CN102288590A - Method for measuring plant stress of heavy metals in soil with laser-induced plant fluorescence - Google Patents
Method for measuring plant stress of heavy metals in soil with laser-induced plant fluorescence Download PDFInfo
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- CN102288590A CN102288590A CN2011101983887A CN201110198388A CN102288590A CN 102288590 A CN102288590 A CN 102288590A CN 2011101983887 A CN2011101983887 A CN 2011101983887A CN 201110198388 A CN201110198388 A CN 201110198388A CN 102288590 A CN102288590 A CN 102288590A
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Abstract
The invention relates to a method for measuring the plant stress of heavy metals in soil with laser-induced plant fluorescence of a He-Cd excitation source. The method comprises the following steps of: selecting normally-growing ryegrasses, cutting young leaves, putting onto a quartz glass plate, and putting a plant to be measured into a photophobic box together with the quartz glass plate; fixing the two ends of a plant sample to be measured on an instrument fixing bracket in sequence with an ester gum substrate, making excitation light pass through a double monochrometer of which the length is 1/4 meters under the condition that He-Cd laser light is taken as an excitation light source, the wave length is 325 nanometers and the slit width is 0.05 millimeter, and recording the photoluminescence intensity at the wave length of 350-800 nanometers; and calculating a result according to a formula A=F450/(F690+F735), wherein F450, F690 and F735 represent the peak heights of a fluorescence intensity spectrogram at the wave lengths of 450 nanometers, 690 nanometers and 735 nanometers respectively; and the value A is larger, the stress of the heavy metals is higher. The method is easy to operate; plant fluorescence is measured in situ on the premise of not damaging the plant growth; and a simple, convenient, quick, economical and dynamic monitoring method is provided for the research of the environmental chemical actions of heavy metals in a soil-plant system.
Description
Technical field:
The present invention relates to a kind of method of measuring heavy metal in soil to plant stress, be specifically related to the method for a kind of induced with laser plant fluorometric assay heavy metal in soil of He-Cd excitaton source plant stress.Belong to heavy metal pollution analytical approach field in the soil environment.
Background technology:
Along with the exploitation utilization of mineral metal products resource, heavy metal element enters the farmland by atmosphere and sewage irrigation, has a strong impact on crop yield and quality of agricultural product, causes serious ecological environment problem.The chemical behavior of heavy metal in soil-plant system is a very complicated process.Different soil typess, different heavy metals have different accumulation patterns at the different growth period of plant the position different with plant, and the extent of injury of bringing thus also is not quite similar.Traditional method has to finish by a large amount of potted plant experiments and loaded down with trivial details chemical analysis when the toxicity mechanism of research heavy metal chemical behavior and heavy metal on plants in soil-plant system.Promptly by adding the heavy metal simulating pollution soil of variable concentrations, potted plant obtains research information by the chemical analysis that has form and content etc. to soil and plant heavy metal of body then.When studying the dynamic law of the migration conversion of heavy metal in soil-plant system, need carry out sample analysis at the different times of plant growth, so require more substantial potted plant experiment and more analysis result.Obviously, traditional research method not only need a large amount of potted plant experiments to obtain a large amount of plant samples, and growing plants is in for destruction.In addition, also to carry out consuming time, effort, the big chemical analysis of cost.Therefore, invent a kind of easy, quick, effective, economic assay method, heavy metal migration under the condition of not damaging the plant growth in the research edaphophyte system transforms dynamically, has important in theory meaning and realistic meaning for the deep development that promotes environmental chemistry study.
When plant stimulates with a shortwave, as ultraviolet ray or blue light (320nm-380nm), will produce four fluorescent bands: blue (F440), green (F520), red (F690) and far infrared (F735).Blue green light (440-520nm) is mainly from the epidermal cell emission of plant, and red and far infrared (690-735nm) can be used for measuring photosynthesis of plants efficient from the chlorophyll a of vegetable protein combination in the chloroplast of plant light and marine alga or green plants cell.Photosynthesis of plant efficient is subjected to arid, cold, light intensity, environmental pollution, cause of disease infringement etc. and often reduces.Heavy metal plays an important role in the plant metabolism process and is reported widely, and heavy metal energy activating enzymes participate in the synthetic of protein and carbohydrates; Can enter plant by the Ca2+ passage, influence plant antioxidant system gas exchange, transpiration and photosynthesis.The extent of injury of heavy metal on plants depends on floristics and gene difference, and the toxicity of heavy metal and the amount etc. that enters the plant heavy metal of body.Therefore, whether plant fluorescence is subjected to the heavy metal stress except being used for direct qualitative plant, perhaps also can be used for reflecting indirectly the amount (amount of plant absorbing heavy metal) of the heavy metal that causes the plant change in fluorescence.
Forefathers are mainly used in floristics with the method for induced with laser plant fluorescence and identify with the remote sensing monitoring of upgrowth situation etc. or contact coercing plant such as determination of plant moisture, illumination, nutrient, temperature.At present, the induced with laser plant chlorophyll fluorescence (LICF) that don't work still reflects that with the method for induced with laser plant fluorescence (LIF) research of coercing of heavy metal mostly is qualitatively.Also do not have coercing of quantitative reaction heavy metal on plants, cause the variation of plant fluorescence intensity and the amount of plant absorbing heavy metal to connect.
At present, used excitaton source is diversified when measuring chlorophyll fluorescence, and excitation wavelength also differs widely.Claus group (1996) is with CW He-Ne lasing light emitter (The Karlsuhe CCD-OMA LIDAR-Fluorosensor) excitaton source, and excitation wavelength is to measure the influence of coercing of plant under the condition of 632.8nm.Gopal research group is that 448nm measures the influence of heavy metal to corn growth with the Argon ion laser excitation wavelength.Because excitation wavelength is longer, energy is lower, so only obtained 690nm and two fluorescence wavestrips of 735nm.We use the He-Cd laser instrument under study for action, and excitation wavelength is 325nm, have obtained 450nm, 690nm and three plant fluorescence of 735nm wavestrip.At present with the He-Cd laser instrument as excitation source, the research that obtains 3 plant fluorescence wavestrips does not simultaneously appear in the newspapers.
Chlorophyll fluorescence ratio F690/F735 has been used to indicate plant physiology to coerce.Ratio F690/F735 increases along with photosynthetic minimizing, reduces along with the increase of chlorophyll concentration.Ratio F450/F690 and F450/F735 also can characterize the stress physiology phenomenon of plant.Also useful F-v/F-m or Fo/Fv characterize the susceptibility of plant to heavy metal.It is generally acknowledged that Lan Hong and bluish-green fluorescence ratio are the most responsive and only indications of coercing.
In sum, traditional research method is in the research soil-plant system during environmental chemistry behavior of heavy metal, and consuming time, the effort of existence, sample size is big, cost is big, be difficult to subject matter such as dynamic monitoring.The research of coercing of the heavy metal of the method for the induced with laser that adopts reflection at present mostly is qualitatively, does not also have the method for coercing of quantitative reaction heavy metal on plants.
Summary of the invention:
The objective of the invention is to overcome the shortcoming of prior art, the induced with laser plant fluorescent in situ that aims to provide and a kind ofly do not destroy plant growth, is convenient to dynamic monitoring is measured the new method that plant absorbs heavy metal.The inventive method is: the live plant exciting light method with the He-Cd excitaton source is used for measuring heavy metal in soil coercing plant as a kind of instrument.The quantitative basis that as plant heavy metal is absorbed with F450/ (F690+F735) ratio.Spectral signature and influence factor by plant fluorescence in different plants, different heavy metal, the different soils, find out the relation between plant fluorescence intensity and the plant absorbing heavy metal, carry out quantitative formula correction by the mathematics match, measure the new method that plant absorbs heavy metal thereby invent the induced with laser plant fluorescent in situ that does not a kind ofly destroy plant growth, is convenient to dynamic monitoring.
In order to realize the foregoing invention purpose, the operation steps of the inventive method is as follows:
(1) collection of plant sample to be measured and pre-service
Select the rye grass of normal growth, cut the 4th young leaves, be placed on the quartz glass plate and (must guard against and be placed on the copy paper with the stainless steel scissors, in case interference measurement), brushes away surface dust on the blade face gently, if surperficial surface dust is more with banister brush, the wiping gently of available wet gauze must not the water flushing.To be with measuring plants together to be placed in the lucifuge box then with quartz glass plate, to be measured.
(2) mensuration of induced with laser plant fluorescence
Get plant sample to be measured stage casing, cut about 5cm blade sections respectively, two is fixed on the instrument fixed support successively with the ester gum base, notes trying not to touch zone to be measured.
At wavelength 325nm, slit width 0.05mm, exciting light arrive 800nm wavelength recording light photoluminescence intensity by the double monochromator (Oriel 77225) of 1/4m length with 350 to excitation source with He-Cd laser (Kimmon IK series).
(3) result calculates
A＝F450/(F690+F735)
In the formula: F450, F690, F735 be respectively the fluorescence intensity spectrogram at 450nm, the peak height at 690nm and 735nm place.The A value that calculates is big more, and coercing of heavy metal is heavy more.
(4) points for attention
1. measure to be preferably in 30 minutes and finish, if sample size is excessive, can sample before mensuration, sampling in batches be must guard against a large amount of samplings and is put into the refrigerator preservation.
2. measure environmental requirement: keep room temperature at 25 ℃, lucifuge or under ruddiness, operate.
This method is with short-wave laser source excitation plant fluorescence, proposition utilizes induced with laser plant fluorescence, the quantitative basis that as plant heavy metal is absorbed with F450/ (F690+F735) ratio, under the prerequisite of not damaging the plant growth, in-site detecting plant fluorescence reflects the method that the migration of heavy metal in the soil-plant system transforms indirectly; For the research of the environmental chemistry behavior of heavy metal in the soil-plant system provide a kind of easy, quick, economical, do not destroy the plant growth, can dynamic monitoring method, have important significance for theories and realistic meaning for the deep development that promotes environmental chemistry study and the in good time dynamic monitoring in heavy metal pollution farmland.
Description of drawings:
Fig. 1 coerces down the fluorescence intensity spectrogram of rye grass for the Cu of variable concentrations.
Fig. 2 coerces down the fluorescence intensity spectrogram of rye grass for the Pb of variable concentrations.
Fig. 3 coerces down the fluorescence intensity spectrogram of rye grass for the Cd of variable concentrations.
Fig. 4 is the fluorescence intensity spectrogram under the different rye grass blade heavy metal stress.
Fig. 5 is the fluorescence intensity spectrogram under the same blade different parts heavy metal stress.
Embodiment:
Below by specific embodiment the inventive method is further elaborated.
The realization of the inventive method comprises that the spectral characteristic, instrumentation condition of excitation source determine; The spectral signature parameter of plant leaf blade under different plants, the different heavy metal stress; Induced with laser plant fluorometric assay heavy metal in soil is to influence factor of the method for plant stress etc.
Embodiment 1:
(1), the spectral signature parameter study of plant leaf blade under different plants, the different heavy metal stress
Take by weighing soil (wind desiceted soil) that 1.00kg crosses 20 mesh sieves in polypots, add 0,50,100 respectively, 150mg.kg
-1Cu
2+(CuSO
4); 0,500,1000,1500mg.kg
-1Pb
2+(PbCl
2); 0,5,10,15mg.kg
-1Cd
2+(CdCl
2), indoor cultivation two months, heavy metal each form in soil distribute reach balance after, every alms bowl is executed potassium dihydrogen phosphate 0.3333g, urea 0.3477g; Sow plant then, growth in the greenhouse.Keep soil moisture at 60%～70% of field capacity in the incubation.36 processing are set on three kinds of soil altogether, and each is handled and repeats 3 times.
(1) collection of plant sample to be measured and pre-service
Select the rye grass of normal growth, cut the 4th young leaves, be placed on the quartz glass plate and (must guard against and be placed on the copy paper with the stainless steel scissors, in case interference measurement), brushes away surface dust on the blade face gently, if surperficial surface dust is more with banister brush, the wiping gently of available wet gauze must not the water flushing.To be with the side plant with together being placed on as the quartz glass in the lucifuge box then, to be measured.
(2) mensuration of induced with laser plant fluorescence
Get plant sample to be measured stage casing, cut about 5cm blade sections respectively, two is fixed on the instrument fixed support successively with the ester gum base, notes trying not to touch zone to be measured.
At wavelength 325nm., slit width 0.05mm, exciting light arrive 800nm wavelength recording light photoluminescence intensity by the double monochromator (Oriel 77225) of 1/4m length with 350 to excitation source with He-Cd laser (Kimmon IK series).
(3) interpretation of result
The Cu of variable concentrations, Pb and Cd coerce down the fluorescence intensity of rye grass at 450nm, and the peak all appears in 690nm and 735nm place.These three peak heights and peak shape are all along with Cu, and Pb coerces the different and different of concentration with Cd.In general, along with Cu, the concentration of Pb and Cd increases, and the peak height at 690nm and 750nm place is reduction trend, and the peak height at 450nm place increases (Fig. 1-Fig. 3).
Among Fig. 1, CK-does not add the processing of Cu; Cu1-adds 50mg Cu kg
-1Handle; Cu2-adds 100mg Cu kg
-1Handle; Cu3-adds 150mg Cu kg
-1
Among Fig. 2, CK-does not add the processing of Pb; Pb1-adds 500mg Pbkg
-1Handle; Pb2-adds 1000mgPb kg
-1Handle; Pb3-adds 1500mg Pb kg
-1
Among Fig. 3, CK-does not add the processing of Cd; Cd1-adds 5.0mg Cdkg
-1Handle; Cd2-adds 10.0mg Cdkg
-1Handle; Cd3-adds 15.0mg Cdkg
-1
The fluorescence intensity spectrogram (Fig. 4, Fig. 5) of the different rye grass individual plant of same processing and the rye grass of same rye grass blade zones of different.At 450nm, the peak appears in 690nm and 735nm place respectively, and this is similar to Fig. 1-Fig. 3 result displayed.As can be seen, in same processing in the peak shape of different rye grass individual plants and the same rye grass blade peak shape of zones of different be similar.
(2), measurement result study on characterization
In order to seek fluorescence intensity and rye grass, calculate F450/F690 respectively according to peak height, F450/F735, the ratio of F690/F735 and F450/ (F690+F735) at three peaks to the relation between the absorption of heavy metal.The result of table 1 shows: add Cu, and in the processing of Pb and Cd, F450/F690, F450/F735 and F450/ (F690+F735) ratio all increases with the increase of heavy metal stress concentration.
Table 1Cu, Pb and Cd coerce the influence to fluorescence intensity and F450/F690, F450/F735, F690/F735, F450/ (F690+F735)
(3), method of sampling research
The different fluorescence intensity ratios (F450/F690, F450/F735, F690/F735 and F450/ (F690+F735)) in zone of measuring with same rye grass blade of the rye grass plant that same processing is different are to understand the precision of fluorescence intensity spectrum assay method.The measurement result of the rye grass plant that same processing is different shows: compare with F450/F735 with F450/F690, F690/F735 and F450/ (F690+F735) have little standard deviation, and relative differences numerical value is respectively 3.34% and 3.69%.And for the different mensuration of same rye grass blade is regional, F450/F690, F450/F735, the standard deviation of four ratios of F690/F735 and F450/ (F690+F735) is all less, relative differences is respectively 3.36%, 5.79%, 4.44% and 4.14% (table 2).
The relative differences of the different regional fluorescence intensities of mensuration of the same processing different plants of table 2 and F450/F690, F450/F735, F690/F735, F450/ (F690+F735) with same blade
(4), compare with classic method
Respectively four the fluorescence intensity ratios and the content of the rye grass heavy metal of body that records with chemical method are done the value of the coefficient R that Linear correlative analysis obtains.In the processing of Cu and Pb, F450/F690, F450/F735 and F450/ (F690+F735) all concentration in the rye cursive script are linear positive correlation, and the facies relationship number average is greater than 0.97.During Cd handled, the related coefficient of the Cd content that these three ratios and rye cursive script are interior also was linear positive correlation, but coefficient R is 0.90 only, is lower than the processing of Cu and Pb. but F690/F735 and rye grass bulk concentration does not all have a correlativity (table 3).
Table 3.F450/F690, F450/F735, F690/F735, F450/ (F690+F735) and the interior Cu that absorbs of rye cursive script, the related coefficient between Pb, the Cd (n=4, P＜0.05, r=0.95)
Claims (3)
1. an induced with laser plant fluorometric assay heavy metal in soil is to the method for plant stress, it is characterized in that operating as follows: the first step, the collection of plant sample to be measured and pre-service: the rye grass of selecting normal growth, cut the 4th young leaves with the stainless steel scissors, be placed on the quartz glass plate, brush away or wipe surface dust on the blade face gently, will be with measuring plants together to be placed in the lucifuge box to be measured then with quartz glass plate; Second step, the mensuration of induced with laser plant fluorescence: get plant sample to be measured stage casing, cut about 5cm blade sections respectively, two is fixed on the instrument fixed support successively with the ester gum base, excitation source He-Cd laser, at wavelength 325nm, slit width 0.05mm, exciting light is by the long double monochromator of 1/4m, with 350-800nm wavelength recording light photoluminescence intensity; In the 3rd step, the result calculates: A=F450/ (F690+F735), in the formula, F450, F690, F735 be respectively the fluorescence intensity spectrogram at 450nm, the peak height at 690nm and 735nm place, the A value that calculates is big more, coercing of heavy metal is heavy more.
2. a kind of induced with laser plant fluorometric assay heavy metal in soil according to claim 1 is characterized in that being determined in 30 minutes and finishes the method for plant stress.
3. a kind of induced with laser plant fluorometric assay heavy metal in soil according to claim 1 is to the method for plant stress, it is characterized in that measuring environment and keeps room temperature at 25 ℃, lucifuge or operate under ruddiness.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104568887A (en) * | 2015-01-16 | 2015-04-29 | 山东师范大学 | Method or measuring stress of heavy metals on plants by using plant micro-domain chlorophyll fluorescence method |
| CN104597107A (en) * | 2014-12-31 | 2015-05-06 | 中国矿业大学 | Field controlled experiment method for simulating soil-crop heavy metal migration |
| RU2610521C1 (en) * | 2015-11-02 | 2017-02-13 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Московский государственный технический университет имени Н.Э. Баумана" (МГТУ им. Н.Э. Баумана) | Method of distant trass detection of vegetation plots under stress |
| CN109239032A (en) * | 2018-09-10 | 2019-01-18 | 西南科技大学 | Using the method for chlorophyll fluorescence parameters plant identification strontium accumulation ability |
| CN109374586A (en) * | 2018-09-28 | 2019-02-22 | 西南科技大学 | With the method for quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104597107A (en) * | 2014-12-31 | 2015-05-06 | 中国矿业大学 | Field controlled experiment method for simulating soil-crop heavy metal migration |
| CN104597107B (en) * | 2014-12-31 | 2019-03-01 | 中国矿业大学 | A kind of field controllable experimental method for simulating soil-crop heavy metal transformation |
| CN104568887A (en) * | 2015-01-16 | 2015-04-29 | 山东师范大学 | Method or measuring stress of heavy metals on plants by using plant micro-domain chlorophyll fluorescence method |
| RU2610521C1 (en) * | 2015-11-02 | 2017-02-13 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Московский государственный технический университет имени Н.Э. Баумана" (МГТУ им. Н.Э. Баумана) | Method of distant trass detection of vegetation plots under stress |
| CN109239032A (en) * | 2018-09-10 | 2019-01-18 | 西南科技大学 | Using the method for chlorophyll fluorescence parameters plant identification strontium accumulation ability |
| CN109239032B (en) * | 2018-09-10 | 2021-11-02 | 西南科技大学 | Method for identifying strontium enrichment capacity of plants by using chlorophyll fluorescence parameters |
| CN109374586A (en) * | 2018-09-28 | 2019-02-22 | 西南科技大学 | With the method for quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability |
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Application publication date: 20111221 |