Screening method of mangrove plant species with heavy metal resistance

Abstract

The invention discloses a method for screening mangrove plant species with heavy metal resistance, which comprises the following steps: dividing physiological indexes of mangrove plants into positive correlation indexes and negative correlation indexes, and quantitatively analyzing and evaluating the heavy metal resistance of different local mangrove plant species by applying a membership function method. And mangrove plant species seedlings with excellent heavy metal pollution resistance and tolerance can be screened as key planting tree species of a protected land.

Description

Technical Field

[0001] The present invention relates to the technical field of plant selection for heavy metal resistance, and particularly to a method for screening mangrove plant species with heavy metal resistance performance. Background Art

[0002] Mangroves are wetland woody plant communities composed of evergreen trees or shrubs mainly of mangrove plants, growing in the intertidal zones of tropical and subtropical coasts. They play an important role in purifying water quality, preventing wind and dissipating waves, promoting sedimentation, sequestering carbon and enhancing sinks, and maintaining biodiversity. With the development of urbanization and industrialization, serious environmental problems have been brought to mangrove wetlands, which have also become the main factors restricting the survival and development of mangroves in coastal areas. Under the stress of heavy metals, it leads to an increase in the membrane permeability of plant cells, a decrease in the photosynthetic metabolism rate, a reduction in the respiratory metabolism, a decline in the enzyme metabolism rate, DNA damage, gene mutations, and changes in genetic effects. Some studies have shown that the antioxidant defense system participates in the adaptive response of plants to metal ions under heavy metal stress. They directly or indirectly quench ROS through multiple pathways to avoid oxidative stress on mangrove plants. However, so far, it is impossible to quantitatively analyze the strength of the heavy metal resistance of mangrove plants. Summary of the Invention

[0003] In view of the above problems, the present invention proposes a method for screening mangrove plant species with heavy metal resistance performance, aiming to quantitatively analyze the strength of the heavy metal resistance of mangrove plants.

[0004] To solve the above technical problems, the technical solution of the present invention is as follows:

[0005] A method for screening mangrove plant species with heavy metal resistance performance includes the following steps:

[0006] According to the first preset condition, initially screen out several varieties of one-year-old mangrove plant seedlings;

[0007] Prepare a contaminated solution containing mixed heavy metals according to a preset concentration gradient. For the mangrove plant seedlings of the same variety, plant each mangrove plant seedling in a contaminated solution with a different concentration. During the screening process, continuously replenish water to the contaminated solution to maintain the liquid level scale of the contaminated solution;

[0008] Place all the mangrove plant seedlings participating in the screening into an artificial climate chamber, and set the environmental parameters of the artificial climate chamber according to the second preset condition;

[0009] Measure the physiological indexes of the leaves of all the mangrove plant seedlings participating in the screening according to a preset date, and calculate the comprehensive heavy metal resistance index of the mangrove plant seedlings by using the membership function method;

[0010] Screen out the mangrove plant seedlings with a relatively high comprehensive heavy metal resistance index as the main tree species to be planted in the target restoration area.

[0011] In some embodiments, the calculation method of the comprehensive heavy metal resistance index includes calculating the membership degrees of the positive correlation index and the negative correlation index of the mangrove plant seedlings respectively:

[0012] U(X ijk1 )=(X ijk1 –X min1 ) / (X max1 –X min1 ) (1);

[0013] U(X ijk2 )=1–(X ijk2 –X min2 ) / (X max2 –X min2 ) (2);

[0014] In the formula, U(X ijk1 ) is the membership degree of the k-th positive correlation index of the i-th variety of mangrove plant seedlings in the j-th time period, and U(X ijk1 ) ∈ [0, 1], X ijk1 represents the measured value of the k-th positive correlation index of the i-th variety of mangrove plant seedlings in the j-th time period, X max1 , X min1 are the maximum and minimum values of the k-th positive correlation index among all experimental species; U(X ijk2 ) is the membership degree of the k-th negative correlation index of the i-th variety of mangrove plant seedlings in the j-th time period, and U(X ijk2 ) ∈ [0, 1], X ijk2 represents the measured value of the k-th negative correlation index of the i-th variety of mangrove plant seedlings in the j-th time period, X max2 , X min2 are the maximum and minimum values of the k-th negative correlation index among all experimental species;

[0015] The sum of the average values of the membership degrees of each index of each variety of mangrove plant seedlings is used as the comprehensive heavy metal resistance index.

[0016] In some embodiments, the positive correlation index includes at least CAT, POD, and SOD.

[0017] In some embodiments, the negative correlation index includes at least protein, MDA, and H2O2.

[0018] The beneficial effects of the present invention are as follows: By classifying the physiological indexes of mangrove plants into positive correlation indexes and negative correlation indexes, and applying the membership function method to quantitatively analyze and evaluate the heavy metal resistance of different native mangrove plant species, it is possible to screen out the seedlings of mangrove plant species with excellent heavy metal pollution tolerance as the key tree species for planting in protected areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a flowchart of a method for screening mangrove plant species with heavy metal resistance performance disclosed in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] To make the objectives, technical solutions and advantages of the present invention clearer and more explicit, the content of the present invention will be further described in detail below with reference to the drawings and specific embodiments. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention. Additionally, it should be noted that for the sake of description, only parts related to the present invention are shown in the drawings, rather than all the content.

[0021] This embodiment provides a method for screening mangrove plant species with heavy metal resistance performance, as Figure 1 shown, including the following steps:

[0022] Step 1: Initially screen out several varieties of one-year-old mangrove plant seedlings according to the first preset condition.

[0023] In one example, in Step 1, 5 species of one-year-old mangrove plant seedlings (Bruguiera gymnorrhiza, Rhizophora stylosa, Avicennia marina, Aegiceras corniculatum, and Kandelia candel) are initially screened out. Under the condition of ensuring sufficient sunlight supply and nutrient solution irrigation, individuals without mechanical damage and pest and disease infestation are selected for the heavy metal resistance screening experiment.

[0024] In Step 1, there are several reasons for choosing one-year-old seedlings as experimental subjects, mainly including the following points:

[0025] (1) Strong cell division ability: The cambium cells of the branches of one-year-old seedlings have strong cell division ability and develop rapidly. This means that under experimental conditions, these seedlings can quickly respond to various treatment measures, making it easier to observe the experimental effects. This rapid growth and development characteristic makes one-year-old seedlings an ideal material for studying plant physiological and biochemical processes.

[0026] (2) Easy to survive and take root: One-year-old seedlings have strong vitality and are easy to survive and take root. During the experiment, even if they are damaged or treated to a certain extent, they may still recover and continue to exhibit the performance required for the experiment. This makes the experimental results more reliable and stable.

[0027] (3) Reducing individual differences: Selecting one-year-old seedlings with the same age and similar growth conditions for the experiment can minimize the influence of individual differences on the experimental results. This helps to more accurately evaluate the effects of different treatment measures on plant performance, thus obtaining more reliable conclusions.

[0028] (4) Easy to control experimental conditions: Seedlings of one-year-old are relatively small and are easy to cultivate and manipulate in the laboratory. This enables researchers to more precisely control experimental conditions such as light, temperature, water, and nutrients, thereby more accurately studying the effects of these conditions on plant performance.

[0029] Step 2: Prepare contaminated solutions containing mixed heavy metals according to a preset concentration gradient. For seedlings of the same variety of mangrove plants, plant each mangrove plant seedling in contaminated solutions of different concentrations, and continuously replenish water to the contaminated solutions during the screening process to maintain the liquid level scale of the contaminated solutions.

[0030] Based on Step 1, plant the cultivated seedlings of 5 mangrove plant species in contaminated solutions containing mixed heavy metals (copper, zinc, and lead in sequence) at different concentrations (mg / L). The concentration gradients are set as: T0 (0, 0, 0), T1 (2, 5, 1), T2 (10, 25, 5), T3 (20, 50, 10), and T4 (30, 75, 15), and continuously replenish water to the composite heavy metal solution to maintain the original solution scale.

[0031] Step 3: Place all mangrove plant seedlings participating in the screening into an artificial climate chamber and set the environmental parameters of the artificial climate chamber according to the second preset conditions.

[0032] In one example, the environmental parameters are set as: temperature 25°C, light time 14 / 10 h (day / night), light intensity 20000 Lx, and humidity 75%.

[0033] Step 4: Measure the physiological indexes of the leaves of all mangrove plant seedlings participating in the screening according to the preset date, and calculate the comprehensive heavy metal resistance index of the mangrove plant seedlings by using the membership function method.

[0034] In one example, under heavy metal stress treatment, measure the contents of protein, MDA, and H2O2 and the activities of SOD, POD, and CAT in the leaves of mangrove plants at 0 d, 3 d, 7 d, 14 d, and 28 d respectively.

[0035] The calculation method of the comprehensive heavy metal resistance index includes calculating the membership degrees of the positive correlation indexes and negative correlation indexes of the mangrove plant seedlings respectively:

[0036] U(X ijk1 )=(X ijk1 –X min1 ) / (X max1 –X min1 ) (1);

[0037] U(X ijk2 )=1–(X ijk2 –X min2 ) / (Xmax2 –X min2 ) (2);

[0038] Wherein, U(X ijk1 ) is the membership degree of the i-th variety of mangrove plant seedlings for the k-th positive correlation index in the j-th time period, and U(X ijk1 ) ∈ [0, 1], X ijk1 represents the measured value of the k-th positive correlation index of the i-th variety of mangrove plant seedlings in the j-th time period, X max1 , X min1 are the maximum and minimum values of the k-th positive correlation index among all experimental species; U(X ijk2 ) is the membership degree of the i-th variety of mangrove plant seedlings for the k-th negative correlation index in the j-th time period, and U(X ijk2 ) ∈ [0, 1], X ijk2 represents the measured value of the k-th negative correlation index of the i-th variety of mangrove plant seedlings in the j-th time period, X max2 , X min2 are the maximum and minimum values of the k-th negative correlation index among all experimental species;

[0039] In formulas (1) and (2), the positive correlation indexes at least include CAT, POD, and SOD, and the negative correlation indexes at least include protein, MDA, and H2O2.

[0040] Protein in leaves: Leaves are the main sites for plants to carry out photosynthesis and synthesize proteins. The leaves of many cereal and legume crops contain 2% - 4% protein. Leaf protein is extracted from plant stems and leaves through juicing. It is a grayish-white or green powder, with a protein content generally around 60% and an unsaturated fatty acid content of 20% - 30%. Leaf protein products contain 55% - 72% protein and include 18 kinds of amino acids, among which 8 are essential amino acids for the human body, and it has various physiological functions such as preventing and treating diseases, anti-aging, and strengthening the body.

[0041] Content of malondialdehyde (MDA): MDA is one of the main products of membrane lipid peroxidation, and its content can reflect the degree of stress damage suffered by plants. When plants are subjected to stresses such as drought, high temperature, low temperature, and salt stress, the balance between the generation and scavenging of free radicals in cells is disrupted, leading to the accumulation of free radicals, which in turn triggers membrane lipid peroxidation and produces MDA. Therefore, the MDA content can be an important indicator for evaluating plant stress resistance. For example, in the study of the drought resistance physiology of fruit trees, it was found that when the relative water content of leaves decreases, the content of MDA in cells will increase.

[0042] Content of hydrogen peroxide (H2O2): H2O2 is an important reactive oxygen species molecule in plant cells and plays an important role in plant growth, development and stress resistance. Under normal circumstances, the content of H2O2 in plant cells remains at a relatively low level, but when plants are subjected to stress, the content of H2O2 will increase. H2O2 can participate in the process of plant response and adaptation to stress as a signaling molecule. At the same time, H2O2 is also one of the products of membrane lipid peroxidation, and the increase in its content can reflect the degree of damage to plant cell membranes. There are various methods for measuring the content of H2O2, such as the KI method, etc.

[0043] Superoxide dismutase (SOD) activity: SOD is an important protective enzyme in plants. It can catalyze the dismutation reaction of superoxide anion radicals (O2-.) to generate H2O2 and O2, thereby scavenging free radicals in cells and protecting the integrity of cell membranes and organelles. SOD activity is closely related to the stress resistance of plants. When plants are subjected to stress, the SOD activity will change to adapt to the stress environment. For example, under stress conditions such as high temperature, drought, and salt stress, the SOD activity in plant leaves may increase to improve the stress resistance of plants.

[0044] Peroxidase (POD) activity: POD is another important protective enzyme in plants. It can catalyze the oxidation-reduction reaction with H2O2 as the oxidant, and while oxidizing other substances, reduce H2O2 to H2O, thereby scavenging H2O2 in cells. POD activity is related to plant respiration, photosynthesis, oxidation of auxin, and formation of lignin, etc. Its activity changes with the growth and development process of plants and the change of environmental conditions. Therefore, measuring POD activity can reflect the changes in metabolism and stress resistance in plants at a certain period.

[0045] Catalase (CAT) activity: CAT is an enzyme scavenger. It can catalyze the decomposition of hydrogen peroxide into oxygen and water, thereby protecting plants from the damage of peroxides. CAT is widely present in plants, especially under stress, its activity may increase to improve the stress resistance of plants. The method for measuring CAT activity is usually based on the characteristic absorption peak of H2O2 at 240 nm, and the CAT activity is calculated by monitoring the change in absorbance of the reaction solution.

[0046] The sum of the average membership degrees of each index of the mangrove plant seedlings of each variety was used as the comprehensive heavy metal resistance index, and the value of the comprehensive heavy metal resistance index was used as the basis for screening the strong and weak heavy metal resistance of mangrove plants, as shown in Table 1.

[0047] Table 1 Comprehensive evaluation results of heavy metal resistance of different mangrove plant species using the membership function method

[0048]

[0049]

[0050] Step 5: Screen the mangrove plant seedlings with relatively high comprehensive heavy metal resistance index as the main tree species to be planted in the target restoration area.

[0051] In this embodiment, the mangrove plant seedlings with relatively high comprehensive heavy metal resistance index refer to the mangrove plant seedlings whose comprehensive heavy metal resistance index performance meets the expectations of the experimenter in the same batch of experiments. For example, the screening results in Step 4 show that the antioxidant enzyme systems of the mangrove plant species Avicennia marina, Bruguiera gymnorrhiza, Rhizophora stylosa, and Kandelia obovata have good heavy metal resistance, while the comprehensive heavy metal resistance index of Aegiceras corniculatum is significantly worse than that of other mangrove plant seedlings. Among them, the antioxidant enzyme system of Avicennia marina has the best heavy metal resistance, and the 4 mangrove plant species can be used for the construction of anti-pollution coastal mangroves and near-natural precise cultivation. Specifically, plant the mangrove plants (or mature hypocotyls, 3 for each sampling point to ensure the survival rate) with a plant spacing and row spacing of 0.5 - 1.5 m, and conduct the management and replanting of mangrove seedlings in the afforestation area for more than 2 consecutive years after afforestation. After 3 years, the acceptance survival rate is above 80%. Before screening, the experimenter can set a quantitative index. For example, for the 5 mangrove plant seedlings in the same batch of experiments, 2, 3, or 4 species with the highest comprehensive heavy metal resistance index can be selected as the main tree species in the target restoration area. Or, eliminate the tree species whose comprehensive heavy metal resistance index is significantly lower than that of other mangrove plant seedlings.

[0052] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those of ordinary skill in the art to understand the content of the present invention and implement it accordingly. It should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the essence of the content of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for screening mangrove plant species with heavy metal resistance, characterized in that: The following steps are involved: Preliminarily select a number of species and one-age mangrove plant seedlings according to the first preset condition; A polluted solution containing mixed heavy metals is prepared according to a preset concentration gradient. For mangrove plant seedlings of the same species, each mangrove plant seedling is planted in a polluted solution of different concentrations. During the screening process, water is continuously added to the polluted solution to maintain the liquid level of the polluted solution; Placing all the mangrove plant seedlings involved in the screening into an artificial climate chamber, and setting the environmental parameters of the artificial climate chamber according to the second preset conditions; The physiological indexes of leaves of all mangrove seedlings participating in the screening were measured on the preset dates, and the comprehensive index of heavy metal resistance of mangrove seedlings was calculated by using the membership function method; Mangrove seedlings with relatively high comprehensive heavy metal resistance index were selected as the main tree species planted in the target restoration areas.

2. The method for screening mangrove plant species with heavy metal resistance according to claim 1, characterized in that: The method for calculating the comprehensive heavy metal resistance index includes respectively calculating the membership degrees of the positive correlation index and the negative correlation index of the mangrove plant seedlings: U(X ijk1 )=(X ijk1 –X min1 ) / (X max1 –X min1 ) (1); U(X ijk2 )=1–(X ijk2 –X min2 ) / (X max2 –X min2 ) (2); In the formula, U(X ijk1 ) is the membership degree of the kth positive correlation index of the i-th species of mangrove seedlings in the j-th time period, and U(X ijk1 )∈[0,1],X ijk1 represents the measured value of the kth positive correlation index of the i-th species of mangrove plant seedlings in the j-th time period, X max1 , X min1 is the maximum and minimum value of the kth positive correlation index in all experimental species; U(X ijk2 ) is the membership degree of the kth negative correlation index of the i-th species of mangrove seedlings in the j-th time period, and U(X ijk2 )∈[0,1],X ijk2 represents the measured value of the kth negative correlation index of the i-th species of mangrove seedlings in the j-th time period, X max2 , X min2 is the maximum and minimum value of the kth negative correlation index in all experimental species; The sum of the average values ​​of the degrees of membership of various indicators of each variety of mangrove plant seedlings is used as the comprehensive heavy metal resistance index.

3. The method for screening mangrove plant species with heavy metal resistance according to claim 1, characterized in that: The positive correlation indexes at least include CAT, POD and SOD.

4. The method for screening mangrove plant species with heavy metal resistance according to claim 1, characterized in that: The negatively correlated indicators at least include protein, MDA and H2O2.

Images