wrong elimination of potentially valuable materials. Second, the screening indicators are singular, resulting in a high false positive rate. Most existing methods rely solely on single morphological indicators such as germination rate. However, germination rate is easily affected by seed vigor and cannot truly reflect the plant's intrinsic physiological drought resistance mechanism, leading to a high false positive rate in screening results and unstable drought resistance performance of the screened materials in subsequent growth stages.

[0004] Space-induced mutation breeding is an advanced breeding technology that utilizes the unique environment of space (such as microgravity, strong radiation, and high vacuum) to induce genetic variations in plant seeds. It boasts advantages such as high mutation frequency, broad mutation spectrum, and numerous beneficial mutations, providing an effective approach for creating groundbreaking new germplasm. However, the core challenge in applying this technology to drought-resistant breeding lies in efficiently and accurately screening for truly valuable drought-resistant mutants from the vast population of space-induced mutation progeny. Current technologies lack a standardized and systematic screening scheme that integrates space-induced mutation with verification of germination, seedling, and physiological mechanisms. Summary of the Invention

[0005] The purpose of this invention is to provide a method for screening drought-resistant rice mutants. This method integrates initial screening at the germination stage, secondary screening at the seedling stage, and in-depth verification of key physiological indicators to form a progressive, multi-dimensional, standardized screening system. This solves the problems of inaccurate screening pressure, high false positive rate, low screening efficiency, and inability to reveal the internal drought resistance mechanism of mutants in existing technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for screening drought-resistant rice mutants, comprising the following steps:

[0007] (1) Initial screening of drought resistance during germination: Using the SP2 generation of rice obtained by space mutagenesis as material, under the conditions of temperature of 26±1℃, photoperiod of 9 hours of light / 15 hours of darkness and relative humidity of 60%, a 22% (w / v) polyethylene glycol-6000 solution was used to conduct drought stress treatment during germination for 7 days. The germination rate was calculated based on the standard that the length of the plumule reaches half the length of the seed and the length of the radicle is equal to that of the seed. Plants with a germination rate significantly higher than that of the wild-type control without mutagenesis were selected as the initial drought-resistant germplasm.

[0008] (2) Re-screening of drought resistance in seedlings: The drought-resistant germplasm obtained in step (1) was cultivated to the three-leaf and one-heart stage. The seedlings were subjected to drought stress treatment for 7 days using a 24% polyethylene glycol-6000 solution. The chlorophyll content of the stress treatment group and the unstressed control group was measured. The percentage ratio of chlorophyll content in the stress group to that in the control group was calculated. Plants with a ratio significantly higher than that in the wild-type control were screened.

[0009] (3) Verification of drought-resistant physiological indicators: For the plants screened in step (2), under the drought stress conditions during the germination period described in step (1), the malondialdehyde content, proline content, superoxide dismutase activity, peroxidase activity and catalase activity were measured. Plants that simultaneously meet the following conditions were selected as drought-resistant rice mutants:

[0010] Malondialdehyde (MDA) content was significantly lower than that of the wild-type control;

[0011] The proline content was significantly higher than that of the wild-type control;

[0012] The activities of superoxide dismutase, peroxidase, and catalase were all significantly higher than those of the wild-type control.

[0013] Preferably, the germination rate in step (1) is significantly higher than that of the wild-type control, the ratio in step (2) is significantly higher than that of the wild-type control, the malondialdehyde content in step (3) is significantly lower than that of the wild-type, and the proline and enzyme activities are significantly higher than those of the wild-type, all of which refer to the statistical significance p-value being greater than 0.05 through Dunnett's multiple comparison test.

[0014] A drought-resistant rice mutant obtained by the above method is characterized in that: after being treated with a 22% (w / v) polyethylene glycol-6000 solution for 7 days during the germination period, the germination rate of the mutant is not less than 30%; after being treated with a 24% (w / v) polyethylene glycol-6000 solution for 7 days during the seedling stage, the percentage ratio of chlorophyll content in the stressed group to that in the control group is not less than 75%.

[0015] Preferably, under drought stress during the germination period, the mutant has a malondialdehyde content not higher than 70% of the wild-type control, a proline content not lower than 150% of the wild-type control, and the activities of superoxide dismutase, peroxidase, and catalase not lower than 130% of the wild-type control.

[0016] Preferably, the mutant is K609, K1238, K1273, K1280, K1296 or K1300.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1. This invention establishes a standardized drought stress concentration system for space-induced mutagenesis rice populations. Through gradient experiments, the optimal screening concentrations of 22% PEG-6000 during the germination stage and 24% PEG-6000 during the seedling stage were determined, solving the problems of inconsistent stress concentrations and unsuitable screening pressures in existing technologies, and ensuring the accuracy and reproducibility of the screening results.

[0019] 2. This invention constructs a progressive screening system from germination stage to seedling stage. First, drought-resistant materials are rapidly enriched from a large-scale mutagenesis population through initial screening at the germination stage. Then, the drought resistance of the materials during the seedling stage is verified through secondary screening at the seedling stage. This effectively eliminates false positive materials that only show drought resistance at the germination stage, improving the screening efficiency by more than 3 times.

[0020] 3. This invention establishes a comprehensive evaluation system for phenotypic and physiological indicators. Based on phenotypic identification, by measuring malondialdehyde (MDA) and proline content and the activities of three antioxidant enzymes, the drought resistance physiological mechanism of mutants was revealed from three aspects: cell membrane stability, osmotic regulation capacity, and antioxidant capacity. This ensures that the screened materials have stable drought resistance and provides a scientific basis for subsequent breeding applications.

[0021] 4. This invention screened and obtained six rice mutant materials with excellent drought resistance. These materials showed significant drought resistance advantages in both the germination and seedling stages and have a clear physiological basis. They can be directly used as parents in drought-resistant rice breeding, thus accelerating the breeding process of drought-resistant rice varieties. Attached Figure Description

[0022] Figure 1This is a schematic diagram of the overall process of the comprehensive screening method for drought-resistant mutants of the present invention;

[0023] Figure 2 The results of determining the PEG-6000 stress concentration during germination are shown in Figure A, where A is the germination potential and germination rate curves of wild-type seeds under different concentrations of PEG-6000 treatment, and B is a schematic diagram of the germination appearance of wild-type seeds under different concentrations of PEG-6000 treatment.

[0024] Figure 3 A schematic diagram showing the germination potential and germination rate curves of wild-type seeds treated with different concentrations of PEG-6000;

[0025] Figure 4 A schematic diagram showing the comparison of chlorophyll content in seedling drought resistance after secondary screening.

[0026] Figure 5 This is a schematic diagram showing the results of the drought resistance physiological index measurement. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The core technical concept of this invention is to construct a progressive multi-index screening system for the SP2 generation of space-mutated rice, consisting of "primary phenotypic screening at germination stage – secondary phenotypic screening at seedling stage – verification of physiological mechanisms." Through standardized PEG-6000 stress concentrations and clearly defined screening indicators, rice mutants with stable drought resistance can be efficiently and accurately screened. The overall technical process of this invention is as follows: Figure 1-5 As shown, the present invention provides a technical solution: a method for screening drought-resistant rice mutants, comprising the following steps:

[0029] (1) Initial screening of drought resistance during germination: Using the SP2 generation of rice obtained by space mutagenesis as material, under the conditions of temperature of 26±1℃, photoperiod of 9 hours of light / 15 hours of darkness and relative humidity of 60%, a 22% (w / v) polyethylene glycol-6000 solution was used to conduct drought stress treatment during germination for 7 days. Germination rate was calculated based on the standard that the length of the embryo reaches half the length of the seed and the length of the radicle is equal to that of the seed. Plants with a germination rate significantly higher than that of the wild-type control without mutagenesis were selected as the initial drought-resistant germplasm. The germination rate significantly higher than that of the wild-type control was defined as a statistical significance p-value greater than 0.05 by Dunnett's multiple comparison test.

[0030] (2) Re-screening of drought resistance in seedlings: The drought-resistant germplasm obtained in step (1) was cultivated to the three-leaf and one-heart stage. The seedlings were subjected to drought stress treatment for 7 days using a 24% polyethylene glycol-6000 solution. The chlorophyll content of the stress treatment group and the unstressed control group were measured respectively. The percentage ratio of chlorophyll content in the stress group to that in the control group was calculated. Plants with a ratio significantly higher than that in the wild-type control were selected. The ratio significantly higher than that in the wild-type control means that the statistical significance p value is greater than 0.05 through Dunnett's multiple comparison test.

[0031] The chlorophyll content was determined using ultraviolet spectrophotometry. Specifically, fresh leaf samples were extracted with a mixture of ethanol, acetone, and water in the dark (4.5:4.5:1). The absorbance was measured at wavelengths of 645 nm and 663 nm, and the chlorophyll content was calculated according to the Arnon formula.

[0032] (3) Verification of drought-resistant physiological indicators: For the plants screened in step (2), under the drought stress conditions during the germination period in step (1), the malondialdehyde content, proline content, superoxide dismutase activity, peroxidase activity and catalase activity were measured. Plants that simultaneously meet the following conditions were screened as drought-resistant rice mutants:

[0033] Malondialdehyde (MDA) content was significantly lower than that of the wild-type control;

[0034] The proline content was significantly higher than that of the wild-type control;

[0035] The activities of superoxide dismutase, peroxidase, and catalase were all significantly higher than those of the wild-type control.

[0036] The significantly lower malondialdehyde content compared to the wild type and the significantly higher proline and enzyme activity compared to the wild type were statistically significant (p-value greater than 0.05) according to Dunnett's multiple comparison test.

[0037] The malondialdehyde content was determined using the thiobarbituric acid method, and the malondialdehyde assay kit (catalog number A003-1-1) from Nanjing Jiancheng Bioengineering Institute was used to measure the absorbance at wavelengths of 532 nm and 600 nm.

[0038] The proline content was determined using the acidic ninhydrin method, employing the Nanjing Jiancheng Bioengineering Institute proline assay kit (catalog number A107-1-1), with absorbance measured at a wavelength of 520 nm.

[0039] Superoxide dismutase activity was determined by the photochemical reduction method of nitroblue tetrazolium using the Nanjing Jiancheng Bioengineering Institute Superoxide Dismutase Assay Kit (catalog number A001-1) at a wavelength of 560 nm.

[0040] Peroxidase activity was determined using the guaiacol method, employing the Nanjing Jiancheng Bioengineering Institute Peroxidase Assay Kit (catalog number A084-3), with absorbance measured at a wavelength of 470 nm.

[0041] Catalase activity was determined by ultraviolet absorption method using the catalase assay kit (catalog number A007-1-1) from Nanjing Jiancheng Bioengineering Institute, with absorbance measured at a wavelength of 405 nm.

[0042] A drought-resistant rice mutant obtained by the above method has the following characteristics: after being treated with a 22% (w / v) polyethylene glycol-6000 (PEG-6000) solution for 7 days during the germination period, its germination rate is not less than 30%; after being treated with a 24% (w / v) polyethylene glycol-6000 solution for 7 days during the seedling stage, the percentage ratio of chlorophyll content in the stressed group to that in the control group is not less than 75%; under drought stress conditions during the germination period, the mutant's malondialdehyde content is not higher than 70% of that in the wild-type control, its proline content is not lower than 150% of that in the wild-type control, and the activities of superoxide dismutase, peroxidase, and catalase are all not lower than 130% of those in the wild-type control. The mutants are K609, K1238, K1273, K1280, K1296, or K1300.

[0043] Example 1: Preliminary screening of drought-resistant mutants during germination.

[0044] 1.1 Experimental Materials

[0045] This embodiment uses the SP2 generation of "Hangjuxiangsimiao" rice, which underwent space-induced mutagenesis on the Chang'e 5 lunar probe, as experimental material, comprising 2250 lines. Unmutated original "Hangjuxiangsimiao" seeds were used as a wild-type (WT) control. All seeds were provided by the Rice Research Institute of Guangdong Academy of Agricultural Sciences, exhibiting uniform maturity, freedom from pests and diseases, and full grains.

[0046] 1.2 Determination of the optimal concentration for drought stress during the germination period

[0047] Accurately weigh PEG-6000 powder (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), and prepare solutions with concentrations of 0%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, and 30% (w / v) using distilled water for later use.

[0048] Select plump, uniformly sized wild-type seeds. First, disinfect them by soaking in 75% (v / v) alcohol for 5 minutes, then rinse 5-7 times with sterile water to remove residual alcohol. Line each 90mm diameter sterile petri dish with double layers of qualitative filter paper, neatly placing 50 seeds in each dish. Add 12mL of PEG-6000 solution of each concentration mentioned above. Set up three biological replicates for each concentration.

[0049] Place the culture dish in an LRH-250-G light incubator and set the culture conditions as follows: temperature 26±1℃, light cycle 9h light / 15h dark, light intensity 2000lx, and relative humidity 60%. Change the PEG-6000 solution of the appropriate concentration daily to maintain a stable osmotic potential.

[0050] Germination was defined as the embryo reaching half the seed length and the radicle being equal in length to the seed. Germination was observed and recorded daily, and germination potential was calculated on day 3. The final germination rate was calculated on day 7. Germination potential = (number of germinated seeds on day 3 / total number of seeds tested) × 100%; Germination rate = (number of germinated seeds on day 7 / total number of seeds tested) × 100%.

[0051] Experimental results are as follows Figure 2 As shown, with increasing PEG-6000 concentration, the germination potential and germination rate of wild-type seeds showed a significant decreasing trend. Within the concentration range of 14%-18%, the germination rate decreased relatively slowly, remaining above 80%. When the concentration reached 22%, the germination rate of wild-type seeds dropped to 8.7% on day 7, and significant differences in germination began to appear among different lines. At concentrations above 24%, germination was severely inhibited, with a germination rate below 2%. Therefore, a 22% PEG-6000 solution was determined to be the optimal concentration for screening drought resistance during the germination period in this invention.

[0052] 1.3SP2 generation large-scale initial screening

[0053] Based on the screening criteria established above, 2250 SP2 generation mutant materials were systematically screened. For each material, 50 healthy, plump seeds were taken, treated according to the sterilization procedure in 1.2, and then placed in a petri dish containing 12 mL of 22% PEG-6000 solution for germination testing, with culture conditions identical to those in 1.2.

[0054] After 7 days of cultivation, the germination rate of each material was recorded. Data analysis was performed using SPSS 20.0 software, and the data are expressed as mean ± standard error. Multiple comparisons were performed using the Dunnett method. The screening criteria were: the germination rate of the mutant was significantly higher than that of the wild-type control (p<0.05), and the germination rate was more than three times the standard deviation of the wild-type germination rate.

[0055] After systematic screening, 98 mutants showing significant drought resistance during germination were initially obtained from 2250 SP2 generation materials, accounting for 4.3% of all screened materials. These initially selected drought-resistant germplasms will proceed to the next stage of seedling rescreening.

[0056] 1.4 Phenotypic Validation

[0057] Phenotypic observation of the 98 drought-resistant mutants selected revealed that under 22% PEG-6000 stress, the plumule elongation rate of the drought-resistant mutants was significantly faster than that of the wild type, with more and longer radicles, and their overall growth was significantly better than that of the wild-type control. For example, the germination rate of mutant K1300 reached 42.3% after 7 days of stress, while that of the wild type was only 8.7%, a highly significant difference (p<0.001). This result validates the reliability of the germination screening system at the phenotypic level.

[0058] Example 2: Secondary screening of drought-resistant mutants during the seedling stage

[0059] 2.1 Experimental Materials and Seedling Cultivation

[0060] The experimental materials were 98 initially selected drought-resistant mutants obtained in Example 1 and wild-type "Hangjuxiangsimiao".

[0061] Soak the seeds of each material in distilled water for 48 hours, changing the water twice during this period. Then, place them in a 30℃ constant temperature incubator for 24 hours to germinate. Once the seeds show signs of germination, sow them into 96-well PCR plates with holes at the bottom, one seed per well, two plates per material, and 48 seeds per plate. Place the sown PCR plates into a culture box containing standard nutrient solution from the International Rice Research Institute (IRRI). The nutrient solution formula is shown in Table 1.

[0062] Table 1. Standard Nutrient Solution Formula of the International Rice Research Institute

[0063]

[0064] The culture boxes were placed in an MGC-350HP artificial climate chamber, with the following environmental conditions: temperature 26±1℃, photoperiod 9h light / 15h darkness, light intensity 3000lx, and relative humidity 60%. The nutrient solution was changed every 3 days, and the pH of the nutrient solution was adjusted to 5.5±0.1 with 1mol / L NaOH or 1mol / L HCl. Stress treatment was applied to the seedlings when they reached the three-leaf stage (approximately 14 days after sowing).

[0065] 2.2 Determination of the optimal concentration for drought stress during the seedling stage

[0066] Using wild-type seedlings as material, six concentration gradients of PEG-6000 were set up: 0%, 20%, 22%, 24%, 26%, and 28% (w / v). Fifteen seedlings of uniform growth were treated with each concentration and subjected to stress for 7 days, during which the stress nutrient solution was changed daily.

[0067] After the stress treatment ended, the seedling growth phenotype was observed, and the chlorophyll content of the second-to-last leaves was determined using the ethanol-acetone mixture method. The results are as follows: Figure 3 As shown: at a concentration of 20%, slight yellowing appeared at the leaf tips; at a concentration of 22%, leaf wilting and yellowing worsened, and the roots turned noticeably yellow; at a concentration of 24%, leaves showed obvious wilting, and chlorophyll content decreased by 44% compared to the control group; at a concentration of 28%, plants showed severe yellowing and wilting, with a mortality rate exceeding 50%. Considering both stress intensity and plant survival rate, 24% PEG-6000 solution was determined to be the optimal concentration for screening seedling drought resistance in this invention.

[0068] 2.3 Stress Treatment and Chlorophyll Content Measurement

[0069] Ninety-eight initially selected mutant and wild-type seedlings were randomly divided into two groups: the control group was cultured in standard nutrient solution throughout the entire process; the stress group had its nutrient solution replaced with a stress nutrient solution containing 24% PEG-6000 when the seedlings reached the three-leaf-one-heart stage. Each group had three biological replicates, with 15 seedlings in each replicate. The stress treatment lasted for 7 days, during which other culture conditions remained unchanged.

[0070] After the stress treatment, 10 seedlings of uniform growth were taken from each of the control and stress groups. 0.1g of the middle portion of the second-to-last leaf (midrib removed) was cut, chopped, and added to 2mL of extraction buffer (ethanol:acetone:water = 4.5:4.5:1). Extraction was carried out at 4℃ in the dark for 12 hours. Using the extract as a blank control, absorbance was measured at 645nm and 663nm using a UV-1800 spectrophotometer. The total chlorophyll content was calculated using the modified Arnon formula.

[0071] Total chlorophyll content (mg / g) = (20.29 × A) 645 +8.05×A 663 )×V / (W×1000)

[0072] Where V is the volume of the extraction liquid (mL) and W is the fresh weight of the sample (g).

[0073] 2.4 Results Analysis and Screening

[0074] The seedling drought tolerance of each material was assessed by calculating the ratio of "chlorophyll content in the stress group / chlorophyll content in the control group × 100%". The results are as follows: Figure 4 As shown, where Figure 4The chlorophyll content ratios of the wild type and each initially selected mutant under 24% PEG-6000 stress were shown, revealing significant differences in drought tolerance among the different mutants at the seedling stage. Mutant K1300 exhibited the best performance, with a chlorophyll content ratio of 96.5%, significantly higher than the wild type (82.9%, p<0.001). Mutants K1280 and K609, with ratios of 52.9% and 58.2% respectively, showed the greatest sensitivity.

[0075] Based on chlorophyll content ratio and plant phenotypic observations (leaf wilting degree, leaf color change, survival rate, etc.), six core mutants that maintained strong drought resistance during the seedling stage were further screened from 98 initial materials: K609, K1238, K1273, K1280, K1296, and K1300. These materials will proceed to the next stage of physiological indicator verification.

[0076] Example 3: Determination and Analysis of Drought-Related Physiological Indicators

[0077] 3.1 Experimental Materials and Sample Preparation

[0078] The experimental materials were the six core drought-resistant mutants obtained in Example 2 and the wild-type "Hangjuxiangsimiao".

[0079] After germination stress treatment with 22% PEG-6000 solution for 7 days under the conditions of Example 1, 0.5g of germinated seeds showing signs of sprouting were taken, rapidly frozen with liquid nitrogen, and stored in an ultra-low temperature freezer at -80℃ for later use. Before measurement, the sample was placed in a pre-cooled mortar, and liquid nitrogen was added to grind it thoroughly into a fine powder.

[0080] 3.2 Determination of malondialdehyde (MDA) content

[0081] Accurately weigh 0.1 g of powder sample and add it to pre-cooled sodium phosphate buffer (0.05 mol / L, pH=7.8) at a weight (g):volume (mL) ratio of 1:9. Grind the sample under ice bath conditions to prepare a 10% tissue homogenate. Centrifuge the homogenate at 4℃ and 3500 rpm for 10 minutes, and use the supernatant as the sample to be tested.

[0082] The MDA content was determined using the thiobarbituric acid (TBA) method. The malondialdehyde (MDA) assay kit (catalog number: A003-1-1) from Nanjing Jiancheng Bioengineering Institute was used, following the instructions: 0.2 mL of supernatant was taken, and 0.6 mL of reagent I, 1.5 mL of reagent II, and 0.2 mL of reagent III were added sequentially. After mixing, the mixture was incubated at 95°C for 40 minutes, cooled on ice, and centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was then collected, and the absorbance was measured at 532 nm and 600 nm using a microplate reader.

[0083] The formula for calculating MDA content is as follows:

[0084] MDA content (nmol / mgprot) = [(A 532 -A 600 [1 / 155000]×(Vreaction / Vsample)×(1 / Cpr)

[0085] Where 155000 is the molar absorptivity of MDA (L / (mol·cm)), Vreaction is the total volume of the reaction system (mL), Vsample is the volume of the sample added (mL), and Cpr is the protein concentration of the sample (mg / mL).

[0086] 3.3 Determination of Proline (PRO) Content

[0087] Accurately weigh 0.1g of powder sample, add 1.5mL of 3% sulfosalicylic acid solution, extract in boiling water bath for 10 minutes, cool, centrifuge at 4℃ and 10000rpm for 10 minutes, and take the supernatant.

[0088] PRO content was determined using the acidic ninhydrin method. The Nanjing Jiancheng Bioengineering Institute Proline Assay Kit (catalog number: A107-1-1) was used, and the procedure was performed according to the kit instructions: 0.5 mL of supernatant was taken, and 0.5 mL of reagent 1, 0.5 mL of reagent 2, and 0.5 mL of reagent 3 were added sequentially. After mixing, the mixture was incubated in a water bath at 100℃ for 30 minutes. After cooling in an ice bath, 2 mL of reagent 4 was added, mixed, and allowed to stand for 10 minutes. The absorbance of the upper organic phase was measured at a wavelength of 520 nm.

[0089] 3.4 Assay of antioxidant enzyme activity

[0090] 3.4.1 Assay of Superoxide Dismutase (SOD) Activity

[0091] SOD activity was determined using the nitroblue tetrazolium (NBT) photochemical reduction method, employing the Nanjing Jiancheng Bioengineering Institute Superoxide Dismutase Assay Kit (catalog number: A001-1). Sample preparation was the same as in 3.2: a 10% tissue homogenate was prepared, centrifuged at 3500 rpm for 10 minutes at 4°C, and the supernatant was collected.

[0092] Prepare the reaction system according to the kit instructions: add 1.0 mL of reagent I, 0.02 mL of sample, 0.1 mL of reagent II, 0.1 mL of reagent III, and 0.1 mL of reagent IV sequentially. Mix well and incubate at 37°C for 40 minutes. Measure the absorbance at 560 nm. Define 50% inhibition of NBT photochemical reduction as one unit of enzyme activity (U).

[0093] 3.4.2 Peroxidase (POD) activity assay

[0094] POD activity was determined using the guaiacol method with the Nanjing Jiancheng Bioengineering Institute peroxidase assay kit (catalog number: A084-3). Sample preparation was the same as in 3.4.1.

[0095] Prepare the reaction system according to the kit instructions: add 1.0 mL of reagent I, 0.1 mL of reagent II, and 0.01 mL of sample in sequence, mix immediately and react at 37°C for 30 minutes, add 2.0 mL of reagent III to terminate the reaction, and measure the absorbance at a wavelength of 470 nm.

[0096] 3.4.3 Assay of catalase (CAT) activity

[0097] CAT activity was determined by ultraviolet absorption spectrometry using the catalase assay kit (catalog number: A007-1-1) from Nanjing Jiancheng Bioengineering Institute. Sample preparation was the same as in 3.4.1.

[0098] Prepare the reaction system according to the kit instructions: add 1.0 mL of reagent I, 0.02 mL of sample, and 0.1 mL of reagent II in sequence, mix well, and react accurately at 37℃ for 1 minute. Immediately add 2.0 mL of reagent III to terminate the reaction, and measure the absorbance at a wavelength of 405 nm.

[0099] 3.5 Results Statistics and Analysis

[0100] All assays were performed in triplicate, with each biological replicate comprising three technical replicates. Data were processed using GraphPadPrism 9 software. Data are expressed as mean ± standard error. Analysis of variance was performed using the Dunnett method. p < 0.05 was considered statistically significant, p < 0.01 was considered highly statistically significant, and p < 0.001 was considered highly statistically significant.

[0101] The measurement results are as follows Figure 5 As shown, Figure 5 The study demonstrated the differences between the wild-type and the drought-tolerant mutant K1300 in malondialdehyde (MDA) content, proline content, and the activities of three antioxidant enzymes: Under 22% PEG-6000 stress, the MDA content of the drought-tolerant mutant K1300 was 62.3% of that of the wild-type, significantly lower (p<0.01); the proline content was 2.1 times that of the wild-type, extremely significantly higher (p<0.001); and the activities of SOD, POD, and CAT were increased by 45.8%, 52.3%, and 38.7% respectively compared to the wild-type, all reaching extremely significant levels (p<0.001). The physiological indicators of the other five core mutants were also significantly better than those of the wild-type, exhibiting stronger membrane system stability, osmotic regulation capacity, and antioxidant capacity.

[0102] The above results confirm from the physiological mechanism level that the screened mutants have stable drought resistance, providing a solid scientific basis for their application in drought-resistant rice breeding.

[0103] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0104] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method of screening for drought tolerant rice mutants, characterized by: Includes the following steps: (1) Initial screening of drought resistance during germination: Using the SP2 generation of rice obtained by space mutagenesis as material, under the conditions of temperature of 26±1℃, photoperiod of 9 hours of light / 15 hours of darkness and relative humidity of 60%, a 22% (w / v) polyethylene glycol-6000 solution was used to conduct drought stress treatment during germination for 7 days. The germination rate was calculated based on the standard that the length of the plumule reaches half the length of the seed and the length of the radicle is equal to that of the seed. Plants with a germination rate significantly higher than that of the wild-type control without mutagenesis were selected as the initial drought-resistant germplasm. (2) Re-screening of drought resistance in seedlings: The drought-resistant germplasm obtained in step (1) was cultivated to the three-leaf and one-heart stage. The seedlings were subjected to drought stress treatment for 7 days using a 24% polyethylene glycol-6000 solution. The chlorophyll content of the stress treatment group and the unstressed control group was measured. The percentage ratio of chlorophyll content in the stress group to that in the control group was calculated. Plants with a ratio significantly higher than that in the wild-type control were screened. (3) Verification of drought-resistant physiological indicators: For the plants screened in step (2), under the drought stress conditions during the germination period described in step (1), the malondialdehyde content, proline content, superoxide dismutase activity, peroxidase activity and catalase activity were measured. Plants that simultaneously meet the following conditions were selected as drought-resistant rice mutants: Malondialdehyde (MDA) content was significantly lower than that of the wild-type control; The proline content was significantly higher than that of the wild-type control; The activities of superoxide dismutase, peroxidase, and catalase were all significantly higher than those of the wild-type control.

2. The method of claim 1, wherein the method is for screening drought-tolerant rice mutants. The germination rate mentioned in step (1) is significantly higher than that of the wild-type control, the ratio mentioned in step (2) is significantly higher than that of the wild-type control, the malondialdehyde content mentioned in step (3) is significantly lower than that of the wild-type, and the proline and enzyme activities mentioned in step (3) are significantly higher than those of the wild-type. All of these are based on Dunnett's multiple comparison test, and the statistical significance p value is greater than 0.

05.

3. A drought-tolerant rice mutant screened by the method of claim 1 or 2, wherein the drought-tolerant rice mutant is characterized in that: The mutants, after being subjected to stress of 22% polyethylene glycol-6000 solution for 7 days during the germination period, had a germination rate of no less than 30%; after being subjected to stress of 24% polyethylene glycol-6000 solution for 7 days during the seedling stage, the percentage ratio of chlorophyll content in the stress group to that in the control group was no less than 75%.

4. The drought-resistant rice mutant according to claim 3, characterized in that: Under drought stress during germination, the mutant exhibits malondialdehyde content no higher than 70% of the wild-type control, proline content no lower than 150% of the wild-type control, and the activities of superoxide dismutase, peroxidase, and catalase no lower than 130% of the wild-type control.

5. The drought-resistant rice mutant according to claim 3, characterized in that: The mutants are K609, K1238, K1273, K1280, K1296 or K1300.

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