CN111122461B - Monitoring Cd 2+ Method for aggregation in plant leaves - Google Patents
Monitoring Cd 2+ Method for aggregation in plant leaves Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention discloses a method for monitoring Cd 2+ A method for gathering in plant leaves, belonging to the field of heavy metal pollution monitoring; to non-glandular epidermis of plant leafCd in wool 2+ The monitoring is carried out, which is 3-7 days earlier than the conventional monitoring method, thereby being more beneficial to the prevention and treatment of heavy metal pollution in plants.
Description
Technical Field
The invention relates to the field of heavy metal pollution monitoring, in particular to monitoring Cd 2+ A method for aggregation in plant leaves.
Background
At present, heavy metal pollution in soil and underground water is increasingly serious due to the problems of massive application of pesticides and fertilizers, metal exploitation, solid waste infiltration and the like; heavy metals are difficult to biodegrade, and pollution is long-term and irreversible; therefore, people pay high attention to the treatment and monitoring of heavy metal pollution. The accumulation of heavy metals in crops such as grains, vegetables, oil and Chinese herbs can be transmitted to human body through food chain, which is harmful to human health. In the early stage of planting grains, vegetables, oil plants and Chinese herbal medicines, the problem that the content of heavy metals in agricultural products exceeds the standard can be prevented by monitoring the accumulation of the heavy metals.
Cadmium ions are not a nutrient element required by plants and can be gathered at edible parts such as plant roots, leaves, seeds and the like; if entering the human body along with the food chain, the food chain will damage the nervous system and cause severe softening of the skeleton. Therefore, there is a need to find an efficient and convenient method for monitoring the accumulation of heavy metals in plants at an early stage, and providing a better support for preventing heavy metal pollution in agricultural products such as vegetables, grains, oil plants and Chinese herbs.
Disclosure of Invention
In view of the above, the present invention provides a method for monitoring Cd 2+ Compared with the conventional monitoring method, the method for gathering in the plant leaves is improved by 3-7 days, thereby being more beneficial to the prevention and treatment of heavy metal pollution in plants.
In order to achieve the purpose, the invention adopts the following technical scheme:
monitoring Cd 2+ Method for gathering Cd in non-glandular epidermal hair of plant leaf 2+ And (5) monitoring.
The non-glandular epidermal hair is positioned on the outer layer of the leaf and is easy to observe and analyze in the growth and development process; cd [ Cd ] 2+ Preferentially aggregate in non-glandular epidermal hairs, and further contribute to Cd in plants 2+ Early monitoring of (2).
The method is suitable for crops such as grains, vegetables, oil plants, chinese herbal medicines and the like with non-gland epidermal hair distributed on leaves of Chinese cabbage, corn, soybean and the like. The non-glandular epidermal hair structures of plants such as arabidopsis thaliana and Chinese cabbage can be seen by naked eyes, and are convenient for external force touch.
Preferably, the Cd is accelerated by touching non-glandular epidermal hairs on the plant leaves by an external force 2+ The concentration of Cd in the leaf is accelerated 2+ And (5) monitoring.
Further preferably, the Cd is accelerated by touching the non-glandular epidermal hair on the leaves of the plant with an external force 2+ Accumulation in non-glandular epidermal hair of leaf, and subsequent treatment of Cd in non-glandular epidermal hair 2+ And (5) monitoring.
Preferably, the non-glandular epidermal hairs can be touched directly by hands or by using a brush or a brush.
Preferably, a method of monitoring Cd 2+ A method of aggregation within a plant leaf comprising the steps of:
(1) Touching non-glandular epidermal hairs of the plant leaves before collecting the plant leaves;
(2) Collecting plant leaves, and dyeing by using a metal chelating agent;
(3) Observing Cd in non-glandular epidermal hair by using microscope 2+ Aggregation conditions.
Preferably, after the step (1) of touching the non-glandular epidermal hair of the plant leaf, the step of waiting for Cd 2+ They were collected in the leaves of the plants and were then sampled for subsequent staining.
Preferably, the non-glandular epidermal hairs of the plant leaves are touched 10-30 times before the plant leaves are collected in the step (1), the touching frequency is not too high, and otherwise the shape and the structure of the non-glandular epidermal hairs are damaged excessively.
Preferably, the post-touch latency depends on the general condition of the heavy metal contamination, and for heavy contamination, the latency may be reduced.
Preferably, the waiting time is 1-2h.
Preferably, in the step (2), the plant leaves are soaked and dyed for 2-4 hours by using a dithizone dye solution, and then are rinsed after dyeing; the preparation method of the dithizone dye solution comprises the following steps:
1) Dissolving dithizone powder in a mixed solution of acetone and distilled water;
2) After being mixed evenly, the pH value is adjusted;
3) The ultrasonic treatment completely dissolves the dithizone powder.
Preferably, the volume ratio of acetone to distilled water in step 1) is 2; the addition amount of the dithizone powder in the mixed solution is 0.325-0.425mg/mL;
adding glacial acetic acid solution to adjust the pH value to 6-7 in the step 2); the slightly acidic condition is more favorable for dyeing;
the ultrasonic treatment time in the step 3) is 5-15s, and the prepared dithizone dye liquor is accelerated in the oxidation process if the ultrasonic treatment time is too long, depending on the dissolution condition of the dithizone powder.
The method has simple and convenient sample treatment, does not need pretreatment such as digestion and fixation, and can be operated under non-laboratory conditions such as fields, greenhouses and the like. Non-glandular epidermal hair aggregates Cd earlier than 3-7d, compared to aggregation of conventional leaf cells 2+ (ii) a The Cd can be further accelerated by the non-glandular epidermal hair touched by external force 2+ To (3) is performed.
Above-mentioned monitoring Cd 2+ The method of aggregation in plant leaves is applied to early monitoring of heavy metal pollution of plants.
According to the technical scheme, compared with the prior art, the invention discloses and provides a method for monitoring Cd 2+ The method for gathering in the plant leaves has the advantages of tissue positioning, convenient and quick operation, low requirement on samples and capability of realizing early monitoring of heavy metal pollution of plants.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings required for the embodiments will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and it is also possible for those skilled in the art to obtain other drawings based on the provided drawings without inventive effort.
FIG. 1 is a graph showing CdCl in different concentrations 2 After application of the solution to a substrate for Arabidopsis plant growth, cd 2+ Observation of aggregation in non-glandular epidermal hairs of the leaf;
wherein (A) no CdCl is applied 2 A solution control group;
(B) Application of 1nM CdCl 2 A solution test group;
(C) Application of 1. Mu.M CdCl 2 A solution test group;
(D) Application of 5. Mu.M CdCl 2 Solution test group.
FIG. 2 is a drawing showing Cd in non-glandular epidermal hair cells of Arabidopsis leaves in example 2 2+ Distribution conditions;
wherein A, C and E are applied with 1nM, 1. Mu.M and 5. Mu.M CdCl, respectively 2 A stressed control group;
B. d and F are the application of 1nM, 1. Mu.M and 5. Mu.M CdCl, respectively 2 Test group stressed and touched non-glandular epidermal hairs using a writing brush.
FIG. 3 is a graph showing CdCl in different concentrations 2 After the solution is applied to a matrix for growth of Chinese cabbage plants, cd 2+ Observation of aggregation in non-glandular epidermal hairs of the leaf;
wherein (A) no CdCl is applied 2 A solution control group;
(B) Application of 1nM CdCl 2 A solution test group;
(C) Application of 1. Mu.M CdCl 2 A solution test group;
(D) Application of 5. Mu.M CdCl 2 Solution test group.
FIG. 4 is the drawing of Cd in non-glandular epidermal hair cells of Chinese cabbage leaves in example 5 2+ Distribution conditions;
wherein A is CdCl applied at 5 μ M 2 A stressed control group;
b is the application of 5. Mu.M CdCl 2 Test group stressed and touched non-glandular epidermal hair with a writing brush.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 heavy Metal cadmium preferentially accumulates in non-glandular epidermal hairs of Arabidopsis leaves
1. Planting of arabidopsis thaliana:
planting arabidopsis thaliana in a matrix with a nutrient soil-vermiculite ratio of 4 (V: V), performing vernalization in a refrigerator at 4 ℃ for 3d, and culturing under the conditions of 16h of illumination for 8h and darkness and 20-25 ℃;
2. heavy metal stress of arabidopsis thaliana:
culturing Arabidopsis plant for 15d (the first pair of true leaves are fully extended), and adding CdCl with final concentration of 1nM, 1 μ M (lower than the standard value of national soil environment quality) or 5 μ M (higher than the standard value of soil environment quality) into the matrix 2 Solution as test group, and no CdCl addition was set 2 Control group of solution; the culture is continued for 8 days under 16h illumination and 8h darkness and 20-25 ℃.
CdCl 2 Preparing a solution: adding CdCl 2 The powder was prepared as a 0.1mol/L solution and diluted before use.
3. Dyeing:
shearing each group of plants to be detected, immersing the plants in the prepared dithizone dye solution, and dyeing for 2 hours;
preparing a dithizone dye solution: dissolving 15mg of dithizone powder into a mixed solution of 30mL of acetone and 10mL of distilled water; after mixing, 300. Mu.L of glacial acetic acid solution is added; and (5) carrying out ultrasonic treatment for 5s to obtain the dithizone dye solution.
4. Rinsing:
rinsing the dyed plants with pure water for 3 times, and then placing the plants in 50% ethanol for 2 hours to remove impurities adhered to the surface of non-glandular epidermal hair, so that the results are easier to observe; and rinsing with sterile water for 3 times to prepare the packaged chip.
5. And (3) microscopic observation:
observation of Arabidopsis thaliana non-glandular epidermal hairs under optical microscopeAnd Cd in leaf epidermal and mesophyll cells 2+ The aggregate condition of (1).
The results are shown in FIG. 1, without CdCl 2 No complex precipitates in the leaf epidermis, mesophyll cells and non-glandular epidermal hairs of the stressed control group; cdCl of different concentrations 2 Complex precipitation can be observed in non-glandular epidermal hairs of a stress test group, and complex precipitation can not be observed in leaf epidermis and mesophyll cells; it can be seen that heavy metal cadmium accumulates preferentially in the non-glandular epidermal hairs of Arabidopsis thaliana.
Example 2 Effect of touching non-glandular epidermal hairs on monitoring of heavy cadmium
1. Planting of arabidopsis thaliana:
planting arabidopsis thaliana in a matrix with a nutrient soil-vermiculite ratio of 4 (V: V), performing vernalization in a refrigerator at 4 ℃ for 3d, and culturing under the conditions of 16h of illumination for 8h and darkness and 20-25 ℃;
2. heavy metal stress of arabidopsis thaliana:
culturing Arabidopsis thaliana plant for 15d (the first pair of true leaves is fully extended), and adding CdCl with final concentration of 1nM, 1 μ M (lower than the first-class standard value of soil environment quality in China) or 5 μ M (higher than the first-class standard value of soil environment quality) into the matrix 2 A solution;
CdCl 2 preparing a solution: adding CdCl 2 The powder was prepared as a 0.1mol/L solution and diluted before use.
3. Touching non-glandular epidermal hair:
CdCl is selected 2 The plants after being stressed for 3 days are touched with a writing brush for 20 times and then are kept stand for 2 hours to serve as test groups; and a control group without touch was set.
4. Dyeing:
shearing a plant to be detected, immersing the plant in the prepared dithizone dye solution, and dyeing for 2 hours;
preparing a dithizone dye solution: dissolving 15mg of dithizone powder into a mixed solution of 30mL of acetone and 10mL of distilled water; after mixing, 300. Mu.L of glacial acetic acid solution is added; and (5) carrying out ultrasonic treatment for 5s to obtain the dithizone dye liquor.
5. Rinsing:
rinsing the dyed plants with pure water for 3 times, and then placing the plants in 50% ethanol for 2 hours to remove impurities adhered to the surface of non-glandular epidermal hairs, so that the results are easier to observe; and rinsing with sterile water for 3 times to prepare the packaged chip.
6. And (3) microscopic observation:
observing Cd in arabidopsis non-glandular epidermal hair, leaf epidermis and mesophyll cells under an optical microscope 2+ The aggregate condition of (2).
The results of the test are shown in FIG. 2, applying 1nMCdCl 2 The stressed control group has no obvious heavy metal aggregation, and the bottom of the non-glandular epidermal hair of the test group has complex precipitation; application of 1. Mu.MCdCl 2 The stressed control group non-glandular epidermal hair has no obvious heavy metal accumulation, while the test group non-glandular epidermal hair obviously has large-area complex precipitation; application of 5. Mu.MCdCl 2 The stressed control group non-glandular epidermal hair has trace complex precipitation, while the test group non-glandular epidermal hair complex precipitation has larger area, and heavy metal aggregation also occurs in mesophyll cells.
The visible touch on non-glandular epidermal hair can increase the sensitivity of heavy metal aggregation and accelerate Cd 2+ Accumulation in the leaves, especially in non-glandular epidermal hairs.
Example 3 Effect of touching non-glandular epidermal hairs on heavy Metal aggregation time
1. Planting and heavy metal stress of arabidopsis thaliana:
arabidopsis thaliana was planted in MS medium (solaribio, cat # M8521, supplemented with CdCl at a final concentration of 5. Mu.M 2 Solution), culturing under the conditions of 16h of light/8 h of dark and 20-25 ℃.
2. Touching non-glandular epidermal hair:
CdCl is selected 2 Stressing plants after different time (13 d and 15 d), touching the non-glandular epidermal hair of the arabidopsis leaves with a writing brush for 20 times, and standing for 2h to serve as a test group; and control groups (13, 15, 21 d) not touched are provided.
4. Staining, rinsing, microscopic examination were as in example 1.
As shown in Table 1, the complex precipitation was observed in the non-glandular epidermal hairs of the test group 13d, while the complex precipitation was observed in the non-glandular epidermal hairs of the control group 21d, and it was found that the contact with the non-glandular epidermal hairs accelerated the accumulation of heavy metal cadmium in the non-glandular epidermal hairs.
TABLE 1 Cd 2+ Aggregation in the skin and coat
'-' No Cd detected 2+ Accumulation in epidermal hair cells, '+' Cd 2+ Aggregation in epidermal hair cells, '/' not detected.
Example 4 heavy metal cadmium preferentially accumulates in non-glandular epidermal hairs of Chinese cabbage leaves
1. Planting Chinese cabbage:
planting Chinese cabbage in nutrient soil, and culturing under 16h light/8 h dark at 20-25 deg.C.
2. Stress of the Chinese cabbage with heavy metals:
adding CdCl with final concentration of 1nM, 1 μ M (lower than the standard value of soil environmental quality in China) or 5 μ M (higher than the standard value of soil environmental quality) into the matrix after Chinese cabbage plants are cultured for about 5 weeks 2 Solution as test group, and no CdCl addition was set 2 Control group of solution; the culture is continued for 8 days under 16h of light and 8h of darkness and at 20-25 ℃.
CdCl 2 Preparing a solution: adding CdCl 2 The powder was prepared as a 0.1mol/L solution and diluted before use.
3. Dyeing:
shearing a first true pair of leaves of each group of plants, immersing the leaves in the prepared dithizone dye solution, dyeing for 4 hours, and replacing the dithizone dye solution once in the process: dissolving 15mg of dithizone powder into a mixed solution of 30mL of acetone and 10mL of distilled water; after mixing, 300. Mu.L of glacial acetic acid solution is added; and (5) carrying out ultrasonic treatment for 5s to obtain the dithizone dye solution.
4. Rinsing:
the dyed leaves are rinsed 3 times with sterile water (the difference between the coat structure and the wax layer structure of different plants, ethanol easily changes the observation result of Chinese cabbage, so the leaves are directly rinsed with water) to prepare the slices.
5. And (3) microscopic observation:
observing Cd in brassica rapa pekinensis non-gland epidermal hair, leaf epidermal and mesophyll cells under an optical microscope 2+ The aggregate condition of (2).
The results are shown in FIG. 3, in which CdCl was not used 2 No complex precipitates in the leaf epidermis, mesophyll cells and non-glandular epidermal hairs of the stressed control group; and through CdCl 2 Stress test group with CdCl 2 The increase of the concentration causes the complex precipitation in non-glandular epidermal hairs to be more obvious, and the addition of the complex is 5 mu M CdCl 2 And (3) outside the stress test group, mesophyll cells of other groups do not have complex precipitation, and the accumulation of heavy metal cadmium is prior to the accumulation of non-glandular epidermal hairs of the Chinese cabbage.
Example 5 Effect of touch on monitoring of heavy cadmium Metal by non-glandular epidermal Hair
1. Planting Chinese cabbage:
planting Chinese cabbage in nutrient soil, and culturing under 16h light/8 h dark at 20-25 deg.C.
2. Stress of the Chinese cabbage with heavy metals:
culturing Chinese cabbage for about 5 weeks, adding CdCl with final concentration of 5 μ M into the matrix 2 A solution;
CdCl 2 preparing a solution: adding CdCl 2 The powder was prepared as a 0.1mol/L solution and diluted before use.
3. Touching non-glandular epidermal hair: cdCl is selected 2 Stressing the plants after 3d, touching the first pair of true leaf non-glandular epidermal hair of the Chinese cabbage with a writing brush for 20 times, and standing for 2h to serve as a test group; and a control group without touch was set.
4. Dyeing:
shearing a first true pair of leaves of each group of plants, immersing the leaves in the prepared dithizone dye solution, dyeing for 4 hours, and replacing the dithizone dye solution once in the process: dissolving 15mg of dithizone powder into a mixed solution of 30mL of acetone and 10mL of distilled water; after mixing, 300. Mu.L of glacial acetic acid solution is added; and (5) carrying out ultrasonic treatment for 5s to obtain the dithizone dye solution.
5. Rinsing:
the dyed leaves are rinsed 3 times with sterile water (the difference between the coat structure and the wax layer structure of different plants, ethanol easily changes the observation result of Chinese cabbage, so the leaves are directly rinsed with water) to prepare the slices.
6. And (3) microscopic observation:
observing Cd in non-glandular epidermal hair, leaf epidermis and mesophyll cells of the Chinese cabbage under an optical microscope 2+ The aggregate condition of (1).
The test results are shown in fig. 4, and the control group which is not touched has no heavy metal aggregation, while the non-glandular epidermal hair of the test group has obvious complex precipitation. Visible contact with non-glandular epidermal hair to accelerate Cd 2+ Accumulation in non-glandular epidermal hairs.
Further, in another preferred embodiment, plants (such as arabidopsis thaliana, chinese cabbage and the like) with non-glandular epidermal hairs are planted in the soil to be monitored, the non-glandular epidermal hairs of plant leaves are touched after the plants are cultured for a period of time, the plants are stained and rinsed after the plants are kept still for a period of time, microscopic observation is carried out, and the heavy metal pollution condition in the soil to be monitored is qualitatively analyzed according to the existence or nonexistence of a complex or the area size of the complex.
In the description, each embodiment is mainly described as different from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (4)
1. Monitoring Cd 2+ A method of aggregation within a plant leaf, comprising the steps of:
(1) Touching non-gland epidermal hair of plant leaf by external force before collecting plant leaf, waiting for Cd 2+ Build up within the plant leaf;
(2) Collecting plant leaves, and dyeing by using a metal chelating agent;
(3) Observing Cd by using microscope 2+ Accumulation in non-glandular epidermal hairs of plant leaves.
2. The method of claim 1 for monitoring Cd 2+ A method of aggregation in a plant leaf, characterized in that,
soaking and dyeing the plant leaves in the step (2) for 2-4h by using a dithizone dye solution, and rinsing after dyeing; the preparation method of the dithizone dye solution comprises the following steps:
1) Dissolving dithizone powder in a mixed solution of acetone and distilled water;
2) After mixing uniformly, adjusting the pH value;
3) The ultrasonic treatment completely dissolves the dithizone powder.
3. The method of claim 2, wherein the method comprises monitoring Cd 2+ A method of aggregation in a plant leaf, characterized in that,
the volume ratio of acetone to distilled water in the step 1) is 2; the addition amount of the dithizone powder in the mixed solution is 0.325-0.425mg/mL;
adding glacial acetic acid solution to adjust the pH value to 6-7 in the step 2);
the ultrasonic treatment time in the step 3) is 5-15s.
4. A method of monitoring Cd as claimed in any one of claims 1 to 3 2+ The method of aggregation in plant leaves is applied to early monitoring of heavy metal pollution of plants.
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