Method for comprehensively evaluating microplastic poison resistance of corn seedlings and identifying microplastic poison resistance corn genotype

Abstract

The invention provides a comprehensive evaluation method for microplastic poison resistance of corn seedlings and an evaluation method for microplastic poison resistance corn genotypes. Under the treatment of PS micro-plastics with different concentrations, 12 characters of corn seedlings are measured, the toxicity resistance index (TRI) is calculated through a membership function method, the total comprehensive resistance (TCTR) is comprehensively evaluated, and it is determined that the toxicity is the most serious when the PS micro-plastics have the concentration of 0.50%. Seedlings of different genotypes are treated with 0.00% and 0.50% PS microplastics, characters are measured, a TRI value is calculated, and the microplastic poison resistance of the seedlings is objectively evaluated. The method is good in operability and small in test error, can objectively, scientifically and accurately determine the concentration of the corn seedlings when the PS microplastic poison degree of the corn seedlings is the highest from the overground and underground of the seedlings in an omnibearing, multi-index and multi-time dimension manner, and can efficiently and accurately judge the microplastic poison resistant corn genotype. The method serves in breeding of new varieties of maize resistant to microplastic poisoning.

Description

Technical Field

[0001] The present invention belongs to the technical field of breeding methods for new stress-resistant maize varieties, and specifically relates to a comprehensive evaluation method for the resistance of maize seedlings to microplastic toxicity and a method for identifying maize genotypes resistant to microplastic toxicity. Background Art

[0002] In modern society, plastics have become the most commonly used materials, existing in many aspects of human life. Due to different uses, the demand for various types of plastics such as polystyrene (PS), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyamide (PA), and polycarbonate (PC) has increased rapidly. According to statistics, the global plastic production has increased from 1.5 million tons in 1950 to 368 million tons in 2019, and the annual plastic production is still increasing. However, only 9% of the plastics are recycled after use, and most plastics are discarded into the environment, not only causing "white pollution" to the environment, but also forming microplastic particles (MPs) with a size less than 5 mm during the degradation process of these plastic waste products in the environment (Thompson et al., 2024), which will further cause toxicity to humans, animals, plants, and microorganisms.

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

[0004] Maize (Zea mays L.) is the most multi-purpose food crop in China, and its production safety plays an important role in ensuring national food security, the development of animal husbandry, and industrial processing. Maize is extremely sensitive to drought, and it has a large demand for water throughout its growth and development process. In China, the reduction in maize production due to drought and water shortage ranges from 20% to 50% (Zhao et al. 2018). In production practice, although people use plastic mulch to inhibit soil evaporation, thereby achieving the purpose of increasing temperature and preserving soil moisture, ultimately improving the drought resistance of maize, ensuring food security, and increasing farmers' income (Yu Junping 2024). However, due to the long-term and extensive use of plastic materials such as plastic mulch, a large amount of microplastics will inevitably accumulate continuously in the soil. We speculate that, like the above-mentioned field crops and vegetables such as wheat, peanut, soybean, and cucumber, these microplastics will also affect the healthy growth, development, yield formation, grain quality, senescence, etc. of maize.

[0005] Problems existing in the prior art: Microplastics remaining in the soil first affect the morphological construction and development of maize seedlings. Then, will microplastic pollution cause toxicity to maize seedlings? What kind of toxicity will microplastics cause to maize seedlings? What is the toxicity mechanism of microplastics to maize seedlings? Which concentration of microplastics has the greatest toxicity to maize seedlings? What indicators can be used as the evaluation indicators for microplastic toxicity in maize seedlings, and what methods can be used to scientifically, objectively, and accurately evaluate the microplastic toxicity resistance performance of different maize genotype seedlings? Can excellent maize genotypes resistant to microplastic toxicity be screened and identified, and then applied to the breeding of new maize varieties resistant to microplastic toxicity? All of these are still unclear and require more in-depth and systematic research. Summary of the Invention

[0006] To solve the above problems, the present invention provides a comprehensive evaluation method for the resistance of maize seedlings to microplastic toxicity. That is, through a soil culture experiment, the present invention systematically analyzes the effects of six concentrations of polystyrene (PS) microplastics on 12 growth phenotypes, chlorophyll accumulation levels, and root activity traits of maize genotype seedlings at the 7th and 21st days of cultivation, and further reveals the internal causes of the toxicity of different concentrations of PS microplastics to maize seedlings at different cultivation time periods and the response mechanism of maize seedlings to microplastic toxicity. Furthermore, we innovatively propose the biological concept and calculation formula of the toxicity resistance index (TRI) of maize seedlings to different concentrations of PS microplastics, and calculate the TRI values of the corresponding 12 traits of maize genotype seedlings at these two cultivation time periods; then, taking these TRI values as the measurement and evaluation indicators of the resistance of maize genotype seedlings to different concentrations of PS microplastic toxicity at the two cultivation time periods, the membership function method is used to comprehensively, objectively, and quantitatively evaluate the comprehensive toxicity resistance (CTR) / total comprehensive toxicity resistance (TCTR) of maize genotype seedlings to different concentrations of PS microplastics at each / all cultivation time periods; and combined with the clustering evaluation results of the microplastic toxicity degree of maize genotype seedlings among different concentrations of PS microplastic treatments at each cultivation time period, finally, the PS microplastic concentration at which the microplastic toxicity of maize seedlings is the most severe at all cultivation time periods is screened out to be 0.50%. On this basis, we further use 0.50% concentration of PS microplastics as the screening concentration for evaluating the resistance of maize to microplastic toxicity, measure the above 12 traits of different maize genotype seedlings at the 7th and 21st days of maize seedling cultivation, and calculate the TRI of the corresponding traits at 0.50% concentration of PS microplastic toxicity, and then use the membership function method to comprehensively evaluate the anti-PS microplastic toxicity performance of different maize genotype seedlings, and finally screen out the resistant maize genotypes with strong resistance to PS microplastic toxicity and apply them to maize breeding, so as to effectively solve the adverse effects of soil microplastics on maize and ensure the safe and efficient production of maize.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions:

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

[0009] (1) Preparation of high-quality maize seeds: Prepare new high-quality maize genotype seeds harvested in the current year, which are plump, uniform in size, strong in vitality, and high in purity, for standby.

[0010] (2) Hydroponic experiment of corn with different concentrations of polystyrene microplastics: Disinfect 10 high-quality corn seeds in step (1) with 70% ethanol (v / v) for 10 min, and rinse the seeds 5 times with ddH 2 O water to remove the residual ethanol on the seed surface, and dry the attached water on the seed surface with sterilized filter paper to obtain the corresponding disinfected corn genotype seeds. Prepare 6 kinds of PS microplastic solutions with concentrations of 0.00%, 0.05%, 0.25%, 0.50%, 0.75% and 1.00% in advance, and prepare them freshly for use. Then soak the disinfected corn genotype seeds in 6 kinds of PS microplastic solutions for 24 h. Prepare 6 kinds of PS microplastic hydroponic substrates, including 0.00% PS microplastic hydroponic substrate, 0.05% PS microplastic hydroponic substrate, 0.25% PS microplastic hydroponic substrate, 0.50% PS microplastic hydroponic substrate, 0.75% PS microplastic hydroponic substrate, 1.00% PS microplastic hydroponic substrate. Then sow 10 corn genotype seeds soaked in the corresponding concentration of PS microplastic solution into flower pots filled with the same concentration of PS microplastic hydroponic substrate, and place them in an artificial climate chamber for cultivation. During the cultivation period, the relative humidity is set to 65%, the temperature is set to 25±0.5 / 20±0.5 °C for 12 h each in an alternating cycle, the photoperiod is set to 16 / 8 h light / dark, and the light intensity is set to 300 μM m -2 s -1 , and the CO 2 concentration is 450 PPM. There are a total of 6 treatments in the hydroponic experiment, namely CK control treatment (0.00% PS microplastic hydroponic substrate), 0.05% PS treatment (0.05% PS microplastic hydroponic substrate), 0.25% PS treatment (0.25% PS microplastic hydroponic substrate), 0.50% PS treatment (0.50% PS microplastic hydroponic substrate), 0.75% PS treatment (0.75% PS microplastic hydroponic substrate) and 1.00% PS treatment (1.00% PS microplastic hydroponic substrate), and each treatment has 4 biological replicates. At the same time, during the cultivation period, pour 50 mL of ddH 2 O water evenly into each flower pot every 3 d to supply water to the seedlings in time.

[0011] (3) Data collection: Measure the 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 strong seedling index (SSI) of corn genotype seedlings cultivated in 6 kinds of PS microplastic hydroponic substrates on the 7th day and the 21st day respectively. The specific calculation of SSI is shown in formula (1): SSI = (SD / SL) × PDW (1). In the formula: SSI is the strong seedling index, SD is the stem diameter, SL is the seedling length, and PDW is the total plant dry biomass.

[0012] (4) Establish a data statistical model: Use Excel 2013 software to calculate the mean and standard deviation of each trait of maize genotype seedlings at the 7th day and the 21st day of cultivation in the soil culture maize experiment, and draw a bar chart. Use IBM-SPSS Statistics 19 software to analyze the combined variance of each trait of maize genotype among the two cultivation time periods and six concentrations of PS microplastics in the soil culture maize experiment. Use IBM-SPSS Statistics 19 software to analyze the Dunnett's significant difference of each trait of maize genotype seedlings at each cultivation time period among six concentrations of PS microplastics in the soil culture maize experiment at the P<0.05 level. Use the GENESCLOUD online software to analyze the Pearson correlation coefficient diagram of all traits among six concentrations of PS microplastics for maize genotype seedlings at each cultivation time period in the soil culture maize experiment. Use the LOG10 function to standardize the average values of all traits among six concentrations of PS microplastics for maize genotype seedlings at two cultivation time periods in the soil culture maize experiment, and then use the Between-groups Linkage clustering method of IBM-SPSS Statistics 16.0 software to conduct a scientific, objective, and qualitative clustering analysis on the microplastic toxicity degree of maize genotype seedlings among six concentrations of PS microplastics at each cultivation time period in the soil culture maize experiment.

[0013] (5) Toxicity resistance index of maize seedlings to different concentrations of polystyrene (PS) microplastics: In order to scientifically, objectively, and accurately reflect the resistance ability of each single trait of maize genotype seedlings to different concentrations of PS microplastic toxicity at each cultivation time period in the soil culture maize experiment, the present invention newly defines the toxicity resistance index (TRI) of maize seedlings to different concentrations of PS microplastics, as shown in formula (2) specifically: In the formula: is the toxicity resistance index of the jth trait of maize genotype seedlings at the kth concentration of PS microplastics at the ith cultivation time period in the soil culture maize experiment, is the measured value of the jth trait of maize genotype seedlings under the CK control treatment at the ith cultivation time period in the soil culture maize experiment, is the measured value of the jth trait of maize genotype seedlings under the kth concentration of PS microplastic treatment at the ith cultivation time period in the soil culture maize experiment. i is the 7th day or the 21st day of cultivation in the soil culture maize experiment, and k-PS is the PS microplastic treatment at the concentrations of 0.05%, 0.25%, 0.50%, 0.75%, or 1.00% in the soil culture maize experiment. The larger the value, the stronger the resistance of the jth trait of maize genotype seedlings to the toxicity of the k-PS concentration microplastics at the ith cultivation time period in the soil culture maize experiment.

[0014] (6) Evaluation of the comprehensive resistance ability of maize seedlings to the toxicity of different concentrations of polystyrene (PS) microplastics: The TRI values of all traits of the maize genotype seedlings obtained in step (4) were used as the measurement and evaluation indicators for the resistance ability of the maize genotype seedlings to the toxicity of different concentrations of PS microplastics at two culture time periods in the soil-cultivated maize experiment. The membership function method was used to comprehensively, objectively, and quantitatively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of the maize genotype seedlings to the toxicity of different concentrations of PS microplastics at each / all culture time periods in the soil-cultivated maize experiment. See specific formulas (3), (4), and (5): and In the formula: is the resistance membership value of the jth trait of the maize genotype seedlings to the toxicity of the kth concentration of PS microplastics at the ith culture time period in the soil-cultivated maize experiment, is the resistance index of the jth trait of the maize genotype seedlings to the toxicity of the kth concentration of PS microplastics at the ith culture time period in the soil-cultivated maize experiment, is the minimum TRI value of the jth trait of the maize genotype seedlings among all concentrations of PS microplastic treatments at the ith culture time period in the soil-cultivated maize experiment, is the maximum TRI value of the jth trait of the maize genotype seedlings among all concentrations of PS microplastic treatments at the ith culture time period in the soil-cultivated maize experiment, is the value of the comprehensive resistance of the maize genotype seedlings to the toxicity of the kth concentration of PS microplastics at the ith culture time period in the soil-cultivated maize experiment. m is the m (m = 12) traits measured for the maize genotype seedlings at the ith culture time period in the soil-cultivated maize experiment. TCTR (k-PS) is the value of the total comprehensive resistance of the maize genotype seedlings to the toxicity of the kth concentration of PS microplastics at n (n = 2, two culture time periods) culture time periods in the soil-cultivated maize experiment. i is the 7th day or the 21st day of the soil-cultivated maize experiment. k-PS is the PS microplastic treatment at the concentrations of 0.05%, 0.25%, 0.50%, 0.75%, or 1.00% in the soil experiment. The larger the value, the stronger the comprehensive resistance of the maize genotype seedlings to the toxicity of the kth concentration of PS microplastics at the ith culture time period in the soil-cultivated maize experiment. TCTR (k-PS) The larger the value, the stronger the total comprehensive resistance of the maize genotype seedlings to the toxicity of the kth concentration of PS microplastics at two culture time periods in the soil-cultivated maize experiment.

[0015] (7) Comprehensive measurement of the concentration of polystyrene (PS) microplastics when the toxicity of PS microplastics to maize seedlings is the most severe: By comparing the measurement results of the comprehensive clustering of between-groups linkage of maize genotype seedlings under different treatments at two culture time periods in the soil culture maize experiment in step (3), and the evaluation results of the total comprehensive resistance of maize genotype seedlings to the toxicity of PS microplastics at corresponding concentrations at two culture time periods in the soil culture maize experiment in step (5), a combined multi-method comparison and evaluation of the toxicity of different concentrations of PS microplastics to maize genotype seedlings at multiple culture time periods is carried out, and finally the concentration of PS microplastics when the toxicity of PS microplastics to maize genotype seedlings is the most severe is comprehensively measured.

[0016] 2. A method for identifying maize genotypes resistant to microplastic toxicity, the specific steps are as follows:

[0017] (1) Preparation of high-quality maize seeds: Prepare new high-quality maize seeds of different genotypes with plump grains, uniform size, strong vitality, and high purity harvested in the same ecological point in the current year for standby.

[0018] (2) Soil culture maize experiment with 0.50% concentration of polystyrene (PS) microplastics: Disinfect the high-quality maize seeds of different genotypes in step (1) with 70% ethanol (v / v) for 10 min, and rinse the seeds 5 times with ddH 2 O water to remove the residual ethanol on the seed surface, and dry the water attached to the seed surface with sterile filter paper to obtain the corresponding disinfected maize genotype seeds. Soak the disinfected maize genotype seeds with 2 kinds of PS microplastic solutions with concentrations of 0.00% and 0.50% for 24 h, and then sow 10 seeds of different maize genotypes soaked with these 2 kinds of PS microplastic solutions into flower pots with 0.00% and 0.50% concentration of PS microplastic soil culture substrates respectively, and place them in an artificial climate chamber for cultivation. During the cultivation period, the relative humidity is set at 65%, the temperature is set at 25±0.5 / 20±0.5 °C for 12 h each in an alternating cycle, the photoperiod is set at 16 / 8 h light / dark, and the light intensity is set at 300 μM m -2 s -1 ,CO 2 concentration of 450 PPM. There are a total of 2 kinds of treatments in the soil culture experiment, namely CK control treatment (0.00% PS microplastic soil culture substrate) and 0.50% PS treatment (0.50% PS microplastic soil culture substrate), and each treatment is set with 4 biological replicates. At the same time, during the cultivation period, 50 mL of ddH 2 O water is evenly poured into each flower pot every 3 d to replenish water for the seedlings in time. Measure 12 traits of different maize genotype seedlings cultured in these 2 kinds of PS microplastic soil culture substrates on the 7th day and the 21st day.

[0019] (3) Data statistical analysis: Using Excel 2013 software, calculate the mean and standard deviation of each trait of different maize genotypes at the 7th day and the 21st day of cultivation under each treatment in the soil cultivation maize experiment. Using IBM-SPSS Statistics 19 software, analyze the combined variance of each trait of all maize genotypes between the two concentrations of PS microplastics at these two cultivation time periods.

[0020] (4) Toxicity resistance index of 0.50% concentration of polystyrene (PS) microplastics for different genotypes of maize seedlings: According to the means of 12 traits of different maize genotype seedlings measured at the 7th day and the 21st day of cultivation in the soil cultivation maize experiment with two concentrations of PS microplastics in step (3), further calculate the toxicity resistance index (TRI) value of each trait of each maize genotype seedling at each cultivation time period under 0.50% concentration of PS microplastics, as shown in formula (6) specifically: In the formula: is the toxicity resistance index of the jth trait of the pth maize genotype seedling at the ith cultivation time period under 0.50% concentration of PS microplastics in the soil cultivation maize experiment, is the measured value of the jth trait under the CK control treatment of the pth maize genotype seedling at the ith cultivation time period in the soil cultivation maize experiment, is the measured value of the jth trait under the 0.50% concentration of PS microplastics treatment of the pth maize genotype seedling at the ith cultivation time period in the soil cultivation maize experiment. i is the 7th day or the 21st day of cultivation in the soil cultivation maize experiment, and 0.50% PS is the 0.50% concentration of PS microplastics treatment in the soil cultivation maize experiment. The larger the value, the stronger the resistance of the jth trait of the pth maize genotype seedling at the ith cultivation time period to the toxicity of 0.50% concentration of microplastics in the soil cultivation maize experiment.

[0021] (5) Evaluation of the comprehensive resistance ability of different genotypes of maize seedlings to the toxicity of 0.50% concentration of polystyrene (PS) microplastics: Using the TRI values of all traits of different maize genotype seedlings calculated in step (4) as the measurement and evaluation indicators of the toxicity resistance ability of different maize genotype seedlings to 0.50% concentration of PS microplastics at the two cultivation time periods in the soil cultivation maize experiment, and using the membership function method to comprehensively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) value of different maize genotype seedlings to the toxicity of 0.50% concentration of PS microplastics at each / all cultivation time periods in the soil cultivation maize experiment, specifically: and In the formula: is the membership value of the resistance of the jth trait of the pth maize genotype seedling at the ith cultivation time period to the toxicity of 0.50% concentration of PS microplastics, It is the toxicity resistance index of the j-th trait of the p-th maize genotype seedling at the i-th cultivation time period in the soil cultivation maize experiment under 0.50% concentration of PS microplastics. It is the minimum TRI value of the j-th trait of all maize genotype seedlings at the i-th cultivation time period in the soil cultivation maize experiment. It is the maximum TRI value of the j-th trait of all maize genotype seedlings at the i-th cultivation time period in the soil cultivation maize experiment. It is the comprehensive resistance value of the p-th maize genotype seedling to the toxicity of 0.50% concentration of PS microplastics at the i-th cultivation time period in the soil cultivation maize experiment. m are the m (m = 12) traits measured for each maize genotype seedling at the i-th cultivation time period in the soil cultivation maize experiment, TCTR p(0.50％PS) It is the total comprehensive resistance value of the p-th maize genotype seedling to the toxicity of 0.50% concentration of PS microplastics at n (n = 2, two cultivation time periods) cultivation time periods in the soil cultivation maize experiment. i is the 7th day or the 21st day of the soil cultivation maize experiment, and 0.50% PS is the treatment of 0.50% concentration of PS microplastics in the soil cultivation experiment. The larger the value, the stronger the comprehensive resistance of the p-th maize genotype seedling to the toxicity of 0.50% concentration of PS microplastics at the i-th cultivation time period in the soil cultivation maize experiment, TCTR (0.50％PS) The larger the value, the stronger the total comprehensive resistance of the p-th maize genotype seedling to the toxicity of 0.50% concentration of PS microplastics at two cultivation time periods in the soil cultivation maize experiment. According to the TCTR values of each maize genotype seedling, the anti-microplastic toxicity performance of different maize genotypes is divided into 5 levels, namely: 0 ≤ TCTR < 0.400, highly sensitive maize genotype to microplastic toxicity; 0.400 ≤ TCTR < 0.500, sensitive maize genotype to microplastic toxicity; 0.500 ≤ TCTR < 0.600, weakly anti-microplastic toxicity maize genotype; 0.600 ≤ TCTR < 0.700, moderately anti-microplastic toxicity maize genotype; 0.700 ≤ TCTR ≤ 1.000, highly anti-microplastic toxicity maize genotype.

[0022] 3. Application of 0.50% polystyrene (PS) microplastic toxicity in the evaluation of anti-microplastic toxicity maize genotypes, specifically:

[0023] Soak high-quality maize seeds of different genotypes disinfected with 70% ethanol (v / v) for 24 h in two PS microplastic solutions with concentrations of 0.00% and 0.50%, and then sow 10 seeds of each of the different maize genotypes soaked in these two concentrations of PS microplastic solutions into flower pots with two PS microplastic soil culture substrates with concentrations of 0.00% and 0.50%, and place them in an artificial climate chamber for cultivation. During the cultivation period, the relative humidity is set at 65%, the temperature is set at 25±0.5 / 20±0.5 °C for 12 h each in an alternating cycle, the photoperiod is set at 16 / 8 h light / dark, and the light intensity is set at 300 μM m -2 s -1 , CO 2 concentration of 450 PPM. There are a total of two treatments in the soil culture experiment, namely the CK control treatment (0.00% PS microplastic soil culture substrate) and the 0.50% PS treatment (0.50% PS microplastic soil culture substrate). During the cultivation period, 50 mL of ddH 2 O water is evenly poured into each flower pot every 3 days to supply water to the seedlings in a timely manner. Then, 12 traits of different maize genotype seedlings cultured in these two PS microplastic soil culture substrates on the 7th day and the 21st day are measured. Using ExCel 2013 software, calculate the mean and standard deviation of each trait of different maize genotype seedlings cultured on the 7th day and the 21st day in the soil culture maize experiment; using IBM-SPSS Statistics 19 software, analyze the combined variance of each trait of all maize genotypes between the two concentrations of PS microplastic treatments at these two cultivation time periods; calculate the toxicity resistance index (TRI) value of each trait of each maize genotype seedling at a concentration of 0.50% PS microplastic at each cultivation time period, and then use the membership function method to comprehensively evaluate the total comprehensive resistance (TCTR) of each genotype of maize seedlings to the toxicity of 0.50% concentration PS microplastic, and further evaluate the anti-microplastic toxicity performance of each genotype of maize seedlings. According to the TCTR values of each maize genotype seedling, the anti-microplastic toxicity performance of different maize genotypes is divided into 5 grades, namely: 0≤TCTR<0.400, highly sensitive maize genotype to microplastic toxicity, 0.400≤TCTR<0.500, sensitive maize genotype to microplastic toxicity, 0.500≤TCTR<0.600, weakly anti-microplastic toxicity maize genotype, 0.600≤TCTR<0.700, moderately anti-microplastic toxicity maize genotype, 0.700≤TCTR≤1.000, highly anti-microplastic toxicity maize genotype.

[0024] Beneficial effects of the present invention: Microplastics are extremely difficult to decompose in the natural environment and can persist in the environment for hundreds of years. With the continuous and massive use of plastic products, the microplastic crisis has become increasingly serious. Therefore, starting from the actual situation of microplastic pollution and harm, the present invention deeply elucidates the internal reasons for the toxicity of PS microplastics to maize seedlings and the adaptive mechanism of maize seedlings to respond to the toxicity of PS microplastics by systematically comparing the effects of different concentrations of PS microplastics on 12 traits of maize genotype seedlings at different culture time periods. At the same time, in order to more scientifically, objectively, simply, and accurately evaluate the resistance of maize genotype seedlings to the toxicity of different concentrations of PS microplastics at each / all culture time periods, we also innovatively proposed the biological concept and calculation formula of the toxicity resistance index (TRI) of maize seedlings to different concentrations of PS microplastics. Using the TRI value, the membership function method was used to scientifically, objectively, accurately, and quantitatively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of maize genotype seedlings to the toxicity of different concentrations of PS microplastics at each / all culture time periods. This comprehensive evaluation result was also comprehensively compared with the clustering evaluation results of the microplastic toxicity degree of maize genotype seedlings among different concentrations of PS microplastic treatments at each culture time period in multiple methods and multiple time dimensions, thereby verifying that the TRI we proposed can scientifically, objectively, and accurately evaluate the comprehensive resistance of maize genotype seedlings to the toxicity of different concentrations of PS microplastics at each / all culture time periods. Finally, the PS microplastic concentration at which the toxicity of PS microplastics to maize seedlings is the most severe was screened out as 0.50%. On this basis, we further used 0.00% and 0.50% concentrations of PS microplastics as the screening concentrations for evaluating the resistance of maize to microplastic toxicity. At the 7th day and the 21st day of maize seedling cultivation, 12 traits of different maize genotype seedlings were measured to comprehensively evaluate the resistance performance of different maize genotype seedlings to the toxicity of PS microplastics, and then maize genotype with strong resistance to the toxicity of PS microplastics was screened out and applied to maize breeding, so as to effectively solve the adverse effects of soil microplastics on maize and ensure the safe and efficient production of maize. The present invention has the advantages of simple experimental operation, good repeatability, and the PS microplastic concentration at which the toxicity of PS microplastics to maize seedlings is the most severe can be used to accurately evaluate the resistance of different maize genotypes to the toxicity of microplastics, providing a set of simple, reliable, fast, and standardized methods for maize breeders to evaluate the maize genotype resistant to microplastic toxicity, and then serving the application of maize breeding resistant to microplastic toxicity, with great breeding application value. Description of the Drawings

[0025] Figure 1 Calculation formula (1) for the vigorous seedling index of maize genotype seedlings and the toxicity resistance index of individual traits of maize genotype seedlings to different concentrations of PS microplastics during the corresponding culture time period in the soil cultivation of maize Calculation formula (2), membership value of the resistance of single traits of maize genotype seedlings to the toxicity of different concentrations of PS microplastics during the corresponding cultivation period in the soil cultivation maize experiment Calculation formula (3), comprehensive resistance of maize genotype seedlings to the toxicity of different concentrations of PS microplastics during the corresponding cultivation period in the soil cultivation maize experiment Calculation formula (4), total comprehensive resistance of maize genotype seedlings to the toxicity of different concentrations of PS microplastics during two cultivation periods in the soil cultivation maize experiment Calculation formula (5), resistance index of each maize genotype seedling to the toxicity of 0.50% concentration PS microplastics for each trait during the corresponding cultivation period in the soil cultivation maize experiment Calculation formula (6), membership value of the resistance of single traits of each maize genotype seedling to the toxicity of 0.50% concentration PS microplastics during the corresponding cultivation period in the soil cultivation maize experiment Calculation formula (7), comprehensive resistance of each maize genotype seedling to the toxicity of 0.50% concentration PS microplastics during the corresponding cultivation period in the soil cultivation maize experiment Calculation formula (8), total comprehensive resistance (TCTR) of each maize genotype seedling to the toxicity of 0.50% concentration PS microplastics during two cultivation periods in the soil cultivation maize experiment p(0.50％PS) ) Calculation formula (9).

[0026] Figure 2 Growth conditions of TS163 maize genotype seedlings cultivated for 7 days under the treatment of 6 concentrations of PS microplastics in the soil cultivation maize experiment. Among them, CK is the control treatment (i.e., 0.00% PS microplastic soil cultivation substrate treatment), 0.05% PS is 0.05% PS microplastic soil cultivation substrate treatment, 0.25% PS is 0.25% PS microplastic soil cultivation substrate treatment, 0.50% PS is 0.50% PS microplastic soil cultivation substrate treatment, 0.75% PS is 0.75% PS microplastic soil cultivation substrate treatment, and 1.00% PS is 1.00% PS microplastic soil cultivation substrate treatment.

[0027] Figure 3 Growth conditions of TS163 maize genotype seedlings cultivated for 21 days under the treatment of 6 concentrations of PS microplastics in the soil cultivation maize experiment. Among them, CK is the control treatment (i.e., 0.00% PS microplastic soil cultivation substrate treatment), 0.05% PS is 0.05% PS microplastic soil cultivation substrate treatment, 0.25% PS is 0.25% PS microplastic soil cultivation substrate treatment, 0.50% PS is 0.50% PS microplastic soil cultivation substrate treatment, 0.75% PS is 0.75% PS microplastic soil cultivation substrate treatment, and 1.00% PS is 1.00% PS microplastic soil cultivation substrate treatment.

[0028] Figure 4Phenotypic analysis of 12 traits of TS163 maize genotype seedlings cultured for 7 days and 21 days under 6 concentrations of PS microplastics in soil culture maize experiment. Among them, 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 strong seedling index, and RV is root activity. CK is the control treatment (i.e., 0.00% PS microplastic soil culture substrate treatment), 0.05% PS is 0.05% PS microplastic soil culture substrate treatment, 0.25% PS is 0.25% PS microplastic soil culture substrate treatment, 0.50% PS is 0.50% PS microplastic soil culture substrate treatment, 0.75% PS is 0.75% PS microplastic soil culture substrate treatment, and 1.00% PS is 1.00% PS microplastic soil culture substrate treatment. Different lowercase letters indicate significant differences in the corresponding traits of TS163 maize genotype seedlings among different concentrations of PS microplastic treatments at the P<0.05 level for a single culture period.

[0029] Figure 5 Pearson correlation coefficient diagram of 12 traits of TS163 maize genotype seedlings cultured for 7 days under 6 concentrations of PS microplastics in soil culture maize experiment. Among them, 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 strong seedling index, and RV is root activity. * indicates significant Pearson correlation between two traits at the P<0.05 level.

[0030] Figure 6 Pearson correlation coefficient diagram of 12 traits of TS163 maize genotype seedlings cultured for 21 days under 6 concentrations of PS microplastics in soil culture maize experiment. Among them, 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 strong seedling index, and RV is root activity. * indicates significant Pearson correlation between two traits at the P<0.05 level.

[0031] Figure 7Cluster evaluation of the toxicity of microplastics on TS163 maize genotype seedlings cultured for 7 days under 6 concentrations of PS microplastics based on 12 traits by the Between-groups Linkage clustering method in the soil culture maize experiment. Among them, CK is the control treatment (i.e., 0.00% PS microplastic soil culture substrate treatment), 0.05% PS is the 0.05% PS microplastic soil culture substrate treatment, 0.25% PS is the 0.25% PS microplastic soil culture substrate treatment, 0.50% PS is the 0.50% PS microplastic soil culture substrate treatment, 0.75% PS is the 0.75% PS microplastic soil culture substrate treatment, and 1.00% PS is the 1.00% PS microplastic soil culture substrate treatment. Type A is the type not poisoned by PS microplastics, type B is the type with weak PS microplastic poisoning; type C is the type with severe PS microplastic poisoning.

[0032] Figure 8 Cluster evaluation of the toxicity of microplastics on TS163 maize genotype seedlings cultured for 21 days under 6 concentrations of PS microplastics based on 12 traits by the Between-groups Linkage clustering method in the soil culture maize experiment. Among them, CK is the control treatment (i.e., 0.00% PS microplastic soil culture substrate treatment), 0.05% PS is the 0.05% PS microplastic soil culture substrate treatment, 0.25% PS is the 0.25% PS microplastic soil culture substrate treatment, 0.50% PS is the 0.50% PS microplastic soil culture substrate treatment, 0.75% PS is the 0.75% PS microplastic soil culture substrate treatment, and 1.00% PS is the 1.00% PS microplastic soil culture substrate treatment. Type A is the type not poisoned by PS microplastics, type B is the type with weak PS microplastic poisoning; type C is the type with severe PS microplastic poisoning.

[0033] Figure 9 Analysis of the comprehensive resistance value CTR of TS163 maize genotype seedlings cultured for 7 days and 21 days to the toxicity of different concentrations of PS microplastics in the soil culture maize experiment. Among them, 0.05% PS is the 0.05% PS microplastic soil culture substrate treatment, 0.25% PS is the 0.25% PS microplastic soil culture substrate treatment, 0.50% PS is the 0.50% PS microplastic soil culture substrate treatment, 0.75% PS is the 0.75% PS microplastic soil culture substrate treatment, and 1.00% PS is the 1.00% PS microplastic soil culture substrate treatment. 7d is the 7th day of the soil culture maize experiment, and 21d is the 21st day of the soil culture maize experiment.

[0034] Figure 10Analysis of the total comprehensive resistance value TCTR of TS163 maize genotypes seedlings to different concentrations of PS microplastics toxicity at two culture time periods in soil-cultivated maize experiments. Among them, 0.05% PS is the treatment of 0.05% PS microplastic soil-cultivated substrate, 0.25% PS is the treatment of 0.25% PS microplastic soil-cultivated substrate, 0.50% PS is the treatment of 0.50% PS microplastic soil-cultivated substrate, 0.75% PS is the treatment of 0.75% PS microplastic soil-cultivated substrate, and 1.00% PS is the treatment of 1.00% PS microplastic soil-cultivated substrate.

[0035] Figure 11 Violin plots of 12 traits of 10 maize genotypes seedlings at the 7th day and the 21st day of culture under two concentrations of PS microplastics treatment in soil-cultivated maize experiments. Among them, 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 whole-plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is strong seedling index, and RV is root activity. CK is the control treatment (i.e., 0.00% PS microplastic soil-cultivated substrate treatment), and 0.50% PS is the treatment of 0.50% PS microplastic soil-cultivated substrate. 7d is the 7th day of the soil-cultivated maize experiment, and 21d is the 21st day of the soil-cultivated maize experiment.

[0036] Figure 12 Analysis of the comprehensive resistance value CTR of 10 maize genotypes seedlings at the 7th day and the 21st day of culture to 0.50% concentration of PS microplastics toxicity in soil-cultivated maize experiments. Among them, 7d is the 7th day of the soil-cultivated maize experiment, and 21d is the 21st day of the soil-cultivated maize experiment.

[0037] Figure 13 Identification and evaluation of the total comprehensive resistance value TCTR of 10 maize genotypes seedlings to 0.50% concentration of PS microplastics toxicity at two culture time periods in soil-cultivated maize experiments. The numbers on the columns represent the total comprehensive resistance value TCTR. Specific implementation method

[0039] The usage methods in the following embodiments of the present invention are all conventional methods without special instructions; the equipment and reagents used are all conventional equipment and reagents purchased from reagent companies. Here, it should also be noted that in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only the technical solutions and / or processing steps closely related to the solution according to the present invention are shown in the embodiments, and other details with little relevance are omitted.

[0040] In addition, those skilled in the art are aware that the maize genotype test materials are not limited to 11. The present invention takes these 11 maize genotype test materials as examples only to clearly describe the content of the technical solution of the present invention.

[0041] Example 1

[0042] The present invention provides a comprehensive evaluation method for the resistance of maize seedlings to microplastic toxicity. The specific evaluation method is carried out according to the following steps:

[0043] 1. Preparation of high-quality maize seeds: Prepare new seeds of high-quality maize genotype TS163 with plump grains, uniform size, strong vitality, and high purity, which were planted at the Longxi experimental site (latitude 34.97°, longitude 104.40°, altitude 2074 m) in 2024, strictly bagged for pollination, and harvested, and set aside for use.

[0044] 2. Hydroponic maize experiment with different concentrations of PS microplastics: Put 40 seeds of the above-prepared high-quality TS163 maize genotype into a triangular flask, add 100 mL of 70% ethanol (v / v), place it on a horizontal shaker to disinfect the seeds for 10 min, and rinse the seeds 5 times with 100 mL of ddH 2 O water to remove the residual ethanol on the seed surface, and blot the attached water on the seed surface with sterile filter paper to obtain the corresponding disinfected TS163 maize genotype seeds. Purchase PS microplastic powder (mesh number: 1000 mesh; particle size: 13 μm) from Dongguan Ruixiang Plastic Raw Material Co., Ltd. Weigh 0.00 g, 0.05 g, 0.25 g, 0.50 g, 0.75 g, and 1.00 g of PS microplastics accurately with an electronic balance in advance, and dissolve them in 100 mL of ddH 2 O water respectively to prepare 6 kinds of PS microplastic solutions with concentrations of 0.00%, 0.05%, 0.25%, 0.50%, 0.75%, and 1.00% (PS mass to ddH 2 O mass ratio concentration), and prepare them freshly before use. Then continue to put the disinfected TS163 maize genotype seeds into the corresponding triangular flasks, add 100 mL of 6 kinds of PS microplastic solutions respectively, and place them on a horizontal shaker in a dark indoor environment to soak the seeds for 24 h. Prepare 6 kinds of PS microplastic hydroponic substrates with different concentrations (PS mass to nutrient soil mass ratio concentration), including 0.00% PS microplastic hydroponic substrate (that is, the hydroponic substrate obtained by fully mixing 0.00 g of PS microplastics with 300.00 g of nutrient soil and then adding 250 mL of ddH 2 O water and stirring evenly), 0.05% PS microplastic hydroponic substrate (that is, the hydroponic substrate obtained by fully mixing 0.15 g of PS microplastics with 300.00 g of nutrient soil and then adding 250 mL of ddH 2 O water and stirring evenly), 0.25% PS microplastic hydroponic substrate (that is, the hydroponic substrate obtained by fully mixing 0.75 g of PS microplastics with 300.00 g of nutrient soil and then adding 250 mL of ddH 2Soil culture substrate stirred evenly with water), 0.50% PS microplastic soil culture substrate (i.e., 1.50 g of PS microplastics and 300.00 g of nutrient soil were fully mixed evenly and then 250 mL of ddH 2 Soil culture substrate stirred evenly with water), 0.75% PS microplastic soil culture substrate (i.e., 2.25 g of PS microplastics and 300.00 g of nutrient soil were fully mixed evenly and then 250 mL of ddH 2 Soil culture substrate stirred evenly with water), 1.00% PS microplastic soil culture substrate (i.e., 3.00 g of PS microplastics and 300.00 g of nutrient soil were fully mixed evenly and then 250 mL of ddH 2 Soil culture substrate stirred evenly with water). Then, 10 seeds of TS163 maize genotype after soaking in PS microplastic solution of corresponding concentration were sown into flower pots (height 11.0 cm, diameter 12.5 cm) filled with PS microplastic soil culture substrate of the same concentration, and placed in an artificial climate chamber for cultivation. During the cultivation period, the relative humidity was set at 65%, the temperature was set at 25±0.5 / 20±0.5 °C for 12 h each in an alternating cycle, the photoperiod was set at 16 / 8 h light / dark, and the light intensity was set at 300 μM m -2 s -1 , CO 2 concentration 450 PPM. There were a total of 6 treatments in the soil culture experiment, namely CK control treatment (0.00% PS microplastic soil culture substrate), 0.05% PS treatment (0.05% PS microplastic soil culture substrate), 0.25% PS treatment (0.25% PS microplastic soil culture substrate), 0.50% PS treatment (0.50% PS microplastic soil culture substrate), 0.75% PS treatment (0.75% PS microplastic soil culture substrate), and 1.00% PS treatment (1.00% PS microplastic soil culture substrate). Each treatment had 4 biological replicates. At the same time, during the cultivation period, 50 mL of ddH 2 O water was evenly poured into each flower pot every 3 days to supply water to the seedlings in a timely manner. Then, the 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) of maize genotype seedlings cultured in 6 kinds of PS microplastic soil culture substrates for 7 days and 21 days were measured respectively. The chlorophyll SPAD value (SPAD) of the leaves was measured using a SPAD-502Plus chlorophyll meter produced in Japan. The root vigor (RV) was detected using a plant root vigor detection kit (naphthylamine method) provided by Beijing Solarbio Science & Technology Co., Ltd. ( LIFE SCIENCES). The strong seedling index (SSI) was calculated according to the formula (1) attached Figure 1 . In the formula: SSI is the strong seedling index, SD is the stem diameter, SL is the seedling length, and PDW is the total plant dry biomass.

[0045] 3. Data statistical analysis: For the 12 traits of TS163 maize genotype seedlings measured on the 7th day and the 21st day in the above-mentioned hydroponic maize experiment with different concentrations of PS microplastics, the Excel 2013 software was used to calculate the mean and standard deviation of each trait of TS163 maize genotype seedlings cultured on the 7th day and the 21st day in the hydroponic maize experiment, and a bar chart was drawn. The IBM-SPSS Statistics 19 software was used to analyze the combined variance of each trait of TS163 maize genotype among the 2 culture time periods and 6 concentrations of PS microplastic treatments in the hydroponic maize experiment, including the F-values and significance levels of the correction model (CM), intercept (IC), culture time period treatment (CT), PS microplastic concentration treatment (PSC), and the interaction between the culture time period and PS microplastic concentration treatment (CT×PSC). The IBM-SPSS Statistics 19 software was used to analyze the Dunman significance of each trait of TS163 maize genotype seedlings in each culture time period among the 6 concentrations of PS microplastic treatments at the P<0.05 level. The GENESCLOUD online software was used to analyze the Pearson correlation coefficient diagram of all traits among the 6 concentrations of PS microplastic treatments of TS163 maize genotype seedlings in each culture time period in the hydroponic maize experiment. The LOG10 function was used to standardize the average values of all traits among the 2 culture time periods and 6 concentrations of PS microplastic treatments of TS163 maize genotype seedlings in the hydroponic maize experiment, and then the Between-groups Linkage clustering method of the IBM-SPSS Statistics 16.0 software was used to conduct a scientific, objective, and qualitative clustering analysis of the microplastic toxicity degree of TS163 maize genotype seedlings among the 6 concentrations of PS microplastic treatments in each culture time period in the hydroponic maize experiment.

[0046] 4. Toxicity resistance index of maize seedlings to different concentrations of PS microplastics: In order to scientifically, accurately, and objectively reflect the resistance of each single trait of TS163 maize genotype seedlings to the toxicity of different concentrations of PS microplastics in each culture time period in the hydroponic maize experiment, the present invention newly defines the toxicity resistance index (TRI) of maize seedlings to different concentrations of PS microplastics, which is specifically calculated according to the formula (2) in the appendix. In the formula: Figure 1 is the toxicity resistance index of the jth trait of maize genotype seedlings at the kth concentration of PS microplastics in the ith culture time period in the hydroponic maize experiment, is the measured value of the jth trait of maize genotype seedlings under the CK control treatment in the ith culture time period in the hydroponic maize experiment, $X_{ijk}$ is the measured value of the $j$-th trait of the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment under the $k$-th concentration of PS microplastics treatment. $i$ is the 7th day or the 21st day of the soil cultivation maize experiment, and $k$-PS is the PS microplastics treatment at the concentrations of 0.05%, 0.25%, 0.50%, 0.75% or 1.00% in the soil cultivation maize experiment. The larger the value, the stronger the resistance of the $j$-th trait of the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment to the toxicity of the $k$-PS concentration of microplastics.

[0047] 5. Evaluation of the comprehensive resistance ability of maize seedlings to the toxicity of different concentrations of PS microplastics: The TRI values of all traits of the maize genotype seedlings calculated above are used as the measurement and evaluation indicators of the resistance ability of the maize genotype seedlings to the toxicity of different concentrations of PS microplastics at 2 cultivation time periods in the soil cultivation maize experiment. Further, the membership function method is used to comprehensively, objectively and quantitatively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of the maize genotype seedlings to the toxicity of different concentrations of PS microplastics at each / all cultivation time periods in the soil cultivation maize experiment, specifically calculated according to Figure 1 Formulas (3), (4) and (5) in the appendix. Where: $u_{ijk}$ is the membership value of the resistance of the $j$-th trait of the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment to the toxicity of the $k$-th concentration of PS microplastics, $x_{ijk}$ is the resistance index of the $j$-th trait of the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment to the toxicity of the $k$-th concentration of PS microplastics, $x_{ijmin}$ is the minimum TRI value of the $j$-th trait of the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment among all concentrations of PS microplastics treatments, $x_{ijmax}$ is the maximum TRI value of the $j$-th trait of the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment among all concentrations of PS microplastics treatments, $CTR_{ik}$ is the value of the comprehensive resistance of the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment to the toxicity of the $k$-th concentration of PS microplastics. $m$ is the $m$ ($m = 12$) traits measured for the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment. TCTR (k-PS) $TCTR_{k}$ is the value of the total comprehensive resistance of the maize genotype seedlings at $n$ ($n = 2$, 2 cultivation time periods) cultivation time periods in the soil cultivation maize experiment to the toxicity of the $k$-th concentration of PS microplastics. $i$ is the 7th day or the 21st day of the soil cultivation maize experiment, and $k$-PS is the PS microplastics treatment at the concentrations of 0.05%, 0.25%, 0.50%, 0.75% or 1.00% in the soil cultivation experiment. The larger the value, the stronger the comprehensive resistance of the maize genotype seedlings at the $i$-th cultivation time period in the soil cultivation maize experiment to the toxicity of the $k$-th concentration of PS microplastics. TTR(k-PS) The larger the value, the stronger the overall comprehensive resistance of maize genotype seedlings to the toxicity of the k-th concentration of PS microplastics during the two cultivation periods in the soil cultivation of maize experiments.

[0048] 7. Comprehensive measurement of the PS microplastic concentration at the time of the most severe toxicity to maize seedlings: By comparing the measurement results of the between-groups linkage comprehensive clustering of the PS microplastic toxicity of maize genotype seedlings among all treatments during the two cultivation periods in the above-mentioned soil cultivation of maize experiments, and the evaluation results of the overall comprehensive resistance of maize genotype seedlings to the toxicity of the corresponding concentration of PS microplastics during the two cultivation periods in the above-mentioned soil cultivation of maize experiments, a combined multi-method comparison and evaluation of the toxicity of different concentrations of PS microplastics to maize genotype seedlings over multiple cultivation periods is carried out. Finally, the PS microplastic concentration at the time of the most severe toxicity to maize genotype seedlings is comprehensively measured.

[0049] Example 2

[0050] The present invention provides a comprehensive evaluation result of the resistance of maize seedlings to microplastic toxicity. The specific evaluation results are as follows:

[0051] 1. Joint variance analysis of measured traits of maize under different concentrations of PS microplastics at different culture time periods in soil culture experiment: Joint variance analysis was performed on 12 traits of TS163 maize genotype seedlings under 6 concentrations of PS microplastics at 2 culture time periods in the soil culture experiment (Table 1). The results showed that the correction models (CM) of these 12 measured traits were all significantly different at the P<0.001 level, indicating that there were significant overall differences among these 12 traits in the joint variance analysis. Therefore, it was effective to perform variance analysis on these 12 traits among PS microplastic concentration treatments (PSC), among culture time period treatments (CT), and the interaction between culture time period and PS microplastic concentration treatments (CT×PSC). Further analysis found that all these 12 traits were significantly different among PS microplastic concentration treatments (P<0.05 or P<0.01 or P<0.001), and the order of the influence of PS microplastic concentration treatments on these 12 traits was root vigor > leaf chlorophyll SPAD value > root dry weight > root fresh weight > shoot fresh weight > root length > stem diameter > shoot dry weight > strong seedling index > total plant dry biomass > root diameter > shoot length, and their F-values decreased in turn; similarly, all these 12 traits were also significantly different among culture time period treatments (P<0.001), and the order of the influence of culture time period treatments on these 12 traits was shoot fresh weight > shoot dry weight > stem diameter > total plant dry biomass > shoot length > root fresh weight > root dry weight > strong seedling index > leaf chlorophyll SPAD value > root vigor > root length > root diameter, and their F-values decreased in turn; in addition, except for shoot length, root diameter, and total plant dry biomass, the remaining 9 traits were significantly different in the interaction between culture time period and PS microplastic concentration treatments (P<0.05 or P<0.01 or P<0.001), indicating that 9 traits such as shoot fresh weight, shoot dry weight, root length, root fresh weight, root dry weight, stem diameter, leaf chlorophyll SPAD value, strong seedling index, and root vigor were jointly regulated by PS microplastic concentration treatments, culture time period treatments, and the interaction between culture time period and PS microplastic concentration treatments.

[0052] Table 1 F-values of joint variance analysis of measured traits of maize among 6 concentrations of PS microplastics at 2 culture time periods in soil culture experiment

[0053]

[0054] Note: SL is shoot length, SFW is shoot fresh weight, SDW is shoot 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 strong seedling index, RV is root vigor. * or ** or *** indicates significant difference at the P<0.05 or P<0.01 or P<0.001 level, respectively.

[0055] 2. Effects of 6 concentrations of PS microplastics on 12 traits of maize seedlings at the 7th and 21st days of cultivation in the soil culture maize experiment: As attached Figure 2 、 3As shown in Figures 4 and 6, the effects of six concentrations of PS microplastics on 12 traits of TS163 maize genotype seedlings at the 7th and 21st days of cultivation were different. Specifically, compared with the control CK, at the 7th and 21st days of cultivation, different concentrations of PS microplastics inhibited the shoot length of TS163 seedlings, with the shoot length decreasing by 6.91% (1.00% PS microplastic soil culture substrate treatment) - 15.74% (0.75% PS microplastic soil culture substrate) and 4.22% (1.00% PS microplastic soil culture substrate treatment) - 4.64% (0.50% PS microplastic soil culture substrate treatment), respectively; compared with the control CK, except that the shoot fresh weight of TS163 seedlings increased by 4.48% under the 0.25% PS microplastic soil culture substrate treatment at the 7th day of cultivation, the shoot fresh weight of TS163 seedlings under the treatment of the remaining all concentrations of PS microplastic soil culture substrate decreased by 22.45% (0.05% PS microplastic soil culture substrate treatment) - 50.36% (0.50% PS microplastic soil culture substrate treatment) and 3.38% (1.00% PS microplastic soil culture substrate treatment) - 21.96% (0.25% PS microplastic soil culture substrate treatment) at these two cultivation time periods; compared with the control CK, at the 7th and 21st days of cultivation, different concentrations of PS microplastics decreased the shoot dry weight of TS163 seedlings, with the shoot dry weight decreasing by 21.03% (0.05% PS microplastic soil culture substrate treatment) - 46.87% (0.50% PS microplastic soil culture substrate) and 7.61% (1.00% PS microplastic soil culture substrate treatment) - 18.29% (0.25% PS microplastic soil culture substrate treatment), respectively; compared with the control CK, except that the root length of TS163 seedlings elongated by 5.66% under the 0.75% PS microplastic soil culture substrate treatment at the 7th day of cultivation, the root length of TS163 seedlings under the treatment of the remaining all concentrations of PS microplastic soil culture substrate shortened by 8.35% (0.05% PS microplastic soil culture substrate treatment) - 13.44% (1.00% PS microplastic soil culture substrate treatment) and 16.74% (0.05% PS microplastic soil culture substrate treatment) - 40.24% (0.50% PS microplastic soil culture substrate treatment) at these two cultivation time periods; compared with the control CK, at the 7th and 21st days of cultivation, different concentrations of PS microplastics decreased the root fresh weight of TS163 seedlings, with the root fresh weight decreasing by 9.31% (0.25% PS microplastic soil culture substrate treatment) - 32.98% (0.75% PS microplastic soil culture substrate) and 11.80% (0.75% PS microplastic soil culture substrate treatment) - 28.00% (0.25% PS microplastic soil culture substrate treatment), respectively; compared with the control CK, at the 7th and 21st days of cultivation, different concentrations of PS microplastics decreased the root dry weight of TS163 seedlings, with the root dry weight decreasing by 0.70% (0.25% PS microplastic soil culture substrate treatment) - 27.85% (0.75% PS microplastic soil culture substrate) and 2.99% (0.25% PS microplastic soil culture substrate treatment) - 25.95% (0.75% PS microplastic soil culture substrate treatment); Compared with the control CK, on the 7th and 21st days of cultivation, different concentrations of PS microplastic treatments would all reduce the stem diameter of TS163 seedlings, and the stem diameters decreased by 7.80% (1.00% PS microplastic soil culture substrate treatment) - 19.08% (0.75% PS microplastic soil culture substrate) and 7.72% (1.00% PS microplastic soil culture substrate treatment) - 10.41% (0.75% PS microplastic soil culture substrate treatment) respectively; Compared with the control CK, on the 7th and 21st days of cultivation, different concentrations of PS microplastic treatments would all reduce the root diameter of TS163 seedlings, and the root diameters decreased by 6.54% (0.25% PS microplastic soil culture substrate treatment) - 33.64% (1.00% PS microplastic soil culture substrate) and 2.89% (0.50% PS microplastic soil culture substrate treatment) - 8.67% (1.00% PS microplastic soil culture substrate treatment) respectively; Compared with the control CK, on the 7th and 21st days of cultivation, different concentrations of PS microplastic treatments would all reduce the total plant dry biomass of TS163 seedlings, and the total plant dry biomasses decreased by 11.33% (0.25% PS microplastic soil culture substrate treatment) - 34.26% (0.75% PS microplastic soil culture substrate treatment) and 6.19% (1.00% PS microplastic soil culture substrate treatment) - 14.88% (0.25% PS microplastic soil culture substrate treatment) respectively; Compared with the control CK, on the 7th and 21st days of cultivation, different concentrations of PS microplastic treatments would all reduce the chlorophyll SPAD value of TS163 seedlings' leaves, and the chlorophyll SPAD values of the leaves decreased by 13.66% (1.00% PS microplastic soil culture substrate treatment) - 25.97% (0.25% PS microplastic soil culture substrate treatment) and 2.07% (1.00% PS microplastic soil culture substrate treatment) - 16.55% (0.25% PS microplastic soil culture substrate treatment) respectively; Compared with the control CK, on the 7th and 21st days of cultivation, different concentrations of PS microplastic treatments would all reduce the strong seedling index of TS163 seedlings, and the strong seedling indices decreased by 10.30% (0.05% PS microplastic soil culture substrate treatment) - 36.16% (0.75% PS microplastic soil culture substrate treatment) and 9.37% (1.00% PS microplastic soil culture substrate treatment) - 16.06% (0.50% PS microplastic soil culture substrate treatment) respectively; Compared with the control CK, on the 7th and 21st days of cultivation, different concentrations of PS microplastic treatments would all reduce the root activity of TS163 seedlings, and the root activities decreased by 58.78% (0.05% PS microplastic soil culture substrate treatment) - 77.75% (0.50% PS microplastic soil culture substrate treatment) and 25.31% (1.00% PS microplastic soil culture substrate treatment) - 68.14% (0.Treatment with 50% PS microplastic soil culture substrate).

[0056] 3. Pearson correlation analysis among 12 traits of maize seedlings at the 7th and 21st days of cultivation under 6 concentrations of PS microplastics in the soil culture maize experiment: As attached Figure 5 and 6 shown, Pearson correlation analysis was performed among 12 traits of TS163 maize genotype seedlings at the 7th and 21st days of cultivation under 6 concentrations of PS microplastics in the soil culture maize experiment. The results showed that the Pearson correlations of the 12 traits of TS163 maize genotype seedlings under 6 concentrations of PS microplastics were exactly the same at the two cultivation time periods, that is, there was a significant positive correlation between seedling length and seedling fresh weight, seedling dry weight, root fresh weight, stem diameter, total plant dry biomass, leaf chlorophyll SPAD value, strong seedling index, and root activity; there was a significant positive correlation between seedling fresh weight and seedling dry weight, root fresh weight, root dry weight, root diameter, total plant dry biomass, strong seedling index, and root activity; there was a significant positive correlation between seedling dry weight and root fresh weight, root dry weight, stem diameter, root diameter, total plant dry biomass, strong seedling index, and root activity; there was a significant positive correlation between root fresh weight and root dry weight, root diameter, total plant dry biomass, strong seedling index, and root activity; there was a significant positive correlation between root dry weight and root diameter, total plant dry biomass, strong seedling index, and root activity; there was a significant positive correlation between stem diameter and total plant dry biomass, leaf chlorophyll SPAD value, strong seedling index, and root activity; there was a significant positive correlation between root diameter and total plant dry biomass, strong seedling index, and root activity; there was a significant positive correlation between total plant dry biomass and strong seedling index, and root activity; there was a significant positive correlation between leaf chlorophyll SPAD value and root activity; there was a significant positive correlation between strong seedling index and root activity. It shows that these traits of maize seedlings at the corresponding cultivation time periods interact with each other and jointly determine their growth and development and biomass accumulation under different concentrations of PS microplastics.

[0057] 4. Cluster analysis of the toxicity of six concentrations of PS microplastics on maize seedlings at the 7th and 21st days of cultivation in soil culture maize experiment: Since the effects of six concentrations of PS microplastics on 12 traits of TS163 maize genotype seedlings at the 7th and 21st days of cultivation in soil culture maize experiment are not the same, and each trait under each cultivation time period can only reflect a certain aspect of the performance of TS163 genotype seedlings under the corresponding concentration of PS microplastics treatment, it cannot comprehensively and objectively reflect the comprehensive performance of TS163 maize genotype seedlings under each concentration of PS microplastics toxicity under the corresponding cultivation time period. Based on this, we used the LOG10 function to standardize 12 traits of TS163 maize genotype seedlings at the 7th and 21st days of cultivation under six concentrations of PS microplastics in the soil culture maize experiment, and then used the Between-groups Linkage clustering method to cluster and evaluate the toxicity degree of PS microplastics on TS163 maize genotype seedlings among six concentrations of PS microplastics under each cultivation time period in the soil culture maize experiment. As shown in the appendix Figure 7 As shown in Figure 7 , when the Euclidean distance is 2, at the 7th day of cultivation, the TS163 maize genotype seedlings under six concentrations of PS microplastics treatment can be divided into three types. Type A only includes the TS163 genotype seedlings grown under the CK control treatment, which are not poisoned by PS microplastics and belong to the type not poisoned by PS microplastics; Type B includes the TS163 genotype seedlings grown under the soil culture substrates of 0.05% and 0.25% PS microplastics, with poor performance in multiple traits and weak toxicity by PS microplastics, belonging to the type with weak PS microplastics toxicity; Type C includes the TS163 genotype seedlings grown under the soil culture substrates of 0.50%, 0.75% and 1.00% PS microplastics, with the worst performance in multiple traits and severe toxicity by PS microplastics, belonging to the type with severe PS microplastics toxicity. Different from this, as shown in the appendix Figure 8 As shown in Figure 8 , when the Euclidean distance is 7, at the 21st day of cultivation, the TS163 maize genotype seedlings under six concentrations of PS microplastics treatment can be divided into three types. Type A only includes the TS163 genotype seedlings grown under the CK control treatment, which are not poisoned by PS microplastics and belong to the type not poisoned by PS microplastics; Type B includes the TS163 genotype seedlings grown under the soil culture substrates of 0.75% and 1.00% PS microplastics, with poor performance in multiple traits and weak toxicity by PS microplastics, belonging to the type with weak PS microplastics toxicity; Type C includes the TS163 genotype seedlings grown under the soil culture substrates of 0.05%, 0.50% and 0.25% PS microplastics, with the worst performance in multiple traits and severe toxicity by PS microplastics, belonging to the type with severe PS microplastics toxicity. Although these results can roughly distinguish the approximate degree of toxicity of six concentrations of PS microplastics on maize seedlings at different cultivation time periods, they cannot clearly distinguish the size of the toxicity of each concentration of PS microplastics on maize seedlings at each cultivation time period.

[0058] 5. Toxicity resistance index of single traits of maize seedlings at the 7th and 21st days of cultivation in soil culture maize experiment: To scientifically, accurately, and objectively reflect the resistance of single traits of TS163 maize genotype seedlings to different concentrations of PS microplastics toxicity in each cultivation period of the soil culture maize experiment, we newly defined the toxicity resistance index (TRI) of different concentrations of PS microplastics for maize seedlings. The advantage of the TRI value is that it can not only objectively and accurately quantify the resistance of single traits of TS163 maize genotype seedlings to different concentrations of PS microplastics toxicity in each cultivation period of the soil culture maize experiment, but also provide a scientific basis for the comprehensive evaluation of the toxicity resistance of maize seedlings to PS microplastics. Therefore, according to the 12 traits of TS163 maize genotype seedlings at the 7th and 21st days of cultivation in the soil culture maize experiment, we calculated the TRI values of the 12 traits of TS163 maize genotype seedlings at the 7th day (Table 2) and the 21st day (Table 3) of cultivation in the soil culture maize experiment according to the formula (2) attached Figure 1 in the appendix.

[0059] Table 2 Toxicity resistance index (TRI) values of different concentrations of PS microplastics for 12 traits of TS163 maize genotype seedlings at the 7th day of cultivation in the soil culture maize experiment

[0060]

[0061] 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 strong seedling index, and RV is root activity. 0.05% PS is the treatment of 0.05% PS microplastic soil culture substrate, 0.25% PS is the treatment of 0.25% PS microplastic soil culture substrate, 0.50% PS is the treatment of 0.50% PS microplastic soil culture substrate, 0.75% PS is the treatment of 0.75% PS microplastic soil culture substrate, and 1.00% PS is the treatment of 1.00% PS microplastic soil culture substrate.

[0062] Table 3 Toxicity resistance index (TRI) values of different concentrations of PS microplastics for 12 traits of TS163 maize genotype seedlings at the 21st day of cultivation in the soil culture maize experiment

[0063]

[0064] 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 strong seedling index, and RV is root activity. 0.05% PS is the treatment of 0.05% PS microplastic soil culture substrate, 0.25% PS is the treatment of 0.25% PS microplastic soil culture substrate, 0.50% PS is the treatment of 0.50% PS microplastic soil culture substrate, 0.75% PS is the treatment of 0.75% PS microplastic soil culture substrate, and 1.00% PS is the treatment of 1.00% PS microplastic soil culture substrate.

[0065] 6. Evaluation of the comprehensive resistance ability of maize seedlings cultured on the 7th and 21st days to the toxicity of different concentrations of PS microplastics in the soil culture experiment: We further used the TRI values of 12 traits of the TS163 maize genotype seedlings cultured on the 7th and 21st days in the soil culture experiment as the measurement and evaluation indicators for the resistance ability of maize genotype seedlings to the toxicity of different concentrations of PS microplastics during these two culture periods in the soil culture experiment. Using the membership function method, according to the Figure 1 formula (3) attached, calculate the membership values U(TRI) of the 12 traits of the TS163 maize genotype seedlings cultured on the 7th and 21st days in the soil culture experiment to the toxicity of different concentrations of PS microplastics (Tables 4 and 5). Then, according to the Figure 1 formula (4) attached, calculate the comprehensive resistance CTR values of the TS163 maize genotype seedlings cultured on the 7th and 21st days in the soil culture experiment to the toxicity of the corresponding concentrations of PS microplastics. Finally, according to the Figure 1 formula (5) attached, calculate the total comprehensive resistance TCTR values of the TS163 maize genotype seedlings to the toxicity of the corresponding concentrations of PS microplastics during the two culture periods in the soil culture experiment. The results are as shown in the Figure 9 attachment, indicating that the CTR values of the TS163 maize genotype seedlings cultured on the 7th day in the soil culture experiment to the toxicity of 0.05%, 0.25%, 0.50%, 0.75%, and 1.00% concentrations of PS microplastics are 0.631, 0.655, 0.175, 0.264, and 0.409 respectively, while the CTR values of the TS163 maize genotype seedlings cultured on the 21st day to the toxicity of 0.05%, 0.25%, 0.50%, 0.75%, and 1.00% concentrations of PS microplastics are 0.425, 0.323, 0.371, 0.497, and 0.760 respectively. It shows that the comprehensive resistance of maize seedlings to the toxicity of different concentrations of PS microplastics is different at different culture periods. Further, as shown in the Figure 10As shown, in the soil-cultured corn experiment, the TCTR of TS163 corn seedlings to 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, in the two cultivation time periods. This shows that the total comprehensive resistance of corn seedlings to 0.50% concentration of PS microplastics in the two cultivation time periods is the smallest at 0.273, while the total comprehensive resistance of corn seedlings to 1.00% concentration of PS microplastics in the two cultivation time periods is the largest at 0.584.

[0066] Table 4 Membership values ​​U(TRI) of resistance of 12 traits of TS163 maize seedlings to different concentrations of PS microplastics on the 7th day of soil-cultured maize experiment

[0067]

[0068] 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 plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling index, RV is root activity. 0.05% PS is 0.05% PS microplastic soil culture substrate treatment, 0.25% PS is 0.25% PS microplastic soil culture substrate treatment, 0.50% PS is 0.50% PS microplastic soil culture substrate treatment, 0.75% PS is 0.75% PS microplastic soil culture substrate treatment, 1.00% PS is 1.00% PS microplastic soil culture substrate treatment.

[0069] Table 5 Membership values ​​U(TRI) of resistance of 12 traits of TS163 maize seedlings to different concentrations of PS microplastics on the 21st day of soil-cultured maize experiment

[0070]

[0071]

[0072] 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 plant dry biomass, SPAD is leaf chlorophyll SPAD value, SSI is seedling index, RV is root activity. 0.05% PS is 0.05% PS microplastic soil culture substrate treatment, 0.25% PS is 0.25% PS microplastic soil culture substrate treatment, 0.50% PS is 0.50% PS microplastic soil culture substrate treatment, 0.75% PS is 0.75% PS microplastic soil culture substrate treatment, 1.00% PS is 1.00% PS microplastic soil culture substrate treatment.

[0073] 7. Comprehensive measurement of the PS microplastic concentration at the most severe degree of PS microplastic toxicity to maize seedlings: We further compared the comprehensive clustering results of the PS microplastic toxicity of TS163 maize genotype seedlings among all PS microplastic treatments at two culture time periods in the above soil-cultivated maize experiment (attached Figure 7 and 8 ), as well as the evaluation results of the total comprehensive resistance of TS163 maize genotype seedlings to the toxicity of all PS microplastic concentrations at two culture time periods in the above soil-cultivated maize experiment (attached Figure 10 ). Through comprehensive comparative analysis of multiple traits of maize seedlings at multiple culture time periods by multiple methods, we finally determined that the PS microplastic concentration at the most severe degree of PS microplastic toxicity to maize seedlings was 0.50%. Therefore, in the future, we can use 0.50% concentration of PS microplastics to cultivate different maize genotype materials, measure the above 12 traits on the 7th and 21st days of cultivation, and comprehensively identify excellent maize genotype materials resistant to PS microplastic toxicity for application in field production, so as to reduce the adverse effects of microplastic pollution on maize and ensure national food production safety.

[0074] As can be seen from the above embodiments, the system of the present invention comprehensively reveals the effects of 6 concentrations of PS microplastics on 12 phenotypic and physiological metabolic traits above / below ground, such as the growth phenotype, strong seedling index, chlorophyll accumulation level, and root activity of maize genotype seedlings at 2 culture time periods, and objectively analyzes the toxicity mechanism of different concentrations of PS microplastics on maize seedlings. The standardized data of these 12 traits are used to conduct a clustering evaluation among all concentrations of PS microplastic treatments for maize genotype seedlings at each culture time period, comprehensively and objectively reflecting the toxicity degree of different concentrations of PS microplastic treatments on maize genotype seedlings. On this basis, we also innovatively proposed the toxicity resistance index (TRI) of a single trait of maize genotype seedlings at different concentrations of PS microplastics at each culture time period, and used the membership function method to comprehensively and quantitatively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) values of maize genotype seedlings to the toxicity of corresponding concentrations of PS microplastics at each / all culture time periods. In addition, we also compared the results of the clustering evaluation of maize genotype seedlings at all concentrations of PS microplastic treatments at each culture time period with the evaluation results of the total comprehensive resistance of maize genotype seedlings to the toxicity of corresponding concentrations of PS microplastics at all culture time periods, verifying that the TRI biological concept and calculation formula proposed by us for comprehensively evaluating the toxicity degree of different concentrations of PS microplastics on maize genotype seedlings are scientific, accurate, and reliable. Therefore, we finally determined that the PS microplastic concentration when the toxicity of maize genotype seedlings to PS microplastics is the most severe is 0.50%. Therefore, in the future, we can use 0.50% concentration of PS microplastics to treat and culture different maize genotype materials for 7 days and 21 days, and then screen excellent maize genotypes (inbred lines / lines / varieties) resistant to microplastic toxicity for application in breeding or production practice.

[0075] Example 3

[0076] The present invention provides a method for evaluating maize genotypes resistant to microplastic toxicity. The specific evaluation method is carried out according to the following steps:

[0077] 1. Preparation of high-quality maize seeds: Prepare 10 new high-quality maize genotype seeds independently selected by our team, which were planted at the Longxi test site (latitude 34.97°, longitude 104.40°, altitude 2074 m) in 2024, strictly bagged for pollination, and harvested with plump grains, uniform size, strong vitality, and high purity, for standby.

[0078] 2. 0.50% concentration of polystyrene (PS) microplastic soil culture maize experiment: Put 40 seeds of each of the 10 high-quality maize genotype seeds independently selected by our team prepared above into a triangular flask, add 100 mL of 70% ethanol (v / v), place it on a horizontal shaker to disinfect the seeds for 10 min, and use 100 mL of ddH 2Wash the seeds with water 5 times to remove the residual ethanol on the seed surface, and dry the attached water on the seed surface with a sterilized filter paper to obtain the corresponding disinfected maize genotype seeds. Purchase PS microplastic powder (mesh number: 1000 mesh; particle size: 13 μm) from Ruixiang Plastic Raw Materials Co., Ltd., Dongguan City. Weigh 0.00 g and 0.50 g of PS microplastics accurately with an electronic balance in advance, and dissolve them in 100 mL of ddH 2 O water respectively to prepare 2 kinds of concentrations of 0.00% and 0.50% (mass ratio concentration of PS microplastics to ddH 2 O) PS microplastic solutions, and prepare them freshly before use. Then continue to put 10 portions of the disinfected maize genotype seeds into the corresponding Erlenmeyer flasks, add 100 mL of these 2 kinds of concentration PS microplastic solutions respectively, and soak the seeds on a horizontal shaker in a dark environment indoors for 24 h. Prepare 2 kinds of concentrations (mass ratio concentration of PS microplastics to nutrient soil) of PS microplastic soil culture substrates, namely 0.00% PS microplastic soil culture substrate (that is, the soil culture substrate prepared by fully mixing 0.00 g of PS microplastics with 300.00 g of nutrient soil and then adding 250 mL of ddH 2 O water and stirring evenly) and 0.50% PS microplastic soil culture substrate (that is, the soil culture substrate prepared by fully mixing 1.50 g of PS microplastics with 300.00 g of nutrient soil and then adding 250 mL of ddH 2 O water and stirring evenly). Then sow 10 seeds of each of the 10 portions of maize genotype seeds soaked in these 2 kinds of concentration PS microplastic solutions into flower pots (height 11.0 cm, diameter 12.5 cm) filled with the same concentration PS microplastic soil culture substrate, and place them in an artificial climate chamber for cultivation. During the cultivation period, the relative humidity is set at 65%, the temperature is set at 25±0.5 / 20±0.5 °C for 12 h each in an alternating cycle, the photoperiod is set at 16 / 8 h light / dark, and the light intensity is set at 300 μM m -2 s -1 , and the CO 2 concentration is 450 PPM. There are a total of 2 kinds of treatments in the soil culture experiment, namely CK control treatment (0.00% PS microplastic soil culture substrate) and 0.50% PS treatment (0.50% PS microplastic soil culture substrate), and each treatment has 4 biological replicates. At the same time, during the cultivation period, pour 50 mL of ddH 2 O water evenly into each flower pot every 3 d to supply water to the seedlings in time. Measure the 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 strong seedling index (SSI) of these 10 portions of maize genotype seedlings cultured in these 2 kinds of PS microplastic soil culture substrates on the 7th d and 21st d respectively. Among them, the strong seedling index is calculated according to the appendixFigure 1 Calculate according to formula (1) in which SSI is the strong seedling index, SD is the stem diameter, SL is the seedling length, and PDW is the total dry biomass of the plant.

[0079] 3. Data statistical analysis: For the 12 traits of 10 maize genotype seedlings measured on the 7th day and the 21st day in the soil culture experiment of maize with 2 concentrations of PS microplastics, use Excel 2013 software to calculate the mean and standard deviation of each trait of these 10 maize genotype seedlings cultured on the 7th day and the 21st day under each treatment in the soil culture experiment of maize. Use IBM-SPSS Statistics 19 software to analyze the combined variance analysis of each trait of all maize genotypes between the two treatments of 2 concentrations of PS microplastics at these two culture time periods, including the F-values and significance levels of differences of the correction model (CM), intercept (IC), genotype (G), culture time period treatment (CT), PS microplastic concentration treatment (PSC), interaction between genotype and culture time period treatment (G×CT), interaction between genotype and PS microplastic concentration treatment (G×PSC), interaction between culture time period and PS microplastic concentration treatment (CT×PSC), and interaction between the three (G×CT×PSC).

[0080] 4. Toxicity resistance index of 0.50% concentration of polystyrene (PS) microplastics for maize seedlings of different genotypes: According to the means of the 12 traits of these 10 maize genotype seedlings measured on the 7th day and the 21st day of the germ in the soil culture experiment of maize with 2 concentrations of PS microplastics, calculate the toxicity resistance index (TRI) value of each trait of each maize genotype seedling at 0.50% concentration of PS microplastics for each culture time period according to formula (6) in the appendix. Figure 1 In the formula: is the toxicity resistance index of the jth trait of the pth maize genotype seedling at the ith culture time period at 0.50% concentration of PS microplastics in the soil culture experiment of maize, is the measured value of the jth trait under the CK control treatment of the pth maize genotype seedling at the ith culture time period in the soil culture experiment of maize, is the measured value of the jth trait under the 0.50% concentration of PS microplastic treatment of the pth maize genotype seedling at the ith culture time period in the soil culture experiment of maize. i is the 7th day or the 21st day of the soil culture experiment of maize, and 0.50% PS is the 0.50% concentration of PS microplastic treatment in the soil culture experiment of maize. The larger the value, the stronger the resistance of the jth trait of the pth maize genotype seedling at the ith culture time period to the toxicity of 0.50% concentration of microplastics in the soil culture experiment of maize.

[0081] 5. Evaluation of the comprehensive resistance ability of maize seedlings with different genotypes to the toxicity of 0.50% polystyrene (PS) microplastics: The TRI values of all traits of the 10 maize genotype seedlings calculated above were used as the measurement and evaluation indicators of the resistance ability of all maize genotype seedlings to the toxicity of 0.50% PS microplastics at two culture time periods in the soil-cultivated maize experiment. Further, the membership function method was used to comprehensively, integrally, and quantitatively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of the 10 maize genotype seedlings to the toxicity of 0.50% PS microplastics at each / all culture time periods in the soil-cultivated maize experiment. Specifically, it was calculated according to the formulas (7), (8), and (9) attached in Figure 1 : The formulas are as follows:. Wherein: is the membership value of the resistance of the jth trait of the pth maize genotype seedling at the ith culture time period to the toxicity of 0.50% PS microplastics in the soil-cultivated maize experiment, is the resistance index of the jth trait of the pth maize genotype seedling at the ith culture time period to the toxicity of 0.50% PS microplastics in the soil-cultivated maize experiment, is the minimum TRI value of the jth trait of all maize genotype seedlings at the ith culture time period in the soil-cultivated maize experiment, is the maximum TRI value of the jth trait of all maize genotype seedlings at the ith culture time period in the soil-cultivated maize experiment, is the value of the comprehensive resistance of the pth maize genotype seedling at the ith culture time period to the toxicity of 0.50% PS microplastics in the soil-cultivated maize experiment. m is the m (m = 12) traits measured for each maize genotype seedling at the ith culture time period in the soil-cultivated maize experiment. TCTR p(0.50％PS) is the value of the total comprehensive resistance of the pth maize genotype seedling at n (n = 2, two culture time periods) culture time periods to the toxicity of 0.50% PS microplastics in the soil-cultivated maize experiment. i is the 7th day or the 21st day of the cultivation of the soil-cultivated maize experiment. 0.50% PS is the treatment of 0.50% concentration of PS microplastics in the soil-cultivated experiment. The larger the value, the stronger the comprehensive resistance of the pth maize genotype seedling at the ith culture time period to the toxicity of 0.50% PS microplastics. TCTR (0.50％PS)The larger the value is, the stronger the overall comprehensive resistance of the seedlings of the p-th maize genotype to the toxicity of 0.50% concentration of PS microplastics in the soil cultivation maize experiment. According to the TCTR values of the seedlings of different maize genotypes, the anti-microplastic toxicity performance of maize genotypes is divided into 5 grades, namely: 0≤TCTR<0.400, highly sensitive maize genotype to microplastic toxicity; 0.400≤TCTR<0.500, sensitive maize genotype to microplastic toxicity; 0.500≤TCTR<0.600, weakly anti-microplastic toxicity maize genotype; 0.600≤TCTR<0.700, moderately anti-microplastic toxicity maize genotype; 0.700≤TCTR≤1.000, highly anti-microplastic toxicity maize genotype.

[0082] Example 4

[0083] The present invention provides a result for evaluating maize genotypes resistant to microplastic toxicity, and the specific results are as follows:

[0084] 1. Combined analysis of variance for 12 traits of all maize seedlings under different concentrations of PS microplastics at different culture time periods in the soil culture maize experiment: A combined analysis of variance was conducted on 12 traits of 10 maize genotype seedlings under 2 concentrations of PS microplastics at 2 culture time periods in the soil culture maize experiment (Table 6). The results showed that the correction models (CM) of these 12 measured traits were all significantly different at the P<0.001 level, indicating that there were significant overall differences among these 12 traits in the combined analysis of variance. Therefore, it was effective to conduct an analysis of variance on these 12 traits for differences among genotypes (G), among PS microplastic concentration treatments (PSC), among culture time period treatments (CT), for the interaction between genotype and PS microplastic concentration treatment (G×PSC), for the interaction between genotype and culture time period treatment (G×CT), for the interaction between culture time period and PS microplastic concentration treatment (CT×PSC), and for the three-way interaction (G×CT×PSC). Further analysis found that these 12 traits were all significantly different at the P<0.001 level among genotypes, among PS microplastic concentration treatments, and among culture time period treatments, indicating that these 12 traits of maize seedlings were significantly affected by their own genetic basis, PS microplastic concentration treatment, and culture time period treatment. In addition, except for seedling length, root length, root fresh weight, and stem diameter, the other 8 traits were also significantly different in the interaction between genotype and PS microplastic concentration treatment (P<0.05 or P<0.01 or P<0.001); except for the strong seedling index, the other 11 traits were also significantly different in the interaction between genotype and culture time period treatment (P<0.05 or P<0.01 or P<0.001); except for seedling length, seedling fresh weight, stem diameter, and leaf chlorophyll SPAD value, the other 8 traits were also significantly different in the interaction between culture time period and PS microplastic concentration treatment (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 were also significantly different in the three-way interaction (P<0.05 or P<0.01 or P<0.001), indicating that multiple traits of maize genotype seedlings were also jointly regulated by the interactions of different factors, and ultimately manifested as significant differences among the 12 traits of different maize genotypes under different PS microplastic concentration treatments at different culture time periods.

[0085] Table 6 F-values of combined analysis of variance for 12 traits of 10 maize genotype seedlings under 2 concentrations of PS microplastics at 2 culture time periods in the soil culture maize experiment

[0086]

[0087] 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 strong seedling index, and RV is root activity. *, **, or *** indicate significant differences at the P<0.05, P<0.01, or P<0.001 level, respectively.

[0088] 2. Overall effects of two concentrations of PS microplastics on 12 traits of 10 maize seedlings at the 7th and 21st days of cultivation in the soil culture maize experiment: As shown Figure 11 in the figure, compared with the control CK, the effects of 0.50% concentration of PS microplastics on 12 traits of 10 maize genotype seedlings were different at the two cultivation time periods. Overall, compared with the control CK, at the 7th day of cultivation, the average of 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, strong seedling index, and root activity of 10 maize genotype seedlings treated with 0.50% concentration of PS microplastics decreased by 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% respectively; while with the growth of maize seedlings, at the 21st day of cultivation, the average of 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, strong seedling index, and root activity of 10 maize genotype seedlings treated with 0.50% concentration of PS microplastics decreased by 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% respectively. It shows that the sensitivities and tolerances of 0.50% concentration of PS microplastics to these 12 traits of 10 maize genotype seedlings are different at different cultivation time periods. Therefore, we can fully use these 12 traits as evaluation indicators for maize resistance to PS microplastic toxicity to comprehensively evaluate the PS microplastic toxicity resistance performance of different maize genotypes.

[0089] 3. Toxicity resistance index of 0.50% concentration of PS microplastics for individual traits of maize seedlings at the 7th and 21st days of cultivation in the soil culture maize experiment: According to the attached Figure 1For formula (6), we further calculated the toxicity resistance index (TRI) values of single traits of these 10 maize genotype seedlings under 2 cultivation time periods for 0.50% concentration of PS microplastics. That is, on the 7th day of cultivation, the TRI values of the shoot length of these 10 maize genotype seedlings ranged from 0.641 (maize genotype GTX-21-2) to 0.934 (maize genotype GTX-21-3), the TRI values of the shoot fresh weight ranged from 0.045 (maize genotype GTX-21-10) to 0.685 (maize genotype GTX-21-6), the TRI values of the shoot dry weight ranged from 0.306 (maize genotype GTX-21-2) to 0.740 (maize genotype GTX-21-5), the TRI values of the root length ranged from 0.652 (maize genotype GTX-21-4) to 0.909 (maize genotype GTX-21-3), the TRI values of the root fresh weight ranged from 0.436 (maize genotype GTX-21-4) to 0.688 (maize genotype GTX-21-6), the TRI values of the root dry weight ranged from 0.079 (maize genotype GTX-21-3) to 0.898 (maize genotype GTX-21-5), the TRI values of the stem diameter ranged from 0.740 (maize genotype GTX-21-4) to 0.919 (maize genotype GTX-21-6), the TRI values of the root diameter ranged from 0.545 (maize genotype GTX-21-9) to 0.899 (maize genotype GTX-21-3), the TRI values of the whole-plant dry biomass ranged from 0.049 (maize genotype GTX-21-10) to 0.789 (maize genotype GTX-21-5), the TRI values of the leaf chlorophyll SPAD value ranged from 0.726 (maize genotype GTX-21-4) to 0.910 (maize genotype GTX-21-5), the TRI values of the strong seedling index ranged from 0.051 (maize genotype GTX-21-10) to 0.817 (maize genotype GTX-21-5), and the TRI values of the root activity ranged from 0.203 (maize genotype GTX-21-2) to 0.591 (maize genotype GTX-21-3) (Table 7). It shows that the TRI of different traits of these 10 maize genotype seedlings is different on the 7th day of cultivation.In addition, on the 21st day of cultivation, the TRI values of the seedling lengths of these 10 maize genotypes ranged from 0.714 (maize genotype GTX-21-9) to 0.935 (maize genotype GTX-21-3), the TRI values of the fresh seedling weights ranged from 0.747 (maize genotype GTX-21-10) to 0.887 (maize genotype GTX-21-7), the TRI values of the dry seedling weights ranged from 0.670 (maize genotype GTX-21-2) to 0.902 (maize genotype GTX-21-5), the TRI values of the root lengths ranged from 0.571 (maize genotype GTX-21-1) to 0.865 (maize genotype GTX-21-6), the TRI values of the fresh root weights ranged from 0.671 (maize genotype GTX-21-1) to 0.864 (maize genotype GTX-21-10), the TRI values of the dry root weights ranged from 0.060 (maize genotype GTX-21-5) to 0.1494 (maize genotype GTX-21-6), the TRI values of the stem diameters ranged from 0.816 (maize genotype GTX-21-9) to 0.988 (maize genotype GTX-21-3), the TRI values of the root diameters ranged from 0.780 (maize genotype GTX-21-9) to 0.982 (maize genotype GTX-21-6), the TRI values of the dry biomass of the whole plant ranged from 0.263 (maize genotype GTX-21-6) to 2.032 (maize genotype GTX-21-4), the TRI values of the SPAD values of the leaf chlorophyll ranged from 0.693 (maize genotype GTX-21-2) to 0.904 (maize genotype GTX-21-5), the TRI values of the strong seedling index ranged from 0.259 (maize genotype GTX-21-5) to 2.027 (maize genotype GTX-21-4), and the TRI values of the root activity ranged from 161 (maize genotype GTX-21-6) to 0.608 (maize genotype GTX-21-5) (Table 8). This shows that similar to the 7th day of cultivation, the TRI values of different traits of these 10 maize genotype seedlings were also different on the 21st day of cultivation.

[0090] Table 7 TRI values of the resistance of 12 traits of 10 maize genotype seedlings to PS microplastic toxicity at a concentration of 0.50% on the 7th day of soil cultivation of maize

[0091]

[0092] Note: SL is the seedling length, SFW is the fresh seedling weight, SDW is the dry seedling weight, RL is the root length, RFW is the fresh root weight, RDW is the dry root weight, SD is the stem diameter, RD is the root diameter, PDW is the dry biomass of the whole plant, SPAD is the SPAD value of the leaf chlorophyll, SSI is the strong seedling index, and RV is the root activity. Table 8 TRI values of the resistance of 12 traits of 10 maize genotype seedlings to PS microplastic toxicity at a concentration of 0.50% on the 21st day of the soil cultivation experiment of maize

[0093]

[0094] 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 strong seedling index, and RV is root activity.

[0095] 4. Evaluation of the comprehensive resistance of different maize seedlings to the toxicity of 0.50% concentration PS microplastics on the 7th and 21st days of cultivation in the soil culture maize experiment: We further used the TRI values of 12 traits of 10 maize genotype seedlings cultivated on the 7th and 21st days in the soil culture maize experiment as the measurement and evaluation indicators for the resistance of the corresponding maize genotype seedlings to the toxicity of 0.50% concentration PS microplastics in these two cultivation time periods in the soil culture maize experiment. Using the membership function method, according to the Figure 1 formula (7) attached, calculate the membership values U(TRI) of the 12 traits of 10 maize genotype seedlings cultivated on the 7th and 21st days in the soil culture maize experiment to the toxicity of 0.50% concentration PS microplastics (Tables 9 and 10). Then, according to the Figure 1 formula (8) attached, calculate the comprehensive resistance CTR values of 10 maize genotype seedlings cultivated on the 7th and 21st days in the soil culture maize experiment to the toxicity of 0.50% concentration PS microplastics respectively. Finally, according to the Figure 1 formula (9) attached, calculate the total comprehensive resistance TCTR values of 10 maize genotype seedlings to the toxicity of 0.50% concentration PS microplastics in the two cultivation time periods in the soil culture maize experiment. The results are as shown in the Figure 12 attachment, indicating that the CTR of 10 maize genotype seedlings cultivated on the 7th day in the soil culture maize experiment to the toxicity of 0.05% concentration PS microplastics ranges from 0.212 (maize genotype GTX-21-4) to 0.839 (maize genotype GTX-21-5), while the CTR of 10 maize genotype seedlings cultivated on the 21st day to the toxicity of 0.50% concentration PS microplastics ranges from 0.347 (maize genotype GTX-21-1) to 0.682 (maize genotype GTX-21-3). It shows that the comprehensive resistance of different maize genotype seedlings to the toxicity of 0.50% concentration PS microplastics varies at different cultivation time periods. Further as shown in the Figure 13As shown in the figure, in the soil cultivation maize experiment, the TCTR of the seedlings of these 10 maize genotypes to the toxicity of 0.50% concentration of PS microplastics at 2 cultivation time periods ranged from 0.354 (maize genotype GTX-21-2) to 0.705 (maize genotype GTX-21-5). This indicates that the GTX-21-5 genotype has the strongest ability to resist microplastic toxicity, while the GTX-21-2 genotype has the weakest ability to resist microplastic toxicity. Further, according to the classification of the anti-microplastic toxicity performance of maize genotypes by TCTR, GTX-21-2 and GTX-21-4 are highly sensitive genotypes to microplastic toxicity, accounting for 20.0% of the test materials; GTX-21-1, GTX-21-9 and GTX-21-10 are sensitive genotypes to microplastic toxicity, accounting for 30.0% of the test materials; GTX-21-7 and GTX-21-8 are weakly anti-microplastic toxicity genotypes, accounting for 20.0% of the test materials; GTX-21-3 and GTX-21-6 are moderately anti-microplastic toxicity genotypes, accounting for 20.0% of the test materials; GTX-21-5 is a highly anti-microplastic toxicity genotype, only accounting for 10.0% of the test materials (Table 11). Therefore, the highly anti-microplastic toxicity genotypes identified by us provide reliable gene resources for the cultivation of maize varieties resistant to microplastic toxicity and provide technical support for ensuring the safe production of maize.

[0096] Table 9 Subordinate values U(TRI) of the resistance of 12 traits of the seedlings of 10 maize genotypes to the toxicity of 0.50% concentration of PS microplastics on the 7th day of soil cultivation maize experiment

[0097]

[0098] 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 SPAD value of leaf chlorophyll, SSI is strong seedling index, RV is root activity. Table 10 Subordinate values U(TRI) of the resistance of 12 traits of the seedlings of 10 maize genotypes to the toxicity of 0.50% concentration of PS microplastics on the 21st day of soil cultivation maize experiment

[0099]

[0100] 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 SPAD value of leaf chlorophyll, SSI is strong seedling index, RV is root activity.

[0101] Table 11 Classification results of the anti-microplastic toxicity performance of 10 maize genotypes based on the classification standard of total comprehensive resistance to microplastic toxicity (TCTR)

[0102]

[0103] As can be seen from the above embodiments, in the soil cultivation of corn experiments, we used PS microplastics at concentrations of 0.00% and 0.50% to treat different maize genotype seedlings at the 7th and 21st days of cultivation. There were significant differences in the growth phenotypes, vigorous seedling indexes, chlorophyll accumulation levels, and root activity traits of the seedlings poisoned by PS microplastics. We innovatively proposed the toxicity resistance index (TRI) of 0.50% concentration of PS microplastics for each single trait in each cultivation period as a measure and evaluation index for the resistance of corresponding maize genotype seedlings to 0.50% concentration of PS microplastics toxicity in soil cultivation of corn experiments. With the help of the membership function method, it was possible to comprehensively, multi-angularly, multi-time-dimensionally, and comprehensively evaluate the comprehensive toxicity resistance (CTR) / total comprehensive toxicity resistance (TCTR) values of each / all maize genotype seedlings to 0.50% concentration of PS microplastics toxicity at each cultivation period, and classify different maize genotypes into different types of resistance to microplastic toxicity according to the microplastic toxicity resistance performance of the TCTR maize genotypes. These studies provided scientific and technological references for the future evaluation of excellent maize genotypes (inbred lines / lines / varieties) resistant to microplastic toxicity, and thus ensured the safe production of corn, with important breeding and practical application values in production.

[0104] Example 5

[0105] The present invention provides the application of 0.50% polystyrene (PS) microplastic toxicity in the evaluation of maize genotypes resistant to microplastic toxicity, specifically as follows:

[0106] On a horizontal shaker, first soak 40 high-quality maize seeds of different genotypes disinfected with 70% ethanol (v / v) in 100 mL of 2 kinds of PS microplastic solutions with concentrations of 0.00% and 0.50% (mass ratio concentration of PS to ddH 2 O) for 24 h. At the same time, prepare 2 kinds of PS microplastic soil cultivation substrates with concentrations of 0.00% and 0.50% (mass ratio concentration of PS to nutrient soil). Then, sow 10 seeds of different maize genotypes soaked in these 2 kinds of PS microplastic solutions into flower pots filled with the same concentration of PS microplastic soil cultivation substrates and place them in an artificial climate chamber for cultivation. During the cultivation period, the relative humidity is set at 65%, the temperature is set at 25±0.5 / 20±0.5 °C for 12 h each in an alternating cycle, the photoperiod is set at 16 / 8 h light / dark, and the light intensity is set at 300 μM m - 2 s -1 ,CO 2Concentration: 450 PPM. There were a total of 2 treatments in the soil culture experiment, namely the CK control treatment (0.00% PS microplastic soil culture substrate) and the 0.50% PS treatment (0.50% PS microplastic soil culture substrate). During the cultivation period, 50 mL of ddH 2 O water was evenly poured into each flower pot every 3 days to timely supply water to the seedlings. Then, for different maize genotype seedlings cultured in these 2 PS microplastic soil culture substrates on the 7th day and the 21st day, 12 traits were measured, including 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), strong seedling index (SSI), leaf chlorophyll SPAD value (SPAD), and root activity (RV). Then, using Excel 2013 software, the mean value and standard deviation of each trait of different maize genotype seedlings cultured on the 7th day and the 21st day in the soil culture maize experiment were calculated; using IBM-SPSS Statistics 19 software, the combined variance of each trait of all maize genotypes between the 2 PS microplastic treatments at these 2 cultivation time periods was analyzed, including the F-values and significance levels of differences of the correction model (CM), intercept (IC), genotype (G), cultivation time period treatment (CT), PS microplastic concentration treatment (PSC), interaction between genotype and cultivation time period treatment (G×CT), interaction between genotype and PS microplastic concentration treatment (G×PSC), interaction between cultivation time period and PS microplastic concentration treatment (CT×PSC), and interaction among the three (G×CT×PSC); the toxicity resistance index (TRI) value of each maize genotype seedling for each trait at a 0.50% concentration of PS microplastic was calculated at each cultivation time period, and then using the membership function method, the total comprehensive resistance (TCTR) of each genotype of maize seedlings to the toxicity of 0.50% concentration of PS microplastic was comprehensively evaluated, and further the anti-microplastic toxicity performance of each genotype of maize seedlings was evaluated. According to the TCTR values of each maize genotype seedling, the anti-microplastic toxicity performance of different maize genotypes was divided into 5 grades, namely: 0≤TCTR<0.400, highly sensitive maize genotype to microplastic toxicity; 0.400≤TCTR<0.500, sensitive maize genotype to microplastic toxicity; 0.500≤TCTR<0.600, weakly anti-microplastic toxicity maize genotype; 0.600≤TCTR<0.700, moderately anti-microplastic toxicity maize genotype; 0.700≤TCTR≤1.000, highly anti-microplastic toxicity maize genotype.

Claims

1. A comprehensive evaluation method for corn seedlings' resistance to microplastic toxicity, characterized in that The method comprises the following steps: (1) preparing high-quality corn seeds; (2) testing soil-grown corn with different concentrations of polystyrene (PS) microplastics; (3) collecting data; (4) establishing a data statistical model; (5) establishing a resistance index of corn seedlings to the toxicity of polystyrene (PS) microplastics at different concentrations; (6) evaluating the comprehensive resistance of corn seedlings to the toxicity of polystyrene (PS) microplastics at different concentrations; and (7) comprehensively measuring the PS microplastic concentration when the toxicity of polystyrene (PS) microplastics to corn seedlings is the most serious.

2. According to claim 1, a comprehensive evaluation method for the resistance of corn seedlings to microplastic toxicity is characterized in that The (1) high-quality corn seed preparation includes: preparing high-quality corn genotype new seeds with full grains, uniform size, strong vitality and high purity for future use.

3. According to claim 1, a comprehensive evaluation method for the resistance of corn seedlings to microplastic toxicity is characterized in that The (2) experiment of cultivating corn with polystyrene (PS) microplastics in soil at different concentrations includes: sterilizing high-quality corn seeds with 70% ethanol for 10 minutes, washing the seeds with ddH2O water for 5 times to remove the ethanol remaining on the surface of the seeds, and absorbing the water attached to the surface of the seeds with sterilized filter paper to obtain the corresponding sterilized corn genotype seeds; preparing 6 concentrations of PS microplastic solutions of 0.00%, 0.05%, 0.25%, 0.50%, 0.75% and 1.00% in advance, and preparing them on the spot; soaking the sterilized corn genotype seeds with 6 concentrations of PS microplastic solutions for 24 hours respectively; and preparing 6 concentrations of PS microplastic soil culture substrates, including 0 0.00% PS microplastic soil culture substrate, 0.05% PS microplastic soil culture substrate, 0.25% PS microplastic soil culture substrate, 0.50% PS microplastic soil culture substrate, 0.75% PS microplastic soil culture substrate, and 1.00% PS microplastic soil culture substrate; then 10 maize seeds of each genotype soaked in PS microplastic solution of corresponding concentration were sown in pots filled with PS microplastic soil culture substrate of the same concentration and placed in an artificial climate chamber for culture; during the culture period, the relative humidity was set at 65%, the temperature was set at 25±0.5 / 20±0.5℃ alternating cycles of 12h each, the photoperiod was set at 16 / 8h light / darkness, and the light intensity was set at 300μM m -2 s -1 , CO2 concentration 450PPM; there were 6 treatments in the soil culture test, namely CK control treatment, 0.05% PS treatment, 0.25% PS treatment, 0.50% PS treatment, 0.75% PS treatment and 1.00% PS treatment, and each treatment had 4 biological replicates; at the same time, 50mL of ddH2O water was evenly poured into each pot every 3 days during the culture period to replenish water to the seedlings in time.

4. According to claim 1, a comprehensive evaluation method for the resistance of corn seedlings to microplastic toxicity is characterized in that The (3) data collection includes: measuring the 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), whole plant dry biomass (PDW), leaf chlorophyll SPAD value (SPAD), root activity (RV) and seedling strength index (SSI) of corn seedlings cultured on the 7th and 21st days in the six PS microplastic soil culture media; SSI is specifically calculated according to formula (1): SSI = (SD / SL) × PDW (1); where: SSI is the seedling strength index, SD is the stem diameter, SL is the seedling length, and PDW is the whole plant dry biomass.

5. According to claim 1, a comprehensive evaluation method for the resistance of corn seedlings to microplastic toxicity is characterized in that The (4) data statistical model was established: using Excel 2013 software to calculate the mean and standard deviation of each trait of corn seedlings on the 7th and 21st days of cultivation in the soil-cultured corn experiment, and to draw a bar graph; using IBM-SPSS Statistics 19 software to analyze the joint variance of each trait of corn genotypes in the two cultivation time periods and the six PS microplastic concentrations treatments in the soil-cultured corn experiment; using IBM-SPSS Statistics 19 software to analyze the Dunman significance of each trait of corn seedlings in each cultivation time period in the soil-cultured corn experiment among the six PS microplastic concentrations treatments at the P<0.05 level; using GENESCLOUD online software to analyze the Pearson correlation coefficient graph of all traits of corn seedlings in each cultivation time period among the six PS microplastic concentrations treatments in the soil-cultured corn experiment; using the LOG10 function to standardize the mean values ​​of all traits of corn seedlings in the soil-cultured corn experiment among the six PS microplastic concentrations treatments in the two cultivation time periods, and then using IBM-SPSS Statistics 19 software to analyze the Dunman significance of each trait of corn seedlings in each cultivation time period among the six PS microplastic concentrations treatments. The Between-groups Linkage clustering method of Statistics16.0 software was used to conduct a scientific, objective and qualitative cluster analysis on the degree of microplastic toxicity of corn genotype seedlings under six PS microplastic concentrations treatments in each cultivation period in the soil-cultured corn experiment.

6. According to claim 1, a comprehensive evaluation method for the resistance of corn seedlings to microplastic toxicity is characterized in that (5) Establishment of the resistance index of corn seedlings to the toxicity of polystyrene (PS) microplastics at different concentrations: In order to scientifically, objectively and accurately reflect the resistance of individual traits of corn genotype seedlings to the toxicity of PS microplastics at different concentrations in each culture period in the soil-cultured corn experiment, the present invention newly defines the resistance index (TRI) of corn seedlings to the toxicity of PS microplastics at different concentrations, as shown in formula (2): (2); where: is the resistance index of PS microplastic toxicity of the jth trait and the kth concentration of corn seedlings in the soil-cultured corn experiment during the i-th cultivation period, is the measured value of the jth trait of corn seedlings of the genotype in the ith cultivation period under the CK control treatment in the soil-cultured corn experiment, is the measured value of the jth trait of the maize seedlings of the genotype under the kth concentration of PS microplastic treatment in the i-th cultivation period in the soil-cultured maize experiment, i is the 7th day or 21st day of cultivation in the soil-cultured maize experiment, and k-PS is the 0.05%, 0.25%, 0.50%, 0.75% or 1.00% concentration of PS microplastic treatment in the soil-cultured maize experiment; The larger the value, the stronger the resistance of the jth trait of the corn seedlings of the soil-cultured corn experiment to the toxicity of microplastics at the k-PS concentration in the i-th cultivation period.

7. According to claim 1, a comprehensive evaluation method for the resistance of corn seedlings to microplastic toxicity is characterized by (6) Evaluation of the comprehensive resistance of corn seedlings to the toxicity of polystyrene (PS) microplastics at different concentrations: The TRI values ​​of all traits of the corn seedlings calculated in step (5) are used as evaluation indicators for the resistance of the corn seedlings to the toxicity of PS microplastics at different concentrations in two cultivation time periods in the soil-cultured corn experiment. The membership function method is used to comprehensively, objectively and quantitatively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) of the corn seedlings to the toxicity of PS microplastics at different concentrations in each / all cultivation time periods of the soil-cultured corn experiment. For details, see formulas (3), (4) and (5): and Where: is the membership value of the resistance of the jth trait of corn seedlings to the kth concentration of PS microplastic toxicity in the i-th cultivation period in the soil-cultured corn experiment, is the resistance index of PS microplastic toxicity of the jth trait and the kth concentration of corn seedlings in the soil-cultured corn experiment during the i-th cultivation period, is the minimum TRI value of the jth trait of corn seedlings of the genotype in the i-th culture period among all concentrations of PS microplastic treatments in the soil-cultured corn experiment, is the maximum TRI value of the jth trait of corn seedlings of the genotype in the i-th culture period among all concentrations of PS microplastic treatments in the soil-cultured corn experiment, is the comprehensive resistance value of corn seedlings of the genotype to the toxicity of PS microplastics at the kth concentration in the i-th cultivation period in the soil-cultured corn experiment, m is the m=12 traits measured in the corn seedlings of the genotype in the i-th cultivation period in the soil-cultured corn experiment, TCTR (k-PS) is the total comprehensive resistance value of corn genotype seedlings to the toxicity of PS microplastics at the kth concentration in the n cultivation time periods in the soil-cultured corn experiment, i is the 7th day or the 21st day of cultivation in the soil-cultured corn experiment, and k-PS is the PS microplastic treatment with a concentration of 0.05%, 0.25%, 0.50%, 0.75% or 1.00% in the soil culture experiment; The larger the value, the stronger the comprehensive resistance of the maize genotype seedlings to the toxicity of the kth concentration of PS microplastics in the i-th cultivation period in the soil-cultured maize experiment. (k-PS) The larger the value, the stronger the overall comprehensive resistance of the corn genotype seedlings to the toxicity of PS microplastics at the kth concentration in the two cultivation time periods in the soil-cultured corn experiment.

8. According to claim 1, a comprehensive evaluation method for the resistance of corn seedlings to microplastic toxicity is characterized in that The (7) comprehensive measurement of the concentration of polystyrene (PS) microplastics when the PS microplastic toxicity of corn seedlings is the most serious: by comparing the measurement results of the Between-groups Linkage comprehensive clustering of PS microplastic toxicity of corn genotype seedlings in all treatments under two culture time periods in the soil-cultured corn experiment in step (4), and the evaluation results of the overall comprehensive resistance of corn genotype seedlings to the toxicity of PS microplastics of corresponding concentrations under two culture time periods in the soil-cultured corn experiment in step (6), a combined multi-method comparative evaluation of the toxicity of PS microplastics of different concentrations in corn genotype seedlings under multiple culture time periods is performed, and finally a comprehensive measurement of the PS microplastic concentration when the PS microplastic toxicity of corn genotype seedlings is the most serious.

9. A method for evaluating corn genotypes resistant to microplastic toxicity, characterized in that The method comprises the following steps: (1) Preparation of high-quality corn seeds: Prepare high-quality new corn seeds of different genotypes with full, uniform grain size, strong vitality and high purity that will be harvested at the same ecological point in the same year for future use; (2) 0.50% polystyrene (PS) microplastic soil culture corn experiment: high-quality corn seeds of different genotypes were disinfected with 70% ethanol for 10 min, and the seeds were rinsed with ddH2O water for 5 times to remove the ethanol residue on the seed surface. The sterilized filter paper was used to absorb the water attached to the seed surface to obtain the corresponding disinfected corn seeds of the genotypes; the corn seeds of the genotypes disinfected with 0.00% and 0.50% PS microplastic solutions were soaked for 24 h, and then 10 seeds of the different genotypes of corn soaked with the two concentrations of PS microplastic solutions were sown in flower pots with 0.00% and 0.50% PS microplastic soil culture substrates, respectively, and placed in an artificial climate chamber for culture; during the culture period, the relative humidity was set at 65%, the temperature was set at 25±0.5 / 20±0.5℃ for 12 h each, the photoperiod was set at 16 / 8 h light / dark, and the light intensity was set at 300 μM m -2 s -1 , CO2 concentration 450PPM; There were two treatments in the soil culture test, namely CK control treatment and 0.50% PS treatment, and each treatment had 4 biological replicates; At the same time, 50mL of ddH2O water was evenly poured into each flower pot every 3 days during the culture period to replenish water for the seedlings in time; Twelve traits of different maize seedlings of different genotypes cultured in these two PS microplastic soil culture substrates on the 7th and 21st days were measured; (3) Data statistical analysis: Excel 2013 software was used to calculate the mean and standard deviation of each trait of seedlings of different maize genotypes at the 7th and 21st days of cultivation under each treatment in the soil-cultured maize experiment; IBM-SPSS Statistics 19 software was used to analyze the joint variance of each trait of all maize genotypes under the two concentrations of PS microplastic treatments in these two cultivation time periods; (4) Resistance index of corn seedlings of different genotypes to 0.50% concentration of polystyrene (PS) microplastics: According to the mean values ​​of 12 traits of corn seedlings of different genotypes on the 7th and 21st days of cultivation in the soil-cultured corn experiment with two concentrations of PS microplastics in step (3), the resistance index (TRI) value of each trait of each corn seedling of each genotype in each cultivation period to 0.50% concentration of PS microplastics was further calculated, as shown in formula (6): Where: is the resistance index of 0.50% concentration of PS microplastics to the jth trait of the pth corn genotype seedlings in the i-th culture period in the soil-cultured corn experiment, is the measured value of the jth trait of the pth corn genotype seedlings under the CK control treatment in the i-th culture period in the soil-cultured corn experiment, is the measured value of the jth trait under the treatment of 0.50% concentration of PS microplastics in the i-th culture period of the p-th corn genotype seedlings in the soil-cultured corn experiment, i is the 7th day or the 21st day of the soil-cultured corn experiment, and 0.50% PS is the treatment of 0.50% concentration of PS microplastics in the soil-cultured corn experiment; The larger the value, the stronger the resistance of the jth trait to the toxicity of 0.50% concentration of microplastics in the i-th culture period of the p-th corn genotype seedlings in the soil-cultured corn experiment; (5) Evaluation of the comprehensive resistance of corn seedlings of different genotypes to the toxicity of 0.50% polystyrene (PS) microplastics: The TRI values ​​of all traits of corn seedlings of different genotypes calculated in step (4) were used as evaluation indicators for the resistance of corn seedlings of different genotypes to the toxicity of 0.50% PS microplastics in two culture time periods in the soil-cultured corn experiment. The membership function method was used to comprehensively evaluate the comprehensive resistance (CTR) / total comprehensive resistance (TCTR) values ​​of corn seedlings of different genotypes to the toxicity of 0.50% PS microplastics in each / all culture time periods of the soil-cultured corn experiment, specifically: and Where: is the membership value of the resistance of the jth trait to the toxicity of 0.50% concentration of PS microplastics in the i-th culture period of the p-th corn genotype seedlings in the soil-cultured corn experiment, is the resistance index of 0.50% concentration of PS microplastics to the jth trait of the pth corn genotype seedlings in the i-th culture period in the soil-cultured corn experiment, is the minimum TRI value of the jth trait of all maize genotype seedlings in the soil-cultured maize experiment at the i-th culture period, is the maximum TRI value of the jth trait of all maize genotype seedlings in the soil-cultured maize experiment at the i-th cultivation period, is the comprehensive resistance value of the pth corn genotype seedlings to 0.50% concentration of PS microplastics in the i-th cultivation period in the soil-cultured corn experiment, m is the m=12 traits measured for each corn genotype seedling in the i-th cultivation period in the soil-cultured corn experiment, TCTR p(0.50％PS) is the total comprehensive resistance value of the p-th corn genotype seedlings in the soil-cultured corn experiment for n (n=2, 2 culture time periods) culture time periods to the toxicity of 0.50% concentration PS microplastics, i is the 7th day or 21st day of the soil-cultured corn experiment, and 0.50% PS is the 0.50% concentration PS microplastic treatment in the soil-cultured experiment; The larger the value, the stronger the comprehensive resistance of the pth corn genotype seedlings to the toxicity of 0.50% concentration of PS microplastics in the i-th culture period in the soil-cultured corn experiment. (0.50％PS) The larger the value, the stronger the overall comprehensive resistance of the pth corn genotype seedlings to the toxicity of 0.50% PS microplastics in the two culture periods in the soil-cultured corn experiment; according to the TCTR value of each corn genotype seedling, the resistance of different corn genotypes to microplastic toxicity is divided into 5 levels, namely: 0≤TCTR<0.400, which is a corn genotype highly sensitive to microplastic toxicity, 0.400≤TCTR<0.500, which is a corn genotype sensitive to microplastic toxicity, 0.500≤TCTR<0.600, which is a corn genotype with weak resistance to microplastic toxicity, 0.600≤TCTR<0.700, which is a corn genotype with moderate resistance to microplastic toxicity, and 0.700≤TCTR≤1.000, which is a corn genotype with high resistance to microplastic toxicity. 10.0.50% polystyrene (PS) microplastic toxicity is used to evaluate corn genotypes resistant to microplastic toxicity, characterized in that The application is specifically as follows: soaking high-quality corn seeds of different genotypes disinfected with 70% ethanol in two PS microplastic solutions with concentrations of 0.00% and 0.50% for 24 hours, and then sowing 10 seeds of different corn genotypes soaked in the two PS microplastic solutions with concentrations of 0.00% and 0.50% respectively into flower pots of two PS microplastic soil culture substrates with concentrations of 0.00% and 0.50%, and placing them in an artificial climate chamber for cultivation; during the cultivation period, the relative humidity is set to 65%, the temperature is set to 25±0.5 / 20±0.5℃ alternating cycles of 12 hours each, the photoperiod is set to 16 / 8h light / darkness, and the light intensity is set to 300μM m -2 s -1 , CO2 concentration 450PPM; There were two treatments in the soil culture experiment, namely CK control treatment and 0.50% PS treatment; During the culture period, 50mL of ddH2O water was evenly poured into each flower pot every 3 days to replenish water for the seedlings in time; Then, the 12 traits of different maize genotypes seedlings cultured on the 7th and 21st days of these two PS microplastic soil culture substrates were measured; The mean and standard deviation of each trait of different maize genotypes seedlings cultured on the 7th and 21st days in the soil culture maize experiment were calculated using Excel 2013 software; IBM-SPSS Statistics 19 software was used to analyze the joint variance of each trait of all maize genotypes under the two concentrations of PS microplastics treatments in the two culture time periods; the resistance index (TRI) value of 0.50% concentration of PS microplastics for each trait of each maize genotype seedling in each culture time period was calculated, and then the membership function method was used to comprehensively evaluate the total comprehensive resistance (TCTR) of each genotype of maize seedlings to 0.50% concentration of PS microplastics, and then the resistance of each genotype of maize seedlings to microplastics was evaluated. The CTR value divides the resistance of different corn genotypes to microplastic toxicity into five levels, namely: 0≤TCTR<0.400, which is a corn genotype highly sensitive to microplastic toxicity; 0.400≤TCTR<0.500, which is a corn genotype sensitive to microplastic toxicity; 0.500≤TCTR<0.600, which is a corn genotype with weak resistance to microplastic toxicity; 0.600≤TCTR<0.700, which is a corn genotype with moderate resistance to microplastic toxicity; 0.700≤TCTR≤1.000, which is a corn genotype with high resistance to microplastic toxicity.

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