Method capable of verifying whether allogenic materials reduce heavy metal content of plants or not

Abstract

The invention belongs to the technical field of agricultural science and environmental science, and discloses a method for verifying whether allogenic materials reduce the heavy metal content of plants or not, which comprises the following steps: in the same controllable environment, setting at least three independent cultivation boxes as a blank control group, a heavy metal stress group and an experimental treatment group respectively; the blank control group adopts a classic total nutrient solution or an improved total nutrient solution; the heavy metal stress group adds a target heavy metal salt solution into the blank control nutrient solution to form a stress nutrient solution; adding a to-be-tested allogenic material solution into the nutrient solution of the heavy metal stress group by the experimental treatment group; plant cultivation, sample treatment and analysis, and if the heavy metal content of an experimental treatment group is significantly lower than that of a heavy metal stress group and no significant accumulation exists in a blank control group, it is judged that the allogenic material has the effect of reducing plant heavy metal accumulation. The method is suitable for agricultural pollution remediation, food safety assessment and plant remediation technology research and development, and provides standardized technical support for screening efficient heavy metal passivators or plant conditioners.

Description

Technical Field

[0001] This invention belongs to the fields of agricultural science and environmental science, and in particular relates to a method for verifying whether exogenous substances reduce the heavy metal content of plants. Background Technology

[0002] The exogenous input of heavy metals into farmland soil has caused severe damage to arable land resources and deterioration of the soil environment, posing a serious threat to agricultural production and food security. This has become a major environmental concern worldwide. Long-term heavy metal toxicity can severely hinder crop growth and development, leading to reduced yields. Cadmium (Cd), in particular, has strong migration capabilities and bioavailability, easily absorbed and accumulated by crops, and transferred to humans through the food chain, causing kidney damage, cardiovascular dysfunction, and reproductive problems, seriously endangering human health. It is the most significant heavy metal pollutant in agricultural soils. Furthermore, Cd is a non-essential toxic heavy metal harmful to plants. Once absorbed, it inhibits plant growth and development, leading to slow root development, leaf curling or chlorosis and yellowing, and in severe cases, plant death, affecting crop yields. Therefore, finding an effective treatment method to reduce Cd accumulation in plants is of great significance.

[0003] Existing methods for assessing the reduction of heavy metal content in plants using exogenous substances lack standardized validation criteria. Significant differences exist in the plant species, growth conditions, heavy metal pollution concentrations, and detection methods used, making cross-sectional comparisons difficult and lacking a universally applicable evaluation system. Furthermore, the effects of exogenous substances during actual cultivation may be influenced by soil physicochemical properties, microbial activity, and environmental variables, and existing methods often fail to systematically eliminate these interferences, leading to insufficient reliability of conclusions. Some experiments lack strict blank or positive controls, making it impossible to quantify the actual contribution of exogenous substances and affecting the reproducibility and application value of the results. Therefore, there is an urgent need to establish a scientific, standardized, and reproducible validation method to accurately assess the effect of exogenous substances on reducing heavy metal content in plants, providing technical support for the safe utilization of polluted farmland. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for verifying whether exogenous substances reduce the heavy metal content of plants.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: A method for verifying whether exogenous substances reduce the heavy metal content of plants includes the following steps: (1) Setting up a box-type hydroponic system: In the same controllable environment, at least three independent cultivation boxes were set up as blank control group (CK), heavy metal stress group (HM) and experimental treatment group (ET), respectively. (2) Preparation of nutrient solution: The blank control group used either classic complete nutrient solution or a modified complete nutrient solution, with the pH adjusted to 5.5-6.5; In the heavy metal stress group, the target heavy metal salt solution was added to the blank control nutrient solution to form a stress nutrient solution containing a set concentration of heavy metal; The experimental treatment group had the exogenous substance solution to be tested added to the nutrient solution of the heavy metal stress group; (3) Plant cultivation: Select seedlings with uniform growth and transplant them into each cultivation box, with no less than 5 seedlings in each box. Cultivate them under constant temperature, constant humidity and light conditions, and replenish the nutrient solution regularly to maintain a constant liquid level and pH and EC values. (4) Sample processing and analysis: Plant tissue samples were collected during the period of significant heavy metal stress or at the end of the experiment. After rinsing and drying, the fresh weight was recorded. After drying to constant weight, the samples were ground, digested, and the heavy metal concentration was determined. (5) Effect verification: If the heavy metal content in the experimental treatment group is significantly lower than that in the heavy metal stress group and there is no significant accumulation in the blank control group, then the exogenous substance is determined to have the effect of reducing the accumulation of heavy metals in plants.

[0006] In the above method, preferably, the target heavy metal is at least one of cadmium, lead, or arsenic; the heavy metal concentration of the heavy metal stress group needs to be set according to the tolerance threshold of the target plant and the pollution background value.

[0007] Preferably, the cultivation conditions described above are in an artificial climate chamber, with the temperature set at 18-25°C, relative humidity at 60-70%, light intensity at 10,000-20,000 Lux, and a photoperiod of 16 h / 8 h. A further preferred temperature is 25°C during the day and 18°C ​​at night.

[0008] In the preferred embodiment of the above method, in step (3), the pH should be adjusted to 5.5-6.5 and the EC value to 1.0-2.0 mS / cm after each replenishment of nutrient solution.

[0009] In the above method, preferably, in step (2), the improved complete nutrient solution contains macro-elements and micro-elements necessary for plant growth, wherein the macro-elements include N, P, and K, and the micro-elements include Fe, Mn, and Zn.

[0010] In the above method, preferably, in step (2), the exogenous substance is a solid or liquid compound, including organic acids, silicates, nanomaterials or microbial preparations, and its concentration is set according to the results of preliminary experiments or the recommended values ​​in the literature.

[0011] In the above method, preferably, in step (4), the plant tissue includes roots, stems, leaves, or edible parts.

[0012] In the above method, preferably, in step (4), the drying refers to drying at a constant temperature of 55°C.

[0013] In the above method, preferably, in step (4), the digestion is carried out by HNO3-H2O2 microwave digestion to extract heavy metals, and the method for determining the concentration of heavy metals is selected from at least one of atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), or atomic fluorescence spectrometry (AFS).

[0014] Compared with the prior art, the advantages of the present invention are as follows: (1) The experimental conditions of the present invention are highly controllable and the results are accurate and reliable: the use of box-type nutrient solution cultivation avoids the influence of differences in the physical and chemical properties of the soil substrate (such as pH, organic matter, microorganisms, etc.) on the experimental results, ensuring that the exposure concentration of heavy metals and exogenous substances is accurately controllable; strict blank control (CK) and heavy metal stress control (HM) are set up to clearly distinguish the effects of exogenous substances and eliminate interference from plant metabolism or environmental factors.

[0015] (2) The standardized operation of the test in this invention ensures repeatability and universality: the nutrient solution formula, heavy metal concentration and culture conditions are unified, avoiding the incomparability of results due to differences in experimental parameters. It is applicable to the verification of different exogenous substances (such as organic acids, nanomaterials, microorganisms, etc.). The heavy metal content is determined by instrumental analysis methods (AAS, ICP-MS, etc.), and the data accuracy is high and meets international testing standards.

[0016] (3) The method of the present invention is flexible and adaptable to different research needs: by adjusting the types of heavy metals (Cd, Pb, As, etc.) and concentration gradients, as well as adjusting the plant varieties, it can simulate the effects of different levels of environmental pollution and different types of crops, support experiments on the concentration gradient of exogenous substances, optimize the best application dosage, and provide a basis for practical applications.

[0017] (4) The method of the present invention that can verify whether exogenous substances reduce the heavy metal content of plants has a wide range of applications: it is applicable to agricultural pollution remediation, food safety assessment and phytoremediation technology research and development, and provides standardized technical support for screening efficient heavy metal passivating agents or plant conditioners. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1This is an observation of the phenotypic characteristics of pepper seedlings under different treatments in Example 1 of the present invention; Figure 2 The Cd content in the roots, stems and fruits of chili peppers under different treatments in Example 1 of this invention; Figure 3 Phenotypic observation of amaranth seedlings under different treatments in Example 2 of this invention; Figure 4 The Cd content of the aboveground parts of amaranth under different treatments in Example 2 of this invention; Figure 5 The Cd content in the underground part of amaranth under different treatments in Example 2 of this invention. Detailed Implementation

[0020] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0021] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0022] Unless otherwise specified, all reagents and raw materials used in this invention are commercially available products or products that can be prepared by known methods.

[0023] A method for verifying whether exogenous substances reduce the heavy metal content of plants includes the following steps: (1) Box-type nutrient solution cultivation: Select cultivation boxes of the same size and material. There are three main treatments: one as a blank control, one as a heavy metal stress control, and one as an experimental treatment. The corresponding incubator can be used for different concentration settings and the treatments are cultivated in the same environment.

[0024] (2) Nutrient solution preparation: The blank control uses a classic complete nutrient solution or a modified nutrient solution formula. The nutrient solution should contain macro-elements (N, P, K, etc.) and micro-elements (Fe, Mn, Zn, etc.) necessary for plant growth. The pH value is adjusted to 5.5~6.5. The heavy metal stress control is to add the target heavy metal to the nutrient solution of the blank control, but not to add the exogenous substance to be tested. The experimental treatment is to add the exogenous substance to be tested to the heavy metal stress solution. (3) Substance weighing: Accurately weigh heavy metal salts, dissolve them in deionized water and add them to the nutrient solution. The concentration of heavy metal stress solution should be set according to the plant's tolerance and pollution threshold. Exogenous substances should be weighed or measured according to their properties (solid or liquid), dissolved and added to the nutrient solution. The concentration should be set according to the values ​​recommended in the preliminary experiment or literature. (4) Plant cultivation: After disinfecting the plant seeds, seedlings are raised in plug trays or floating trays and pre-cultured until the seedling stage. Then, seedlings with uniform growth are selected and transplanted into cultivation boxes with different treatments. Each box contains no less than 5 seedlings to avoid root damage. All seedlings must be cultivated in a pollution-free environment and in a controllable environment (such as an artificial climate chamber or light incubator) to ensure that the temperature, humidity and light conditions are consistent. Nutrient solution is replenished regularly (to keep the liquid level consistent) and the pH and EC values ​​are kept constant to avoid evaporation interference.

[0025] (5) Plant sample collection: Samples were collected during the period of significant heavy metal stress or at the end of the experiment. Phenotypic observation and photography were performed. The roots, stems, leaves or edible parts of the plants were collected. The surface adsorbates were rinsed with deionized water, the water was absorbed by filter paper and the fresh weight was recorded. The samples were then dried at 105 °C to constant weight to avoid heavy metal loss.

[0026] (6) Sample preparation and determination: After drying, the sample was ground and sieved, and heavy metals were extracted by microwave digestion with HNO3-H2O2. The concentration of heavy metals in the digestion solution was determined by atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS) or atomic fluorescence spectrometry (AFS), and the heavy metal accumulation in each tissue (mg / kg DW) was calculated. If the heavy metal content of the experimental treatment group was significantly lower than that of the heavy metal stress control group (*p*<0.05), and the blank control group had almost no heavy metal accumulation, it can be determined that the exogenous substance can effectively reduce the heavy metal accumulation of the crop.

[0027] Example 1: A method for verifying whether exogenous substances reduce the heavy metal content of plants includes the following steps: (1) Setting up the box-type hydroponic system: In the same controllable environment, three independent cultivation boxes were set up as blank control group (CK), heavy metal stress group (HM) and experimental treatment group (ET), respectively. (2) Preparation of nutrient solution: The blank control group used Hoagland nutrient solution, with the pH adjusted to 6.0 and the EC value of 1.2 mS / cm; The heavy metal stress group used a nutrient solution prepared by adding CdCl2·2.5 H2O powder and deionized water to the blank control nutrient solution to make the Cd ion concentration 150 µM. In the experimental treatment group, the exogenous substance solution to be tested was added to the nutrient solution of the heavy metal stress group. Specifically, a solution prepared by adding Na2SiO3·9H2O powder and deionized water was added to the nutrient solution of the heavy metal stress group to make the Si ion concentration 1 mM. (3) Plant cultivation: When the pepper seedlings grow to 4 leaves and 1 heart, select seedlings with the same growth and place them into three treatment nutrient solutions. Each treatment consists of 12 pepper seedlings. The seedlings are cultivated in an artificial climate chamber. The cultivation conditions are set as follows: temperature 25℃ (day) / 18℃ (night), relative humidity 60%, light intensity 20000 Lux, photoperiod 16 h / 8 h. The nutrient solution is replenished regularly to keep the liquid level consistent, the pH value is kept at 6.0, the EC value is kept at 1.2 mS / cm, and evaporation interference is avoided.

[0028] (4) Sample processing and analysis: After 7 days of cultivation, the phenotype of pepper plants was observed and samples were taken. Pepper root, stem and fruit tissues were collected, the surface adsorbate was rinsed with deionized water, filtered and dried in an oven at 55℃ until constant weight, heavy metals were extracted by HNO3-H2O2 microwave digestion method, and the Cd content of each tissue was detected by AAS method.

[0029] The results obtained are as follows: Phenotypic observation results are as follows Figure 1 As shown, the chili seedlings in the blank control group grew normally, while the chili seedlings under heavy metal Cd stress treatment suffered severe leaf drop and some new leaves turned yellow. The chili seedlings in the experimental treatment group had less leaf damage, fewer leaf drop, and no yellowing.

[0030] Cd content test results are as follows Figure 2 As shown, the Cd content in the roots, stems, and fruits of chili peppers under heavy metal stress treatment was the highest among the three treatments. In contrast, the Cd content in the roots, stems, and fruits of chili peppers under exogenous substance treatment decreased by 33.6%, 68.8%, and 69.9%, respectively, all reaching a highly significant level (*p*<0.01). The Cd content in the blank control group was negligible compared to the other two treatments.

[0031] The results of this case study indicate that exogenous Na2SiO3·9H2O can reduce Cd accumulation in chili peppers.

[0032] Example 2: A method for verifying whether exogenous substances reduce the heavy metal content of plants includes the following steps: (1) Box-type hydroponic system setup: Four independent cultivation boxes were set up in the same controllable environment as blank control group (CK), heavy metal stress group (HM) and experimental treatment group (ET), respectively. (2) Preparation of nutrient solution: The blank control group used 1 / 2 garden nutrient solution, with pH adjusted to 6.0 and EC value of 1.2 mS / cm; The heavy metal stress group used a nutrient solution prepared by adding CdCl2·2.5 H2O powder and deionized water to the blank control nutrient solution to make the Cd ion concentration 5 µM. In the experimental treatment groups, the exogenous substance to be tested was added to the nutrient solution of the heavy metal stress group. Experimental treatment group 1 was a Si solution prepared by adding Na2SiO3·9H2O powder and deionized water to the heavy metal stress solution, with a Si concentration of 1 mM. Experimental treatment group 2 was a Si solution prepared by adding Na2SiO3·9H2O powder and deionized water to the heavy metal stress solution, with a Si concentration of 2 mM.

[0033] (3) Plant cultivation: When the amaranth seedlings grow to 4 leaves and 1 heart, select seedlings with the same growth and place them into 4 treatments, with 12 seedlings in each treatment. They are cultivated in an artificial climate chamber with the following conditions: temperature 25℃ (day) / 18℃ (night), relative humidity 60%, light intensity 10000 Lux, photoperiod 16 h / 8 h, nutrient solution is replenished regularly (to keep the liquid level consistent), and the pH value is monitored to be 6.0 and the EC value is 1.2 mS / cm to avoid evaporation interference.

[0034] (4) Sample processing and analysis: After 7 days of cultivation, the phenotype of amaranth plants was observed and samples were taken. The underground and aboveground tissues of amaranth were collected, the surface adsorbate was rinsed with deionized water, filtered and dried, and then placed in an oven at 55 ℃ to constant weight. Heavy metals were extracted by HNO3-H2O2 microwave digestion method, and the Cd content of each tissue was detected by AAS method.

[0035] Phenotypic results are as follows Figure 3 As shown, compared with the blank control, amaranth seedlings under heavy metal stress treatment showed obvious yellowing, and the lateral roots of amaranth were shorter and their elongation was inhibited. In contrast, amaranth seedlings treated with exogenous Si at concentrations of 1 mM and 2 mM showed normal growth and normal lateral root growth.

[0036] Cd content test results are as follows Figure 4 and Figure 5 As shown, under Si treatments of 1 mM and 2 mM, the Cd content in the aboveground parts of amaranth decreased by 42.4% and 37.4%, respectively. Figure 4 The difference between the treatment group and the heavy metal stress group was highly significant (*p*<0.01). The Cd content in the aboveground parts of amaranth treated with 1 mM and 2 mM Si decreased by 63.1% and 60.4% respectively compared to the heavy metal stress group. Figure 5 The difference was also highly significant (*p*<0.01). Compared with the other three treatments, the blank control group had almost no Cd content in amaranth.

[0037] The results of this case study indicate that exogenous Na2SiO3·9H2O can reduce Cd accumulation in amaranth.

Claims

1. A method for verifying whether exogenous substances reduce the heavy metal content of plants, characterized in that, Includes the following steps: (1) Setting up a box-type hydroponic system: In the same controllable environment, at least three independent cultivation boxes were set up as blank control group, heavy metal stress group and experimental treatment group, respectively; (2) Preparation of nutrient solution: The blank control group used either classic complete nutrient solution or a modified complete nutrient solution, with the pH adjusted to 5.5-6.5; In the heavy metal stress group, the target heavy metal salt solution was added to the blank control nutrient solution to form a stress nutrient solution containing a set concentration of heavy metal; The experimental treatment group had the exogenous substance solution to be tested added to the nutrient solution of the heavy metal stress group; (3) Plant cultivation: Select seedlings with uniform growth and transplant them into each cultivation box, with no less than 5 seedlings in each box. Cultivate them under constant temperature, constant humidity and light conditions, and replenish the nutrient solution regularly to maintain a constant liquid level and pH and EC values. (4) Sample processing and analysis: Plant tissue samples were collected during the period of significant heavy metal stress or at the end of the experiment. After rinsing and drying, the fresh weight was recorded. After drying to constant weight, the samples were ground, digested, and the heavy metal concentration was determined. (5) Effect verification: If the heavy metal content in the experimental treatment group is significantly lower than that in the heavy metal stress group, and there is no significant accumulation in the blank control group, then the exogenous substance is determined to have the effect of reducing the accumulation of heavy metals in plants.

2. The method as described in claim 1, characterized in that, The target heavy metal is at least one of cadmium, lead, or arsenic; the heavy metal concentration of the heavy metal stress group needs to be set according to the tolerance threshold of the target plant and the background pollution value.

3. The method as described in claim 1, characterized in that, The cultivation conditions were in an artificial climate chamber, with a temperature of 18-25°C, a relative humidity of 60-70%, a light intensity of 10,000-20,000 Lux, and a photoperiod of 16 h / 8 h.

4. The method as described in claim 1, characterized in that, In step (3), after each replenishment of nutrient solution, the pH should be adjusted to 5.5~6.5 and the EC value to 1.0~2.0 mS / cm.

5. The method as described in claim 1, characterized in that, In step (2), the improved complete nutrient solution contains macro-elements and micro-elements necessary for plant growth. The macro-elements include N, P, and K, and the micro-elements include Fe, Mn, and Zn.

6. The method as described in claim 1, characterized in that, In step (2), the exogenous substance is a solid or liquid compound, including organic acids, silicates, nanomaterials or microbial preparations, and its concentration is set according to the results of preliminary experiments or the recommended values ​​in the literature.

7. The method as described in claim 1, characterized in that, In step (4), the plant tissue includes roots, stems, leaves or edible parts.

8. The method as described in claim 1, characterized in that, In step (4), the drying refers to drying at a constant temperature of 55°C.

9. The method as described in claim 1, characterized in that, In step (4), the method for determining the concentration of heavy metals is selected from at least one of atomic absorption spectrometry, inductively coupled plasma mass spectrometry, or atomic fluorescence spectrometry.

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