Method for analyzing spatial distribution and morphological change of heavy metals in plant rhizosphere region

Abstract

The invention discloses a method for analyzing spatial distribution and morphological change of heavy metals in a plant rhizosphere region, and belongs to the technical field of ecological environment. The method comprises a plant planting procedure, a fluid replacement drying procedure and a resin immersion procedure. Wherein in the fluid replacement drying procedure, graded ethanol is adopted for fluid replacement according to gradients; a mixed solution of ethanol and Surr resin is used as a replacement liquid in the resin immersion procedure. According to the method, indoor plant cultivation, fluid replacement drying and resin immersion embedding technologies are innovatively combined for the first time, and the method is applied to in-situ preparation of the plant rhizosphere region sample. The method can be used for accurately researching the behavior of the heavy metal in the rhizosphere region, especially the spatial distribution and morphological change of the heavy metal near the plant root in the soil.

Description

Technical Field

[0001] The present invention belongs to the technical field of ecological environment, and particularly relates to a method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants. Background Art

[0002] The rhizosphere is a unique "microenvironment" where the interaction between plant roots and soil is relatively strong. There are significant differences in the physical, chemical, and biological properties of rhizosphere soil and non-rhizosphere soil. Plant roots secrete organic molecules such as organic acids, amino acids, and sugars, which change the pH value, redox potential, etc. of the rhizosphere soil, thereby affecting the bioavailability, chemical form, and spatial distribution of heavy metals. The growth and metabolic activities of plant roots also reduce the soil capacity in the rhizosphere, exacerbating the differences between rhizosphere soil and non-rhizosphere soil. Although the influence of the rhizosphere environment on the behavior of heavy metals has been widely recognized, the specific influence of plant roots on the bioavailability and distribution of metals in soil, especially the changes in the behavior and fate of heavy metals in the rhizosphere region, is still insufficiently studied.

[0003] Heavy metal pollution in soil is one of the important environmental problems faced globally. It not only affects the health of the soil ecosystem but also poses a threat to human health through the food chain. Plant roots, as the interface between soil and plants, play a key role in the process of heavy metal migration, transformation, and accumulation. However, most current studies on the behavior of metals in soil mainly focus on non-rhizosphere soil, ignoring this important "microenvironment" of the rhizosphere. The particularity of the rhizosphere makes the behavior of heavy metals in it may be significantly different from that in non-rhizosphere soil. For example, the morphological transformation, mobility, and bioavailability of metals may change significantly due to different chemical and biological conditions in the rhizosphere. Therefore, it is difficult to comprehensively reflect the actual environmental risks of heavy metals in soil based only on the research results of non-rhizosphere soil.

[0004] Currently, there are still many technical challenges in the study of heavy metal behavior in the rhizosphere. First, the physical separation of rhizosphere soil and plant roots is extremely difficult, especially when the rhizosphere region is very narrow (usually in millimeters). Traditional soil sampling methods are difficult to accurately capture the metal distribution and morphological evolution in the rhizosphere region. Second, existing research methods, such as the rhizobox system, although able to simulate the rhizosphere environment to a certain extent, its soil sampling scheme is usually carried out at the millimeter level, and the resolution is not sufficient to reveal the fine distribution of metals in the rhizosphere region. Moreover, the fine nylon mesh often used in the rhizobox system, although able to separate the roots and soil, also hinders the natural interaction between the roots and soil, which may cause the loss of some key information on metal behavior. This design limitation will lead to bias in the risk assessment of heavy metals in soil.

[0005] Therefore, most of the existing studies have not fully revealed the mechanism of metal behavior changes in the rhizosphere region, especially the spatial distribution and morphological changes of contaminated metals near plant roots in the soil. This knowledge gap limits the in-depth understanding of rhizosphere metal behavior and also affects the scientific decision-making of soil pollution remediation and agricultural safety production. In order to more accurately evaluate the environmental risks of heavy metals in the soil and optimize phytoremediation and agricultural planting strategies, there is an urgent need to develop a new method that can analyze the distribution and morphology of metals in the rhizosphere region. Summary of the Invention

[0006] In view of the above technical problems, the present invention proposes a method for analyzing the spatial distribution and morphological changes of heavy metals in the plant rhizosphere region, which can be used to accurately study the behavior of heavy metals in the rhizosphere region, especially the spatial distribution and morphological changes of heavy metals near plant roots in the soil. By solving the problems such as the difficulty in separating rhizosphere soil from roots, low sampling resolution, and limited root-soil interaction, the present invention provides more reliable technical support for the environmental risk assessment of metals in the soil and provides a scientific basis for its application in pollution remediation and agricultural safety.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for analyzing the spatial distribution and morphological changes of heavy metals in the plant rhizosphere region, comprising a plant planting procedure, a fluid replacement drying procedure, and a resin immersion procedure; wherein,

[0009] In the fluid replacement drying procedure, graded ethanol is used for fluid replacement according to a gradient;

[0010] In the resin immersion procedure, a mixed solution of ethanol and Spurr resin is used as the replacement liquid.

[0011] The present invention first uses an ethanol solution to gradually displace the fluid in the soil pores in gradients, which can effectively avoid the problem of soil sample cracking caused by traditional physical drying methods such as air drying or oven drying, thereby maintaining the integrity and original state of the soil structure. Then, through a gradient-by-gradient replacement of the ethanol residue in the soil pore fluid with an ethanol-Spurr resin mixed solution, it ensures that the soil structure is always in a fluid-supported state, avoiding structural collapse or deformation caused by liquid loss, and maximizing the maintenance of the original state of the root-soil interface. Moreover, the present invention selects a Spurr resin with low viscosity and high permeability that is prepared and used immediately, which can achieve efficient immersion and saturation of the soil mass with minimal disturbance to the soil structure, providing stable and uniform basic conditions for subsequent solidification treatment. In addition, the pre-setting of the aluminum mesh lining is not only corrosion-resistant but also enables the interference-free transfer of soil samples, effectively supporting the soil skeleton and avoiding the influence of external disturbances. This invention can effectively solve the problem that it is difficult to accurately separate rhizosphere soil and non-rhizosphere soil in traditional methods, and at the same time can avoid problems such as cracking of test samples caused by physical drying. During the entire sample preparation process, the soil structure is always supported by the fluid in the pores, maximizing the preservation of the original state of the sample and providing high-fidelity detailed information for subsequent analysis.

[0012] Further, the specific operation steps of the plant planting program include: cultivating plant shoots in the soil of a seedling-raising pot, harvesting the above-ground parts of the plant shoots after the height of the above-ground parts of the plant shoots no longer changes during growth, and retaining the whole part of the soil and the plant rhizosphere area.

[0013] Furthermore, a drain outlet is provided at the bottom of the seedling-raising pot. A polypropylene geotextile is laid above the drain outlet, and an aluminum mesh lining is laid above the polypropylene geotextile and on the inner wall of the seedling-raising pot. The aluminum mesh lining is provided with a handle at the edge of the seedling-raising pot, so that the aluminum mesh lining containing soil can be taken out separately from the main body of the seedling-raising pot.

[0014] Furthermore, during the growth period of the plant shoots, the soil moisture content only needs to meet the growth requirements of the plants. In the present invention, the soil moisture content is set at 35%.

[0015] Further, the specific operation steps of the fluid displacement drying program include: putting the whole part of the soil and the plant rhizosphere area obtained after being processed by the plant planting program into another container, and injecting graded ethanol in gradients using a syringe for fluid displacement.

[0016] Furthermore, the grades of the graded ethanol are respectively: an ethanol solution with a concentration of 50 wt%, an ethanol solution with a concentration of 70 wt%, an ethanol solution with a concentration of 95 wt%, and an ethanol solution with a concentration of 100 wt%; each stage of the ethanol fluid displacement program lasts for 1 hour, and the ethanol solution fluid displacement program with a concentration of 100 wt% is repeated 3 times.

[0017] Further, the specific operation steps of the resin immersion procedure include: soaking the whole part of the soil and the rhizosphere of plants obtained after the fluid replacement drying procedure in a mixed solution, and after the soaking is completed, taking out the whole part of the soil and the rhizosphere of plants for solidification treatment, slicing, grinding and polishing to obtain a sample to be analyzed.

[0018] Furthermore, the soaking is a graded soaking, specifically: putting the whole part of the soil and the rhizosphere of plants into mixed solutions of different levels for soaking, and the soaking time for each level of the mixed solution is 24 hours.

[0019] Furthermore, the mixed solution is a mixed solution of ethanol and Spurr resin;

[0020] The levels of the mixed solution are: a mixed solution with a mass ratio of ethanol to Spurr resin of 2:1, a mixed solution with a mass ratio of ethanol to Spurr resin of 1:1, a mixed solution with a mass ratio of ethanol to Spurr resin of 1:3, and a mixed solution with a mass ratio of ethanol to Spurr resin of 0:1.

[0021] Furthermore, the slicing includes transverse slicing and longitudinal slicing;

[0022] The specific operation steps of the grinding are: grinding the surface of the slice with silicon sandpaper with decreasing particle size;

[0023] The specific operation steps of the polishing treatment are: polishing the surface of the slice with anhydrous oil-based diamond gypsum of 3μm, 1μm and 0.25μm respectively.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] The present invention innovatively combines indoor plant cultivation, fluid replacement drying and resin immersion embedding technology for the first time and applies it to the in-situ preparation of rhizosphere samples of plants, filling the blank of the prior art. This method can be used to accurately study the behavior of heavy metals in the rhizosphere region, especially the spatial distribution and morphological changes of heavy metals near the roots of plants in the soil.

[0026] The present invention can use indoor pot experiments to prepare rhizosphere samples of plants with different growth cycles for studying the influence of the interaction between plant roots and farmland soil over time. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0028] Figure 1 This is the flowchart for preparing in-situ rhizosphere samples in the embodiments of the present invention;

[0029] Figure 2 This is the rhizosphere soil sample of spring barley prepared in Example 1 of the present invention;

[0030] Figure 3 This is the microscopic structure A) of the rhizosphere soil sample of spring barley prepared in Example 1 of the present invention, and the distribution of Ca-Al-Si-Zn elements in the rhizosphere regions B) and C);

[0031] Figure 1 In the figure, 1 - plant bud; 2 - aluminum mesh lining; 3 - seedling raising pot; 4 - polypropylene geotextile; 5 - soil; 6 - drainage hole; 7 - handle; 8 - polypropylene plastic cylindrical container; 9 - graded ethanol solution; 10 - mixed solution of ethanol and Spurr resin in different ratios. Detailed implementation manners

[0032] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0033] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are also obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0036] The terms "comprising", "including", "having", "containing", etc. used in this text are all open-ended terms, meaning including but not limited to.

[0037] An embodiment of the present invention provides a method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants, including a plant cultivation procedure, a fluid replacement drying procedure, and a resin immersion procedure. The specific technical solution is as follows (see the flowchart in Figure 1 ):

[0038] (1) Use a seedling-raising pot 3 to cultivate the plant shoots 1. The bottom of the seedling-raising pot 3 is provided with a drain hole 6. Two layers of polypropylene geotextiles 4 are laid above the drain hole 6 (i.e., the inner bottom of the seedling-raising pot 3) to prevent the loss of soil 5; an aluminum mesh lining 2 (i.e., the inner wall of the seedling-raising pot 3, including the bottom and side walls) is closely attached above the polypropylene geotextile 4 and to the inner wall of the seedling-raising pot 3. The soil 5 is filled into the seedling-raising pot 3 with the aluminum mesh lining 2 set; the plant shoots 1 are cultivated into the soil 5, and after the height of the above-ground part of the plant no longer changes, the above-ground part of the plant shoots 1 is harvested.

[0039] (2) Slowly lift the handle 7 of the aluminum mesh lining 2, take out the soil 5 containing the roots of the plant shoots 1 from the seedling-raising pot 3 and transfer it to a polypropylene plastic cylindrical container 8. Use a syringe to slowly inject a graded ethanol solution 9 into the polypropylene plastic cylindrical container 8 in gradients to gradually replace the pore fluid in the soil 5.

[0040] (3) Suck out the excess ethanol solution in the polypropylene plastic cylindrical container 8 that has not entered the interior of the soil 5, and then add a mixed solution 10 to soak the soil 5 containing the roots of the plant shoots 1 processed in step (2) in the mixed solution 10. This soaking is a step-by-step soaking to gradually replace the residual ethanol solution 9 in the soil 5, and obtain the soil 5 containing the roots of the plant shoots 1 embedded with resin.

[0041] (4) Transfer the soil 5 containing the roots of the plant shoots 1 embedded with resin obtained in step (3) to an oven for curing treatment. After curing is completed, take it out for sectioning, grinding, and polishing treatment to obtain the sample to be analyzed.

[0042] In the following optional embodiment, in step (1), after planting the plant shoots 1, adjust the moisture content of the soil 5 to meet its growth requirements. In the present invention, it is set to 35%, and this moisture content is maintained throughout the growth cycle of the plant shoots 1.

[0043] In the following optional embodiments, in step (2), the grades of the graded ethanol solution 9 are: an ethanol solution with a concentration of 50 wt%, an ethanol solution with a concentration of 70 wt%, an ethanol solution with a concentration of 95 wt%, and an ethanol solution with a concentration of 100 wt%. Each replacement procedure lasts for 1 hour, and the replacement procedure of the ethanol solution with a concentration of 100 wt% is repeated 3 times.

[0044] In the following optional embodiments, in step (3), the step-by-step soaking means soaking in different grades of the mixed solution 10 respectively. The grades of the mixed solution 10 are: a mixed solution with a mass ratio of ethanol to Spurr resin of 2:1, a mixed solution with a mass ratio of ethanol to Spurr resin of 1:1, a mixed solution with a mass ratio of ethanol to Spurr resin of 1:3, and a mixed solution with a mass ratio of ethanol to Spurr resin of 0:1 (i.e., 100% Spurr resin). Each replacement procedure lasts for 24 hours, and the 100% resin replacement step is repeated 3 times to completely remove the residual graded ethanol solution 9.

[0045] In the following optional embodiments, in step (4), the conditions for the curing treatment are: curing at 70 °C for 12 hours.

[0046] In the following optional embodiments, in step (4), the sectioning can be vertical sectioning or horizontal sectioning to obtain a transverse section or a longitudinal section that fully exposes the root-soil contact surface.

[0047] In the following optional embodiments, in step (4), the conditions for the grinding are: grinding the surface of the section with silicon sandpapers with decreasing particle sizes (P600 and P1200).

[0048] In the following optional embodiments, in step (4), the conditions for the polishing treatment are: polishing the surface of the section with anhydrous oil-based diamond pastes of 3 μm, 1 μm, and 0.25 μm respectively.

[0049] All raw materials used in the present invention are obtained by purchasing in the market.

[0050] The technical solutions of the present invention are further described below through embodiments.

[0051] Example 1

[0052] This example takes spring barley (variety: Hordeum vulgare) as an example for the experiment. The specific steps include:

[0053] (1) Use multiple groups of seedling-raising pots 3 (top surface: 2 cm × 2 cm; bottom surface: 1 cm × 1 cm; height: 3 cm) to cultivate plant sprouts 1 (spring barley). A drainage hole 6 (diameter: 0.6 cm) is provided at the bottom of the seedling-raising pot 3. Two layers of polypropylene geotextiles 4 are laid above the drainage hole 6 to prevent soil 5 from flowing away; an aluminum mesh lining 2 is closely attached to the upper part of the polypropylene geotextile 4 and the inner wall of the seedling-raising pot 3;

[0054] (2) Put 7.5 g of soil 5 with an initial moisture content of 11.6% into the seedling-raising pot 3 with the aluminum mesh lining 2 set; plant the plant sprouts 1 in the soil 5, adjust the moisture content of the soil 5 to 35%, and keep this moisture content throughout the growth period;

[0055] (3) After the plant sprouts 1 grow for 6 weeks, harvest the above-ground part of the plant sprouts 1;

[0056] (4) Slowly lift the handle 7 of the aluminum mesh lining 2, take out the soil 5 containing the roots of the plant sprouts 1 from the seedling-raising pot 3 and transfer it to a polypropylene plastic cylindrical container 8. Slowly inject a graded ethanol solution 9 into the polypropylene plastic cylindrical container 8 in gradients using a syringe (the grades of the graded ethanol solution 9 are: ethanol solution with a concentration of 50 wt%, ethanol solution with a concentration of 70 wt%, ethanol solution with a concentration of 95 wt%, and ethanol solution with a concentration of 100 wt%. Each replacement procedure lasts for 1 hour, and the replacement procedure of the ethanol solution with a concentration of 100 wt% is repeated 3 times), and gradually replace the pore fluid in the soil 5; each replacement procedure lasts for 1 hour, and the replacement procedure of the ethanol solution with a concentration of 100 wt% is repeated 3 times;

[0057] (5) Suck out the excess ethanol solution in the polypropylene plastic cylindrical container 8 that has not entered the interior of the soil 5, and then add a mixed solution 10 to immerse the soil 5 containing the roots of the plant sprouts 1 treated in step (4) in the mixed solution 10. This immersion is a step-by-step immersion (the grades of the mixed solution 10 are: mixed solution with a mass ratio of ethanol to Spurr resin of 2:1, mixed solution with a mass ratio of ethanol to Spurr resin of 1:1, mixed solution with a mass ratio of ethanol to Spurr resin of 1:3, and mixed solution with a mass ratio of ethanol to Spurr resin of 0:1). Gradually replace the residual ethanol solution 9 in the soil 5. Each replacement procedure lasts for 24 hours, and the replacement step of the mixed solution with a mass ratio of ethanol to Spurr resin of 0:1 is repeated 3 times to completely remove the residual graded ethanol solution 9 and obtain a resin-embedded soil 5 containing the roots of the plant sprouts 1;

[0058] (6) Transfer the resin-embedded soil 5 containing the roots of the plant sprouts 1 obtained in step (3) to an oven and cure it at 70 °C for 12 hours;

[0059] (7) After curing is completed, take out the samples and perform vertical and horizontal slicing respectively to obtain longitudinal and transverse slices that fully expose the root-soil contact surface;

[0060] (8) Grind the surface of the slices with silicon sandpapers of decreasing particle size (P600 and P1200), and then polish the surface of the slices with anhydrous oil-based diamond pastes of 3μm, 1μm, and 0.25μm respectively to obtain a transverse section and a longitudinal section (see Figure 2 ), that is, the rhizosphere soil sample of spring barley.

[0061] Figure 3 The microstructure A) of the rhizosphere soil sample of spring barley prepared in this example, and the distribution of Ca-Al-Si-Zn elements in the rhizosphere B) and C). It can be seen from the figure the spatial distribution characteristics of heavy metals near the roots of spring barley in the soil.

[0062] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants, characterized in that: It includes plant planting procedure, fluid displacement drying procedure and resin immersion procedure; among which, In the fluid replacement drying procedure, graded ethanol is used to perform fluid replacement according to a gradient; The resin immersion procedure uses a mixed solution of ethanol and Spurr resin as the replacement liquid.

2. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 1, characterized in that: The specific operation steps of the plant planting procedure include: planting plant sprouts in the soil of a seedling pot, harvesting the above-ground part of the plant sprouts after the plant sprouts grow to a height that no longer changes, and retaining the soil and the entire rhizosphere of the plant.

3. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 2, characterized in that: A drainage outlet is provided at the bottom of the seedling pot, a polypropylene geotextile is laid above the drainage outlet, an aluminum mesh lining is laid above the polypropylene geotextile and on the inner wall of the seedling pot, and a handle is provided on the edge of the seedling pot so that the aluminum mesh lining containing soil can be separated from the seedling pot body and taken out separately.

4. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 2, characterized in that: During the growth period of the plant sprouts, the soil moisture content is controlled to be 35%.

5. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 1, characterized in that: The specific operation steps of the fluid replacement drying procedure include: placing the soil obtained after the plant planting procedure and the entire plant rhizosphere zone into another container, and injecting graded ethanol solution in a gradient manner using a syringe to perform fluid replacement.

6. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 5, characterized in that: The levels of the graded ethanol are: 50wt% ethanol solution, 70wt% ethanol solution, 95wt% ethanol solution and 100wt% ethanol solution; each level of ethanol fluid replacement procedure lasts for 1 hour, and the 100wt% ethanol solution fluid replacement procedure is repeated 3 times.

7. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 1, characterized in that: The specific operation steps of the resin immersion procedure include: immersing the soil and the entire plant rhizosphere obtained after the fluid displacement drying procedure in a mixed solution, and after the immersion is completed, taking out the soil and the entire plant rhizosphere for solidification treatment, slicing, grinding and polishing to obtain a sample to be analyzed.

8. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 7, characterized in that: The soaking is graded soaking, specifically: the soil and the entire plant rhizosphere are placed in mixed solutions of different levels for soaking, and the soaking time of each level of mixed solution is 24 hours.

9. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 7, characterized in that: The mixed solution is a mixed solution of ethanol and Spurr resin; The levels of the mixed solution are: a mixed solution with a mass ratio of ethanol to Spurr resin of 2:1, a mixed solution with a mass ratio of ethanol to Spurr resin of 1:1, a mixed solution with a mass ratio of ethanol to Spurr resin of 1:3 and a mixed solution with a mass ratio of ethanol to Spurr resin of 0:

1.

10. The method for analyzing the spatial distribution and morphological changes of heavy metals in the rhizosphere of plants according to claim 7, characterized in that: The slices include transverse slices and longitudinal slices; and / or The specific operation steps of the grinding are: grinding the slice surface with silicon sandpaper of decreasing particle size; and / or The specific operation steps of the polishing treatment are: polishing the slice surface with 3 μm, 1 μm and 0.25 μm anhydrous oil-based corundum respectively.

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