CN109374586A - With the method for quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability - Google Patents
With the method for quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability Download PDFInfo
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Abstract
The invention discloses a kind of method with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability, this method includes 1) plant cultivating: the nutrient solution containing caesium of plant various concentration to be determined is handled；2) quick chlorophyll fluorescence kinetics curve and fluorescence parameter of the plant under different caesium concentration for the treatment of after dark adaptation the measurement of chlorophyll fluorescence parameters: are measured with continuous agitation formula chlorophyll fluorescence instrument；3) PI is calculated according to the measurement result of step 2)ABSValue, and calculate the PI of caesium processing groupABSChanging value；4) according to the PI of the plant leaf blade of step 3)ABSThe accumulation ability of changing value identification plant pair caesium.The disadvantages of cumbersome, the period is long, low efficiency the present invention overcomes existing method processing, there are the advantages such as non-destructive testing, sensitive, quick, instant in-site detecting can be carried out to fresh plant sample, had the characteristics that timely, sensitive, quick, efficient.
Description
Technical field
The invention belongs to remediation contaminated soil fields, and in particular to a kind of to be identified with quick chlorophyll fluorescence kinetics parameters
The method of plant caesium accumulation ability.
Background technique
The development and utilization of nuclear technology also produce a large amount of while bringing great economic benefit and social benefit to the mankind
Radioactive pollutant.Radioactive pollutant especially can cause radioactive pollution to soil and water body to ecological environment, this is just
It is nuclear pollution.After radionuclide enters soil and environment, remove extremely difficult.Although conventional physics and chemical method
Can be used to handle in soil water curve it is low put nucleic, but it is at high cost, easily cause secondary pollution.Phytoremediation technology is to plant
Object restrain oneself with based on excessive accumulaiton certain or the theory of certain chemical elements, using plant and its microorganism system coexisted,
Remove an environmental pollution treatment technology of pollutant in environment.It is low with maintenance cost, easy to operate, environmental-friendly with investing
And the advantages that cleaning, and certain economic benefit is obtained possibly through resource utilization, it has broad application prospects, by state
Inside and outside scholar is considered to administer low in large area soil and water body put and extremely low one of the most effective means for putting nucleic pollution environment.
But the research both at home and abroad about the nucleic polluted soil phyto reparation such as strontium, caesium, uranium is still in the primary stage at present, mainly super rich
Collect the screening of plant.It is current research shows that section not of the same race, the plant of category are different from accumulation to the absorption of the nucleic such as strontium, caesium, uranium,
And the Different Organs of kindred plant are not also identical to the accumulation of strontium, caesium, uranium.The phytoremediation technology of nucleic pollution environment is ground
Study carefully, find and screen more nucleic super enriching plants by be improve phytoremediation technology efficiency most important material base and must
Want premise.
The screening and identification of nucleic or heavy metal super-enriched plant mainly follows following procedure progress both at home and abroad at present: sample is adopted
Collect -- sample drying -- Specimen eliminating, then will the sample liquid handled well of resolution by Atomic Absorption Spectrometer or inductive coupling etc. from
Daughter emission spectrometer carries out constituent content analysis, the finally concentration coefficient according to plant to some nucleic or heavy metal and transhipment
Coefficient judges the accumulation ability size with plant identification to nucleic and heavy metal.Whole flow process almost needs several days time,
And sample handling processes are very complicated, and efficiency is very low.Therefore, find one quickly, be easy to measure, can examine in a short time
That surveys a large amount of samples judges that the method for plant nucleic or heavy metal accumulation ability relative size is super for screening nucleic and heavy metal
Enriching plant has important application value and prospect.
The photosynthesis of plant is that an absorbing carbon dioxide and water convert light energy into chemical energy and discharges the mistake of oxygen
Journey.Contain photosystemⅰ (Photosystem I, PS I) and lightsystemⅡ (Photosystem II, PS II) two in chloroplaset
Photosynthesis reaction center for absorbing, transmitting the chlorophyll of luminous energy is a kind of fluorescent material in chloroplaset, when green plants into
It usually can be along with the generation of fluorescence phenomenon when row photosynthesis.As shown in Figure 1, the luminous energy master that antenna beam molecule will be absorbed
It is used for the photochemical reaction of reaction center, excessive excitation can be then dissipated in the form of thermal energy, and a small amount of excitation
Can just it be emitted in the form of fluorescence.It is dissipated since the luminous energy of plant absorption only has sub-fraction in the form of fluorescence
(be usually no more than incident optical energy 5%) and the fluorescence signal of chlorophyll is fainter, therefore the wavelength of fluorescence is than the wavelength of absorption
Longer, Kautsky and Hirsh most just have realized that when 1931 close between photosynthetic primary reaction and chlorophyll fluorescence
Relationship is cut, they point out the plant after abundant dark adaptation in report, its chlorophyll fluorescence can be first under illumination condition
A maximum value is rapidly risen to, is then gradually reduced, can finally be stablized in some fixed value.
As that studies chlorophyll fluorescence deepens continuously, fluorescence induction kinetics curve and its rapid induction dynamics institute
The abundant information contained is fully realized, so that more deep to II donor side of PS and by the research of side electron transmission.It is typical
Fast chlorophyll fluorescence induction dynamics curve have O, J, I, P etc. mutually as shown in Figure 2.Under normal circumstances, light will be just exposed to
Minimum fluorescence when lower is defined as O point, and the peak-peak of fluorescence is defined as P point, and quick chlorophyll fluorescence kinetics curve refers to
From O point to the change in fluorescence process of P point, the primary photochemical reaction of PS II and the structure of Photosynthetic and state are mainly reflected
Deng situation of change.The O-J stage is the photochemical reaction stage, is by electronics after II reaction center of PS is excited via going magnesium leaf green
Plain (Pheo) passes to QA, and by QAIt is reduced into QA -Process, the stage is especially high to the sensibility of light intensity；The J-I stage is PQ
The stage of library reduction, the fluorescence in the stage be completely reduced by the library fast reduced form PQ in electron transfer process it is caused, it
It can reflect the heterogeneity in the library PQ；The fluorescence in I-P stage is caused by the library slow reduced form PQ is reduced, it can react redox egg
White reduction situation.When the oxygen evolution complex (Oxygen-evolving complex, OEC) of II donor of PS comes to harm, leaf
Green element fluorescence intensity will rise, and K phase (300 μ s or so after illumination) occur, and multiphase fluorescence O-J-I-P then becomes O-K-J-I-P,
Even there are more inflection points to occur.The available a large amount of initial data from quick chlorophyll fluorescence kinetics curve, in order to
It can preferably reflect the relationship of kinetic curve and sample material, nineteen ninety-five, Strasser and Strasser were with biomembrane energy
Based on amount flowing, the specimen material interior change under given physical state is measured by calculating energy stream and energy ratio,
Establish highly simplified energy flow illustraton of model (Fig. 3).The energy absorbed according to energy flow model, antenna beam (Chl)
(ABS) a part is dissipated in the form of thermal energy and fluorescence (F), and another part is then by reaction center (RC, in JIP- measurement
RC refers to active reaction center) (TR) is captured, heart excitation can be converted into also proper energy in the reaction, by QARestore QA -, after
Person can be reoxidized again, to generate electron transmission (electron transport, ET), the electronics of transmitting is used for solid
Determine CO2Or other approach.The data processing (table 1) to grow up on this basis is known as " JIP- measurement "." JIP- measurement " can
To provide the change of the structure and function of the bulk information of sample material, especially photosynthetic organs under difficult environmental conditions
Change.
Term and formula used in the analysis of 1 fast chlorophyll fluorescence induction dynamics curve (O-J-I-P) of table
The photosynthesis of plant is very sensitive to various environment-stress, and quickly chlorophyll fluorescence kinetics analytical technology is made
To evaluate the sensitive probe that the adverse circumstances such as plant photosynthesis efficiency and moisture, temperature, salt stress influence photosynthesis of plant,
It is widely applied to the multiple fields such as agricultural production, ecological protection and environmental monitoring.Through the inventors of the present invention discovered through researches that,
The nucleic such as strontium, caesium, uranium significantly affect the photosynthetic efficiency of plant, and the photosynthesis of the different plant of accumulation ability is to nucleic
Stress also show different response differences, and quickly chlorophyll fluorescence kinetics parameters very sensitive, can be supervised quickly
Survey the variation of this response difference, thus present inventor attempt to look for it is a kind of using caesium to the quick chlorophyll of plant leaf blade
Change of Chlorophyll Fluorescence Kinetics Parameters influences to come plant identification to the method for the accumulation ability difference of caesium.
Summary of the invention
The present inventor analyzes influence of the caesium to the not quick chlorophyll fluorescence kinetics parameters of kindred plant blade,
And establish corresponding database, the results showed that quick chlorophyll fluorescence kinetics parameters PIABSVariation tendency can and plant
Corresponding relationship is established to the size of caesium accumulation ability, therefore can be with quick chlorophyll fluorescence kinetics technology come plant identification pair
The size of caesium accumulation ability.
Based on this, the present invention provides a kind of with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability
Method, method includes the following steps:
1) plant cultivating: the nutrient solution containing caesium of plant various concentration to be determined is handled；
2) chlorophyll fluorescence kinetics parameters measure: measuring different caesium concentration for the treatment of with continuous agitation formula chlorophyll fluorescence instrument
Under quick chlorophyll fluorescence kinetics curve and fluorescence parameter of the plant after dark adaptation；
3) PI is calculated according to the measurement result of step 2)ABSValue, and calculate the PI of caesium processing groupABSChanging value；
4) according to the PI of the plant leaf blade of step 3)ABSThe accumulation ability of changing value identification plant pair caesium.
Further, the above-mentioned method with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability, step
5) identification method is, by PIABSFall more than or equal to 20% foliage filter screening be corresponding concentration under caesium accumulation ability it is strong
Plant；By PIABSFall less than 20% foliage filter screening be the weak plant of caesium accumulation ability under corresponding concentration.
Further, the above-mentioned method with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability, step
It 1) is to use the nutrient solution containing caesium of various concentration to handle again to plant length to 6 leaf phases.If handling plant with nutrient solution containing caesium too early,
Plant Tolerance ability is weak, it will be difficult to filter out satisfactory plant, if handling plant with nutrient solution containing caesium too late, and miss
The best period of plant strain growth and absorption nucleic, therefore, the present invention selects 6 leaf phase plant to handle.
Further, the above-mentioned method with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability, step
2) before the assay first by 20~25min of plant dark adaptation.
Some specific embodiments according to the present invention, it is above-mentioned rich with quick chlorophyll fluorescence kinetics parameters plant identification caesium
The method of collection ability, comprising the following steps:
1) plant cultivating: choose plant to be determined in quartz sand and Hoagland nutrient solution cultivation system nursery to 4 leaves
Phase chooses the consistent healthy plant of growing way and transplants into the plastic tub containing 10 kilograms/basin quartz sand, 6 plants of every pot transplanting, wait grow
Handled to 6 leaf phases with the Hoagland nutrient solution of various concentration caesium (CsCl), caesium concentration is respectively 0,0.1,0.5,1,
5mmol·L-1, 5 repetitions；
2) quickly chlorophyll fluorescence kinetics parameters measurement: using continuous agitation formula chlorophyll fluorescence instrument, (Britain Lufthansa is public
The Handy PEA or M-PEA of department), it has chosen and has been measured in the middle part of the mature leaf being just unfolded entirely, dark adaptation before measuring
Then 20-25min measures the quick chlorophyll fluorescence kinetics curve of dark adaptation blade, each processing measures 6 repetitions, and
Obtain minimum fluorescence Fo, maximum fluorescence Fm, K phase fluorescence (fluorescence when 300 μ s) FK, J phase fluorescence (fluorescence when 20ms) FJEtc. glimmering
Optical parameter；
3) PI is calculated according to the measurement result of step 2)ABSValue (calculation formula is as shown in table 1), and calculate caesium processing
The PI of groupABSChanging value；
4) PI of the plant leaf blade obtained according to step 3)ABSChanging value identifies the accumulation ability of plant pair caesium, PIABSUnder
The foliage filter screening that range of decrease degree is 20% or more is the plant that caesium accumulation ability is strong under corresponding concentration；By PIABSFall be less than
20% foliage filter screening is the plant that caesium accumulation ability is weak under corresponding concentration.
The beneficial effects of the present invention are: overcome existing method screening caesium enriching plant existing for sample treatment it is cumbersome,
The disadvantages of period is long, low efficiency, quick chlorophyll fluorescence kinetics analytical technology of the present invention have non-destructive testing, spirit
The advantages such as quick, quick can carry out instant in-site detecting to fresh plant sample, without carrying out again after sampling, drying, resolution
Instrument analysis test, has the characteristics that timely, sensitive, quick, efficient.
Detailed description of the invention
Fig. 1 is the schematic diagram that chlorophyll fluorescence generates；
Fig. 2 is typical fast chlorophyll fluorescence induction dynamics curve, left: linear time base, right: logarithmic time axis；
Flow model of Fig. 3 energy in photosynthetic organs；
Fig. 4 is the comparison figure of Four Plants caesium enrichment content after handling 7d；
Comparison figure of the Four Plants to the concentration coefficient of caesium after Fig. 5 is processing 7d；
Fig. 6 is Four Plants PI after processing 7dABSThe comparison figure of parameter.
Specific embodiment
It is illustrated below by way of specific test example is further to summary of the invention of the invention, but should not be construed as the present invention
Range be only limitted to example below, invention thinking according to the present invention and entire contents, can will be each in following instance
Technical characteristic makes combination/replacement/adjustment/modification appropriate etc., this is will be obvious to those skilled in the art that still
Belong to the scope that the present invention protects.
Materials and methods
The present embodiment selects the speed of growth fast, the big several plant of biomass, the respectively Brassica rapa L of Cruciferae, white
The semen viciae fabae and pea of dish and pulse family.See Table 1 for details for the specific section's category of plant of participating in the experiment.
The test of table 2 selects vegetable formal name and section to belong to
Experimental design and plant cultivating
Test is using Brassica rapa L, Chinese cabbage, semen viciae fabae and pea as material to be tested, nursery to 4 leaf phases in the soil, chooses growing way one
The healthy plant of cause is transplanted into the plastic tub of the quartz sand of basin containing 10kg/, 6 plants of every pot transplanting.To long to 6 leaf phase various concentrations
The Hoagland nutrient solution of caesium (CsCl) is handled, and caesium concentration for the treatment of is respectively 0,0.1,0.5,1,5mmolL-1, each place
Manage 5 repetitions.
Data acquisition
7d acquisition sample after treatment carries out caesium content analysis and the analysis of quick chlorophyll fluorescence kinetics.
Caesium assay: the whole strain plant that takes that treated, with after rinsing with ruinning water, by plant root in 20mmolL-1's
Na230min is impregnated in-EDTA solution, to chelate the caesium for being adsorbed in root system surface.Then it is washed with deionized water, drains away the water,
By the aerial part and under ground portion of plant, finish 30min at 105 DEG C, and it is finely ground that drying to constant weight at 75 DEG C, takes dry powder respectively
0.1g carries out micro-wave digestion using microwave dissolver (Mars, U.S. CE M company).Using atomic absorption method (AA700, U.S. PE
Company) measurement Plant aboveground and under ground portion Cs content, each sample duplicate measurements 3 times.
Caesium content in caesium content/cultivating system in concentration coefficient (BCFs)=plant body.
Quick chlorophyll fluorescence kinetics analysis: different disposal plant chooses same leaf position, the mature leaf being fully deployed
Middle part, dark adaptation 20-25min before the assay, using continuous agitation formula chlorophyll fluorescence instrument (M-PEA, Britain Hansatech
Company), the rapid fluorescence kinetics curve of blade is measured, each processing measures 6 repetitions.It is carried out according to the formula of table 1
JIP-test analysis, needs to use: fluorescence (O phase, F when 20 μ so), 300 μ s when fluorescence (K phase, Fk), 2ms when fluorescence (J phase,
FJ), 30ms when fluorescence (I phase, FI) and maximum fluorescence (P phase, Fm).Variable fluorescence FkAccount for FJ-FoThe ratio W of amplitudek；Variable fluorescence
FIAnd FJAccount for Fp-FoThe ratio V of amplitudeIAnd VJ；The exciton of capture Q into electron transport chain by electron transmissionAOther electricity in downstream
The probability Ψ of sub- receptoro；Photochemical properties indices P I based on absorbing luminous energyABSEtc. parameters.
Data processing and interpretation of result
Make the variance analysis of correlation test data with statistical analysis software SPSS PASW Statistics 18.0
(ANOVA), compare the difference between processing, and mapped with Excel 2016.
Caesium assay data are as shown in table 3, and Four Plants caesium is enriched with comparision contents result such as Fig. 4, and Four Plants are to caesium
Concentration coefficient such as Fig. 5.
Leaf chlorophyll fluorescence parameter PIABSAnd its variation is as shown in table 4, Four Plants chlorophyll fluorescence parameters PIABSThan
Compared with result such as Fig. 6.
In Fig. 4~6, every four column diagrams are one group of same treatment concentration from left to right, successively respectively represent Brassica rapa L,
Chinese cabbage, semen viciae fabae and pea Four Plants, duplicate four groups of column diagrams represent 4 concentration for the treatment of (0.1,0.5,1,5mmolL-
1)。
Enrichment difference of the Four Plants to caesium after the processing of table 3 7d
Table 4 handles the quick chlorophyll fluorescence parameters PI of Four Plants blade after 7dABSAnd its variation
The caesium accumulation ability of conventional method identification Four Plants
Traditional method usually uses concentration coefficient (concentration coefficient=aerial part or under ground portion Cs+Content (g/kg)/
Cs in cultivating system+Concentration (g/L)) size judge that plant is strong and weak to the accumulation ability of caesium, concentration coefficient is smaller, shows
Plant is weaker to the adsorption capacity of nucleic and heavy metal, and concentration coefficient shows that more greatly plant is stronger to the absorption accumulation ability of caesium.
It is shown according to the data of Fig. 4~5 and table 3, under identical nuclide concentration, in the Four Plants of tested person, accumulation in Brassica rapa L body
Caesium it is most, concentration coefficient is maximum, has strongest accumulation ability, and the caesium cumulant and accumulation ability of Chinese cabbage occupy intermediate, and
Semen viciae fabae and the caesium of pea accumulation are minimum, and concentration coefficient is also minimum, and accumulation ability is most weak.
The caesium accumulation ability of the method for the present invention identification Four Plants
According to Fig. 6 and table 4, the processing of various concentration caesium can cause quick chlorophyll fluorescence kinetics parameters i.e. based on absorption
The performance parameter PI of reaction centerABSDecline, the PI of the strongest Brassica rapa L of accumulation abilityABSFall it is maximum, the lower range of decrease
Degree is greater than 20% or more, and Chinese cabbage is less than Brassica rapa L to the accumulation ability of caesium and is greater than semen viciae fabae and pea, PIABSFall occupy
Between semen viciae fabae and pea, and semen viciae fabae and pea are minimum to the enriching quantity of caesium, PIABSFall it is minimum, in various concentration
Under processing, PIABSFall be respectively less than 20%, semen viciae fabae is similar with accumulation ability of the pea to caesium, PIABSFall
Also similar.The method of the present invention is consistent with conventional method conclusion, and has the advantages that quick, efficient, lossless.
The plant that caesium accumulation ability is strong under a certain concentration can be nondestructively filtered out rapidly using method of the invention, and
It is different according to accumulation ability of the plant under various concentration, plant can be effectively chosen according to the caesium concentration of contaminated soil to be processed
Strain.For example, in 0.1~1.0mmol/L, Brassica rapa L can be selected, and then can choose Brassica rapa L or white in 5.0mmol/L or so
Dish.
Claims (5)
1. a kind of method with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability, which is characterized in that including
Following steps:
1) plant cultivating: the nutrient solution containing caesium of plant various concentration to be determined is handled；
2) plant under different caesium concentration for the treatment of the measurement of chlorophyll fluorescence parameters: is measured with continuous agitation formula chlorophyll fluorescence instrument
Quick chlorophyll fluorescence kinetics curve and fluorescence parameter after dark adaptation；
3) PI is calculated according to the measurement result of step 2)ABSValue, and calculate the PI of caesium processing groupABSChanging value；
4) according to the PI of the plant leaf blade of step 3)ABSThe accumulation ability of changing value identification plant pair caesium.
2. a kind of side with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability according to claim 1
Method, which is characterized in that the identification method of step 4) is, by fluorescence parameter PIABSFall be 20% or more foliage filter screening
For the strong plant of caesium accumulation ability under corresponding concentration；By PIABSFoliage filter screening of the fall less than 20% be under corresponding concentration
The weak plant of caesium accumulation ability.
3. the method according to claim 1 with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability,
It is characterized in that, step 1) uses the nutrient solution containing caesium of various concentration to handle again to plant length to 6 leaf phases.
4. the method according to claim 1 with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability,
It is characterized in that, step 2) is before the assay first by 20~25min of plant dark adaptation.
5. the method according to claim 1 with quick chlorophyll fluorescence kinetics parameters plant identification caesium accumulation ability,
Characterized by comprising the following steps:
1) it plant cultivating: chooses plant to be determined nursery in quartz sand and nutrient solution and chooses the consistent health of growing way to 4 leaf phases
In plantlet of transplant to the plastic tub containing 10 kilograms/basin quartz sand, 6 plants of every pot transplanting, to long to 6 leaf phases various concentration caesium
Nutrient solution is handled, and caesium concentration is respectively 0,0.1,0.5,1.0,5.0mmolL-1, 5 repetitions；
2) chlorophyll fluorescence kinetics parameters measure: continuous agitation formula chlorophyll fluorescence instrument are used, after the processing of nutrient solution containing caesium
It 7th day, has chosen and has been measured in the middle part of the mature leaf being just fully deployed, dark adaptation 20-25min, is then surveyed before measuring
Determine the quick chlorophyll fluorescence kinetics curve of dark adaptation blade, each processing measures 6 repetitions, and obtains fluorescence parameter；
3) PI is calculated according to the measurement result of step 2)ABSValue, and calculate the PI of caesium processing groupABSChanging value；
4) PI of the plant leaf blade obtained according to step 3)ABSChanging value identifies the accumulation ability of plant pair caesium, PIABSThe lower range of decrease
The foliage filter screening that degree is 20% or more is the plant that caesium accumulation ability is strong under corresponding concentration；By PIABSFall less than 20%
Foliage filter screening be the weak plant of caesium accumulation ability under corresponding concentration.
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