CN115156286B - Efficient screening method for heavy metal low-accumulation ecological restoration plants/crops - Google Patents
Efficient screening method for heavy metal low-accumulation ecological restoration plants/crops Download PDFInfo
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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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
-
- 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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
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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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2866—Grinding or homogeneising
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- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Engineering & Computer Science (AREA)
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- Mycology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for efficiently screening heavy metal low-accumulation ecological restoration plants/crops, which comprises the following steps: on an explant tissue culture platform conforming to the specification, establishing a characteristic curve for accumulating heavy metals in stems and leaves of a reaction species/variety; according to the soil environment of the target planting area, negative selection pressure is set pertinently, a heavy metal absorption curve based on specific conditions is obtained directionally, low-enrichment ecological restoration plants/crop materials with optimal comprehensive performance are selected, and nutrition bodies are recommended to the specific area to have environment-friendly characteristics, wherein the low-enrichment ecological restoration plants/crop species or crop varieties are selected. The method greatly reduces scientific research cost, efficiently screens high-accumulation and low-accumulation ecological restoration plants/crop varieties, can be freely combined with efficient methods such as gene editing, physicochemical mutagenesis and the like to develop researches, provides a joint port with an efficient breeding system, and greatly reduces pollution to farmland and potting soil, groundwater and surrounding environment during screening.
Description
Technical Field
The invention belongs to the technical field of heavy metal enrichment type screening, and relates to a method for efficiently screening heavy metal low-accumulation ecological restoration plants/crops.
Background
The main effects of plants on heavy metals have been reported to include extraction (Phytoextraction), volatilization (Phytovolatilization), filtration (Rhizofilization), and plant inactivation (Phytostabilization). Accordingly, the relationship between the two is also multiple: first, plants can separate acidic substances through root systems, and activate metal elements (including heavy metals) to be converted from insoluble states to soluble states, so that absorption and enrichment of trace elements are promoted. The capacity size is related to the genotype of the plantable. Therefore, selectively planting plants can reduce the activation of heavy metals in soil caused by plant acidification; secondly, the plants can promote the heavy metal ions in some soil to be converted into gas, volatilize into the air, and influence the air quality and human respiratory system health; thirdly, the plant can promote the transfer of rhizosphere heavy metals by means of waterpower through root holes, so that the safety of a local ecological system and a downstream water system is deeply polluted; fourth, the plants may also promote valence state conversion of heavy metal ions, thereby causing aggravated conversion between non-toxic and toxic states.
Thus, the potential impact of unnecessary plant activation on soil heavy metals in high background or contaminated areas is great, with risks far threatening the safety of the product, and possibly the long-term safety of the ecosystem and the social system in which it is located. Although it is obvious that plants indirectly affect the safety of heavy metals in the surrounding environment, the safety is realized through nutrients (rhizomes and leaves). However, in the past, public attention has focused mainly on agricultural product safety and food safety, and there are studies and reports on the threat of heavy metals entering biosphere through nutrients to ecosystems and future agricultural product safety.
In practice, the need for low enrichment species/varieties is also mainly reflected in the study and application of low enrichment capacity on the product parts of edible plants or on the edible parts of crops/medicinal herbs. Less attention is paid to the action of crop nutrients for enabling organic heavy metals to enter a food chain through the function of a feed, the action of volatilizing the heavy metals of plant leaves, and the action of returning the nutrients such as straw/fallen matters and the like to soil for entering a saprophytic chain and releasing the organic heavy metals for the second time. The plant nutrient effects which are often neglected above have a substantial multiple effect on the heavy metal risk of the ecosystem and even on the entire social-ecosystem of the place of production.
Thus, the value of the nutritionally low enriched species/variety is severely underestimated. In fact, the nutrition weight metal enrichment type is concerned, the nutrition weight metal enrichment type has extremely high correlation with the safety of product parts, and the unnecessary activation of residual heavy metal molecules through interaction of root systems and soil in the growth of high enrichment type plants can be reduced, so that the generation of heavy metal ions with biological effectiveness is reduced; the unnecessary release of gaseous heavy metals to the air by plants through air holes is reduced; the release and transfer of soluble harmful metal ions to a water system and a soil system through water and soil loss are reduced; the organized heavy metal ions are reduced to enter a food amount chain or a soil saprophytic chain; the problem that toxic or more toxic organic heavy metal ions enter the biosphere and further influence the safety of the human society and the ecological system is solved.
The selection of low enrichment species or varieties in the past has generally been based on in situ selection of contaminated sites, contaminated farmland experiments or potted plant experimental performance. Seed selection is carried out on the basis of a polluted site, so that the problems of uneven heavy metal distribution, excessively complex influence factors and the like of the polluted site are difficult to overcome. The environmental stress conditions to which the selected materials are subjected are quite different in spite of their neighboring growth, so that conventional selection methods based on enrichment coefficients face an incomparable embarrassment. It is thus difficult to correctly distinguish between environmental changes and actual gene expression or mutation. The potted plant selection system cannot avoid the problem that single plant seed selection is not representative, and materials with real genetic characteristics cannot be selected without multiple rounds of random observation experiments. Meanwhile, the heavy metal release dosage of potting soil is large, and the overflow result is serious. The whole system has strict control requirements and may cause serious environmental pollution due to slight carelessness. Meanwhile, the artificially added pollutants are not necessarily uniformly distributed in potting soil, and the experimental effect is still limited.
On the other hand, in the front of molecular biological breeding, high and new technologies such as gene editing, accurate mutagenesis and the like face the screening difficulty. For example, all broad-spectrum screens require substantial background knowledge about element absorption, transport, and their regulatory mechanisms. However, since the absorption of heavy metal elements is affected by too many biological and non-biological factors, many factors affecting the absorption of heavy metals are difficult to find major genes and define major influencing mechanisms. The complexity of heavy metal absorption and accumulation problems and the deficiency of current knowledge accumulation are difficult to meet the negative pressure selection requirement after gene editing or accurate mutation breeding.
Based on the above research difficulties, the selection of various enriched varieties or species in the past has been largely ineffective, questionable, or inefficient, long lasting. Traditional breeding emphasizes repeated selfing purification genotypes, but lacks the apparent morphology of the heavy metal low-enrichment genes which can be directly observed, and each generation detects the heavy metal enrichment concentration according to the traditional breeding convention, so that the field difference needs to be overcome, and the sampling amount and the detection number are increased. These operations clearly further make field screening inefficient and expensive. The larger probability can be put into great effort with little effect.
Therefore, there is a need to develop more efficient, low cost, low pollution selection systems that address the low consumption screening problem of low enriched species/varieties. Thereby providing a practical and effective screening technology for agricultural breeding, ecological seed selection and even landscape ecological restoration plant configuration.
Disclosure of Invention
The invention aims to solve the technical problems that: the efficient screening method for selecting the ecological restoration plants/crop species/varieties with low accumulation of heavy metals under the normal growth environment conditions with low effective concentration, which are non-lethal and have no obvious influence, is provided to solve the problem of ineffective screening in the conventional technology, assist the seed selection part of the high-tech breeding technology, compensate the problems of wrong selection and missed selection of DNA molecular identification caused by insufficient background knowledge and the like, and simultaneously reduce the false positives and the like associated with the application of the technologies such as gene editing and the like in breeding.
The technical scheme adopted by the invention is as follows: a method for high-efficiency screening of heavy metal low-accumulation ecological restoration plants/crops comprises the following steps: on an ecological restoration plant/crop explant tissue culture platform meeting the specification, adding heavy metal components of composite or single elements according to concentration gradient, and establishing a characteristic curve of accumulated heavy metal in stems and leaves of a reaction species/variety by carrying out simplified research on two aspects of test tube growth data observation and recording and trace heavy metal content detection methods in a concentration interval in which the additives are completely non-lethal. If necessary, according to the soil environment of a target planting area, selectively setting negative selection pressure on environmental specific elements such as pH value, iron, manganese and the like or nutrient elements such as N, P and the like in a targeted manner, directionally acquiring a heavy metal absorption curve based on specific conditions, selecting low-enrichment ecological restoration plant/crop materials (stem and leaf heavy metal residual quantity is less than or equal to the national standard of product safety) with optimal comprehensive performance of ecological restoration plants/crop nutrient bodies (stems and leaves), and recommending the low-enrichment ecological restoration plant/crop species or crop varieties with environmental friendly characteristics to the specific area. To reduce unwanted activation of soil heavy metals due to improper species/breed selection, and adverse effects of its nutrients entering the local food chain system, or returning to the soil, entering the saprophytic circulation system, on the local agricultural social-ecosystem;
The establishment method of the explant tissue culture platform meeting the specification comprises the following steps: firstly, establishing an aseptic explant with the convergence of maximum growth amount; secondly, the explant needs to be subjected to rooting treatment in advance, and a culture medium suitable for corresponding ecological restoration plants/crop types is selected for rooting treatment for rooting culture; third, long-term treatment coupled heavy metal stress protocols are employed. Short-term treatment focuses on physiological changes and heavy metal absorption laws; long-term treatment is carried out to observe the long-term accumulation characteristic of ecological restoration plants/crops. Note that the processing modes are different, and the homogeneity implementation emphasis strategies are different. The short-term treatment liquid adopts a liquid and homogeneous solid culture medium; the long-term treatment adopts the method that liquid heavy metal solution is injected on the surface of the culture medium at one time, the culture medium is uniformly shaken by hand, and the culture medium is kept stand for 90-120d.
The negative selection pressure setting method comprises the following steps: and setting a heavy metal composite/single treatment negative pressure, specifically combining with pH value change, and/or excessive/insufficient stress of environmental elements such as Fe, mn, ca and the like which influence heavy metal attraction and nutrient elements such as N and the like which influence heavy metal attraction to form screening negative pressure, and selecting low-enrichment type ecological restoration plants/crop species/crop varieties under the condition of excessive or insufficient elements which influence the environmental pH value and the heavy metal absorption.
The heavy metal treatment method comprises the following steps: for the selected explant in the explant tissue culture platform, under the aseptic condition, a10 mL configured Pb solution (Pb standard solution or prepared Pb soluble solution) is sucked by a liquid-transfering gun, and is injected into the surface of about 30-50ml of solid culture medium or liquid culture medium with the same volume, and the solution is slightly shaken to be uniform, so that uniform negative selection pressure is formed. The size of the treatment materials is as follows: seedling or explant materials which grow uniformly and have a height of 4-6cm and a root length of 1-3 cm; aiming at the culture condition of a solid culture medium, forceps are uniformly used for lifting each seedling moderately upwards before injection, so as to ensure that newly generated root tips are fully contacted with the treatment fluid. Meanwhile, a blank control with the same culture condition but the concentration of the treatment fluid is 0 is set, and the same stress treatment is repeated for 3-5 times for 1-110 days.
The method for detecting the trace heavy metals comprises the following steps: I. the preparation method of the trace heavy metal detection sample comprises the steps of preparing a sample: shearing 0.1-0.3g of stem and leaf materials of test-tube seedlings which are not stained with the treatment liquid, putting the materials into a batch-treatment micro-tissue grinder, heating liquid nitrogen to quickly freeze seedling tissues, grinding the materials into powder, transferring the powder into a digestion test tube, putting the digestion test tube into a microwave digestion instrument for treatment, and detecting the current day; II. The preparation method of manual small-scale batch detection samples comprises the following steps: preparing a sample by adopting a batch-processing micro-tissue grinder according to the operation convenience and the limit time limit of liquid nitrogen freeze thawing; III, detecting trace heavy metals: and (3) carrying out multichannel rapid detection on the sample processed in the step (I) by adopting a plasma inductively coupled mass spectrometer (ICP-MS), and carrying out high-flux measurement on relevant elements such As Pb, cd, as, hg, cr, fe, mn, zn and the like, wherein the content detection repetition times are set for 3 times theoretically. The homogeneity is well controlled, 3 times of repetition are detected in the early stage, and the tissue culture platform with the heavy metal detection result error below 5% can accept no repetition under the same condition.
The above comprehensive evaluation and species/variety recommendation: according to the heavy metal risk condition of the planting area, establishing an absorption and accumulation curve of the stem leaves of the species/varieties to be detected on different heavy metal ions under the condition of combined pollution or single pollution; the concentration gradient absorption rule of the unit heavy metal accumulation amount to be detected in each material stem leaf or root system is integrated, the heavy metal accumulation rule aiming at the typical environmental characteristics of a specific area is analyzed by using statistical software such as SPSS (specific surface soil system) and the like, and the influence rule of the environmental pH value, the key element influence rule and the important management means (such as N application, quicklime application and the like) are focused. The correlation between the influence factors and the accumulation of heavy metals is established by utilizing SPSS, the weights of different factors are analyzed, and an element path analysis chart is established; according to different accumulation performances under the set negative pressure, primarily selecting low enrichment type evaluation as a primary selection material; in conjunction with the detection of physiological indicators, it is desirable to combine the identification of key influencing genes (e.g., ASA, GSH, etc. key regulatory genes) and recommend species/varieties suitable for planting for different regions, when conditions warrant.
The ecological planting verification and variety authorization are carried out: the selected material is subjected to multi-point multi-observation verification experiments for more than or equal to 3 years, and the high enrichment or low enrichment species/variety types can be authorized by the verification.
Above-mentioned batch processing micro-tissue grinder, including grinding pestle, EP pipe, grinding heat insulating board and grinding fixer, the grinding fixer is upper end open-ended cavity, and the sealed lid of grinding heat insulating board closes in grinding fixer upper end, is provided with EP pipe through jack and liquid nitrogen access hole on the grinding heat insulating board, and EP pipe lower extreme can stretch into in the grinding fixer through EP pipe through hole, grinds the pestle and arranges in the EP pipe.
Preferably, the bottom of the grinding pestle is spherical and is provided with a grinding layer.
Preferably, a plurality of EP pipe penetrating holes and 1 liquid nitrogen inlet hole are formed in the grinding heat insulation plate.
The invention has the beneficial effects that: compared with the prior art, the invention has the following effects:
1) The low enrichment type crop varieties or grasses selected by the platform are planted in the heavy metal geological high background area (geological source heavy metal), so that the safety requirements of agricultural products, foods and food chains and a local society-ecological system are met; the low-enrichment ecological restoration plant species or variety selected by the planting platform is planted in the ecological landscape land and ecological restoration land with heavy metal pollution, is used for reducing the entry of harmful heavy metal elements in the land into the biosphere, and plays roles of guaranteeing the safety of long-term products and the prevention and control of the risk of an ecological system;
2) The method can be combined with a high-tech breeding method for breeding research. For example, the method can be combined with methods such as gene editing, chemical mutagenesis, radiation mutagenesis, precise mutagenesis and the like, and can also be combined with any traditional negative pressure (saline-alkali stress, hypertonic stress, single metal, composite metal influence and the like) selection method so as to enhance the selection efficiency and the accuracy;
3) The inner diameter of the bottom surface of the grinding pestle of the innovative equipment 6-hole micro-grinder is slightly smaller than the diameter of the 2.5ml EP pipe, and the bottom surface of the grinding pestle is spherical, so that the effective grinding area and the grinding sufficiency can be ensured; thanks to the 2.5ml EP tube ball bottom structure, the extruded sample can slide and collect at the tube bottom by simple knocking or gentle shaking in the grinding process, so that the grinding effect can be further improved; liquid nitrogen is not easy to volatilize in the grinding process, and a portable sample is not easy to splash out; the operator can exert better force by holding the grinding holder; the device is provided with a plurality of EP pipe penetrating holes, can be used for batch processing, and has higher extraction efficiency; after finishing grinding, batch transfer is convenient, and the recovery rate is high; the components of the device are easy to obtain, and the cost is low; liquid nitrogen is poured into the sealing in advance, so that the risk of frostbite during liquid nitrogen pouring is avoided.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a schematic diagram of the structure of the present invention;
FIG. 3 is a schematic top view of a grinding heat shield.
Detailed Description
The invention will be further described with reference to the accompanying drawings and specific examples.
Example 1: as shown in figure 1, the method for efficiently screening the heavy metal low-accumulation type ecological restoration plants/crops comprises the steps of constructing a micro-platform system, covering the ecological restoration plants/crops for cultivation, environment management, constructing a micro-platform system with a composite factor/single factor negative pressure, constructing a composite factor/single factor negative pressure (stress) environment system, completing the preparation and analysis flow of heavy metal micro-detection samples, and completing an evaluation system of the accumulation rule of the ecological restoration plants/crops. Finally screening out ecologically restored plant/crop species/varieties with target enrichment characteristics; the method comprises the following steps: 1) The method is characterized by efficiently and quickly finely selecting different ecological restoration plants/crop types or different varieties and strains of the same type; 2) And (3) adding heavy metals into the environment by adopting a micro research platform, and checking the accumulation rule and influence factors of the ecological restoration plants/crops to be detected. The method can reduce the unavoidable environmental heavy metal pollution in the research, and can comprehensively and systematically understand the influence of various influence factors on the ecological restoration plant/crop accumulation effect; 3) The effective heavy metal absorption rule of ecological restoration plants/crops in the environment is accurately locked, invalid research can be reduced, environmental pollutant detection links can be saved, and scientific research cost is greatly saved; 4) The method changes the limitation based on field or potting selection, adopts a mode of precisely controlling environmental heavy metals, eliminates the influence of environmental decoration change on the expression of key genes, and reduces the working efficiency of breeders or seed selectors; 5) The method can meet the selection of low-accumulation type crops in production and the selection requirement of high-accumulation type ecological species in ecological restoration, and the method comprises the following steps: on an ecological restoration plant/crop explant tissue culture platform meeting the specification, adding heavy metal components of composite or single elements according to concentration gradient, and establishing a characteristic curve of accumulated heavy metal in stems and leaves of a reaction species/variety by carrying out simplified research on two aspects of test tube growth data observation and recording and trace heavy metal content detection methods in a concentration interval in which the additives are completely non-lethal. If necessary, according to the soil environment of a target planting area, selectively setting negative selection pressure on environmental specific elements such as pH value, iron, manganese and the like or nutrient elements such as N, P and the like in a targeted manner, directionally acquiring a heavy metal absorption curve based on specific conditions, selecting low-enrichment ecological restoration plant/crop materials (stem and leaf heavy metal residual quantity is less than or equal to the national standard of product safety) with optimal comprehensive performance of ecological restoration plants/crop nutrient bodies (stems and leaves), and recommending the low-enrichment ecological restoration plant/crop species or crop varieties with environmental friendly characteristics to the specific area. To reduce unwanted activation of soil heavy metals due to improper species/breed selection, and adverse effects of its nutrients entering the local food chain system, or returning to the soil, entering the saprophytic circulation system, on the local agricultural social-ecosystem;
the explant tissue culture platform scheme meeting the specification is established: firstly, establishing an aseptic explant with the convergence of maximum growth amount; secondly, the explant needs to be subjected to rooting treatment in advance, and a culture medium suitable for corresponding ecological restoration plants/crop types is selected for rooting treatment for rooting culture; third, long-term treatment coupled heavy metal stress protocols are employed. Short-term treatment focuses on physiological changes and heavy metal absorption laws; long-term treatment is carried out to observe the long-term accumulation characteristic of ecological restoration plants/crops. Note that the processing modes are different, and the homogeneity implementation emphasis strategies are different. The short-term treatment liquid adopts a liquid and homogeneous solid culture medium; the long-term treatment adopts the method that liquid heavy metal solution is injected on the surface of the culture medium at one time, the culture medium is uniformly shaken by hand, and the culture medium is kept stand for 90-120d.
The above environmental control scheme: 1) And (5) culturing in the early stage. The stage should ensure the homogenization of each step, the consistency of the substrate and the culture condition, and the length of the transfer material should be as consistent as possible. The matrix preparation process and the split charging link should be fully oscillated to ensure the homogeneity of the solution. The temperature and humidity control, the illumination intensity and the duration are required to be accurate and consistent, so that the ecological restoration plant/crop material with unit mass can absorb the same nutrition components as possible, the light energy which is consistent as possible can be obtained, the growth height, the stem leaf thickness and the leaf number of the culture material can be ensured, the leaf number and the leaf size can be ensured as orderly as possible, and the growth can be homogenized as possible. 2) And (5) environmental control in the heavy metal negative pressure treatment stage. The greatest possible homogeneity is also required in the formulation of the environmental matrix at this stage to ensure the reliability of the results from the details. Preparing mother liquor for heavy metals which are difficult to dissolve so as to ensure the homogeneity of the solution and avoid overlarge local concentration difference; the growth influence of the solvent on ecological restoration plants/crops is considered for liquid heavy metals, and chlorinated heavy metal chemical reagents are avoided for chlorine-bearing crops. After the heavy metal solution is added, the change of the pH value of the culture medium is observed through a pre-test so as to avoid the excessive influence on the growth of ecological restoration plants/crops and avoid the influence of heavy metals; 3) And (5) accumulating stage environment control. This stage should be emphasized to avoid contamination. The use of aseptic seedlings is thus very advantageous for refining environmental control schemes.
The negative pressure setting and treating scheme comprises the following steps: under the aseptic condition, 10mL prepared Pb solution is sucked by a liquid-transfering gun and injected into the surface of 30-50ml of culture medium, and the size of the treatment material is about: seedling or explant material with uniform growth, height of 4-6 cm and root length of 1-3 cm. If the culture medium is solid, the forceps are uniformly used for lifting each seedling moderately upwards before injection, so that the root system can be fully contacted with the heavy metal treatment liquid. The above test was repeated 3-5 times and the treatment was performed for 1-110 days. And (3) using inductively coupled plasma (Q type) (Thermo SCIENTIFIC ICP-MS-Q) to measure Pb and related element content in high flux, and detecting concentration effect of heavy metal Pb.
The heavy metal treatment scheme comprises the following steps: for the explant selected in the step (2), under aseptic conditions, 10mL configured Pb solution (Pb standard solution or prepared Pb soluble solution) is sucked by a pipette, and is injected into the surface of about 30-50ml of solid culture medium or liquid culture medium with equivalent volume, and the solution is slightly shaken to be uniform, so that uniform negative selection pressure is formed. The size of the treatment materials is as follows: seedling or explant materials which grow uniformly and have a height of 4-6cm and a root length of 1-3 cm; aiming at the culture condition of a solid culture medium, forceps are uniformly used for lifting each seedling moderately upwards before injection, so as to ensure that newly generated root tips are fully contacted with the treatment fluid. Meanwhile, a blank control with the same culture condition but the concentration of the treatment fluid is 0 is set, and the same stress treatment is repeated for 3-5 times for 1-110 days.
The method for detecting the trace heavy metals comprises the following steps: I. the preparation method of the trace heavy metal detection sample comprises the steps of preparing a sample: shearing 0.1-0.3g of stem and leaf materials of test-tube seedlings which are not stained with the treatment liquid, putting the materials into a batch-treatment micro-tissue grinder, heating liquid nitrogen to quickly freeze seedling tissues, grinding the materials into powder, transferring the powder into a digestion test tube, putting the digestion test tube into a microwave digestion instrument for treatment, and detecting the current day; II. The preparation method of manual small-scale batch detection samples comprises the following steps: preparing a sample by adopting a batch-processing micro-tissue grinder according to the operation convenience and the limit time limit of liquid nitrogen freeze thawing; III, detecting trace heavy metals: and (3) carrying out multichannel rapid detection on the sample processed in the step I by adopting a plasma inductively coupled mass spectrometer ICP-MS, carrying out high-flux measurement Pb, cd, as, hg, cr and related elements such as Fe, mn, zn and the like, and carrying out content detection repetition times, wherein the theoretical setting is carried out for 3 times. The homogeneity is well controlled, 3 times of repetition are detected in the early stage, and the tissue culture platform with the heavy metal detection result error below 5% can accept no repetition under the same condition.
The above comprehensive evaluation and species/variety recommendation: according to the heavy metal risk condition of the planting area, establishing an absorption and accumulation curve of the stem leaves of the species/varieties to be detected on different heavy metal ions under the condition of combined pollution or single pollution; the concentration gradient absorption rule of the unit heavy metal accumulation amount to be detected in each material stem leaf or root system is integrated, the heavy metal accumulation rule aiming at the typical environmental characteristics of a specific area is analyzed by using statistical software such as SPSS (specific surface soil system) and the like, and the influence rule of the environmental pH value, the key element influence rule and the important management means (such as N application, quicklime application and the like) are focused. The correlation between the influence factors and the accumulation of heavy metals is established by utilizing SPSS, the weights of different factors are analyzed, and an element path analysis chart is established; according to different accumulation performances under the set negative pressure, primarily selecting low enrichment type evaluation as a primary selection material; in conjunction with the detection of physiological indicators, it is desirable to combine the identification of key influencing genes (e.g., ASA, GSH, etc. key regulatory genes) and recommend species/varieties suitable for planting for different regions, when conditions warrant.
The ecological planting verification and variety authorization are carried out: the selected material is subjected to multi-point multi-observation verification experiments for more than or equal to 3 years, and the high enrichment or low enrichment species/variety types can be authorized by the verification.
After the method is implemented, scientific research cost is greatly reduced, low-accumulation ecological restoration plants/crop varieties can be efficiently screened, and the method can be freely combined with a high-efficiency physicochemical mutagenesis method system or gene editing breeding, so that a joint port with the high-efficiency breeding system is provided, and pollution to farmlands, potting soil, groundwater and surrounding environment during screening can be greatly reduced.
Example 2: as shown in fig. 2 to 3, a simple batch-processing micro-tissue grinder (8-hole micro-grinder) comprises a grinding pestle 1, an EP tube insertion hole 2, an EP tube 3, a liquid nitrogen inlet hole 4, a grinding heat insulation plate 5 and a grinding fixture 6, wherein: the bottom of the grinding pestle 1 (with the height of 29.70mm and the diameter of 5.70 mm) is flat and frosted, the grinding heat insulation plate 5 (with the outer diameter of 137.90mm and the height of 18.18 mm) is provided with an 8 round hole (with the inner diameter of 10.61 mm) for inserting 2.5ml of EP pipe and a larger round hole (with the inner diameter of 40.00 mm) so as to facilitate the inflow of liquid nitrogen, and the grinding fixer (with the height of 76.40mm, the pipe orifice outer diameter of 149.10mm, the pipe orifice inner diameter of 137.90mm, the base outer diameter of 106.84mm and the base inner diameter of 98.76 mm) is connected with the grinding heat insulation plate for fixing and is easy to hold. The grinding fixer 6 is a cavity with an opening at the upper end, the grinding heat insulation plate 5 is sealed to be closed at the upper end of the grinding fixer 6, the grinding heat insulation plate 5 is provided with an EP pipe penetrating hole 2 and a liquid nitrogen inlet hole 4, the lower end of the EP pipe 3 can extend into the grinding fixer 6 through the EP pipe penetrating hole 2, the grinding pestle 1 is arranged in the EP pipe 3, the grinding fixer is made of ceramic, and the inner diameter of a pipe orifice is the same as the outer diameter of the grinding heat insulation plate 5, so that the grinding heat insulation plate 5 is convenient to be sleeved in.
Preferably, the EP tube 3 is 2.5ml in size.
Preferably, the bottom of the grinding pestle 1 is spherical and is provided with a grinding layer, so that the grinding pestle is convenient to fully and effectively contact, and the grinding effect is better.
Preferably, the grinding heat insulation plate 5 is provided with 8 EP pipe penetrating holes 2 and 1 liquid nitrogen inlet hole 4, so that batch grinding treatment can be realized.
Working principle: when the grinding is started, a 2.5ml EP tube 3 filled with ecological restoration plant/crop root/stem/leaf nutrition tissue is inserted into a 2.5ml EP tube penetrating hole 2, a grinding heat insulation plate 5 is embedded into a tube opening of a grinding fixer 6, the grinding fixer 6 is held by hands, a proper amount of liquid nitrogen is sequentially poured into the 2.5ml EP tube, and is injected into the grinding fixer 6 through a grinding liquid nitrogen inlet hole 4 to keep a low-temperature environment, after the tissue is frozen and crisp, part of liquid nitrogen volatilizes, the grinding is started, the upper end of the grinding fixer 6 is held by hands to grind the tissue through a grinding pestle 1, and after the grinding is finished, the 2.5ml EP tube 3 filled with ecological restoration plant/crop tissue is taken out for measurement data, because the diameter of the grinding pestle 1 is similar to the diameter of the 2.5ml EP tube 3 (slightly smaller than the tube diameter and generally 1-2), the grinding is more sufficient, and the 2.5ml EP tube 3 provided with a plurality of grinding tissues on the 1 grinding heat insulation plate is convenient to transfer in batches after the grinding is finished, and the device is simple and has strong practicability.
The advantages of the device are as follows:
(1) The inner diameter of the bottom surface of the grinding pestle is slightly smaller than the diameter of the 2.5ml EP tube, and the bottom surface of the grinding pestle is spherical, so that the effective grinding area and the grinding sufficiency can be ensured; thanks to the 2.5ml EP tube ball bottom structure, the extruded sample can slide and collect at the tube bottom by simple knocking or gentle shaking in the grinding process, so that the grinding effect can be further improved;
(2) Liquid nitrogen is not easy to volatilize in the grinding process, and a portable sample is not easy to splash out;
(3) The operator can exert better force by holding the grinding holder;
(4) The diameter of the grinding pestle is similar to that of a 2.5ml EP pipe, so that the grinding is more complete;
(5) 8 EP pipe penetrating jacks of 2.5ml are provided, batch treatment can be carried out, and the extraction efficiency is high;
(6) After finishing grinding, batch transfer is convenient, and the recovery rate is high;
(7) The components of the device are easy to obtain, and the cost is low;
(8) Liquid nitrogen is poured into the sealing in advance, so that the risk of frostbite during liquid nitrogen pouring is avoided.
The foregoing is merely illustrative of the present invention, and the scope of the present invention is not limited thereto, and any person skilled in the art can easily think about variations or substitutions within the scope of the present invention, and therefore, the scope of the present invention shall be defined by the scope of the appended claims.
Claims (9)
1. A method for efficiently screening heavy metal low-accumulation ecological restoration plants/crops is characterized by comprising the following steps: on an ecological restoration plant/crop explant tissue culture platform conforming to the specifications, adding heavy metal components of composite or single elements according to concentration gradients, and establishing a characteristic curve of accumulated heavy metals in stems and leaves of reactive species/varieties through test researches of test tube growth data observation and recording and trace heavy metal content detection methods in a low concentration interval in which the additives are not lethal at all and do not cause obvious growth manifestation; selectively setting negative selection pressure on pH value, iron and manganese environment specific elements or N and P nutrient elements according to the soil environment and the characteristics of the parent rock of a target planting area, directionally acquiring a heavy metal absorption curve based on specific conditions, selecting low-enrichment ecological restoration plants/crop materials with optimal comprehensive performance of ecological restoration plants/crop nutrients, and recommending low-enrichment ecological restoration plants/crop species or crop varieties with environmental friendly characteristics of the nutrients to the specific area, wherein the residual quantity of the heavy metals of stems and leaves of the low-enrichment ecological restoration plants/crop materials is less than or equal to the national product safety standard; the negative selection voltage setting method comprises the following steps: and setting a heavy metal composite/single treatment negative pressure, specifically combining with pH value change, and/or Fe, mn or Ca to influence environmental elements for heavy metal attraction and N to influence excessive/insufficient stress of nutrient elements for heavy metal attraction to form screening negative pressure, and selecting low-enrichment type ecological restoration plants/crop species/crop varieties under the conditions of excessive or insufficient environmental pH value and elements which have important influence on heavy metal absorption.
2. The method for high-efficiency screening of heavy metal low-accumulation type ecological restoration plants/crops according to claim 1, which is characterized in that: the method for establishing the ecological restoration plant/crop explant tissue culture platform comprises the following steps: firstly, establishing an aseptic explant with the convergence of maximum growth amount; secondly, the explant needs to be subjected to rooting treatment in advance, and a culture medium suitable for corresponding ecological restoration plants/crop types is selected for rooting treatment for rooting culture; thirdly, adopting a long-term and short-term treatment combined heavy metal stress scheme; short-term treatment focuses on physiological changes and heavy metal absorption laws; long-term treatment for observing long-term accumulation characteristics of ecological restoration plants/crops, wherein a liquid and homogeneous solid culture medium is selected as a short-term treatment liquid; the long-term treatment adopts the method that liquid heavy metal solution is injected on the surface of a culture medium at one time, and the culture medium is subjected to shaking manually, standing and culturing.
3. The method for high-efficiency screening of heavy metal low-accumulation type ecological restoration plants/crops according to claim 2, which is characterized in that: for selected explants, under aseptic conditions, 10 mL configured Pb solution is sucked by a pipette and injected into the surface of about 30-50ml of solid culture medium or liquid culture medium, and the solution is slightly shaken to be uniform, so that uniform negative selection pressure is formed, and the size of the treatment material is as follows: seedling or explant materials which grow uniformly and have a height of 4-6cm and a root length of 1-3 cm; aiming at the culture condition of a solid culture medium, before injection, each seedling is lifted up moderately by tweezers, and meanwhile, a blank control with the same culture condition but the concentration of a treatment solution of 0 is arranged, and the same stress treatment is repeated for 3-5 times and is carried out for 1-110 days.
4. The method for high-efficiency screening of heavy metal low-accumulation type ecological restoration plants/crops according to claim 1, which is characterized in that: the method for detecting the content of the trace heavy metals comprises the following steps: I. the preparation method of the trace heavy metal detection sample comprises the steps of preparing a sample: shearing 0.1-0.3g of stem and leaf materials of test-tube seedlings which are not stained with the treatment liquid, putting the materials into a batch-treatment micro-tissue grinder, heating liquid nitrogen to quickly freeze seedling tissues, grinding the materials into powder, transferring the powder into a digestion test tube, putting the digestion test tube into a microwave digestion instrument for treatment, and detecting the current day; II. The preparation method of manual small-scale batch detection samples comprises the following steps: preparing small-scale batch detection samples by adopting a batch processing micro-tissue grinder according to the operation convenience and the limit time limit of liquid nitrogen freeze thawing; III, a trace heavy metal detection method comprises the following steps: carrying out multichannel rapid detection on the sample processed in the step I by adopting a plasma inductively coupled mass spectrometer ICP-MS, carrying out high-flux measurement Pb, cd, as, hg, cr and related elements of Fe, mn and Zn, and setting the content detection repetition times for 3 times; the homogeneity is well controlled, 3 times of repetition are detected in the early stage, and the repeated detection is not carried out on a tissue culture platform with the heavy metal detection result error below 5% under the same condition.
5. The method for high-efficiency screening of heavy metal low-accumulation type ecological restoration plants/crops according to claim 1, which is characterized in that: comprehensive evaluation and species/variety recommendation: according to the heavy metal risk condition of the planting area, establishing an absorption and accumulation curve of the stem leaves of the species/varieties to be detected on different heavy metal ions under the condition of combined pollution or single pollution; integrating the concentration gradient absorption rule of the unit heavy metal accumulation to be detected in each material stem leaf or root system, analyzing the heavy metal accumulation rule aiming at the typical environmental characteristics of a specific area by utilizing the statistical software of SPSS, focusing on the environmental pH value influence rule, the key element influence rule and the important management means influence rule, establishing the correlation between the influence factors and the heavy metal accumulation by utilizing the SPSS, analyzing the weights of different factors, and establishing an element path analysis chart; according to different accumulation performances under the set negative pressure, primarily selecting low enrichment type evaluation as a primary selection material; and detecting the joint physiological indexes, and recommending species/varieties suitable for planting aiming at different areas by combining with identification of key influencing genes.
6. The method for high-efficiency screening of heavy metal low-accumulation type ecological restoration plants/crops according to claim 1, which is characterized in that: ecological planting verification and variety authorization: the selected material is subjected to multi-point multi-observation verification experiments for more than or equal to 3 years, and the high enrichment or low enrichment species/variety types can be authorized by the verification.
7. The method for high-efficiency screening of heavy metal low-accumulation type ecological restoration plants/crops according to claim 4, which is characterized in that: the batch treatment micro-tissue grinder comprises a grinding pestle (1), an EP pipe (3), a grinding heat insulation plate (5) and a grinding fixer (6), wherein the grinding fixer (6) is a cavity with an opening at the upper end, the grinding heat insulation plate (5) is sealed at the upper end of the grinding fixer (6), an EP pipe penetrating hole (2) and a liquid nitrogen inlet hole (4) are formed in the grinding heat insulation plate (5), the lower end of the EP pipe (3) can extend into the grinding fixer (6) through the EP pipe penetrating hole (2), and the grinding pestle (1) is arranged in the EP pipe (3).
8. The method for high-efficiency screening of heavy metal low-accumulation type ecological restoration plants/crops according to claim 7, which is characterized in that: the bottom of the grinding pestle (1) is spherical and is provided with a grinding layer.
9. The method for high-efficiency screening of heavy metal low-accumulation type ecological restoration plants/crops according to claim 7, which is characterized in that: a plurality of EP pipe penetration holes (2) and 1 liquid nitrogen inlet hole (4) are arranged on the grinding heat insulation plate (5).
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