Method for comprehensive evaluation of resistance of corn seedling to microplastic toxicity and method for identifying corn genotype resistant to microplastic toxicity

Abstract

The application provides a comprehensive evaluation method for resistance of corn seedling to microplastic toxicity and a genotype identification method for corn resistant to microplastic toxicity. Under treatment of different concentrations of PS microplastics, 12 traits of corn seedlings are measured, resistance index (TRI) is calculated by a membership function method, and total comprehensive resistance (TCTR) is comprehensively evaluated to determine that the most serious toxicity is at a PS microplastic concentration of 0.50%. Different genotypes of seedlings are treated with PS microplastics at concentrations of 0.00% and 0.50%, traits are measured, and TRI values are calculated to objectively evaluate the performance of the seedlings resistant to microplastic toxicity. The application has good operability and small test error, can not only objectively, scientifically and accurately determine the concentration at which the most serious PS microplastic toxicity of corn seedlings occurs from the perspective of all-around, multi-index and multi-time dimension of the aboveground and underground of the seedlings, but also can efficiently and accurately identify corn genotypes resistant to microplastic toxicity, and serves corn new variety breeding resistant to microplastic toxicity.

Description

Technical Field

[0001] This invention belongs to the technical field of breeding methods for new maize varieties with stress resistance, specifically involving a comprehensive evaluation method for maize seedling resistance to microplastic toxicity and a method for identifying maize genotypes resistant to microplastic toxicity. Background Technology

[0002] In modern society, plastics have become the most common and widely used material, present in many aspects of human life. Due to their diverse applications, the demand for various types of plastics, such as polystyrene (PS), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyamide (PA), and polycarbonate (PC), has grown rapidly. Statistics show that global plastic production increased from 1.5 million tons in 1950 to 368 million tons in 2019, and annual production continues to grow. However, only 9% of plastics are recycled after use, with the majority being discarded into the environment. This not only causes "white pollution," but also, during the degradation process, these plastic wastes form microplastic particles (MPs) smaller than 5 mm in size (Thompson et al. 2024), which can further harm humans, animals, plants, and microorganisms.

[0003] Regarding the harmful effects of microplastics on plants, Yan et al. (2024) showed that 0.001%–0.1% PE microplastics reduced wheat (Triticum aestivum L.) plant height by 0.70%–16.60%, stem thickness by 2.05%–24.10%, and total protein content in grains by 7.98%–16.01%. Jiang et al. (2023) found that adding 0.40% PP microplastics reduced peanut (Arachis hypogaea L.) total biomass, aboveground biomass, relative growth rate, and 100-grain weight by 17.10%, 18.64%, 16.12%, and 11.98%, respectively, while soybean (Glycine max L.) total biomass, aboveground biomass, relative growth rate, and 100-grain weight decreased by 13.61%, 14.07%, 13.23%, and 7.84%, respectively. The study by Liu Xiaohong et al. (2022) also found that treatment with PE microplastics of 13μm, 58μm and 178μm all reduced the germination potential, seed vigor index and sprout length of cucumber (Cucumis sativus L.) seeds to varying degrees.

[0004] Corn (Zea mays L.) is my country's primary multi-purpose food crop, and its production plays a crucial role in ensuring national food security, livestock development, and industrial processing. Corn is extremely sensitive to drought, requiring a large amount of water throughout its growth and development. Drought and water shortages in my country have led to corn yield reductions of 20-50% (Zhao et al. 2018). In production practice, although mulching with plastic film suppresses soil evaporation, thereby increasing soil temperature and moisture, ultimately improving corn's drought resistance, ensuring food security, and increasing farmers' income (Yu Junping 2024), the long-term widespread use of plastic film and other plastic materials inevitably leads to the continuous accumulation of large amounts of microplastics in the soil. We speculate that, like the aforementioned field crops and vegetables such as wheat, peanuts, soybeans, and cucumbers, these microplastics will also affect the healthy growth, development, yield formation, grain quality, and senescence of corn.

[0005] Existing technologies present several challenges: Microplastic residues in the soil primarily affect the morphogenesis and development of maize seedlings. Does microplastic pollution cause toxicity to maize seedlings? What types of toxicity do microplastics cause to maize seedlings? What are the mechanisms of microplastic toxicity in maize seedlings? What concentration of microplastics causes the greatest toxicity to maize seedlings? What indicators can be used to assess the microplastic toxicity of maize seedlings? What methods can scientifically, objectively, and accurately evaluate the microplastic resistance of different maize genotypes? Can superior maize genotypes resistant to microplastic toxicity be screened and identified for application in breeding superior new maize varieties resistant to microplastic toxicity? These questions remain unclear and require further in-depth and systematic research. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a comprehensive evaluation method for the microplastic toxicity resistance of maize seedlings. Specifically, this invention uses soil culture experiments to systematically analyze the effects of six concentrations of polystyrene (PS) microplastic treatments on 12 growth phenotypes, chlorophyll accumulation levels, and root activity traits of maize genotype seedlings at 7 and 21 days of culture. This reveals the underlying reasons for the toxicity of different concentrations of PS microplastics to maize seedlings at different culture time periods and the response mechanism of maize seedlings to microplastic toxicity. Furthermore, we innovatively proposed the biological concept and calculation formula of the toxicity resistance index (TRI) for different concentrations of PS microplastics in maize seedlings, and calculated the TRI values ​​of 12 traits for maize genotype seedlings under these two culture periods. Then, we used this TRI value as an evaluation index to measure the resistance of maize genotype seedlings to different concentrations of PS microplastics under the two culture periods. We employed the membership function method to comprehensively, objectively, and quantitatively evaluate the magnitude of the comprehensive toxicity resistance (CTR) / total comprehensive toxicity resistance (TCTR) of maize genotype seedlings to different concentrations of PS microplastics under each / all culture periods. Combining the cluster evaluation results of the microplastic toxicity levels of maize genotype seedlings among different concentrations of PS microplastics under each culture period, we finally determined that the PS microplastic concentration at which the severity of PS microplastic toxicity in maize seedlings was 0.50% across all culture periods. Based on this, we further used a 0.50% concentration of PS microplastics as the screening concentration for evaluating maize's resistance to microplastic toxicity. We measured the aforementioned 12 traits in maize seedlings of different genotypes on days 7 and 21 of seedling culture, and calculated the corresponding 0.50% PS microplastic toxicity resistance index (TRI). Then, using the membership function method, we comprehensively evaluated the PS microplastic toxicity resistance of different maize genotypes, ultimately screening out resistant maize genotypes with strong resistance to PS microplastic toxicity for application in maize breeding. This effectively addresses the adverse effects of soil microplastics on maize, ensuring safe and efficient maize production.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] 1. A comprehensive evaluation method for the resistance of maize seedlings to microplastic toxicity, the specific steps of which are as follows:

[0009] (1) Preparation of high-quality corn seeds: Prepare new high-quality corn genotype seeds harvested in the current year that are plump, uniform in size, vigorous and pure, for later use.

[0010] (2) Experiment on soil-grown corn with different concentrations of polystyrene microplastics: Disinfected with 70% ethanol (v / v) for 10 min, rinsed with ddH2O water 5 times to remove residual ethanol on the seed surface, and dried with sterile filter paper to obtain the corresponding disinfected corn genotype seeds. Prepare six concentrations of PS microplastic solutions in advance: 0.00%, 0.05%, 0.25%, 0.50%, 0.75% and 1.00%, and prepare them fresh for use. Then soak the disinfected corn genotype seeds in the six concentrations of PS microplastic solutions for 24 h. Then prepare six concentrations of PS microplastic soil substrates, including 0.00% PS microplastic soil substrate, 0.05% PS microplastic soil substrate, 0.25% PS microplastic soil substrate, 0.50% PS microplastic soil substrate, 0.75% PS microplastic soil substrate and 1.00% PS microplastic soil substrate. Ten maize seeds of each genotype, soaked in PS microplastic solution of the corresponding concentration, were then sown into pots containing soil substrate of the same concentration of PS microplastic and placed in an artificial climate chamber for cultivation. During cultivation, the relative humidity was set at 65%, the temperature was alternated between 25±0.5℃ and 20±0.5℃ for 12 hours each, the photoperiod was set at 16 / 8 hours of light / dark, and the light intensity was set at 300 μMm. -2 s -1 The CO2 concentration was 450 PPM. A total of six treatments were used in the soil culture experiment: control (0.00% PS microplastic soil substrate), 0.05% PS treatment (0.05% PS microplastic soil substrate), 0.25% PS treatment (0.25% PS microplastic soil substrate), 0.50% PS treatment (0.50% PS microplastic soil substrate), 0.75% PS treatment (0.75% PS microplastic soil substrate), and 1.00% PS treatment (1.00% PS microplastic soil substrate). Each treatment was replicated four times. During the cultivation period, 50 mL of ddH2O was evenly applied to each pot every 3 days to ensure timely watering of the seedlings.

[0011] (3) Data collection: Seedling length (SL), seedling fresh weight (SFW), seedling dry weight (SDW), root length (RL), root fresh weight (RFW), root dry weight (RDW), stem diameter (SD), root diameter (RD), total plant dry biomass (PDW), leaf chlorophyll SPAD value (SPAD), root activity (RV), and seedling vigor index (SSI) were measured on maize seedlings cultured in six PS microplastic soil substrates on days 7 and 21, respectively. SSI was calculated using formula (1): SSI = (SD / SL) × PDW (1). Where: SSI is the seedling vigor index, SD is the stem diameter, SL is the seedling length, and PDW is the total plant dry biomass.

[0012] (4) Establishing a statistical model: Using ExCel 2013 software, the mean and standard deviation of each trait of maize genotype seedlings at 7 and 21 days of cultivation in the soil-cultured maize experiment were calculated, and bar charts were plotted. IBM-SPSS Statistics 19 software was used to analyze the joint variance of each trait of maize genotypes among the two cultivation periods and the six PS microplastic treatments in the soil-cultured maize experiment. IBM-SPSS Statistics 19 software was used to analyze the Dunman significance of each trait of maize genotype seedlings at each cultivation period among the six PS microplastic treatments at the P<0.05 level in the soil-cultured maize experiment. GENESCLOUD online software was used to analyze the Pearson correlation coefficients of all traits among the six PS microplastic treatments at each cultivation period of maize genotype seedlings in the soil-cultured maize experiment. The LOG10 function was used to standardize the average values ​​of all traits among the six PS microplastic treatments at two different culture time periods in the soil-cultured maize seedling experiment. Then, the Between-groups Linkage clustering method of IBM-SPSS Statistics 16.0 software was used to conduct a scientific, objective, and qualitative cluster analysis on the degree of microplastic toxicity of maize seedlings among the six PS microplastic treatments at each culture time period in the soil-cultured maize experiment.

[0013] (5) Resistance index of maize seedlings to polystyrene (PS) microplastics at different concentrations: In order to scientifically, objectively and accurately reflect the resistance of individual traits of maize seedlings to the toxicity of different concentrations of PS microplastics at each culture period in the soil-cultured maize experiment, this invention defines a new resistance index (TRI) of maize seedlings to the toxicity of different concentrations of PS microplastics, as shown in formula (2): In the formula: The term represents the resistance index to PS microplastic toxicity of maize seedlings with the j-th trait and the k-th concentration during the i-th culture period in a soil-cultured maize experiment. The value of the j-th trait in maize seedlings of genotype i during the i-th culture period in the soil-cultured maize experiment, under the CK control treatment. The value of the j-th trait of maize seedlings with the k-th concentration of PS microplastics is measured at the i-th culture time period in the soil-cultured maize experiment. i represents the 7th or 21st day of soil-cultured maize experiment, and k-PS represents the 0.05%, 0.25%, 0.50%, 0.75%, or 1.00% concentration of PS microplastics in the soil-cultured maize experiment. The larger the value, the stronger the resistance of the j-th genotype of maize seedlings to microplastic toxicity at the k-PS concentration during the i-th culture period in the soil-cultured maize experiment.

[0014] (6) Evaluation of the comprehensive resistance of maize seedlings to different concentrations of polystyrene (PS) microplastic toxicity: The TRI values ​​of all traits of maize genotype seedlings calculated in step (4) were used as evaluation indicators to measure the resistance of maize genotype seedlings to different concentrations of PS microplastic toxicity in the two culture periods of the soil-cultured maize experiment. The membership function method was used to comprehensively, objectively and quantitatively evaluate the magnitude of the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of maize genotype seedlings to different concentrations of PS microplastic toxicity in each / all culture periods of the soil-cultured maize experiment. See formulas (3), (4) and (5) for details: and In the formula: The value represents the membership value of the resistance of maize seedlings with the j-th genotype to the toxicity of the k-th concentration of PS microplastics during the i-th culture period in a soil-cultured maize experiment. The term represents the resistance index to PS microplastic toxicity of maize seedlings with the j-th trait and the k-th concentration during the i-th culture period in a soil-cultured maize experiment. The minimum TRI value for the j-th genotype of maize seedlings under the i-th culture time period in a soil-cultured maize experiment is the minimum TRI value among all PS microplastic treatments. The maximum TRI value of the j-th genotype of maize seedlings under the i-th culture time period in the soil-cultured maize experiment is the maximum value among all concentrations of PS microplastic treatments for the j-th trait. ti represents the overall resistance of maize genotype seedlings to the k-th concentration of PS microplastics during the i-th culture period in the soil-cultured maize experiment; m represents the m (m=12) individual traits measured in maize genotype seedlings during the i-th culture period in the soil-cultured maize experiment; TCTR (k-PS) The value represents the overall resistance of maize seedlings of genotype to the toxicity of PS microplastics at the kth concentration during n (n=2, 2 culture time periods) culture time periods in the soil-cultured maize experiment. i represents the 7th or 21st day of soil-cultured maize experiment, and k-PS represents the PS microplastic treatment at the concentrations of 0.05%, 0.25%, 0.50%, 0.75%, or 1.00% in the soil-cultured experiment. A higher TCTR value indicates a stronger overall resistance of maize seedlings of the i-th culture period to the toxicity of the k-th concentration of PS microplastics. (k-PS) The larger the value, the stronger the overall resistance of maize seedlings of the kth concentration of PS microplastics to the toxicity of the maize genotype under the two culture periods in the soil-cultured maize experiment.

[0015] (7) Comprehensive assessment of PS microplastic concentration at the time of most severe polystyrene (PS) microplastic toxicity in maize seedlings: By comparing the results of the Between-groups Linkage of PS microplastic toxicity in maize genotype seedlings under two cultivation periods in the soil-cultured maize experiment in step (3), and the evaluation results of the overall resistance of maize genotype seedlings to corresponding concentrations of PS microplastic toxicity under two cultivation periods in step (5), the degree of PS microplastic toxicity in maize genotype seedlings under multiple cultivation periods was evaluated by a combination of multiple methods, and finally the PS microplastic concentration at the time of most severe PS microplastic toxicity in maize genotype seedlings was comprehensively assessed.

[0016] 2. A method for identifying microplastic-resistant maize genotypes, comprising the following steps:

[0017] (1) Preparation of high-quality maize seeds: Prepare new high-quality maize seeds of different genotypes that are plump, uniform in size, vigorous and pure, harvested from the same ecological point in the current year, for later use.

[0018] (2) Experiment on soil-grown corn with 0.50% concentration polystyrene (PS) microplastics: Disinfected with 70% ethanol (v / v) for 10 min, rinsed with ddH2O water 5 times to remove residual ethanol on the seed surface, and dried with sterile filter paper to obtain the corresponding disinfected corn genotype seeds. The disinfected corn genotype seeds were soaked in two concentrations of PS microplastic solution (0.00% and 0.50%) for 24 h, and then 10 seeds of each of the different corn genotypes soaked in the two concentrations of PS microplastic solution were sown into flower pots of 0.00% and 0.50% concentration PS microplastic soil substrate and placed in an artificial climate chamber for cultivation. During the cultivation period, the relative humidity was set to 65%, the temperature was set to 25±0.5 / 20±0.5℃ for 12 h alternating, the photoperiod was set to 16 / 8 h light / dark, and the light intensity was set to 300 μM m -2 s -1 The CO2 concentration was 450 PPM. Two treatments were used in the soil culture experiment: a control (0.00% PS microplastic soil substrate) and a 0.50% PS treatment (0.50% PS microplastic soil substrate), with four biological replicates for each treatment. During the cultivation period, 50 mL of ddH2O was evenly applied to each pot every 3 days to ensure timely watering of the seedlings. Twelve traits of different maize genotypes were measured in the two PS microplastic soil substrates at days 7 and 21.

[0019] (3) Statistical analysis: Using ExCel 2013 software, the mean and standard deviation of each trait of different maize genotype seedlings under each treatment on the 7th and 21st days of cultivation were calculated. Using IBM-SPSS Statistics 19 software, the joint variance of each trait of all maize genotypes between the two concentrations of PS microplastics at these two cultivation periods was analyzed.

[0020] (4) Resistance index to toxicity of 0.50% polystyrene (PS) microplastics in maize seedlings of different genotypes: Based on the mean values ​​of 12 traits of different maize genotype seedlings measured on the 7th and 21st days of cultivation in the soil-cultured maize experiment with two concentrations of PS microplastics in step (3), the resistance index (TRI) to toxicity of 0.50% PS microplastics in each genotype seedling for each trait at each cultivation period was further calculated, as shown in formula (6): In the formula: The resistance index to toxicity of 0.50% PS microplastics in the j-th trait of the p-th maize genotype seedling at the i-th culture time period in a soil-cultured maize experiment. The value of the j-th trait under the CK control treatment in the i-th culture time period of the p-th maize genotype seedling in the soil-cultured maize experiment is shown. The value of the jth trait under 0.50% PS microplastic treatment in the i-th culture period of the p-th maize genotype seedling in the soil-cultured maize experiment is given. i represents the 7th or 21st day of soil-cultured maize experiment, and 0.50% PS represents the 0.50% concentration of PS microplastic treatment in the soil-cultured maize experiment. The larger the value, the stronger the resistance of the j-th trait of the p-th maize genotype seedling to 0.50% microplastic toxicity during the i-th culture period in the soil-cultured maize experiment.

[0021] (5) Evaluation of the comprehensive resistance of maize seedlings of different genotypes to 0.50% polystyrene (PS) microplastic toxicity: The TRI values ​​of all traits of maize seedlings of different genotypes calculated in step (4) were used as the evaluation index for the resistance of maize seedlings of different genotypes to 0.50% PS microplastic toxicity at two culture time periods in the soil-cultured maize experiment. The membership function method was used to comprehensively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) values ​​of maize seedlings of different genotypes to 0.50% PS microplastic toxicity at each / all culture time periods in the soil-cultured maize experiment. Specifically: and In the formula: The value represents the membership value of the resistance of the j-th trait of the p-th maize genotype seedling to 0.50% PS microplastic toxicity during the i-th culture period in a soil-cultured maize experiment. The resistance index to toxicity of 0.50% PS microplastics in the j-th trait of the p-th maize genotype seedling at the i-th culture time period in a soil-cultured maize experiment. The minimum TRI value for the j-th trait of all maize genotypes in the soil-cultured maize experiment during the i-th cultivation period is given. The maximum TRI value for the j-th trait of all maize genotypes in the soil-cultured maize seedlings during the i-th culture period is given. ti represents the overall resistance of the p-th maize genotype seedling to 0.50% PS microplastic toxicity during the i-th culture period in the soil-cultured maize experiment; m represents the m (m=12) traits measured in each maize genotype seedling during the i-th culture period in the soil-cultured maize experiment; TCTR p(0.50％PS) The value represents the overall resistance of the p-th maize genotype seedling to 0.50% PS microplastic toxicity during the n (n=2, 2 culture time periods) culture time period in the soil-cultured maize experiment. i represents the 7th or 21st day of soil-cultured maize experiment, and 0.50% PS represents the 0.50% PS microplastic treatment in the soil-cultured experiment. A higher TCTR value indicates a stronger overall resistance to 0.50% PS microplastic toxicity in the p-th maize genotype seedlings during the i-th culture period in a soil-cultured maize experiment. (0.50％PS) A higher value indicates a stronger overall resistance to 0.50% PS microplastic toxicity in the p-th maize genotype seedlings across two culture periods in the soil-cultured maize experiment. Based on the TCTR value of each maize genotype seedling, the resistance to microplastic toxicity of different maize genotypes was divided into five levels: 0 ≤ TCTR < 0.400, highly sensitive to microplastic toxicity; 0.400 ≤ TCTR < 0.500, sensitive to microplastic toxicity; 0.500 ≤ TCTR < 0.600, weakly resistant to microplastic toxicity; 0.600 ≤ TCTR < 0.700, moderately resistant to microplastic toxicity; and 0.700 ≤ TCTR ≤ 1.000, highly resistant to microplastic toxicity.

[0022] The application of 3.0.50% polystyrene (PS) microplastic toxicity in the evaluation of microplastic-resistant maize genotypes is as follows:

[0023] High-quality maize seeds of different genotypes, sterilized with 70% ethanol (v / v), were soaked in two PS microplastic solutions at concentrations of 0.00% and 0.50% for 24 hours. Ten seeds of each genotype were then sown in pots containing the two PS microplastic solutions at concentrations of 0.00% and 0.50%, respectively, and placed in an artificial climate chamber for cultivation. During cultivation, the relative humidity was set at 65%, the temperature was alternated between 25±0.5℃ and 20±0.5℃ for 12 hours, the photoperiod was set at 16 / 8 hours of light / dark, and the light intensity was set at 300 μM m². -2 s -1 The CO2 concentration was 450 PPM. Two treatments were used in the soil culture experiment: a control (0.00% PS microplastic soil substrate) and a 0.50% PS treatment (0.50% PS microplastic soil substrate). During the cultivation period, 50 mL of ddH2O was evenly applied to each pot every 3 days to ensure timely watering of the seedlings. Then, 12 traits of different maize genotypes were measured on days 7 and 21 of cultivation in both PS microplastic soil substrates. The mean and standard deviation of each trait in different maize genotypes on days 7 and 21 of cultivation were calculated using ExCel 2013 software; IBM-SPSS Statistics were used. Software 19 was used to analyze the joint variance of each trait for all maize genotypes across two cultivation periods and two concentrations of PS microplastic treatment. The Trial of Resistance (TRI) to 0.50% PS microplastic toxicity was calculated for each genotype of maize seedling at each cultivation period. Then, the membership function method was used to comprehensively evaluate the total total resistance (TCTR) of each genotype of maize seedling to 0.50% PS microplastic toxicity, thereby assessing the microplastic resistance of each genotype of maize seedling. Based on the TTR of each maize genotype of seedling... The CTR value classifies the resistance of different maize genotypes to microplastic toxicity into five levels: 0 ≤ TCTR < 0.400, indicating a highly sensitive maize genotype to microplastic toxicity; 0.400 ≤ TCTR < 0.500, indicating a sensitive maize genotype to microplastic toxicity; 0.500 ≤ TCTR < 0.600, indicating a weakly resistant maize genotype to microplastic toxicity; 0.600 ≤ TCTR < 0.700, indicating a moderately resistant maize genotype to microplastic toxicity; and 0.700 ≤ TCTR ≤ 1.000, indicating a highly resistant maize genotype to microplastic toxicity.

[0024] The beneficial effects of this invention: Microplastics are extremely difficult to decompose in the natural environment and can persist for hundreds of years. With the continued large-scale use of plastic products, the microplastic crisis has become increasingly serious. Therefore, this invention, starting from the actual situation of microplastic pollution and harm, systematically compares the effects of different concentrations of PS microplastic treatment on 12 traits of maize genotype seedlings under different cultivation periods, and deeply elucidates the intrinsic causes of PS microplastic toxicity in maize seedlings and the adaptive mechanisms of maize seedlings in response to PS microplastic toxicity. Meanwhile, to more scientifically, objectively, simply, and accurately assess the resistance of maize genotype seedlings to different concentrations of PS microplastics at each / all culture time periods, we innovatively proposed the biological concept and calculation formula of the resistance index (TRI) to different concentrations of PS microplastics in maize seedlings. Using the TRI value, we employed the membership function method to scientifically, objectively, accurately, and quantitatively evaluate the magnitude of the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of maize genotype seedlings to different concentrations of PS microplastics at each / all culture time periods. This comprehensive evaluation result was also compared with the cluster evaluation results of the degree of microplastic toxicity of maize genotype seedlings among different concentrations of PS microplastics at each culture time period using multiple methods and multiple time dimensions. This verified that our proposed TRI can scientifically, objectively, and accurately evaluate the comprehensive resistance of maize genotype seedlings to different concentrations of PS microplastics at each / all culture time periods, and finally screened out the PS microplastic concentration at which the degree of PS microplastic toxicity in maize seedlings was 0.50%. Building upon this, we further used 0.00% and 0.50% concentrations of PS microplastics as screening concentrations for evaluating maize's resistance to microplastic toxicity. We measured 12 traits in maize seedlings of different genotypes on days 7 and 21 of seedling cultivation, comprehensively evaluating the resistance of different maize genotypes to PS microplastic toxicity. This allowed us to screen out resistant maize genotypes with strong resistance to PS microplastic toxicity for application in maize breeding, effectively addressing the adverse effects of soil microplastics on maize and ensuring safe and efficient maize production. This invention features simple experimental operation, good repeatability, and the ability to accurately evaluate the resistance of different maize genotypes to microplastic toxicity by identifying the highest PS microplastic toxicity concentration in maize seedlings. It provides maize breeders with a simple, reliable, rapid, and standardized method for evaluating maize genotypes resistant to microplastic toxicity, thus serving the application of maize microplastic resistance breeding and possessing significant breeding application value. Attached Figure Description

[0025] Figure 1 Formula for calculating the seedling vigor index of maize genotype seedlings (1), resistance index of maize genotype seedlings to different concentrations of PS microplastics under corresponding culture time periods in soil-cultured maize experiments. Formula (2) is used to calculate the membership value of the resistance of individual traits of maize seedlings of different genotypes to the toxicity of different concentrations of PS microplastics during the corresponding culture time period in the soil-cultured maize experiment. Formula (3) was used to calculate the comprehensive resistance of maize seedlings of different genotypes to the toxicity of different concentrations of PS microplastics during the corresponding culture time period in the soil-cultured maize experiment. Formula (4) was used to calculate the overall resistance of maize seedlings of different genotypes to different concentrations of PS microplastics during two cultivation periods in the soil-cultured maize experiment. Formula (5) is used to calculate the resistance index of 0.50% concentration PS microplastics to each trait of each maize genotype seedling during the corresponding culture period in the soil-cultured maize experiment. Formula (6) is used to calculate the membership value of the resistance of each maize genotype seedling to 0.50% PS microplastic toxicity during the corresponding culture period in the soil-cultured maize experiment. Formula (7) was used to calculate the overall resistance of each maize genotype seedling to 0.50% PS microplastic toxicity during the corresponding culture period in the soil-cultured maize experiment. Formula (8) is used to calculate the total comprehensive resistance (TCTR) of each maize genotype seedling to 0.50% PS microplastic toxicity during two culture periods in the soil-cultured maize experiment. p(0.50％PS) ) Calculation formula (9).

[0026] Figure 2 The growth of TS163 maize genotype seedlings on day 7 under six concentrations of PS microplastic treatment in a soil-cultured maize experiment. The control treatment (CK) was 0.00% PS microplastic soil substrate treatment, 0.05% PS was 0.05% PS microplastic soil substrate treatment, 0.25% PS was 0.25% PS microplastic soil substrate treatment, 0.50% PS was 0.50% PS microplastic soil substrate treatment, 0.75% PS was 0.75% PS microplastic soil substrate treatment, and 1.00% PS was 1.00% PS microplastic soil substrate treatment.

[0027] Figure 3 The growth of TS163 maize genotype seedlings on day 21 under six concentrations of PS microplastic treatment in a soil-cultured maize experiment. The control treatment (0.00% PS microplastic soil substrate treatment), treatments with 0.05% PS, 0.25% PS, 0.50% PS, 0.75% PS, and 1.00% PS were all treated with 1.00% PS microplastic soil substrate.

[0028] Figure 4Phenotypic analysis of 12 traits in TS163 maize genotype seedlings cultured on days 7 and 21 under six concentrations of PS microplastic treatment in a soil-cultured maize experiment. The phenotypic values ​​are as follows: SL = seedling length, SFW = seedling fresh weight, SDW = seedling dry weight, RL = root length, RFW = root fresh weight, RDW = root dry weight, SD = stem diameter, RD = root diameter, PDW = total plant dry biomass, SPAD = leaf chlorophyll SPAD value, SSI = seedling vigor index, and RV = root activity. CK = control treatment (0.00% PS microplastic soil substrate treatment), 0.05% PS = 0.05% PS microplastic soil substrate treatment, 0.25% PS = 0.25% PS microplastic soil substrate treatment, 0.50% PS = 0.50% PS microplastic soil substrate treatment, 0.75% PS = 0.75% PS microplastic soil substrate treatment, and 1.00% PS = 1.00% PS microplastic soil substrate treatment. Different lowercase letters indicate that the corresponding traits of TS163 maize genotype seedlings treated with different concentrations of PS microplastics during a single culture period showed significant differences at the P<0.05 level.

[0029] Figure 5 Pearson correlation coefficients for 12 traits in TS163 maize genotype seedlings cultured for 7 days under six concentrations of PS microplastic treatment in a soil-cultured maize experiment. SL represents seedling length, SFW represents seedling fresh weight, SDW represents seedling dry weight, RL represents root length, RFW represents root fresh weight, RDW represents root dry weight, SD represents stem diameter, RD represents root diameter, PDW represents total plant dry biomass, SPAD represents leaf chlorophyll SPAD value, SSI represents seedling vigor index, and RV represents root activity. * indicates a significant Pearson correlation between the two traits at the P < 0.05 level.

[0030] Figure 6 Pearson correlation coefficients for 12 traits in TS163 maize genotype seedlings cultured for 21 days under six concentrations of PS microplastic treatment in a soil-cultured maize experiment. SL represents seedling length, SFW represents seedling fresh weight, SDW represents seedling dry weight, RL represents root length, RFW represents root fresh weight, RDW represents root dry weight, SD represents stem diameter, RD represents root diameter, PDW represents total plant dry biomass, SPAD represents leaf chlorophyll SPAD value, SSI represents seedling vigor index, and RV represents root activity. * indicates a significant Pearson correlation between the two traits at the P < 0.05 level.

[0031] Figure 7In a soil-cultured maize experiment, the microplastic toxicity of TS163 maize genotype seedlings treated with six concentrations of PS microplastics on day 7 was evaluated using the Between-groups Linkage clustering method based on 12 traits. The control treatment (0.00% PS microplastic soil substrate treatment), treatments with 0.05% PS, 0.25% PS, 0.50% PS, 0.75% PS, and 1.00% PS were all treated with 1.00% PS microplastic soil substrate. Type A represented no PS microplastic toxicity, Type B represented weak PS microplastic toxicity, and Type C represented severe PS microplastic toxicity.

[0032] Figure 8 In a soil-cultured maize experiment, the microplastic toxicity of TS163 maize genotype seedlings treated with six concentrations of PS microplastics on day 21 was evaluated using the Between-groups Linkage method based on 12 traits. The control treatment (0.00% PS microplastic soil substrate treatment), treatments with 0.05% PS, 0.25% PS, 0.50% PS, 0.75% PS, and 1.00% PS were all treated with 1.00% PS microplastic soil substrate. Type A represented no PS microplastic toxicity, Type B represented weak PS microplastic toxicity, and Type C represented severe PS microplastic toxicity.

[0033] Figure 9 CTR analysis was performed on the overall resistance of TS163 maize genotype seedlings to different concentrations of PS microplastic toxicity at 7 and 21 days of soil-cultured maize experiments. The treatments included 0.05% PS, 0.25% PS, 0.50% PS, 0.75% PS, and 1.00% PS. Day 7 and day 21 of soil-cultured maize seedlings were used in the experiment.

[0034] Figure 10Total Comprehensive Resistance Test (TCTR) was analyzed for TS163 maize genotype seedlings to different concentrations of PS microplastics during two cultivation periods in a soil-cultured maize experiment. The treatments included 0.05% PS, 0.25% PS, 0.50% PS, 0.75% PS, and 1.00% PS.

[0035] Figure 11 Violin plots of 12 traits in 10 maize seedlings of 10 genotypes cultured under two concentrations of PS microplastics at days 7 and 21 of cultivation in a soil-cultured maize experiment. In the figures, SL represents seedling length, SFW represents seedling fresh weight, SDW represents seedling dry weight, RL represents root length, RFW represents root fresh weight, RDW represents root dry weight, SD represents stem diameter, RD represents root diameter, PDW represents total plant dry biomass, SPAD represents leaf chlorophyll SPAD value, SSI represents seedling vigor index, and RV represents root activity. CK represents the control treatment (0.00% PS microplastic soil substrate treatment), and 0.50% PS represents the 0.50% PS microplastic soil substrate treatment. 7d represents day 7 of the soil-cultured maize experiment, and 21d represents day 21 of the soil-cultured maize experiment.

[0036] Figure 12 CTR analysis of the combined resistance of 10 maize genotype seedlings at 7 and 21 days of soil-grown maize to 0.50% PS microplastic toxicity. 7 days refers to the 7th day of soil-grown maize cultivation, and 21 days refers to the 21st day of soil-grown maize cultivation.

[0037] Figure 13 The Total Comprehensive Resistance Test (TCTR) values ​​were used to evaluate the overall resistance of 10 maize seedlings of different genotypes to 0.50% PS microplastic toxicity during two culture periods in a soil-cultured maize experiment. The numbers on the columns represent the TCTR values. Specific implementation methods

[0038] Unless otherwise specified, the methods used in the following embodiments of the present invention are conventional methods; the equipment and reagents used are all conventional equipment and reagents purchased from reagent companies. It should also be noted that, in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only technical solutions and / or processing steps closely related to the solution according to the present invention are shown in the embodiments, while other details that are not closely related are omitted.

[0039] Furthermore, those skilled in the art will understand that the maize genotype test materials are not limited to 11 samples. This invention uses these 11 maize genotype test materials as an example only to clearly describe the content of the technical solution of this invention.

[0040] Example 1

[0041] This invention provides a comprehensive evaluation method for the resistance of corn seedlings to microplastic toxicity. The specific evaluation method is carried out according to the following steps:

[0042] 1. Preparation of high-quality maize seeds: Prepare new high-quality maize genotype TS163 seeds that were planted, strictly bagged and pollinated, and harvested in 2024 at the Longxi experimental site (34.97°N, 104.40°E, altitude 2074m), with plump kernels, uniform size, strong vitality, and high purity, for later use.

[0043] 2. Experiment on soil-cultured maize with different concentrations of PS microplastics: Forty high-quality TS163 maize genotype seeds prepared above were placed in Erlenmeyer flasks, and 100 mL of 70% ethanol (v / v) was added. The flasks were placed on a horizontal shaker to sterilize the seeds for 10 min. The seeds were then rinsed five times with 100 mL of ddH2O water to remove residual ethanol from the seed surface. The water adhering to the seed surface was blotted dry with sterile filter paper to obtain the corresponding sterilized TS163 maize genotype seeds. PS microplastic powder (mesh size: 1000 mesh; particle size: 13 μm) was purchased from Dongguan Ruixiang Plastic Raw Materials Co., Ltd. Precisely weigh 0.00g, 0.05g, 0.25g, 0.50g, 0.75g, and 1.00g of PS microplastics using an electronic balance, and dissolve each in 100mL of ddH2O water to prepare six PS microplastic solutions of 0.00%, 0.05%, 0.25%, 0.50%, 0.75%, and 1.00% (PS mass to ddH2O mass ratio). Prepare each solution immediately before use. Then, place the sterilized TS163 maize genotype seeds into the corresponding Erlenmeyer flasks, add 100mL of each of the six PS microplastic solutions, and soak the seeds on a horizontal shaker in a dark indoor environment for 24 hours. Six concentrations (based on the ratio of PS mass to nutrient soil mass) of PS microplastic soil substrate were prepared, including: 0.00% PS microplastic soil substrate (i.e., 0.00g PS microplastic mixed thoroughly with 300.00g nutrient soil, then 250mL ddH2O water was added and stirred thoroughly); 0.05% PS microplastic soil substrate (i.e., 0.15g PS microplastic mixed thoroughly with 300.00g nutrient soil, then 250mL ddH2O water was added and stirred thoroughly); 0.25% PS microplastic soil substrate (i.e., 0.75g PS microplastic mixed thoroughly with 300.00g nutrient soil, then 250mL ddH2O water was added and stirred thoroughly); and 0.50% PS microplastic soil substrate (i.e., 1.50g PS microplastic mixed thoroughly with 300.00g nutrient soil, then 250mL ddH2O water was added and stirred thoroughly); and 0.50% PS microplastic soil substrate (i.e., 1.50g PS microplastic mixed thoroughly with 300.00g nutrient soil, then 1.50g PS microplastic mixed thoroughly with 300.00g nutrient soil). The following are the different types of soil substrates: 1) a soil substrate containing PS microplastics (2.25g PS microplastics mixed thoroughly with 300g nutrient soil and then 250mL ddH2O); and 2) a soil substrate containing 0.75% PS microplastics (2.25g PS microplastics mixed thoroughly with 300g nutrient soil and then 250mL ddH2O); and 3) a soil substrate containing 1.00% PS microplastics (3.00g PS microplastics mixed thoroughly with 300g nutrient soil and then 250mL ddH2O). Ten TS163 maize genotype seeds, each soaked in a PS microplastic solution of the corresponding concentration, were sown in pots (11.0cm high, 12.5cm in diameter) containing the same concentration of PS microplastic soil substrate and then cultured in an artificial climate chamber.During the cultivation period, the relative humidity was set to 65%, the temperature was set to 25±0.5℃ / 20±0.5℃ alternating for 12 hours each, the photoperiod was set to 16 / 8 hours of light / dark, and the light intensity was set to 300 μM m. -2 s -1 The CO2 concentration was 450 PPM. A total of six treatments were used in the soil culture experiment: control (0.00% PS microplastic soil substrate), 0.05% PS treatment (0.05% PS microplastic soil substrate), 0.25% PS treatment (0.25% PS microplastic soil substrate), 0.50% PS treatment (0.50% PS microplastic soil substrate), 0.75% PS treatment (0.75% PS microplastic soil substrate), and 1.00% PS treatment (1.00% PS microplastic soil substrate). Each treatment was replicated four times. During the cultivation period, 50 mL of ddH2O was evenly applied to each pot every 3 days to ensure timely watering of the seedlings. Then, seedling length (SL), seedling fresh weight (SFW), seedling dry weight (SDW), root length (RL), root fresh weight (RFW), root dry weight (RDW), stem diameter (SD), root diameter (RD), and total plant dry biomass (PDW) were measured on maize seedlings cultured in six different PS microplastic soil substrates for 7 days and 21 days, respectively. The chlorophyll SPAD value (SPAD) of leaves was measured using a SPAD-502Plus chlorophyll meter (made in Japan). The chlorophyll content was measured using data from Beijing Solarbio Biotechnology Co., Ltd. The plant root activity assay kit (naphthylamine method) provided by LIFE SCIENCES is used to detect root activity (RV). Follow the instructions in the appendix... Figure 1 Formula (1) is used to calculate the seedling strength index (SSI). Where: SSI is the seedling strength index, SD is the stem diameter, SL is the seedling length, and PDW is the total dry biomass of the plant.

[0044] 3. Statistical Analysis: Twelve traits of TS163 maize genotype seedlings were measured on days 7 and 21 of the soil-cultured maize experiment with different concentrations of PS microplastics. ExCel 2013 software was used to calculate the mean and standard deviation of each trait in TS163 maize genotype seedlings on days 7 and 21 of cultivation, and bar charts were plotted. IBM-SPSSStatistics 19 software was used to analyze the joint variance of each trait of the TS163 maize genotype among the two cultivation time periods and six PS microplastic concentration treatments in the soil-cultured maize experiment, including the corrected model (CM), intercept (IC), cultivation time treatment (CT), PS microplastic concentration treatment (PSC), and the F-value and significance of the interaction between cultivation time and PS microplastic concentration treatment (CT×PSC). IBM SPSS Statistics 19 software was used to analyze the Dunman significance of each trait among the six PS microplastic treatments at each culture time point in soil-cultured maize seedlings of the TS163 genotype at the p<0.05 level. GENESCLOUD online software was used to analyze the Pearson correlation coefficients of all traits among the six PS microplastic treatments at each culture time point in soil-cultured maize seedlings of the TS163 genotype. The LOG10 function was used to standardize the mean values ​​of all traits among the six PS microplastic treatments at two culture time points in soil-cultured maize seedlings of the TS163 genotype. Then, IBM SPSS Statistics 16.0 software's Between-groups Linkage clustering method was used to conduct a scientific, objective, and qualitative cluster analysis of the microplastic toxicity of TS163 maize genotype seedlings among the six PS microplastic treatments at each culture time point in the soil-cultured maize experiment.

[0045] 4. Resistance Index to Different Concentrations of PS Microplastics in Maize Seedlings: To scientifically, accurately, and objectively reflect the resistance of individual traits of TS163 maize genotype seedlings to different concentrations of PS microplastics at each culture period in soil-cultured maize experiments, this invention newly defines the Resistance Index to Different Concentrations of PS Microplastics in Maize Seedlings (TRI), specifically as shown in Appendix. Figure 1 The calculation is performed using formula (2). Where: The term represents the resistance index to PS microplastic toxicity of maize seedlings with the j-th trait and the k-th concentration during the i-th culture period in a soil-cultured maize experiment. The value of the j-th trait in maize seedlings of genotype i during the i-th culture period in the soil-cultured maize experiment, under the CK control treatment. The value of the j-th trait of maize seedlings with the k-th concentration of PS microplastics is measured at the i-th culture time period in the soil-cultured maize experiment. i represents the 7th or 21st day of soil-cultured maize experiment, and k-PS represents the 0.05%, 0.25%, 0.50%, 0.75%, or 1.00% concentration of PS microplastics in the soil-cultured maize experiment. The larger the value, the stronger the resistance of the j-th genotype of maize seedlings to microplastic toxicity at the k-PS concentration during the i-th culture period in the soil-cultured maize experiment.

[0046] 5. Evaluation of the comprehensive resistance of maize seedlings to different concentrations of PS microplastics: The TRI values ​​of all traits of maize genotype seedlings calculated above were used as evaluation indicators to measure the resistance of maize genotype seedlings to different concentrations of PS microplastics at two cultivation time periods in the soil-cultured maize experiment. Furthermore, the membership function method was used to comprehensively, objectively, and quantitatively evaluate the magnitude of the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of maize genotype seedlings to different concentrations of PS microplastics at each / all cultivation time periods in the soil-cultured maize experiment. Specific details are provided in Appendix […]. Figure 1 The calculations are performed using formulas (3), (4), and (5). Where: The value represents the membership value of the resistance of maize seedlings with the j-th genotype to the toxicity of the k-th concentration of PS microplastics during the i-th culture period in a soil-cultured maize experiment. The term represents the resistance index to PS microplastic toxicity of maize seedlings with the j-th trait and the k-th concentration during the i-th culture period in a soil-cultured maize experiment. The minimum TRI value for the j-th genotype of maize seedlings under the i-th culture time period in a soil-cultured maize experiment is the minimum TRI value among all PS microplastic treatments. The maximum TRI value of the j-th genotype of maize seedlings under the i-th culture time period in the soil-cultured maize experiment is the maximum value among all concentrations of PS microplastic treatments for the j-th trait. ti represents the overall resistance of maize genotype seedlings to the k-th concentration of PS microplastics during the i-th culture period in the soil-cultured maize experiment; m represents the m (m=12) individual traits measured in maize genotype seedlings during the i-th culture period in the soil-cultured maize experiment; TCTR (k-PS) The value represents the overall resistance of maize seedlings of genotype to the toxicity of PS microplastics at the kth concentration during n (n=2, 2 culture time periods) culture time periods in the soil-cultured maize experiment. i represents the 7th or 21st day of soil-cultured maize experiment, and k-PS represents the PS microplastic treatment at the concentrations of 0.05%, 0.25%, 0.50%, 0.75%, or 1.00% in the soil-cultured experiment. A higher TTR value indicates a stronger overall resistance of maize seedlings of the i-th culture time period to the toxicity of the k-th concentration of PS microplastics in soil-cultured maize experiments.(k-PS) The larger the value, the stronger the overall resistance of maize seedlings of the kth concentration of PS microplastics to the toxicity of the maize genotype under the two culture periods in the soil-cultured maize experiment.

[0047] 7. Comprehensive assessment of PS microplastic concentration at the point of maximum PS microplastic toxicity in maize seedlings: By comparing the results of the Between-groups Linkage comprehensive clustering of PS microplastic toxicity in maize genotype seedlings under all treatments at two cultivation time periods in the above soil-cultured maize experiment, and the evaluation results of the overall comprehensive resistance of maize genotype seedlings to corresponding concentrations of PS microplastic toxicity at two cultivation time periods in the above soil-cultured maize experiment, the degree of PS microplastic toxicity in maize genotype seedlings at different concentrations under multiple cultivation time periods was evaluated by a combination of multiple methods, and finally the PS microplastic concentration at the point of maximum PS microplastic toxicity in maize genotype seedlings was comprehensively assessed.

[0048] Example 2

[0049] This invention provides a comprehensive evaluation result of the resistance of corn seedlings to microplastic toxicity, and the specific evaluation results are as follows:

[0050] 1. Joint ANOVA of maize traits measured at different cultivation time periods and different concentrations of PS microplastics in soil-cultured maize experiments: Joint ANOVA was performed on 12 traits of TS163 maize genotype seedlings at two cultivation time periods and six concentrations of PS microplastics (Table 1). The results showed that the corrected model (CM) of these 12 traits was significantly different at the P<0.001 level, indicating that the overall differences of these 12 traits were significant in the joint ANOVA. Therefore, it is effective to perform ANOVA on these 12 traits at different PS microplastic concentration treatments (PSC), cultivation time treatments (CT), and the interaction between cultivation time and PS microplastic concentration treatments (CT×PSC). Further analysis revealed significant differences in all 12 traits among the PS microplastic concentration treatments (P<0.05, P<0.01, or P<0.001). The influence of PS microplastic concentration on these 12 traits was in the following order: root activity > leaf chlorophyll SPAD value > root dry weight > root fresh weight > seedling fresh weight > root length > stem diameter > seedling dry weight > seedling vigor index > total plant dry biomass > root diameter > seedling length, with the F-value decreasing in that order. Similarly, all 12 traits showed significant differences among the treatments based on the cultivation time period (P<0.001), and the influence of cultivation time period on these 12 traits was in the following order: seedling fresh weight > seedling dry weight > stem diameter > total plant dry biomass. The order of biomass > seedling length > root fresh weight > root dry weight > seedling vigor index > leaf chlorophyll SPAD value > root activity > root length > root diameter, with the F- value decreasing in that order. In addition, except for seedling length, root diameter, and total dry biomass, the other nine traits showed significant differences in the interaction between the culture time period and the PS microplastic concentration treatment (P<0.05 or P<0.01 or P<0.001), indicating that the nine traits of seedling fresh weight, seedling dry weight, root length, root fresh weight, root dry weight, stem diameter, leaf chlorophyll SPAD value, seedling vigor index, and root activity are simultaneously regulated by the PS microplastic concentration treatment, the culture time period treatment, and the interaction between the culture time period and the PS microplastic concentration treatment.

[0051] Table 1. F-values ​​of joint ANOVA analysis of maize traits measured among six PS microplastic concentrations during two cultivation periods in the soil-grown maize experiment.

[0052]

[0053] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity. *, **, or *** indicate significant differences at the P<0.05, P<0.01, or P<0.001 levels, respectively.

[0054] 2. Effects of six concentrations of PS microplastics on 12 traits of maize seedlings on days 7 and 21 of culture in a soil-cultured maize experiment: (see attached) Figure 2 , 3As shown in Figure 4, the effects of six concentrations of PS microplastic treatment on 12 traits of TS163 maize genotype seedlings at 7 and 21 days of culture were not entirely the same. Specifically, compared with the control (CK), all concentrations of PS microplastic treatment inhibited the seedling growth of TS163 seedlings at 7 and 21 days of culture, with seedling length decreasing by 6.91% (1.00% PS microplastic soil substrate treatment) - 15.74% (0.75% PS microplastic soil substrate treatment) and 4.22% (1.00% PS microplastic soil substrate treatment) - 4.64% (0.50% PS microplastic soil substrate treatment), respectively. Compared with the control (CK), the fresh weight of TS163 seedlings treated with 0.25% PS microplastic soil substrate increased by 4.48% at 7 days of culture, while all other concentrations of PS microplastic soil substrate showed no increase at these two culture time points. The fresh weight of TS163 seedlings decreased by 22.45% (0.05% PS microplastic soil substrate treatment) - 50.36% (0.50% PS microplastic soil substrate treatment) and 3.38% (1.00% PS microplastic soil substrate treatment) - 21.96% (0.25% PS microplastic soil substrate treatment) under the different treatments. Compared with the control (CK), on days 7 and 21 of cultivation, different concentrations of PS microplastic treatments reduced the dry weight of TS163 seedlings, decreasing by 21.03% (0.05% PS microplastic soil substrate treatment) - 46.87% (0.50% PS microplastic soil substrate treatment) and 7.61% (1.00% PS microplastic soil substrate treatment), respectively. The root length of TS163 seedlings treated with 0.75% PS microplastic soil substrate was 18.29% (0.25% PS microplastic soil substrate treatment). Compared with the control (CK), except for a 5.66% increase in root length at day 7 under the 0.75% PS microplastic soil substrate treatment, the root length of TS163 seedlings treated with all other concentrations of PS microplastic soil substrate at these two culture time periods was shortened by 8.35% (0.05% PS microplastic soil substrate treatment) - 13.44% (1.00% PS microplastic soil substrate treatment) and 16.74% (0.05% PS microplastic soil substrate treatment) - 40.24% (0.50% PS microplastic soil substrate treatment), respectively. Compared with the control (CK), on days 7 and 21 of cultivation, different concentrations of PS microplastics treatments reduced the root fresh weight of TS163 seedlings, decreasing by 9.31% (0.25% PS microplastic soil substrate treatment) - 32.98% (0.75% PS microplastic soil substrate treatment) and 11.80% (0.75% PS microplastic soil substrate treatment) - 28.00% (0.25% PS microplastic soil substrate treatment), respectively. Compared with the control (CK), on days 7 and 21 of cultivation, different concentrations of PS microplastics treatments also reduced the root dry weight of TS163 seedlings, decreasing by 0.70% (0.25% PS microplastic soil substrate treatment) - 27.85% (0.25% PS microplastic soil substrate treatment) - 28.00% (0.25% PS microplastic soil substrate treatment), respectively.The treatments used included 75% (0.25% PS microplastic soil substrate) and 2.99% (0.75% PS microplastic soil substrate treatment) – 25.95% (0.75% PS microplastic soil substrate treatment). Compared with the control (CK), on days 7 and 21 of cultivation, all concentrations of PS microplastic treatment reduced the stem diameter of TS163 seedlings, decreasing by 7.80% (1.00% PS microplastic soil substrate treatment) – 19.08% (0.75% PS microplastic soil substrate treatment) and 7.72% (1.00% PS microplastic soil substrate treatment) – 10.41% (0.75% PS microplastic soil substrate treatment), respectively. Compared with the control (CK), on days 7 and 21 of cultivation, all concentrations of PS microplastic treatment reduced the stem diameter. The root diameter of TS163 seedlings decreased by 6.54% (0.25% PS microplastic soil substrate treatment) - 33.64% (1.00% PS microplastic soil substrate treatment) and 2.89% (0.50% PS microplastic soil substrate treatment) - 8.67% (1.00% PS microplastic soil substrate treatment), respectively. Compared with the control (CK), on days 7 and 21 of cultivation, different concentrations of PS microplastic treatments reduced the total dry biomass of TS163 seedlings, decreasing by 11.33% (0.25% PS microplastic soil substrate treatment) - 34.26% (0.75% PS microplastic soil substrate treatment) and 6.19% (1.00% PS microplastic soil substrate treatment), respectively. The chlorophyll SPAD value of TS163 seedlings decreased by 13.66% (1.00% PS microplastic soil substrate treatment) - 25.97% (0.25% PS microplastic soil substrate treatment) and 2.07% (1.00% PS microplastic soil substrate treatment) - 16.55% (0.25% PS microplastic soil substrate treatment) on days 7 and 21 of cultivation, respectively. Compared with the control (CK), the seedling vigor index of TS163 seedlings decreased by 14.88% (0.25% PS microplastic soil substrate treatment) - 25.97% (0.25% PS microplastic soil substrate treatment) and 2.07% (1.00% PS microplastic soil substrate treatment) - 16.55% (0.25% PS microplastic soil substrate treatment) on days 7 and 21 of cultivation, respectively. The seedling index decreased by 10.30% (0.05% PS microplastic soil substrate treatment) - 36.16% (0.75% PS microplastic soil substrate treatment) and 9.37% (1.00% PS microplastic soil substrate treatment) - 16.06% (0.50% PS microplastic soil substrate treatment), respectively. Compared with the control CK, on ​​days 7 and 21 of cultivation, different concentrations of PS microplastic treatment reduced the root activity of TS163 seedlings, with decreases of 58.78% (0.05% PS microplastic soil substrate treatment) - 77.75% (0.50% PS microplastic soil substrate treatment) and 25.31% (1.00% PS microplastic soil substrate treatment) - 68.14%, respectively.(50% PS microplastic soil substrate treatment).

[0055] 3. Pearson correlation analysis among 12 traits of maize seedlings cultured for 7 and 21 days under 6 concentrations of PS microplastic treatment in a soil-cultured maize experiment: (see attached) Figure 5 and 6 As shown, Pearson correlation analysis was performed on 12 traits of TS163 maize genotype seedlings at 7 and 21 days of culture under six concentrations of PS microplastic treatment in a soil-cultured maize experiment. The results showed that the Pearson correlations of the 12 traits of TS163 maize genotype seedlings were completely consistent across the two culture time periods under the six PS microplastic treatments. Specifically, seedling length was significantly positively correlated with seedling fresh weight, seedling dry weight, root fresh weight, stem diameter, total plant dry biomass, leaf chlorophyll SPAD value, seedling vigor index, and root activity; seedling fresh weight was significantly positively correlated with seedling dry weight, root fresh weight, root dry weight, root diameter, total plant dry biomass, seedling vigor index, and root activity; and seedling dry weight was significantly positively correlated with root fresh weight, root dry weight, stem diameter, root diameter, total plant dry biomass, seedling vigor index, and root activity. Significant positive correlations were found between root fresh weight and root dry weight, root diameter, total plant dry biomass, seedling vigor index, and root activity; significant positive correlations were also found between root dry weight and root diameter, total plant dry biomass, seedling vigor index, and root activity; significant positive correlations were also found between stem diameter and total plant dry biomass, leaf chlorophyll SPAD value, seedling vigor index, and root activity; significant positive correlations were also found between root diameter and total plant dry biomass, seedling vigor index, and root activity; significant positive correlations were also found between total plant dry biomass, seedling vigor index, and root activity; significant positive correlations were also found between leaf chlorophyll SPAD value and root activity; and significant positive correlations were also found between seedling vigor index and root activity. This indicates that these traits of maize seedlings at corresponding cultivation time periods synergistically determine their growth, development, and biomass accumulation under different concentrations of PS microplastic treatment.

[0056] 4. Cluster analysis of microplastic toxicity of six concentrations of PS microplastics on maize seedlings at 7 and 21 days of culture in soil-cultured maize experiments: Since the effects of six concentrations of PS microplastics on the 12 traits of TS163 maize genotype seedlings at 7 and 21 days of culture were not entirely consistent, and each trait at each culture time point only reflected one aspect of the TS163 genotype seedlings under the corresponding PS microplastic concentration, it could not comprehensively and objectively reflect the overall performance of TS163 maize genotype seedlings to the toxicity of each PS microplastic concentration at the corresponding culture time point. Therefore, we used the LOG10 function to standardize the 12 traits of TS163 maize genotype seedlings at 7 and 21 days of culture under the six concentrations of PS microplastics in soil-cultured maize experiments. Then, we used the Between-groups Linkage clustering method to evaluate the degree of microplastic toxicity of TS163 maize genotype seedlings among the six PS microplastic concentrations at each culture time point in the soil-cultured maize experiment. (See attached...) Figure 7 As shown, when the Euclidean distance is 2, on day 7 of cultivation, TS163 maize genotype seedlings treated with six concentrations of PS microplastics can be divided into three types: Type A includes only TS163 genotype seedlings grown under the CK control treatment, which are unaffected by PS microplastics; Type B includes TS163 genotype seedlings grown under soil substrate treatments of 0.05% and 0.25% PS microplastics, which exhibit poorer performance in multiple traits and are less affected by PS microplastics; Type C includes TS163 genotype seedlings grown under soil substrate treatments of 0.50%, 0.75%, and 1.00% PS microplastics, which exhibit the worst performance in multiple traits and are severely affected by PS microplastics. The difference is shown in the attached diagram. Figure 8 As shown, when the Euclidean distance is 7, on day 21 of cultivation, TS163 maize genotype seedlings treated with six concentrations of PS microplastics can be divided into three types: Type A includes only TS163 genotype seedlings grown under the CK control treatment, which are not affected by PS microplastics and are therefore unaffected by PS microplastics; Type B includes TS163 genotype seedlings grown under soil substrate treatments of 0.75% and 1.00% PS microplastics, which show poorer performance in multiple traits and are less affected by PS microplastics and are therefore weakly affected by PS microplastics; Type C includes TS163 genotype seedlings grown under soil substrate treatments of 0.05%, 0.50%, and 0.25% PS microplastics, which show the worst performance in multiple traits and are severely affected by PS microplastics and are therefore severely affected by PS microplastics. While these results can roughly distinguish the degree of toxicity of six concentrations of PS microplastics to corn seedlings at different cultivation time periods, they cannot clearly distinguish the extent of toxicity of each concentration of PS microplastics to corn seedlings at each cultivation time period.

[0057] 5. Resistance Index to PS Microplastic Toxicity of Individual Traits in Maize Seedlings at 7 and 21 Days of Cultivation in Soil-Cultivated Maize Experiments: To scientifically, accurately, and objectively reflect the resistance of individual traits of TS163 maize genotype seedlings to different concentrations of PS microplastic toxicity at each cultivation time point in soil-cultivated maize experiments, we newly defined the Resistance Index to PS Microplastic Toxicity of Maize Seedlings at Different Concentrations (TRI). The advantage of the TRI value is that it can not only objectively and accurately quantify the resistance of individual traits of TS163 maize genotype seedlings to different concentrations of PS microplastic toxicity at each cultivation time point in soil-cultivated maize experiments, but also provide a scientific basis for the comprehensive evaluation of PS microplastic toxicity resistance of maize seedlings. Therefore, based on the 12 traits of TS163 maize genotype seedlings at 7 and 21 days of cultivation in soil-cultivated maize experiments, according to the attached... Figure 1 Formula (2) was used to calculate the TRI values ​​of 12 traits of TS163 maize genotype seedlings on the 7th day (Table 2) and the 21st day (Table 3) of soil-cultured maize experiments.

[0058] Table 2. Resistance Index (TRI) values ​​of different concentrations of PS microplastics for 12 traits in TS163 maize genotype seedlings on day 7 of soil-cultured maize experiments.

[0059]

[0060] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity. 0.05% PS is 0.05% PS microplastic soil substrate treatment, 0.25% PS is 0.25% PS microplastic soil substrate treatment, 0.50% PS is 0.50% PS microplastic soil substrate treatment, 0.75% PS is 0.75% PS microplastic soil substrate treatment, and 1.00% PS is 1.00% PS microplastic soil substrate treatment.

[0061] Table 3. Resistance Index (TRI) values ​​of different concentrations of PS microplastics for 12 traits in TS163 maize genotype seedlings on day 21 of soil-cultured maize experiments.

[0062]

[0063] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity. 0.05% PS is 0.05% PS microplastic soil substrate treatment, 0.25% PS is 0.25% PS microplastic soil substrate treatment, 0.50% PS is 0.50% PS microplastic soil substrate treatment, 0.75% PS is 0.75% PS microplastic soil substrate treatment, and 1.00% PS is 1.00% PS microplastic soil substrate treatment.

[0064] 6. Evaluation of the comprehensive resistance of maize seedlings at 7 and 21 days of cultivation to different concentrations of PS microplastics in soil-cultured maize experiments: We further used the TRI values ​​of 12 traits of TS163 maize genotype seedlings at 7 and 21 days of cultivation in soil-cultured maize experiments as the evaluation index for the resistance of maize genotype seedlings to different concentrations of PS microplastics at these two cultivation periods. The membership function method was used, based on the appendix... Figure 1 Formula (3) was used to calculate the resistance membership values ​​(U(TRI)) of 12 traits of TS163 maize genotype seedlings to different concentrations of PS microplastic toxicity in soil-cultured maize seedlings at 7 days and 21 days of culture (Tables 4 and 5). Then, according to the appendix... Figure 1 Formula (4) was used to calculate the CTR values ​​of the comprehensive resistance to PS microplastic toxicity of TS163 maize genotype seedlings on the 7th and 21st days of soil-cultured maize experiments, respectively. Finally, the values ​​were calculated based on the attached formula. Figure 1 Formula (5) was used to calculate the total comprehensive resistance (TCTR) of TS163 maize genotype seedlings to corresponding concentrations of PS microplastics during two culture periods in the soil-cultured maize experiment. The results are attached. Figure 9 The results show that in the soil-cultured maize experiment, the CTRs of TS163 maize genotype seedlings cultured for 7 days to resist PS microplastic toxicity at concentrations of 0.05%, 0.25%, 0.50%, 0.75%, and 1.00% were 0.631, 0.655, 0.175, 0.264, and 0.409, respectively. In contrast, the CTRs of TS163 maize genotype seedlings cultured for 21 days to resist PS microplastic toxicity at the same concentrations were 0.425, 0.323, 0.371, 0.497, and 0.760, respectively. This indicates that the overall resistance of maize seedlings to different concentrations of PS microplastic toxicity varies at different culture time periods. Further details are attached. Figure 10As shown, in the soil-cultured maize experiment, the TCTRs of TS163 maize genotype seedlings against 0.05%, 0.25%, 0.50%, 0.75%, and 1.00% concentrations of PS microplastics were 0.523, 0.489, 0.273, 0.381, and 0.584, respectively, at two different culture periods. This indicates that the overall resistance of maize seedlings to 0.50% PS microplastics was the lowest at 0.273, while the overall resistance to 1.00% PS microplastics was the highest at 0.584.

[0065] Table 4. Membership values ​​(U(TRI)) of 12 traits of TS163 maize genotype seedlings on day 7 of soil-cultured maize experiments to resistance to different concentrations of PS microplastics.

[0066]

[0067] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity. 0.05% PS is 0.05% PS microplastic soil substrate treatment, 0.25% PS is 0.25% PS microplastic soil substrate treatment, 0.50% PS is 0.50% PS microplastic soil substrate treatment, 0.75% PS is 0.75% PS microplastic soil substrate treatment, and 1.00% PS is 1.00% PS microplastic soil substrate treatment.

[0068] Table 5. Membership values ​​(U(TRI)) of 12 traits of TS163 maize genotype seedlings on day 21 of soil-cultured maize experiments to resistance to different concentrations of PS microplastics.

[0069]

[0070]

[0071] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity. 0.05% PS is 0.05% PS microplastic soil substrate treatment, 0.25% PS is 0.25% PS microplastic soil substrate treatment, 0.50% PS is 0.50% PS microplastic soil substrate treatment, 0.75% PS is 0.75% PS microplastic soil substrate treatment, and 1.00% PS is 1.00% PS microplastic soil substrate treatment.

[0072] 7. Comprehensive assessment of PS microplastic concentration at the point of highest PS microplastic toxicity in maize seedlings: We further compared the results of the Between-groups Linkage of PS microplastic toxicity in TS163 maize genotype seedlings under all PS microplastic concentrations during the two culture periods in the above soil-cultured maize experiment (see appendix). Figure 7 and 8 The results of the evaluation of the overall resistance of TS163 maize genotype seedlings to all concentrations of PS microplastic toxicity during the two culture periods in the above-mentioned soil-cultured maize experiment are attached. Figure 10 Through comprehensive comparative analysis of multiple traits in maize seedlings at various cultivation periods using multiple methods, the study ultimately determined that the concentration of PS microplastics causing the most severe toxicity in maize seedlings was 0.50%. Therefore, in the future, we can use a 0.50% concentration of PS microplastics to cultivate different maize genotypes, and measure the aforementioned 12 traits on days 7 and 21 of cultivation. This comprehensive evaluation will identify superior maize genotypes resistant to PS microplastic toxicity, which can then be applied in field production to mitigate the adverse effects of microplastic pollution on maize and ensure national food security.

[0073] As can be seen from the above embodiments, this invention systematically and comprehensively reveals the effects of six concentrations of PS microplastic treatments on 12 aboveground / belowground phenotypes and physiological metabolic traits of maize genotype seedlings at two cultivation time periods, including growth phenotype, seedling vigor index, chlorophyll accumulation level, and root activity. It objectively analyzes the toxic mechanism of different concentrations of PS microplastics on maize seedlings. Furthermore, standardized data of these 12 traits were used to perform cluster evaluation among all concentrations of PS microplastic treatments at each cultivation time period, comprehensively and objectively reflecting the degree of toxicity of different concentrations of PS microplastic treatments to maize genotype seedlings. Based on this, we also innovatively proposed a resistance index (TRI) for individual traits of maize genotype seedlings to different concentrations of PS microplastic toxicity at each cultivation time period, and used the membership function method to comprehensively and quantitatively evaluate the magnitude of the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of maize genotype seedlings to the corresponding concentration of PS microplastic toxicity at each / all cultivation time periods. Furthermore, we compared the cluster evaluation results of maize genotype seedlings to all concentrations of PS microplastic treatment at each culture time period, and the evaluation results of the overall comprehensive resistance of maize genotype seedlings to the corresponding concentrations of PS microplastic toxicity at all culture time periods. This verified that the TRI biological concept and calculation formula we proposed are scientific, accurate, and reliable for comprehensively evaluating the degree of PS microplastic toxicity in maize genotype seedlings at different concentrations. Therefore, we ultimately determined that the PS microplastic concentration at which the degree of PS microplastic toxicity in maize genotype seedlings was the most severe. Thus, in the future, we can treat different maize genotype materials with a 0.50% concentration of PS microplastic for 7 days and 21 days to screen for superior maize genotypes (inbred lines / strains / variety) resistant to microplastic toxicity, for application in breeding or production practices.

[0074] Example 3

[0075] This invention provides a method for identifying microplastic-resistant maize genotypes. The specific identification method is carried out according to the following steps:

[0076] 1. Preparation of high-quality maize seeds: Prepare 10 high-quality maize genotypes independently bred by our team in 2024, which were planted at the Longxi test site (34.97°N, 104.40°E, altitude 2074m), strictly bagged and pollinated, and harvested with plump, uniform-sized, vigorous, and high-purity kernels.

[0077] 2.0.50% Polystyrene (PS) Microplastic Soil-Cultivated Maize Experiment: Forty seeds each of 10 high-quality maize genotypes selected by our team were placed in Erlenmeyer flasks. 100 mL of 70% ethanol (v / v) was added, and the flasks were placed on a horizontal shaker for 10 min to sterilize the seeds. The seeds were then rinsed five times with 100 mL of ddH2O water to remove residual ethanol. The seeds were then dried with sterile filter paper to obtain the corresponding sterilized maize genotype seeds. PS microplastic powder (mesh size: 1000 mesh; particle size: 13 μm) was purchased from Dongguan Ruixiang Plastic Raw Materials Co., Ltd. 0.00 g and 0.50 g of PS microplastic were accurately weighed using an electronic balance and dissolved in 100 mL of ddH2O water to prepare two concentrations (PS microplastic mass to ddH2O mass ratio) of PS microplastic solutions, 0.00% and 0.50%, respectively, which were prepared fresh for each use. Ten sterilized maize genotype seeds were then placed into corresponding Erlenmeyer flasks, and 100 mL of each of the two concentrations of PS microplastic solution was added. The flasks were then placed on a horizontal shaker in a dark indoor environment to soak the seeds for 24 hours. Two concentrations (PS microplastic to nutrient soil mass ratio) of soil-grown PS microplastic substrate were then prepared: a 0.00% PS microplastic substrate (i.e., 0.00 g PS microplastic was thoroughly mixed with 300.00 g nutrient soil, then 250 mL of ddH2O was added and stirred thoroughly) and a 0.50% PS microplastic substrate (i.e., 1.50 g PS microplastic was thoroughly mixed with 300.00 g nutrient soil, then 250 mL of ddH2O was added and stirred thoroughly). Ten maize genotype seeds from each of the ten seeds soaked in the two concentrations of PS microplastic solutions were then sown into pots (11.0 cm high, 12.5 cm in diameter) containing soil substrate of the same concentration of PS microplastic and placed in an artificial climate chamber for cultivation. During cultivation, the relative humidity was set at 65%, the temperature was alternated between 25±0.5℃ and 20±0.5℃ for 12 hours each, the photoperiod was set at 16 / 8 hours of light / dark, and the light intensity was set at 300 μMm. -2 s -1The CO2 concentration was 450 PPM. Two treatments were used in the soil culture experiment: a control (0.00% PS microplastic soil substrate) and a 0.50% PS treatment (0.50% PS microplastic soil substrate), with four biological replicates for each treatment. During the cultivation period, 50 mL of ddH2O was evenly applied to each pot every 3 days to ensure timely watering of the seedlings. On days 7 and 21 of cultivation in either of the two PS microplastic soil substrates, the following parameters were measured for maize genotype seedlings: seedling length (SL), seedling fresh weight (SFW), seedling dry weight (SDW), root length (RL), root fresh weight (RFW), root dry weight (RDW), stem diameter (SD), root diameter (RD), total plant dry biomass (PDW), leaf chlorophyll SPAD value (SPAD), root activity (RV), and seedling vigor index (SSI). The seedling vigor index was calculated according to the attached... Figure 1 The formula (1) is used for calculation, where: SSI is the seedling strength index, SD is the stem diameter, SL is the seedling length, and PDW is the total dry biomass of the plant.

[0078] 3. Statistical Analysis: For the 12 traits of 10 maize genotype seedlings cultured in soil at 7 and 21 days of cultivation under two concentrations of PS microplastics in the soil-cultured maize experiment, ExCel 2013 software was used to calculate the mean and standard deviation of each trait for each genotype seedling under each treatment at 7 and 21 days of cultivation. IBM-SPSSStatistics 19 software was used to analyze the joint ANOVA of each trait for all maize genotypes across the two PS microplastic treatments at these two cultivation time periods, including the corrected model (CM), intercept (IC), genotype (G), cultivation time treatment (CT), PS microplastic concentration treatment (PSC), interactions between genotype and cultivation time treatment (G×CT), interactions between genotype and PS microplastic concentration treatment (G×PSC), interactions between cultivation time and PS microplastic concentration treatment (CT×PSC), and interactions among the three (G×CT×PSC), and the F-values ​​and significance levels of these interactions.

[0079] 4. Toxicity resistance index of 0.50% polystyrene (PS) microplastics in maize seedlings of different genotypes: Based on the mean values ​​of 12 traits of 10 maize genotype seedlings measured on days 7 and 21 of embryonic development in soil-cultured maize experiments with two concentrations of PS microplastics, according to the attached... Figure 1 Formula (6) is used to calculate the TRI value of resistance to toxicity from 0.50% PS microplastics for each genotype of maize seedling at each culture period. Where: The resistance index to toxicity of 0.50% PS microplastics in the j-th trait of the p-th maize genotype seedling at the i-th culture time period in a soil-cultured maize experiment. The value of the j-th trait under the CK control treatment in the i-th culture time period of the p-th maize genotype seedling in the soil-cultured maize experiment is shown. The value of the jth trait under 0.50% PS microplastic treatment in the i-th culture period of the p-th maize genotype seedling in the soil-cultured maize experiment is given. i represents the 7th or 21st day of soil-cultured maize experiment, and 0.50% PS represents the 0.50% concentration of PS microplastic treatment in the soil-cultured maize experiment. The larger the value, the stronger the resistance of the j-th trait of the p-th maize genotype seedling to 0.50% microplastic toxicity during the i-th culture period in the soil-cultured maize experiment.

[0080] 5. Evaluation of the comprehensive resistance of maize seedlings of different genotypes to 0.50% polystyrene (PS) microplastic toxicity: The TRI values ​​of all traits of the 10 maize genotype seedlings calculated above were used as evaluation indicators to measure the resistance of all maize genotype seedlings to 0.50% PS microplastic toxicity at two cultivation time periods in the soil-cultured maize experiment. Furthermore, the membership function method was used to comprehensively, quantitatively, and systematically evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of the 10 maize genotype seedlings to 0.50% PS microplastic toxicity at each / all cultivation time periods in the soil-cultured maize experiment. Specific details are as follows (see Appendix). Figure 1 The calculations are performed using formulas (7), (8), and (9): Where: The value represents the membership value of the resistance of the j-th trait of the p-th maize genotype seedling to 0.50% PS microplastic toxicity during the i-th culture period in a soil-cultured maize experiment. The resistance index to toxicity of 0.50% PS microplastics in the j-th trait of the p-th maize genotype seedling at the i-th culture time period in a soil-cultured maize experiment. The minimum TRI value for the j-th trait of all maize genotypes in the soil-cultured maize experiment during the i-th cultivation period is given. The maximum TRI value for the j-th trait of all maize genotypes in the soil-cultured maize seedlings during the i-th culture period is given. ti represents the overall resistance of the p-th maize genotype seedling to 0.50% PS microplastic toxicity during the i-th culture period in the soil-cultured maize experiment; m represents the m (m=12) traits measured in each maize genotype seedling during the i-th culture period in the soil-cultured maize experiment; TCTR p(0.50％PS) The value represents the overall resistance of the p-th maize genotype seedling to 0.50% PS microplastic toxicity during the n (n=2, 2 culture time periods) culture time period in the soil-cultured maize experiment. i represents the 7th or 21st day of soil-cultured maize experiment, and 0.50% PS represents the 0.50% PS microplastic treatment in the soil-cultured experiment. A higher TCTR value indicates a stronger overall resistance to 0.50% PS microplastic toxicity in the p-th maize genotype seedlings during the i-th culture period in a soil-cultured maize experiment. (0.50％PS) A higher TCTR value indicates a stronger overall resistance to 0.50% PS microplastic toxicity in the p-th maize genotype seedlings across two culture periods in the soil-cultured maize experiment. Based on the TCTR values ​​of different maize genotypes, the resistance to microplastic toxicity was classified into five levels: 0 ≤ TCTR < 0.400, indicating a highly sensitive maize genotype to microplastic toxicity; 0.400 ≤ TCTR < 0.500, indicating a sensitive maize genotype to microplastic toxicity; 0.500 ≤ TCTR < 0.600, indicating a weakly resistant maize genotype to microplastic toxicity; 0.600 ≤ TCTR < 0.700, indicating a moderately resistant maize genotype to microplastic toxicity; and 0.700 ≤ TCTR ≤ 1.000, indicating a highly resistant maize genotype to microplastic toxicity.

[0081] Example 4

[0082] This invention provides the results of evaluating maize genotypes resistant to microplastic toxicity, and the specific results are as follows:

[0083] 1. Joint ANOVA of 12 traits of all maize seedlings under different PS microplastic concentration treatments at different culture time periods in soil-cultured maize experiments: Joint ANOVA was performed on 12 traits of 10 maize seedlings of genotypes under two PS microplastic concentration treatments at two culture time periods in soil-cultured maize experiments (Table 6). The results showed that the corrected model (CM) of these 12 traits was significantly different at the P<0.001 level, indicating that the overall differences of these 12 traits were significant in the joint ANOVA. Therefore, it is effective to perform ANOVA on these 12 traits among genotypes (G), PS microplastic concentration treatments (PSC), culture time treatments (CT), the interaction between genotype and PS microplastic concentration treatments (G×PSC), the interaction between genotype and culture time treatments (G×CT), the interaction between culture time and PS microplastic concentration treatments (CT×PSC), and the interaction among the three (G×CT×PSC). Further analysis revealed that all 12 traits showed significant differences (P < 0.001) among genotypes, PS microplastic concentration treatments, and culture time treatments, indicating that these 12 traits of maize seedlings were significantly influenced by their own genetic basis, PS microplastic concentration treatments, and culture time treatments. Furthermore, except for seedling length, root length, root fresh weight, and stem diameter, the other 8 traits also showed significant differences (P < 0.05, P < 0.01, or P < 0.001) between genotype and PS microplastic concentration treatments; except for the seedling vigor index, the other 11 traits also showed significant differences (P < 0.05, P < 0.01, or P < 0.001) between genotype and culture time treatments; and except for seedling length, seedling fresh weight, stem diameter, and leaf chlorophyll SPAD value, the other 8 traits also showed significant differences (P < 0.05, P < 0.01, or P < 0.001) between culture time and PS microplastic concentration treatments. Significant differences were found among the interactions (P<0.01 or P<0.001). Except for seedling length, seedling fresh weight, seedling dry weight, root fresh weight, stem diameter, and leaf chlorophyll SPAD value, the other 6 traits also showed significant differences among the interactions of the three factors (P<0.05 or P<0.01 or P<0.001). This indicates that multiple traits of maize seedlings of different genotypes are also jointly regulated by the interactions of different factors. Ultimately, this resulted in significant differences among the 12 traits of maize seedlings of different genotypes under different PS microplastic concentration treatments at different culture time periods.

[0084] Table 6. F-values ​​of 12 traits from 10 maize seedlings of genotypes treated with two concentrations of PS microplastics during two different culture periods in the soil-grown maize experiment.

[0085]

[0086] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity. *, **, or *** indicate significant differences at the P<0.05, P<0.01, or P<0.001 levels, respectively.

[0087] 2. The overall effects of two concentrations of PS microplastic treatment on 12 traits of 10 maize seedlings at 7 and 21 days of culture in a soil-cultured maize experiment: (see attached) Figure 11 As shown, compared with the control (CK), the effects of 0.50% PS microplastic treatment on the 12 traits of 10 maize genotype seedlings differed at the two culture time periods. Overall, compared with the control (CK), on day 7 of culture, 0.50% PS microplastic treatment resulted in average decreases of 22.69%, 53.32%, 52.10%, 19.17%, 40.50%, 68.02%, 13.92%, 27.04%, 72.98%, 16.75%, 70.52%, and 65.41% in seedling length, seedling fresh weight, seedling dry weight, root length, root fresh weight, root dry weight, stem diameter, root diameter, total plant dry biomass, leaf chlorophyll SPAD value, seedling vigor index, and root activity in the 10 maize genotype seedlings, respectively. As the maize seedlings grew, by day 21 of cultivation, treatment with 0.50% PS microplastics resulted in average decreases of 13.91%, 18.88%, 21.20%, 25.05%, 24.16%, 74.52%, 12.02%, 12.09%, 46.09%, 16.63%, 40.72%, and 57.07% in seedling length, fresh weight, dry weight, root length, fresh weight, dry weight, stem diameter, root diameter, total plant dry biomass, leaf chlorophyll SPAD value, seedling vigor index, and root activity in 10 maize genotypes. This indicates that the sensitivity and tolerance of 0.50% concentration PS microplastics to the 12 traits of these 10 maize genotype seedlings are not the same under different cultivation periods. Therefore, we can use these 12 traits as evaluation indicators for maize resistance to PS microplastic toxicity and comprehensively evaluate the resistance of different maize genotypes to PS microplastic toxicity.

[0088] 3. Resistance index of 0.50% PS microplastic concentration to individual traits of maize seedlings on days 7 and 21 of soil cultivation in a maize experiment: according to the appendix Figure 1Formula (6) was used to further calculate the resistance index (TRI) values ​​of individual traits of these 10 maize genotype seedlings to 0.50% PS microplastic toxicity at two different culture periods. Specifically, on day 7 of culture, the TRI values ​​for seedling length of these 10 maize genotype seedlings ranged from 0.641 (GTX-21-2 maize genotype) to 0.934 (GTX-21-3 maize genotype), the TRI values ​​for seedling fresh weight ranged from 0.045 (GTX-21-10 maize genotype) to 0.685 (GTX-21-6 maize genotype), and the TRI values ​​for seedling dry weight ranged from 0.306 (GTX-21-2 maize genotype) to 0.740 (GTX-21-3 maize genotype). The root length TRI value ranges from 0.652 (GTX-21-4 maize genotype) to 0.909 (GTX-21-3 maize genotype), the root fresh weight TRI value ranges from 0.436 (GTX-21-4 maize genotype) to 0.688 (GTX-21-6 maize genotype), and the root dry weight TRI value ranges from 0.079 (GTX-21-3 maize genotype) to 0.898 (GTX-21-5 maize genotype). The stem diameter TRI value ranged from 0.740 (GTX-21-4 maize genotype) to 0.919 (GTX-21-6 maize genotype), the root diameter TRI value ranged from 0.545 (GTX-21-9 maize genotype) to 0.899 (GTX-21-3 maize genotype), and the total plant dry biomass TRI value ranged from 0.049 (GTX-21-10 maize genotype) to 0.789 (GTX-21-5 maize genotype). Leaf chlorophyll content... The SPAD and TRI values ​​ranged from 0.726 (GTX-21-4 maize genotype) to 0.910 (GTX-21-5 maize genotype), the seedling vigor index TRI value ranged from 0.051 (GTX-21-10 maize genotype) to 0.817 (GTX-21-5 maize genotype), and the root activity TRI value ranged from 0.203 (GTX-21-2 maize genotype) to 0.591 (GTX-21-3 maize genotype) (Table 7). This indicates that the TRI values ​​for different traits of these 10 maize genotype seedlings differed at day 7 of culture.Furthermore, on day 21 of cultivation, the seedling length TRI values ​​of these 10 maize genotypes ranged from 0.714 (GTX-21-9 maize genotype) to 0.935 (GTX-21-3 maize genotype), the seedling fresh weight TRI values ​​ranged from 0.747 (GTX-21-10 maize genotype) to 0.887 (GTX-21-7 maize genotype), and the seedling dry weight TRI values ​​ranged from 0.670 (GTX-21-2 maize genotype) to 0.902 (GTX-21-3 maize genotype). The root length TRI value ranged from 0.571 (GTX-21-1 maize genotype) to 0.865 (GTX-21-6 maize genotype), the root fresh weight TRI value ranged from 0.671 (GTX-21-1 maize genotype) to 0.864 (GTX-21-10 maize genotype), and the root dry weight TRI value ranged from 0.060 (GTX-21-5 maize genotype) to 0.1.494 (GTX-21-6 maize genotype). The stem diameter TRI value ranged from 0.816 (GTX-21-9 maize genotype) to 0.988 (GTX-21-3 maize genotype), the root diameter TRI value ranged from 0.780 (GTX-21-9 maize genotype) to 0.982 (GTX-21-6 maize genotype), and the total plant dry biomass TRI value ranged from 0.263 (GTX-21-6 maize genotype) to 2.032 (GTX-21-4 maize genotype). The leaf... The SPAD (Special Percutaneous Transformation Index) and TRI (Trial Value) values ​​for chlorophyll ranged from 0.693 (GTX-21-2 maize genotype) to 0.904 (GTX-21-5 maize genotype), the TRI value for seedling vigor ranged from 0.259 (GTX-21-5 maize genotype) to 2.027 (GTX-21-4 maize genotype), and the TRI value for root activity ranged from 161 (GTX-21-6 maize genotype) to 0.608 (GTX-21-5 maize genotype) (Table 8). This indicates that, similar to day 7 of culture, the TRI values ​​for different traits in these 10 maize genotype seedlings also differed at day 21 of culture.

[0089] Table 7. TRI values ​​of 12 traits in 10 maize genotype seedlings from soil-cultured maize on day 7 of cultivation at 0.50% concentration of PS microplastics.

[0090]

[0091] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity. Table 8 shows the TRI values ​​of 12 traits of 10 maize genotype seedlings with 0.50% PS microplastic toxicity on day 21 of soil-cultured maize.

[0092]

[0093] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity.

[0094] 4. Evaluation of the comprehensive resistance of different maize seedlings to 0.50% PS microplastic toxicity at 7 and 21 days of soil-cultured maize experiments: We further used the TRI values ​​of 12 traits of 10 maize genotype seedlings at 7 and 21 days of soil-cultured maize experiments as the evaluation index for the resistance of corresponding maize genotype seedlings to 0.50% PS microplastic toxicity at these two culture time periods. The membership function method was used, based on the appendix... Figure 1 Formula (7) was used to calculate the resistance membership values ​​U(TRI) of 12 traits of 10 maize genotype seedlings from soil-cultured maize at 7 and 21 days of culture to 0.50% PS microplastic toxicity (Tables 9 and 10). Then, according to the appendix... Figure 1 Formula (8) was used to calculate the CTR values ​​of the comprehensive resistance to 0.50% PS microplastic toxicity in 10 maize genotype seedlings cultured on days 7 and 21 of soil-cultured maize experiments. Finally, the values ​​were calculated based on the attached formula. Figure 1 Formula (9) was used to calculate the total comprehensive resistance (TCTR) values ​​of 10 maize genotype seedlings to 0.50% PS microplastic toxicity during two culture periods in a soil-cultured maize experiment. The results are attached. Figure 12 The results show that in the soil-cultured maize experiment, the CTRs of 10 maize genotype seedlings cultured for 7 days to 0.05% PS microplastic toxicity ranged from 0.212 (GTX-21-4 maize genotype) to 0.839 (GTX-21-5 maize genotype), while the CTRs of 10 maize genotype seedlings cultured for 21 days to 0.50% PS microplastic toxicity ranged from 0.347 (GTX-21-1 maize genotype) to 0.682 (GTX-21-3 maize genotype). This indicates that the overall resistance of different maize genotype seedlings to 0.50% PS microplastic toxicity varied at different culture time points. Further details are attached. Figure 13As shown, in the soil-cultured maize experiment, the TCTRs of these 10 maize genotypes' seedlings against 0.50% PS microplastic toxicity at two different culture periods ranged from 0.354 (GTX-21-2 maize genotype) to 0.705 (GTX-21-5 maize genotype). This indicates that the GTX-21-5 genotype showed the strongest resistance to microplastic toxicity, while the GTX-21-2 genotype showed the weakest resistance. Further classification of maize genotypes according to their resistance to microplastic toxicity using the TCTR assay revealed the following: GTX-21-2 and GTX-21-4 were highly sensitive to microplastic toxicity, accounting for 20.0% of the tested materials; GTX-21-1, GTX-21-9, and GTX-21-10 were sensitive to microplastic toxicity, accounting for 30.0%; GTX-21-7 and GTX-21-8 were weakly resistant to microplastic toxicity, accounting for 20.0%; GTX-21-3 and GTX-21-6 were moderately resistant, accounting for 20.0%; and GTX-21-5 was highly resistant, accounting for only 10.0% (Table 11). Therefore, the highly resistant genotypes we identified provide reliable genetic resources for breeding microplastic-resistant maize varieties and offer technical support for ensuring safe maize production.

[0095] Table 9. Membership values ​​(U(TRI)) of the resistance to 0.50% PS microplastic toxicity of 12 traits in 10 maize genotype seedlings from soil-cultured maize seedlings on day 7.

[0096]

[0097] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity. Table 10 shows the membership values ​​(U(TRI)) of 12 traits of 10 maize genotype seedlings from soil-cultured maize on day 21 of culture for resistance to 0.50% PS microplastic toxicity.

[0098]

[0099] Note: SL is seedling length, SFW is seedling fresh weight, SDW is seedling dry weight, RL is root length, RFW is root fresh weight, RDW is root dry weight, SD is stem diameter, RD is root diameter, PDW is total plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling vigor index, and RV is root activity.

[0100] Table 11. Classification of microplastic resistance of 10 maize genotypes based on Total Comprehensive Resistance to Microplastics (TCTR) classification criteria.

[0101]

[0102] As can be seen from the above examples, in the soil-cultured maize experiment, we used 0.00% and 0.50% concentration PS microplastic treatments to cultivate maize seedlings of different genotypes on days 7 and 21 to observe significant differences in growth phenotypes, seedling vigor index, chlorophyll accumulation level, and root activity traits affected by PS microplastic toxicity. We also used our innovative proposed resistance index (TRI) for individual traits to 0.50% PS microplastic toxicity at each cultivation time point as a measure of the resistance of the corresponding maize genotype seedlings to 0.50% PS microplastic toxicity at each cultivation time point in the soil-cultured maize experiment. Using the membership function method, we can comprehensively evaluate the combined resistance (CTR) / total combined resistance (TCTR) of each maize genotype seedling to 0.50% PS microplastic toxicity at each / all cultivation time point from multiple angles and time dimensions. Based on the TCTR, the maize genotypes are classified into different levels of resistance to microplastic toxicity, clearly classifying different maize genotypes into different types of resistance to microplastic toxicity. These studies will provide scientific and technological references for the future evaluation of superior maize genotypes (inbred lines / strains / varieties) resistant to microplastic toxicity, thereby ensuring safe maize production and having important practical application value in breeding and production.

[0103] Example 5

[0104] This invention provides the application of 0.50% polystyrene (PS) microplastic toxicity in the evaluation of microplastic-resistant maize genotypes, specifically:

[0105] Forty high-quality maize seeds of different genotypes, sterilized with 70% ethanol (v / v), were first soaked in 100 mL of two PS microplastic solutions (0.00% and 0.50% concentrations, respectively, as a ddH2O mass ratio) for 24 hours on a horizontal shaker. Simultaneously, two PS microplastic soil substrates (0.00% and 0.50% concentrations, respectively) were prepared. Then, ten seeds of each maize genotype soaked in these two PS microplastic solutions were sown into pots containing soil substrates of the same PS microplastic concentration and placed in an artificial climate chamber for cultivation. During cultivation, the relative humidity was set at 65%, the temperature was alternated between 25±0.5℃ and 20±0.5℃ for 12 hours each, the photoperiod was set at 16 / 8 hours of light / dark, and the light intensity was set at 300 μM m². - 2 s -1The CO2 concentration was 450 PPM. Two treatments were used in the soil culture experiment: a control (CK) treatment (0.00% PS microplastic soil substrate) and a 0.50% PS treatment (0.50% PS microplastic soil substrate). During the cultivation period, 50 mL of ddH2O water was evenly applied to each pot every 3 days to ensure timely watering of the seedlings. Then, on days 7 and 21 of cultivation in both PS microplastic soil substrates, 12 traits of different maize genotypes were measured: seedling length (SL), seedling fresh weight (SFW), seedling dry weight (SDW), root length (RL), root fresh weight (RFW), root dry weight (RDW), stem diameter (SD), root diameter (RD), total plant dry biomass (PDW), seedling vigor index (SSI), leaf chlorophyll SPAD value (SPAD), and root activity (RV). Then, using Excel 2013 software, the mean and standard deviation of each trait of different maize genotype seedlings at 7 and 21 days of cultivation in the soil-cultured maize experiment were calculated. IBM-SPSS Statistics 19 software was used to analyze the joint variance of each trait of all maize genotypes among the two concentrations of PS microplastics at these two cultivation time periods, including the corrected model (CM), intercept (IC), genotype (G), cultivation time treatment (CT), PS microplastic concentration treatment (PSC), interaction between genotype and cultivation time treatment (G×CT), interaction between genotype and PS microplastic concentration treatment (G×PSC), interaction between cultivation time and PS microplastic concentration treatment (CT×PSC), and interaction among the three (G×CT×PSC). The TRI value of the resistance index to 0.50% PS microplastic toxicity was calculated for each trait of each maize genotype seedling at each cultivation time period, and then the membership function method was used. The total total resistance (TCTR) of maize seedlings of each genotype to 0.50% PS microplastic toxicity was comprehensively evaluated to assess the microplastic resistance of each genotype. Based on the TCTR value of each maize genotype, the resistance to microplastic toxicity of different maize genotypes was divided into five levels: 0 ≤ TCTR < 0.400, indicating a highly sensitive maize genotype to microplastic toxicity; 0.400 ≤ TCTR < 0.500, indicating a sensitive maize genotype to microplastic toxicity; 0.500 ≤ TCTR < 0.600, indicating a weakly resistant maize genotype to microplastic toxicity; 0.600 ≤ TCTR < 0.700, indicating a moderately resistant maize genotype to microplastic toxicity; and 0.700 ≤ TCTR ≤ 1.000, indicating a highly resistant maize genotype to microplastic toxicity.

Claims

1. The application of 0.50% polystyrene (PS) microplastic toxicity in evaluating microplastic-resistant maize genotypes, characterized in that... The specific application involves: high-quality maize seeds of different genotypes, sterilized with 70% ethanol, being soaked in 0.00% and 0.50% PS microplastic solutions for 24 hours, respectively. Then, 10 seeds of each genotype soaked in these two concentrations of PS microplastic solutions are sown into pots containing 0.00% and 0.50% PS microplastic soil substrates, respectively, and placed in an artificial climate chamber for cultivation. During cultivation, the relative humidity is set at 65%, the temperature is set at alternating cycles of 25±0.5℃ and 20±0.5℃ for 12 hours each, the photoperiod is set at 16 / 8 hours of light / dark, and the light intensity is set at 300 μM m². -2 s -1 The CO2 concentration was 450 PPM. Two treatments were used in the soil culture experiment: a control (CK) treatment and a 0.50% PS treatment. During the cultivation period, 50 mL of ddH2O was evenly applied to each pot every 3 days to replenish the seedlings' water. Then, 12 traits of different maize genotypes were measured on days 7 and 21 of cultivation in the two PS microplastic soil substrates. Excel 2013 software was used to calculate the mean and standard deviation of each trait for different maize genotypes on days 7 and 21 of cultivation in the soil-cultured maize experiment. IBM-SPSS Statistics was used. Software 19 was used to analyze the joint variance of each trait of all maize genotypes across two cultivation periods and two concentrations of PS microplastic treatment. The Trial of Resistance (TRI) values ​​for each trait of each maize genotype seedling at 0.50% PS concentration was calculated for each cultivation period. Then, the membership function method was used to comprehensively evaluate the Total Total Resistance (TCTR) of each genotype maize seedling to 0.50% PS microplastic toxicity, thereby assessing the microplastic resistance of each genotype. Based on the TCTR values ​​of each maize genotype seedling, the resistance to microplastic toxicity of different maize genotypes was divided into five levels: 0 ≤ TCTR < 0.400, indicating a highly sensitive maize genotype to microplastic toxicity; 0.400 ≤ TCTR < 0.500, indicating a sensitive maize genotype to microplastic toxicity; 0.500 ≤ TCTR < 0.600, indicating a weakly resistant maize genotype to microplastic toxicity; 0.600 ≤ TCTR < 0.5 ... ≤TCTR<0.700 indicates a moderately resistant maize genotype to microplastics, while 0.700 ≤ TCTR ≤ 1.000 indicates a highly resistant maize genotype to microplastics.

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