CN104142382A - Isotope stalk double-tagging tracing method - Google Patents
Isotope stalk double-tagging tracing method Download PDFInfo
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Abstract
The invention belongs to the technical field of isotope tracing methods and particularly relates to an isotope stalk double-tagging tracing method in order to solve the problems that through an original tagging method, simultaneous tracing of N and C is limited, disturbance on plants is large, natural secretion of deposition matter on plant root systems is influenced, the isotope recovery rate is low, and research results are instable. The isotope stalk double-tagging tracing method comprises the steps of (1) preparing materials, (2) conducting tagging, (3) conducting sampling and measuring, and (4) conducting calculation. By means of the method, in-situ tagging can be conducted on the condition of soil cultivation without disturbing space distribution of the root systems, and the relationship between soil and roots can be maintained; moreover, carbon-nitrogen double-tagging can be conducted through a mixed solution containing 13C glucose and 15N urea; compared with other methods, the isotope stalk double-tagging tracing method has the advantages that plant materials are uniformly tagged, and a high recovery rate of 15N and 13C is achieved.
Description
Technical field
The invention belongs to tagging method and technology field, particularly a kind of isotope stem stalk double-tagging tracing method.
Background technology
At present at the main isotope labelling method of rhizodeposition research field, can be divided into gas labelling method, Split-Root Nutrient Solution Method and overground part labelling method, below put up with the maximally related overground part labelling method of the present invention and describe in detail, and analyze its relative merits.
Plant shoot labeling method is used abundant conventionally
15n-urea,
15n-NO
3 -or
15n-NH
4 +solution carries out single or multiple pulse labeling, according to the difference at mark position, can be divided into leaf marking method, petiole labelling method and stem stalk labelling method.
Leaf marking method is mainly to spray, smear or the mode such as blade tip end immersion is carried out isotope and fed by blade.During blade profile elongate (as wheat, oat etc.), conventionally from most advanced and sophisticated or centre, blade is cut off, immersed in the solution that contains label isotope, naturally absorbing solution is sucked in plant body by crop; When blade is comparatively roomy (as soybean, mung bean etc.), conventionally adopt the method for smearing or spraying to carry out mark.For preventing that blade from sustaining damage, generally use the label solution of low concentration, the isotope solution concentration of use is often lower than 1.0%, as 0.25% and 0.40%, 0.5%.
Advantage: blade picked-up solution speed is fast, can be easily for multiple labelling; Method of operating is easier, can carry out in field relatively large leaf marking; Be not subject to the impact in plant growth stage, can carry out mark at any growth time.
Shortcoming: mark is inhomogeneous, the distribution of tagged element is relevant to mark position, and tagged element is more distributed in blade and the overground part of crop, and underground part is relatively less; Khan etc. (2002b) study discovery, and during mark plant radical leaves, root system has higher
15n abundance; The part that adopts label solution to process contains higher isotopic abundance than other parts, causes partly
15n is with the loss of ammonia form, and this is also one of reason that the method recovery is low; Because mark system is open, label solution water clock can contaminated soil, causes the amount being absorbed by plant can not Accurate Determining.
Petiole labelling method and leaf marking are similar, choose a slice blade and cut blade-section, and petiole is inserted in the solution that contains label isotope, utilize nature to absorb and carry out isotope labeling.The tagged element amount that in petiole labelling method, plant absorbs will be lacked compared with leaf marking method, if the petiole that contains high concentration tagged element comes off, and can contaminated soil.Feed and compare with petiole, blade feeding method can cause mung bean and the isotope enrichment of pea root.The transpiration of plant, the position of leaf and environmental baseline around etc. all affect the absorption of plant to solution.Solution absorbs and labeling effciency depends on the growth phase of weather conditions and plant, and this is that the absorption because of solution is that transhipment by transpiration pull and nitrogen promotes.The concentration of passing label solution in time raises gradually, and causing label solution absorptivity on the low side is also a large defect of petiole labelling method.
Stem stalk labelling method mainly contains microinjection.
Microinjection is expelled to label solution in plants stems stalk with micro syringe.The advantage of the method is fast and convenient, and the repeating label time interval is short, and label isotope consumption can accurately control, but conventionally causes above-ground plant parts to be preferentially labeled, and isotope may be highly enriched at mark position.
will by the method Deng (2002)
15nH
4cl (95%) is expelled in cowpea stem stalk, studies three different growth phases
15distribution and the utilization ratio of N between Different Organs, but because the tagged element amount of using in research is extremely low, Wichern etc. (2008) think that the method is not suitable for the research of rhizodeposition nitrogen.
The imperfection of above research method, usually causes the difficulty of crop isotope labeling correlative study, for discipline development has caused very large obstruction.
Summary of the invention
The invention provides a kind of isotope stem stalk double-tagging tracing method, be specially a kind of use
15n and
13c isotope carries out the technology of stem stalk double-tagging spike simultaneously, to solve original labeling method in the limitation aspect N, C tracking simultaneously, larger to plant disturbance, affect the secretion naturally of root system of plant deposited material, the isotope recovery is low, and the problem such as result of study is unstable.
An isotope stem stalk double-tagging tracing method, the method step is as follows:
(1) preparation of material;
(2) carry out mark;
(3) sampling and mensuration;
(4) calculate.
Detailed operating process of the present invention is as follows:
(1) preparation of material:
The material needing has: gas chromatography bottle (1.5mL), degreasing cotton thread, sewing-needle (medium size), needle-threader, thin copper wire, plastic tube (diameter 5mm), the abundance of pre-opening are 99%
15n-urea, abundance are 99%
13c-glucose, the alcohol water blend that volume fraction is 75%, cotton ball soaked in alcohol, tweezers, little watering can, little U-shaped hard wire.
1. degreasing cotton thread:
Buy common thin cotton thread, use deionized water to add liquid detergent and soak 2h～3h, rubbing cotton thread makes its abundant wash clean, sloughs fat, is beneficial to solution and absorbs.After cotton thread is air-dry, be cut into the long segment in 12cm left and right standby.
2. thin copper wire:
Thin copper wire is cut into the long segment in 5cm left and right, standby.
3. plastic tube:
Plastic tube is cut into the long segment in about 8cm, and centre position is cut to U-shaped gap, make fine rule can wear from one end as, in the middle of managing, pass, then penetrate, then pass from the other end.
4. isotope solution:
By mark demand, take a certain amount of
15n urea and
13c glucose, incorporates in a certain amount of sterile purified water, mixes, and is made into isotope mixed solution, is placed in aseptic fuming cupboard standby.Dose of an isotope can regulate according to research purpose, as mark and sampling interval time longer, should suitably increase label isotope consumption.
After above-mentioned thing is ready, all apparatus (except isotope solution) are carried out to steam sterilizing 20min at 121 ℃, be then placed in sterile hood standby.
(2) carry out mark:
1. labeling process general introduction:
In the latter stage of nourishing and growing of plant, carry out mark, (plant is apart from about 3 centimeters in the ground) borings (diameter is 0.25mm) at plant position to be marked, with degreasing cotton thread, through this hole, the two ends of degreasing cotton thread are inserted and are equipped with in the reagent bottle of label solution; Described degreasing cotton thread overcoat has plastic tube, to prevent the loss of label solution; With thin copper wire, fixedly between degreasing cotton thread, plastic tube and plant, do not misplace, and fixation, prevent that gap from causing too greatly the rising loss of solution; Described label solution is that the abundance for preparing is 99%
13c-glucose and abundance are 99%
15n-urea mixed solution, mark 2 times, midfeather 1 time-of-week adds the label solution of 1mL at every turn, after absorption, then adds the deionized water of 1mL～2mL, so that residual solution is all absorbed by plants on degreasing cotton thread; After all labeling process completes, the material that mark is used and device take off from plant gently, Collection and conservation.
2. mark detailed step:
1. by removings such as plant peripheral obstacles, potted plant in this way, should, by basin along cleaning, prevent from operating process making troubles, and pollute;
2. the alcohol water blend that is 75% by volume fraction spray hand, tweezers and the article that around may touch, carry out disinfection, the plant of protecting from infection;
3. with tweezers, clipping cotton balls, to dip volume fraction be 75% alcohol water blend, and at plant position to be marked, (apart from 3cm place, ground) surface is smeared, carried out disinfection;
4. with needle-threader, degreasing cotton thread is penetrated to syringe needle, pin, through plastic flexible pipe, is pulled out from middle osculum, it is slow that action is wanted, and prevents that degreasing cotton thread from skidding off;
5. pin is passed to plant (pin splashes into one or two deionized water immediately through after plant in pinprick porch, prevent mark overlong time, plant injury), from the middle osculum other end of plastic tube, enter, from outlet one end, pull out gently;
6. with thin copper wire, two parts of flexible pipe closed up and be fixed on labeled plant, dwindling as much as possible the space between plant and flexible pipe, reducing rising loss;
7. with a little U-shaped hard wire, insert the two ends of bottom hose, drive flexible pipe through the gas chromatography bottle cap of pre-opening, then take off bottle, take out U-shaped hard wire, cotton thread is chosen to the bottle of then screwing on pin.(cotton thread is also unsuitable oversize, but end must arrive bottle bottom);
8. the syringe that is 1mL with volume adds index liquid in bottle, adds 1mL～2mL deionized water after having absorbed again, and the processing of mark does not add the deionized water of same volume;
9. after solution is completely absorbed, by labelling apparatus, slowly from plant, take off, reduce as far as possible the injury to plant;
10., after mark completes, on plant ground watch glasses, the silk screen in 1mm aperture, collected fallen leaves every several days, prevented that the isotope in fallen leaves from affecting soil.
(3) sampling and mensuration:
In described step (3), sampling is as follows with method for measuring:
A. the preparation of material:
Valve bag, marking pen, pincet, little scoop, sieve (2mm), sample grinding machine, paper bag, Polypropylence Sheet, balance, little paper bag;
B. sample:
1. divide flush with ground to cut plant shoot, by the Different Organs such as cauline leaf of plant, merotomize, pack in paper bag, carry out mark, at 60 ℃, dry 72h, weigh, use bowl mill grind away, to be measured;
2. the soil in earth pillar is taken out, be poured on a clean Polypropylence Sheet, and with scuppit, the soil of earth pillar inwall scraped, get as much as possible all soil;
3. soil is smashed to pieces, whole 2mm that cross sieve, and obtain axial root system, with distilled water, clean, and at 60 ℃, dry 72h, weigh, and grind, to be measured.
4. weigh the quality of whole wet soils, soil is fully mixed and gets 100g left and right, dry and measure water cut;
5. separately get 100g left and right wet soil, by wet screening, use 200 mesh sieve to sieve and wash, obtain the residue radicula in soil, radicula is cleaned and dry 72h at 60 ℃, weigh (not for isotope assay, for estimating the amount of the residual radicula of soil, thereby comprehensively calculate root biomass in soil).
6. get sub-fraction soil sample, at 60 ℃, dry 72h, bowl mill grinds, to be measured;
C. measure:
Use mass spectrometer to measure in Plants and Soils sample
13c with
15the abundance of N, is used the total carbon of carbon blood urea/nitrogen analyzer working sample, full nitrogen.
(4) calculate:
The method proposing according to Janzen and Bruisma (1989) is calculated.
While adding tracer element, the super atom%excess of SI standard unit atom percentage (atom percent excess) usually using represents isotopic relative content.Atom percentage is super to be referred in certain test specimen, the abundance of stable nuclide and its natural abundance poor.The computing formula that nitrogen and carbon isotope atom percentage are super is as follows:
15n atom percentage surpasses (atom%
15n excess) in=sample
15abundance-natural abundance of N (1)
13c atom percentage surpasses (atom%
13c excess) in=sample
13abundance-natural abundance of C (2)
In actual computation process, plant part or soil
15n (
13c) atom percentage super common usage flag part
15n (
13c) abundance deducts unlabelled adjoining tree or this part of soil
15n (
13c) abundance.
Rhizodeposition nitrogen (carbon) number percent refers to that rhizodeposition nitrogen (carbon) accounts for the ratio of total soil nitrogen (total carbon).Conventional method for expressing is respectively NdfR (Nitrogen derived from Rhizodeposition) and CdfR (Carbon derived from Rhizodeposition), and the computing formula of use is as follows:
The computing formula of rhizodeposition nitrogen is:
NdfR＝Ns×％NdfR (5)
CdfR＝Cs×％CdfR (6)
Wherein Ns and Cs are respectively in soil the content of full nitrogen and total carbon.
The accuracy of rhizodeposition nitrogen result of calculation is along with mark plant root
15the raising of N abundance and the reduction of total nitrogen content of soil and improve.In addition, every basin plantation number strain plant can be estimated plant rhizodeposition nitrogen more accurately than only planting a strain.Crucial is to plant unmarked contrast, is used as the super natural abundance of calculating of atom percentage, and should use in atmosphere
15natural abundance (the 0.3663atom% of N
15n).
Beneficial effect of the present invention is:
The inventive method can be carried out original position mark under soil cultivation condition, does not need to upset the space distribution of root system, keeps contacting of soil and root, and can be with containing
13the glucose of C and
15the urea mixed solution of N carries out carbon nitrogen double-tagging.Compare with additive method, should realize in this way the more uniform mark of vegetable material and
15n and
13the high-recovery of C.
Embodiment
The invention provides a kind of isotope stem stalk double-tagging tracing method, below in conjunction with embodiment, the present invention will be further described.
Experimental example 1
Application
15n-
13c stem stalk Double Labelling Technique research soybean rhizosphere deposit carbon, nitrogen:
1. overview experimental field:
Test in 2012 academy of agricultural sciences, Nian Baicheng City in Jilin Province rail mounted net canopies (45 ° of 37'N, 122 ° of 48'E) and carry out.Local climate belongs to the continental monsoon climate in temperate zone, average annual sunshine time 2919.4h, 4.9 ℃ of average annual temperature, frostless season 157d, average annual precipitation 407.9mm.Potted plant soil is taken from academy of agricultural sciences experimental field, the organic 12.4g/kg of topsoil soils, and full nitrogen 1.17g/kg, alkali-hydrolyzable nitrogen 88.6mg/kg, available phosphorus 12.3mg/kg, effective potassium 71.8mg/kg, pH is 6.86.Preceding crop is No. 2 oats of white swallow.Weather data is provided by Baicheng City weather bureau.
2. earth pillar is prepared to process with test:
Using height is 50cm, internal diameter be 37cm bucket as Potted-plant container, get topsoil soil in above-mentioned plot, air-dry, crossing aperture is 3mm sieve, and every barreled enters 20kg, water all wetting to the soil of whole bucket, wait showing native water cut, drop to after suitable humidity, sow, crop moisture content in the time of infertility remains on 40% left and right of maximum water holding capacity (quality).
For examination soybean (Glycine max L), be No. 10, white agriculture, academy of agricultural sciences, Baicheng City in Jilin Province provides; After vernalization, sow, on May 29 2012 sowing time, after emerging, thinning is to every barrel of two strains, and test arranges 6 repetitions; Use potted plant simulation land for growing field crops environment, by
13c with
15the N stem stalk double-tagging isotope tracer technique of feeding, total amount and the distribution transfer case of research rhizodeposition carbon, nitrogen.
3. labeling method:
Within after soybean planting the 5th week, bring into use original position cotton core feeding method to carrying out mark, a Zhou Houzai carries out mark one time.Labeling method: the hole of boring a diameter 0.25mm apart from about three centimeters in ground plant,, connects plants stems and a bottle that is filling label solution through this eye with a cotton thread.The outer soft plastic tube of surface cover of cotton thread, to prevent the loss of label solution.With a thin copper wire, fixedly between cotton thread, plastic tube and plant, do not misplace.Label solution is to use quality volume fraction 4% (g/mL, refers to the quality volume fraction in mixed solution, lower same)
13the glucose solution of C mark (atom percentage surpasses 99.9%) and quality volume fraction are 0.2% (g/mL's)
15the mixed aqueous solution of the urea liquid of N mark (atom percentage surpasses 99.9%).Mark 2 times, each consumption is 1mL.Used tool is at 121 ℃ of steam sterilizing 20min.After solution is all absorbed, then add 1mL～2mL deionized water, so that residual solution is all absorbed by plants on cotton core.At later stages, the silk screen that aperture is 1mm in soil upper cover, regularly collects fallen leaves, prevents that the isotope in fallen leaves from affecting soil.
4. sample and assay method:
During sampling, along earth's surface, plant overground part is cut.Soybean is divided into stem, leaf, pod, seed by organ, and earth pillar is dug out, and all soil is crossed the sieve that aperture is 2mm and collects by hand all visible root systems in soil layer, clean by a small amount of washed with de-ionized water; Claim soil heavy, and measure water cut.Get 200g wet soil, put into 200 mesh sieves and swing and wash wet screening at water, with tweezers, pick fine root, as supplementing the manual root system that can not all sort out; Finally in the soil of having crossed 2mm sieve, get a part and cross 1mm sieve, air-dry weighing as rhizosphere soil.All plant samples and 60 ℃ of low temperature dryings of soil sample are at least weighed after 72h, and sample is used in mass spectrometer working sample after pulverizing with bowl mill
13c with
15the abundance of N, is used carbon blood urea/nitrogen analyzer working sample organic carbon, total nitrogen content.
5. computing formula
While adding tracer element, the SI standard unit usually using is the super a%e of atom percentage (atom percent excess).
Abundance-the natural abundance of stable nuclide in super (the atom%excess)=test specimen of atom percentage
Rhizodeposition nitrogen (carbon) number percent refers to that rhizodeposition nitrogen (carbon) accounts for the ratio of total soil nitrogen (total carbon).Conventional method for expressing is respectively NdfR (Nitrogen derived from Rhizodeposition) and CdfR (Carbon derived from Rhizodeposition), and the computing formula of use is as follows:
The computing formula of rhizodeposition nitrogen is:
NdfR＝Ns×％NdfR (4)
CdfR＝Cs×％CdfR (5)
Wherein Ns and Cs are respectively in soil the content of full nitrogen and total carbon.
6. results and analysis:
6.1 mark nitrogen element recovery rates:
Test findings shows, in soybean, and plant haulm
15n abundance is the highest, follows by seed, blade, beanpod and foot end, plant
15n abundance is between 0.44atom% and 1.21atom%, and isotopic abundance is (table 1) relatively evenly.
15the N recovery has reached 85.1%, far above other labeling method.Reclaim
15n concentrates in seed and beanpod, meets the general distribution rule of nitrogen.In sum, this kind of labeling method is soybean
15in N isotope labeling, application is more successful.
Table 1 soybean plant strain different parts
15n atom percentage is super, the recovery and distributed data table
Plant is used stem stalk double label method to carry out weekly a mark (midfeather 1 week), and totally twice, mark starts from after planting the 5th week (July 4).In table, data are mean value ± standard error (n=6).
6.2 mark nitrogen element recovery rates:
By table 2 result, shown, in soybean, plant haulm
13c abundance is the highest, follows by blade, foot end, beanpod and seed, plant
13c abundance is between 0.04atom% and 0.41atom%, and isotopic abundance is (table 2) relatively evenly.
13the C recovery has reached 70.8%, far above other labeling method, reclaims
13c concentrates in stem stalk.In sum, this kind of labeling method is soybean
13in C isotope labeling, application is more successful.
Table 2 soybean plant strain different parts
13c atom percentage is super, the recovery and distributed data table
Plant is used stem stalk double label method to carry out weekly a mark (midfeather 1 week), and totally twice, mark starts from after planting the 5th week (July 4).In table, data are mean value ± standard error (n=6).
Experimental example 2
Application
15n-
13c stem stalk Double Labelling Technique research Avena sativa rhizosphere deposit carbon, nitrogen:
1. overview experimental field:
With experimental example 1.
2. earth pillar is prepared to process with test:
For examination naked oats (Avena nuda L), be white swallow No. 2, academy of agricultural sciences, Baicheng City in Jilin Province provides; On May 29 2012 sowing time, after emerging, thinning is to every barrel of four strains, and test arranges 6 repetitions; All the other are with experimental example 1.
3. labeling method:
The oat jointing stage brings into use original position cotton core feeding method to carrying out mark, and a Zhou Houzai carries out mark one time.Labeling method is with experimental example 1.
4. sample and assay method:
During sampling, along earth's surface, plant overground part is cut.Oat is divided into seed, stem stalk and blade by organ.All the other are with experimental example 1.
5. computing formula:
With experimental example 1.
6. results and analysis:
6.1 mark nitrogen element recovery rates:
Test findings shows, in oat, and plant seed
15n abundance is the highest, follows by stem stalk, blade and foot end, plant
15n abundance is between 0.55atom% and 2.03atom%, and isotopic abundance is (table 3) relatively evenly.
15the N recovery has reached 83.3%, far above other labeling method.Reclaim
15n concentrates in seed, meets the general distribution rule of nitrogen.In sum, this kind of labeling method is oat
15in N isotope labeling, application is more successful.
Table 3 oat plant different parts
15n atom percentage is super, the recovery and distributed data table
Plant is used stem stalk double label method to carry out weekly a mark (midfeather 1 week), and totally twice, mark starts from after planting the 5th week (July 4).In table, data are mean value ± standard error (n=6).
The 6.2 mark carbon recovery:
By table 2 result, shown, in soybean, plant haulm
13c abundance is the highest, follows by seed, foot end and blade, plant
13c abundance is between 0.09atom% and 0.20atom%, and isotopic abundance is (table 4) relatively evenly.
13the C recovery has reached 50.9%, slightly higher or approaching than other labeling method recovery, reclaims
13c concentrates in stem stalk.In sum, this kind of labeling method is oat
13in C isotope labeling, application is more successful.
Table 4 oat plant different parts
13c atom percentage is super, the recovery and distributed data table
Plant is used stem stalk double label method to carry out weekly a mark (midfeather 1 week), and totally twice, mark starts from after planting the 5th week (July 4).In table, data are mean value ± standard error (n=6).
Experimental example 3
Application
15n-
13c stem stalk Double Labelling Technique research mung bean and oat intercropping rhizodeposition carbon, nitrogen and transfer:
1. overview experimental field:
With experimental example 1.
2. earth pillar is prepared to process with test:
For examination naked oats (Avena nuda L.), being white swallow No. 2 (Baiyan2), is white green No. 11 (Bailv11) for examination mung bean (Vigna radiate), and being academy of agricultural sciences, Baicheng City in Jilin Province provides; On June 29 2011 mung bean (sowing after vernalization) sowing time, the oat sowing time is July 17; Research method is to use under the soil and root growth environment in column simulation land for growing field crops, by
13c with
15the N stem stalk double-tagging isotope tracer technique of feeding, total amount and the distribution transfer case of research rhizodeposition carbon, nitrogen.
By 24 long be 55cm, diameter is that the pvc pipe of 20cm is imbedded in the pond that about 40cm is dark, the plastic pallet of a diameter 25cm of each earth pillar bottom surface pad, pack 19kg into and cross the dry ground that aperture is 3mm sieve, water all wetting to the soil of whole pvc pipe, wait showing native water cut, drop to after suitable humidity, sow.Pond is planted white swallow around and as protection, is gone for No. 2.
Test arranges 2 kinds of intercropping systems: mung bean nonoculture, oat, mung bean intercropping; 2 sub-sampling times and breeding time are respectively: Mung Bean Blooming fruiting period (2011.8.22), mung bean maturity stage (2011.9.19).Each is processed mung bean and all carries out mark, and oat is unmarked.CK1, CK2 are unmarked in contrast, and for measuring mung bean, oat natural abundance, 4 repetitions are established in test.The every post of nonoculture oat 4 caves, the every post of nonoculture mung bean 2 caves, the every post 2 cave oats of intercropping, 2 cave mung beans.4, every cave seed during sowing, after emerging, thinning is to 1 strain of every cave.
Table 5 test is processed tables of data is set
3. labeling method:
During mung bean branching stage, with original position cotton core feeding method, mung bean is carried out to mark.Labeling method is with experimental example 1.
4. sample and assay method:
During sampling, along earth's surface, plant overground part is cut.Mung bean is divided into stem, leaf, pod, seed by organ, and oat is divided into seed, stem stalk and blade.All the other are with experimental example 1.
5. computing formula:
Rhizodeposition carbon, nitrogen computing method are with experimental example 1.
Mung bean is as follows to the transfer amount computing method of oat:
6. results and analysis:
6.1 mark nitrogen element recovery rates:
Test findings shows, Mung Bean Blooming fruiting period (2011-8-24), nonoculture mung bean root
15n abundance is minimum is 0.09atom%, and the abundance of intercropping leaf is minimum is 0.08atom%; When separate room is done, in mung bean stem, abundance is the highest, is respectively 0.20atom%, 0.24atom%; The mung bean maturity stage (2011-9-19), in nonoculture green gram leaf and seed
15n abundance is minimum is 0.07atom%, and during intercropping, the abundance of root is minimum is 0.05atom%, and when separate room is done, in mung bean stem, abundance is the highest, is respectively 0.16atom%, 0.18atom%.
Bloom fruiting period, mark
15the intercopping overall recovery of N element is respectively 61.96%, 68.41%; During the maturity stage, mark
15the overall recovery mung bean nonoculture of N element, intercropping is respectively 63.26%, 81.87%, and the overall recovery of nitrogen element is higher, loses littlely, and loss cause may be solution vapour loss, maturity stage fallen leaves etc.
During fruiting period, each position intercropping overall recovery is all higher than nonoculture (blade intercropping slightly declines compared with nonoculture Mung Bean Blooming), and no matter separate room is done, in beanstalk and blade
15the recovery of N element is the highest, is respectively 46.56%, 41.85%; During the maturity stage, except beans root, soil, all the other each position intercropping overall recoverys are all higher than intercropping, and no matter separate room is done, in beanpod
15the recovery of N element is the highest, is respectively 35.86%, 50.74%;
The nonoculture of table 6 mung bean, mung bean and oat Intercropping System plant different parts
15n atom percentage is super, the recovery and distributed data table
In table, alphabetical implication is as follows: S
p: mung bean nonoculture, the fruiting period of blooming sampling; S
m: mung bean and oat intercropping, Mung Bean Blooming fruiting period sampling; I
p: mung bean nonoculture, maturity stage sampling; I
m: mung bean and oat intercropping, the sampling of mung bean maturity stage.In table, data are mean value ± standard error (n=4).
The 6.2 mark carbon recovery:
Test findings shows, Mung Bean Blooming fruiting period (2011-8-24), in nonoculture mung bean seed
13c abundance is minimum, is 0.04atom%, during intercropping the abundance of oat aerial part minimum be 0.003atom%; When no matter separate room is done, in mung bean stem, abundance is the highest, is 0.25atom%; The mung bean maturity stage (2011-9-19), during mung bean nonoculture in pod
13c abundance is minimum, is 0.02atom%, during intercropping the abundance of oat aerial part minimum be 0.001atom%, when separate room is done, in mung bean stem, abundance is the highest, is respectively 0.20atom%, 0.19atom%.Mark
13the overall recovery of C lower than
15n, this may to participate in respiration relevant with carbon.The a large amount of heterotrophic microorganisms that exist in soil utilize the soil organism as carbon source, and carbon is consumed, and nitrogen element is broken down into gaseous state after only in depositing to soil, just can lose relevant.
The nonoculture of table 7 mung bean, mung bean and oat Intercropping System plant different parts
13c atom percentage is super, the recovery and distributed data table
In table, alphabetical implication is as follows: S
p: mung bean nonoculture, the fruiting period of blooming sampling; S
m: mung bean and oat intercropping, Mung Bean Blooming fruiting period sampling; I
p: mung bean nonoculture, maturity stage sampling; I
m: mung bean and oat intercropping, the sampling of mung bean maturity stage.In table, data are mean value ± standard error (n=4).
Test findings shows, blooms in fruiting period, and mark
13the overall recovery mung bean intercopping of C element is respectively 55.88%, 67.30%; During the maturity stage, mark
13the overall recovery mung bean intercopping of C element is respectively 50.54%, 52.10%.
Each position overall recovery of mung bean while blooming fruiting period, no matter separate room is done, in beanstalk
13the recovery of C element is the highest, is respectively 60.91%, 55.27%; During the maturity stage, each position intercropping overall recovery is all higher than nonoculture (except root system) for mung bean, and no matter separate room is done, in beanstalk
13the recovery of C element is all the highest, is respectively 45.38%, 48.47%;
Mung bean maturity stage overall recovery was compared with the fruiting period of blooming, and intercopping all declines to some extent, and beanstalk, beans leaf decline the most obvious, and in beanpod, wheat root, wheat cauline leaf, soil, overall recovery all raises.
Comprehensive above three experimental example, we are known,
15n-
13c stem stalk double-tagging tracer technique can successfully be applied to the nitrogen carbon markings of soybean, mung bean, oat, and tracks isotope to the transfer in soil, can extrapolate rhizodeposition nitrogen and the carbon of different plants according to formula; In addition, adopting said method can successful mark mung bean and oat Intercropping System in mung bean, and track isotope to the transfer of intercropping oat, by formula to calculating, go out nitrogen and carbon transfer amount.
Claims (8)
1. an isotope stem stalk double-tagging tracing method, is characterized in that, the method step is as follows:
(1) preparation of material;
(2) carry out mark;
(3) sampling and mensuration;
(4) calculate.
2. a kind of isotope stem stalk double-tagging tracing method according to claim 1, is characterized in that, the material of preparing in described step (1) is as follows: gas chromatography bottle, degreasing cotton thread, sewing-needle, needle-threader, copper wire, plastic tube, abundance are 99%
15n-urea, abundance are 99%
13c-glucose, the alcohol water blend that volume fraction is 75%, cotton balls, tweezers, watering can, U-shaped iron wire.
3. a kind of isotope stem stalk double-tagging tracing method according to claim 1, it is characterized in that, the method of carrying out mark in described step (2) is as follows: in the latter stage of nourishing and growing of plant, carry out mark, in plant position to be marked boring, with degreasing cotton thread, through this hole, the two ends of degreasing cotton thread are inserted and are equipped with in the reagent bottle of label solution; Described degreasing cotton thread overcoat has plastic tube, to prevent the loss of label solution; With copper wire, fixedly between degreasing cotton thread, plastic tube and plant, do not misplace, and fixation, prevent that gap from causing too greatly the rising loss of solution; Described label solution is that the abundance for preparing is 99%
13c-glucose and abundance are 99%
15n-urea mixed solution, mark 2 times, midfeather 1 time-of-week adds the label solution of 1mL at every turn, after absorption, then adds the deionized water of 1mL～2mL, so that residual solution is all absorbed by plants on degreasing cotton thread; After all labeling process completes, the material that mark is used and device take off from plant, Collection and conservation.
4. according to a kind of isotope stem stalk double-tagging tracing method claimed in claim 3, it is characterized in that, described in to carry out the concrete steps of mark as follows:
1. plant barrier is around removed, prevented from making troubles in operating process, and pollute;
2. the alcohol water blend that is 75% by volume fraction spray hand, tweezers and the article that can touch, carry out disinfection, the plant of protecting from infection;
3. with tweezers, clipping cotton balls, to dip volume fraction be 75% alcohol water blend, on surface, plant position to be marked, smears, and carries out disinfection;
4. with needle-threader, degreasing cotton thread is penetrated to syringe needle, pin, through plastic tube, is pulled out from plastic tube centre exit, prevent that degreasing cotton thread from skidding off;
5. by pin through plant, from the other end of plastic tube centre exit, enter, from plastic tube not one end outlet of threading pull out;
6. with copper wire, two parts of plastic tube closed up and be fixed on labeled plant;
7. with U-shaped iron wire, drive flexible pipe through the gas chromatography bottle cap of pre-opening, then take off bottle, take off U-shaped iron wire, the bottle of then screwing on, described degreasing cotton thread end will arrive gas chromatography bottle bottom;
8. the syringe that is 1mL with volume adds label solution in bottle, adds 1mL～2mL deionized water after having absorbed again, and the space management of mark does not add the deionized water with the same volume of label solution;
9. after label solution is completely absorbed, the material that mark is used and device take off from plant;
10., after mark completes, the silk screen that aperture is 1mm on the plant ground of institute's mark watch glasses, regularly collects fallen leaves, prevents that the isotope in fallen leaves from affecting soil.
5. a kind of isotope stem stalk double-tagging tracing method according to claim 1, is characterized in that, in described step (3), sampling is as follows with method for measuring:
A. the preparation of material;
B. sample;
C. measure.
6. a kind of isotope stem stalk double-tagging tracing method according to claim 5, is characterized in that, the material of preparing in described step a is as follows: valve bag, marking pen, tweezers, scoop, sieve, sample grinding machine, paper bag, Polypropylence Sheet, balance, paper bag.
7. a kind of isotope stem stalk double-tagging tracing method according to claim 5, is characterized in that, in described step b, sampling procedure is as follows:
1. divide flush with ground to cut the plant shoot of institute's mark, the Different Organs classification by plant, packs in paper bag, dries 72h respectively at 60 ℃, weighs, and uses bowl mill grind away, to be measured;
2. the plant of institute's mark soil used is taken out;
3. soil is smashed to pieces, whole sieves that aperture is 2mm excessively, obtain axial root system, with distilled water, clean, and through gained axial root system, at 60 ℃, dry 72h, weigh, and grind, to be measured;
4. weigh the quality of whole soil, after soil is fully mixed, get 100g, dry and measure water cut;
5. separately get 100g soil, by wet screening, use 200 mesh sieve to sieve and wash, obtain the residue radicula in soil, gained radicula is cleaned and dry 72h at 60 ℃, weigh;
6. get appropriate soil sample, at 60 ℃, dry 72h, bowl mill grinds, to be measured.
8. a kind of isotope stem stalk double-tagging tracing method according to claim 5, is characterized in that, in described step c, assay method is as follows: use mass spectrometer to measure respectively in the plant sample and pedotheque of institute's mark
13c with
15the abundance of N, is used carbon blood urea/nitrogen analyzer plant sample and the total carbon in pedotheque and the total nitrogen content of working sample institute mark respectively.
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| CN106417194A (en) * | 2016-12-09 | 2017-02-22 | 广州微因生物科技有限公司 | N15 stable isotope labeling method for insect protein quantification and tracing |
| CN107047265A (en) * | 2016-12-31 | 2017-08-18 | 中国农业科学院农业资源与农业区划研究所 | A kind of culture high abundance isotope carbon, the circulatory system of nitrogen double labelling plant sample |
| CN111320158A (en) * | 2020-02-25 | 2020-06-23 | 南京林业大学 | An obtaining13C and15n double-labeling plant method and application thereof in preparation of biomass charcoal |
| CN112730346A (en) * | 2020-12-25 | 2021-04-30 | 中国农业科学院茶叶研究所 | Method for effectively determining nitrogen efficiency of perennial woody plants in field |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106417194A (en) * | 2016-12-09 | 2017-02-22 | 广州微因生物科技有限公司 | N15 stable isotope labeling method for insect protein quantification and tracing |
| CN107047265A (en) * | 2016-12-31 | 2017-08-18 | 中国农业科学院农业资源与农业区划研究所 | A kind of culture high abundance isotope carbon, the circulatory system of nitrogen double labelling plant sample |
| CN111320158A (en) * | 2020-02-25 | 2020-06-23 | 南京林业大学 | An obtaining13C and15n double-labeling plant method and application thereof in preparation of biomass charcoal |
| WO2021169154A1 (en) * | 2020-02-25 | 2021-09-02 | 南京林业大学 | Method for obtaining 13c and 15n dual-labeled plant and use thereof in preparation of biomass charcoal |
| CN111320158B (en) * | 2020-02-25 | 2022-03-25 | 南京林业大学 | An obtaining13C and15n double-labeling plant method and application thereof in preparation of biomass charcoal |
| CN112730346A (en) * | 2020-12-25 | 2021-04-30 | 中国农业科学院茶叶研究所 | Method for effectively determining nitrogen efficiency of perennial woody plants in field |
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