Method for rapidly evaluating plant growth capability of transgenic herbicide-resistant cotton in seedling stage
By spraying glyphosate during the cotyledon unfolding stage of cotton and testing the aboveground dry weight and rhizosphere soil indicators, the problem of long evaluation cycles and high costs in existing technologies for evaluating the resistance of transgenic cotton has been solved. This has enabled a rapid and standardized evaluation of plant growth capacity, which is suitable for large-scale screening.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF COTTON RES CHINESE ACAD OF AGRI SCI
- Filing Date
- 2025-11-13
- Publication Date
- 2026-05-01
AI Technical Summary
Existing technologies for evaluating the resistance of genetically modified herbicide-tolerant cotton suffer from long cycles, high costs, significant susceptibility to environmental factors, and limited evaluation indicators, failing to meet the demand for high-throughput, rapid, and precise screening of resistant materials.
The method of spraying 0, 2, or 4 times the concentration of glyphosate when the cotton cotyledons are fully expanded was adopted. After 7 days, the dry weight of the aboveground parts of the cotton, the soil moisture content of the rhizosphere, and the microbial diversity of the rhizosphere were measured. The plant growth capacity was judged by statistical methods. Standardized containers and spraying systems were used to ensure consistency of operation.
It enables rapid, standardized, and repeatable evaluation of plant growth capacity, shortens the identification cycle, improves screening efficiency, reduces external interference, and provides stable and reliable results. It is suitable for multi-material and large-scale testing and is easy to promote and apply.
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Abstract
Description
A method for rapidly evaluating the growth ability of herbicide-tolerant transgenic cotton seedlings Technical Field
[0001] This invention belongs to the field of genetic engineering, specifically relating to a method for evaluating the growth capacity of transgenic herbicide-tolerant cotton seedlings. Background Technology
[0002] Cotton is an important economic crop in my country, and weed infestations consistently reduce cotton yields by 14% to 16% annually, severely hindering high-yield and high-quality cotton production. Chemical weeding is the main method for weed control in cotton fields, with glyphosate being widely used due to its broad-spectrum, high-efficiency, and low-toxicity advantages. However, as a non-selective herbicide, glyphosate can easily cause severe phytotoxicity to non-target crops, especially common cotton varieties, manifesting as inhibited plant height, yellowing leaves, reduced photosynthetic capacity, and impaired reproductive development, even leading to plant death. Furthermore, long-term and excessive use of glyphosate can lead to soil residues, environmental pollution, and increased herbicide resistance in weeds, seriously affecting the safe production and sustainable development of cotton. Therefore, while promoting herbicides such as glyphosate, it is crucial to pay close attention to their potential risks and seek scientific methods for their use and evaluation.
[0003] To address the phytotoxicity of cotton caused by glyphosate, the breeding and promotion of transgenic herbicide-resistant cotton has become an important direction. By introducing exogenous glyphosate-resistant genes (such as gl0-epsps, GR79epsps, and GAT), cotton can acquire tolerance to glyphosate, allowing it to continue growing normally after glyphosate application. This type of transgenic cotton can not only effectively control weeds and reduce the cost of manual weeding, but also improve the safety and efficiency of herbicide use, significantly enhancing the economic and ecological benefits of cotton field management. Currently, several glyphosate-resistant cotton lines have been successfully bred both domestically and internationally. Some materials can tolerate more than four times the recommended dose of glyphosate without significant impact on agronomic traits, demonstrating promising application prospects.
[0004] Currently, the evaluation of herbicide-tolerant transgenic cotton resistance largely relies on field spraying trials or greenhouse cultivation observations. Resistance levels are determined by observing plant survival rates and growth vigor after spraying with a certain concentration of glyphosate. In addition, some studies have used the glyphosate seed soaking method for germination-stage resistance identification. While this method is faster than field trials, it is still limited to the seed germination stage and cannot comprehensively reflect the overall growth capacity and resistance stability of seedlings. Existing methods suffer from problems such as long cycles, high costs, significant susceptibility to environmental factors, and limited evaluation indicators, failing to meet the demand for high-throughput, rapid, and precise screening of resistant materials. Summary of the Invention
[0005] The technical problem to be solved by this invention is to provide a rapid, standardized, and repeatable method for evaluating the growth capacity of seedling plants. This method is particularly suitable for multi-material, large-scale testing and is easy to promote and apply in the agricultural field. It is of urgent and significant practical importance for accelerating the breeding and industrial application of glyphosate-resistant cotton varieties. The technical solution provided by this invention is: a method for rapidly evaluating the growth capacity of transgenic herbicide-resistant cotton seedlings, the method comprising:
[0006] S1 is planted with transgenic cotton seeds to be tested;
[0007] S2 involves spraying glyphosate at 0x, 2x, or 4x the working concentration when the cotton cotyledons are fully expanded. The 0x glyphosate working concentration is the control group, and the other concentrations are the experimental groups. The concentration of glyphosate is the concentration of glyphosate in water.
[0008] Seven days after S3 spraying, the aboveground parts of cotton and rhizosphere soil were taken to test the dry weight of the aboveground parts, the moisture content of the rhizosphere soil, and the microbial diversity of the rhizosphere soil.
[0009] S4 assesses the growth capacity of herbicide-tolerant cotton seedlings by comparing whether there are statistically significant differences in the aboveground dry weight and rhizosphere soil moisture content between the experimental and control groups.
[0010] In some implementations, spraying can be accomplished using a spray tower.
[0011] In some implementations, the spray pressure of the spray tower can be 0.1~0.3 MPa to ensure uniform spraying.
[0012] In some implementations, the sowing of the cotton seeds to be tested can be carried out inside a container.
[0013] In some embodiments, the container can be a polyethylene plastic bowl with a diameter of 18 cm and a height of 20 cm. The bottom of the bowl can have seven water-permeable holes with a diameter of 1 cm, and the water-permeable holes can be covered with a water-permeable material. Preferably, the water-permeable material can be a single layer of filter paper.
[0014] In some embodiments, the glyphosate may be Roundup containing 41% glyphosate isopropylamine salt, which is manufactured by Monsanto, Inc.
[0015] In some implementations, the criteria for judging the growth capacity of transgenic herbicide-tolerant cotton seedlings can be as follows: Seven days after spraying glyphosate at a concentration more than twice the working level, the aboveground dry weight of cotton in the experimental group and the control group is measured. If the experimental group is significantly higher than the control group in a statistically significant manner, then the experimental group has strong growth capacity. The rhizosphere soil moisture content of cotton in the experimental group and the control group is measured 14 days and beyond after spraying glyphosate at a concentration more than twice the working level. If the experimental group is significantly lower than the control group in a statistically significant manner, then the experimental group has strong growth capacity. The rhizosphere soil moisture content of cotton in the experimental group and the control group is measured 7 days after spraying glyphosate at a concentration more than four times the working level. If the experimental group is significantly lower than the control group in a statistically significant manner, then the experimental group has strong growth capacity. In other cases, the experimental group has weak growth capacity.
[0016] The methods for measuring the aboveground dry weight and rhizosphere soil moisture content are common in this field. Moreover, the statistical analysis used to determine whether there is a statistically significant difference in the aboveground dry weight and rhizosphere soil moisture content of cotton between the experimental group and the control group can be performed using multiple comparison analysis software or methods known in this field, such as Duncans' new multiple range method and LSD minimum significant difference method.
[0017] Compared with the prior art, the present invention has the following advantages:
[0018] (1) The spraying time is precisely optimized, which is time-saving and efficient. This invention selects to spray glyphosate when the cotton cotyledons are fully expanded. At this time, the cotton is metabolically active and sensitive to herbicides. It can clearly distinguish between resistant and non-resistant materials in a short period of time. Compared with the traditional method of spraying in the middle and late stages of seedling, it significantly shortens the identification cycle. The entire evaluation process only takes 2-3 weeks, which improves the screening efficiency.
[0019] (2) It occupies little space. The cotton is cultivated in small plastic pots, and each identification material is set up with 3 treatments. Each treatment is repeated 3 times, and each repetition is in 1 pot, requiring only 9 pots.
[0020] (3) Low cost: The determination of the dry weight of the aboveground part of cotton and the soil moisture content of the rhizosphere are routine operations and no additional reagents are required. The soil microbiome determination technology is mature and inexpensive.
[0021] (4) High accuracy and good repeatability, stable and reliable results. The entire evaluation process is carried out in an indoor controllable environment to avoid external interference and eliminate the interference of uncontrollable factors such as climate and soil heterogeneity in field trials, ensuring high consistency and repeatability of the identification results.
[0022] (5) Standardized containers and spraying systems are used to ensure consistent operation and comparable results, effectively reducing errors caused by differences in containers or uneven spraying and improving the reliability of the method.
[0023] (6) The combined evaluation of the aboveground dry weight and rhizosphere soil moisture content, combined with the high-throughput omics data of the rhizosphere soil microbiome, can not only directly reflect the plant biomass accumulation, but also indirectly reflect the root water absorption capacity and soil water use efficiency. It is superior to the single-index evaluation method for comprehensively judging the overall growth capacity of the plant after glyphosate treatment.
[0024] (7) Set up multiple concentration gradients and multiple time points for observation to enhance the dynamism and accuracy of identification and avoid misjudgment caused by single time point observation.
[0025] Therefore, the identification method of the present invention is not limited by season, can be repeated, and has good consistency of results. The technical method of the present invention is particularly suitable for the determination of multiple materials and large quantities, and is easy to promote and apply in the agricultural field. Attached Figure Description
[0026] Figure 1. Morphology of the aboveground parts of cotton after spraying with water.
[0027] Figure 2. Morphology of the aboveground parts of cotton after spraying with 2 times the concentration of glyphosate.
[0028] Figure 3. Morphology of the aboveground parts of cotton after spraying with 4 times the concentration of glyphosate.
[0029] Figure 4. Dry weight of aboveground parts of wild-type and herbicide-tolerant cotton plants at different times after spraying with different concentrations of glyphosate.
[0030] Figure 5. Aboveground dry weight of wild-type and herbicide-tolerant cotton plants under different treatments. In Figure 5, A represents the aboveground dry weight of wild-type and herbicide-tolerant cotton plants 7 days after spraying different concentrations of glyphosate; B represents the aboveground dry weight of wild-type and herbicide-tolerant cotton plants 28 days after spraying different concentrations of glyphosate; C represents the aboveground dry weight of wild-type cotton plants at different times after spraying different concentrations of glyphosate; and D represents the aboveground dry weight of wild-type and herbicide-tolerant cotton plants at different times after spraying with glyphosate at twice the working concentration.
[0031] Figure 6. Rhizosphere soil moisture content of wild-type and herbicide-tolerant cotton at different times after spraying with different concentrations of glyphosate.
[0032] Figure 7. Rhizosphere soil moisture content of wild-type and herbicide-tolerant cotton under different treatments. A. Rhizosphere soil moisture content of wild-type and herbicide-tolerant cotton 0 days after spraying different concentrations of glyphosate; B. Rhizosphere soil moisture content of herbicide-tolerant cotton at different times after spraying different concentrations of glyphosate; C. Rhizosphere soil moisture content of wild-type and herbicide-tolerant cotton 28 days after spraying different concentrations of glyphosate; D. Rhizosphere soil moisture content of wild-type and herbicide-tolerant cotton at different times after spraying 4 times the working concentration of glyphosate.
[0033] Figure 8. Rhizosphere soil microorganisms of wild-type and herbicide-tolerant cotton at different time points after spraying with different concentrations of glyphosate. Diversity.
[0034] Figure 9. Rhizosphere soil microbial β-diversity at different time points after spraying different concentrations of glyphosate on wild-type and herbicide-tolerant cotton. Detailed Implementation
[0035] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.
[0036] I. Materials and Methods
[0037] 1. Materials
[0038] The transgenic herbicide-tolerant cotton material KJDLC517T20506 and its wild-type control, Xinluzao 45 cotton, were both developed by Kejidalong (Beijing) Biotechnology Co., Ltd.
[0039] 2. Experimental Design and Methods
[0040] 2.1 Experimental Design
[0041] Control and transgenic herbicide-tolerant cotton were cultured in polyethylene plastic pots with a diameter of 18 cm, a height of 20 cm, and 7 drainage holes with a diameter of 1 cm at the bottom. Glyphosate at 0, 2, and 4 times the working concentration was sprayed on the experimental materials when the cotyledons were fully expanded. At weeks 0, 1, 2, 3, and 4 after spraying, the aboveground parts of the plants and the rhizosphere soil were collected (a cylinder with a diameter of 8 cm and a depth of 8 cm centered on the taproot) for subsequent testing.
[0042] 2.2 Test Methods
[0043] Cut off the entire above-ground part of the cotton plant and flatten the leaves. Place it next to a steel ruler with a range of one meter and take a picture to record the shape of the above-ground part.
[0044] Weigh the fresh weight of the above-ground part of the cotton, then place it in an 80-degree Celsius oven and weigh it every hour until the weight no longer changes; this is the dry weight of the above-ground part.
[0045] A random sample of soil from the cotton rhizosphere was placed in an aluminum box with a diameter of 46 mm and a height of 25 mm. The total weight was recorded, and the weight of the aluminum box was subtracted from the total weight to obtain the wet weight of the soil. The soil was then placed in a well-ventilated and dry indoor environment, and its weight was recorded every 3 days until the weight no longer changed. The weight of the aluminum box was then subtracted from the wet weight to obtain the dry weight of the soil. Soil moisture content = (wet weight of soil - dry weight of soil) / dry weight of soil 100%; an additional portion of rhizosphere soil was used for microbiome sequencing, with 3-5 replicates per sample.
[0046] II. Results and Analysis
[0047] 1. Effects of glyphosate spraying on the aboveground morphology of cotton
[0048] Seven days after spraying control and transgenic herbicide-tolerant cotton with 0 times the working concentration of glyphosate, there were no significant differences in the morphology of cotton leaves and stems, and they continued to grow normally without significant differences until 28 days later (Figure 1).
[0049] Seven days after spraying with glyphosate at twice the working concentration, the control cotton plants showed obvious wilting of a few leaves and slight yellowing of the stems. By day 28, the plants had died completely. In contrast, the herbicide-tolerant cotton plants maintained normal growth under the same concentration of glyphosate (Figure 2).
[0050] Seven days after spraying with 4 times the working concentration of glyphosate, the control cotton plants showed significant wilting of many leaves and most of the stems turned yellowish-brown. By day 28, the plants had completely died. In contrast, the herbicide-tolerant cotton plants maintained normal growth under the same concentration of glyphosate (Figure 3). This demonstrates that spraying with 2 times the working concentration of glyphosate had an impact on cotton within a week, while the transgenic herbicide-tolerant cotton plants could tolerate at least 4 times the working concentration of glyphosate, and their growth remained unaffected up to four weeks after spraying.
[0051] 2. Effects of glyphosate application on the aboveground dry weight of cotton
[0052] The aboveground dry weights of the control group sprayed with water and the transgenic herbicide-tolerant cotton plants sprayed with 0, 2, and 4 times the working concentration of glyphosate were similar. One week after spraying, the average aboveground dry weights were 1.63, 1.40, 1.68, and 1.14 g / plant, respectively. Three weeks after spraying, the aboveground dry weights of the transgenic herbicide-tolerant cotton plants treated with 2 and 4 times the working concentration of glyphosate were 1.87 and 2.00 g / plant, respectively, which were 32%-48% heavier than the control group and the transgenic herbicide-tolerant cotton plants that were not sprayed with glyphosate at the same time point, and 164% and 300% heavier than the control group treated with the same concentration of glyphosate, respectively. Four weeks after spraying, the aboveground dry weight of the control cotton plants treated with 4 times the working concentration of glyphosate was not significantly different from that three weeks after spraying and could be considered to have died completely. The dry weights of the transgenic herbicide-tolerant cotton plants treated with the same concentration of glyphosate were similar to those treated with other concentrations of glyphosate and showed no significant difference (Figure 4). Overall, the aboveground dry weight of wild-type cotton decreased over time under different working concentrations of glyphosate treatment; while the aboveground dry weight of genetically modified herbicide-tolerant cotton increased over time.
[0053] As shown in Figure 5A, the aboveground dry weight of wild-type and herbicide-tolerant cotton showed a highly significant difference after 7 days of application of glyphosate at 2x and 4x the working concentrations, increasing by 109.21% and 62.16%, respectively; while the aboveground dry weight of cotton without glyphosate application showed no significant difference at the same time. This phenomenon became more pronounced after 28 days of application, with the highly significant differences in aboveground dry weight between wild-type and herbicide-tolerant cotton at 2x and 4x the working concentrations of glyphosate increasing by 371.41% and 291.15%, respectively (Figure 5B). For wild-type cotton, the aboveground dry weight increased with increasing application time when glyphosate was not applied; however, when glyphosate was applied at 2x and 4x the working concentrations, the aboveground dry weight decreased with increasing time, and a significant difference appeared from 7 days after application (Figure 5C). Taking the application of glyphosate at twice the working concentration as an example, on the 7th day after application, there were extremely significant differences in the aboveground dry weight of wild-type and herbicide-tolerant cotton, which increased by 108.98%, 283.05%, 192.26%, and 371.41%, respectively (D in Figure 5).
[0054] In summary, after spraying glyphosate at 2 times or higher working concentrations for 7 days, the aboveground dry weight of transgenic herbicide-tolerant cotton can be measured to determine whether its seedling growth capacity is significantly higher than that of wild type.
[0055] 3. Effects of glyphosate spraying on rhizosphere soil moisture content in cotton
[0056] One week after spraying different working concentrations of glyphosate, the rhizosphere soil moisture content of the control and transgenic herbicide-tolerant cotton was 57.17%, 51.38%, 51.89%, 56.34%, 52.38%, and 55.89%, respectively, with no significant difference. Three weeks after spraying, the rhizosphere soil moisture content of the control cotton sprayed with 0 times the working concentration of glyphosate was 67.43%, slightly lower than that of the transgenic herbicide-tolerant cotton at the same concentration by 7.84%. The rhizosphere soil moisture content of the control cotton sprayed with 2 times and 4 times the working concentration of glyphosate was 25.61% and 16.92% lower than that of the transgenic herbicide-tolerant cotton at the same concentration, respectively (Figure 6). Overall, the rhizosphere soil moisture content of wild-type cotton decreased with increasing time under different working concentrations of glyphosate treatment; the rhizosphere soil moisture content of herbicide-tolerant cotton also decreased with increasing time.
[0057] As shown in Figure 7A, without glyphosate application, there was no significant difference in rhizosphere soil moisture content between wild-type and transgenic herbicide-tolerant cotton. However, 28 days after applying 2x and 4x the working concentration of glyphosate, the rhizosphere soil moisture content of transgenic herbicide-tolerant cotton showed a highly significant difference compared to the wild-type, decreasing by 30.67% and 41.25%, respectively (Figure 7B). For transgenic herbicide-tolerant cotton, there was no significant difference in rhizosphere soil moisture content from 0 to 21 days after application of different working concentrations of glyphosate. It wasn't until day 28 that the rhizosphere soil moisture content decreased with increasing glyphosate working concentration, decreasing by 8.03% and 27.31% at 2x and 4x the working concentrations, respectively (Figure 7C). Taking the application of glyphosate at 4 times the working concentration as an example, on the 7th day after application, there were extremely significant differences in the rhizosphere soil moisture content of wild-type and herbicide-tolerant cotton, which decreased by 16.38%, 41.59%, 38.98% and 41.25%, respectively (D in Figure 7).
[0058] In summary, the seedling growth capacity of herbicide-tolerant cotton can be determined by measuring whether the rhizosphere soil moisture content of the transgenic herbicide-tolerant cotton is significantly lower than that of the wild type 14 days after spraying with 2 times the working concentration or 7 days after spraying with 4 times the working concentration.
[0059] 4. Effects of glyphosate spraying on rhizosphere microbial diversity in cotton rhizosphere soil
[0060] Rhizosphere soil microorganisms of control and transgenic herbicide-tolerant cotton on day 1 after application of different working concentrations of glyphosate. Diversity (Shannon index) was similar, approximately 2.5–2.6. On day 7 post-spraying, the Shannon index of rhizosphere soil microorganisms in transgenic herbicide-tolerant cotton sprayed with 2x and 4x the working concentration of glyphosate peaked, reaching a maximum of 3.0; while the control remained stable at 2.6–2.8. 14 and 21 days after spraying with 2x the working concentration of glyphosate, the Shannon index of rhizosphere soil microorganisms in transgenic herbicide-tolerant cotton fluctuated more significantly (2.7–3.0), showing a wave-like decline, reaching approximately 2.5 by day 28 post-spraying; the control remained relatively stable, with a difference exceeding 15% (Figure 8).
[0061] Simultaneously, β-diversity analysis was performed on rhizosphere soil microorganisms of wild-type and herbicide-tolerant cotton at different time points after spraying with different concentrations of glyphosate. On days 1 and 7 post-spraying, the ellipses showed complete overlap with 95% confidence, but separation became apparent from day 14 onwards: the ellipse representing transgenic herbicide-tolerant cotton shifted positively along PC1, while the wild-type showed negative expansion. By days 21 and 28 post-spraying, the ellipses showed significant differentiation (PERMANOVA, genotype × time, pseudo-F = 2.34, P = 0.079), indicating that glyphosate-driven transgenic composition turnover was achieved through selective species replacement, while the wild-type retained core stability (Figure 9).
[0062] In summary, after spraying glyphosate at twice the working concentration for 7 days, the rhizosphere soil microorganisms of herbicide-resistant transgenic cotton can be determined by measuring these microorganisms. Whether the diversity is significantly increased compared to the wild type can be used to determine the seedling growth capacity; or, 14 days after spraying glyphosate at 2 / 4 times the working concentration, whether the rhizosphere soil microbial β diversity of transgenic herbicide-tolerant cotton is significantly differentiated compared to the wild type can be used to determine its seedling growth capacity.
Claims
1. A method for rapidly evaluating the growth capacity of transgenic herbicide-tolerant cotton seedlings, the method comprising the following steps: S1 sowing transgenic cotton seeds to be tested; S2 spraying glyphosate at 0 times, 2 times, or 4 times the working concentration when the cotton cotyledons are fully expanded, wherein the 0 times glyphosate working concentration is the control group, and the other concentrations are the experimental groups; the concentration of glyphosate is the concentration of glyphosate in water; S3 seven days after spraying, taking samples of the aboveground parts and rhizosphere soil of the cotton, and detecting the aboveground dry weight, rhizosphere soil moisture content, and microbiome; S4 judging the growth capacity of transgenic herbicide-tolerant cotton seedlings by comparing whether there are statistically significant differences in the aboveground dry weight, rhizosphere soil moisture content, and microbial diversity between the experimental group and the control group.
2. The method as described in claim 1, characterized in that, Spraying is accomplished using a spray tower.
3. The method as described in claim 2, characterized in that, The spray pressure of the spray tower is 0.1~0.3 MPa to ensure uniform spraying.
4. The method as described in claim 1, characterized in that, The cotton seeds to be tested were sown inside a container.
5. The method as described in claim 4, characterized in that, The container is a polyethylene plastic bowl with a diameter of 18cm and a height of 20cm. The bottom of the bowl is provided with water-permeable holes, which can be covered with water-permeable material. Preferably, the water-permeable material is a single layer of filter paper.
6. The method as described in claim 1, characterized in that, The glyphosate may be Roundup containing 41% glyphosate isopropylamine salt.
7. The method as described in claim 1, characterized in that, The criteria for judging the growth ability of genetically modified herbicide-tolerant cotton seedlings are as follows: Seven days after spraying glyphosate at a concentration more than twice the working level, the aboveground dry weight of cotton in the experimental and control groups is measured. If the experimental group is statistically significantly higher than the control group, the experimental group is considered to have strong growth ability. The rhizosphere soil moisture content of cotton in the experimental and control groups is measured 14 days and beyond after spraying glyphosate at a concentration more than twice the working level. If the experimental group is statistically significantly lower than the control group, the experimental group is considered to have strong growth ability. The rhizosphere soil moisture content of cotton in the experimental and control groups is measured 7 days after spraying glyphosate at a concentration more than four times the working level. If the experimental group is statistically significantly lower than the control group, the experimental group is considered to have strong growth ability. Finally, the rhizosphere soil microorganisms of cotton in the experimental and control groups are measured 7 days after spraying glyphosate at a concentration more than twice the working level. Diversity was assessed by measuring the β-diversity of rhizosphere soil microorganisms in cotton plants in the experimental and control groups 14 days after spraying with 2 / 4 times the working concentration of glyphosate. If the experimental group showed significant differentiation from the control group in a statistically significant manner, the experimental group was considered to have strong growth capacity; otherwise, the experimental group was considered to have weak growth capacity.