CN104142382B - A kind of isotope stem stalk double-tagging tracing method - Google Patents
A kind of isotope stem stalk double-tagging tracing method Download PDFInfo
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Abstract
The invention belongs to tagging method and technology field, particularly a kind of isotope stem stalk double-tagging tracing method.For solving the limitation of original labeling method in N, C simultaneously tracking, comparatively large to plant disturbance, affect naturally secreting of root system of plant deposited material, the isotope recovery is low, and the problem such as result of study is unstable, the invention provides a kind of isotope stem stalk double-tagging tracing method.The inventive method comprises the following steps: the preparation of (1) material; (2) mark; (3) sampling and mensuration; (4) calculate.The inventive method can carry out labeled in situ under soil cultivation condition, does not need to upset the space distribution of root system, keeps contacting of soil and root, and can with containing
13the glucose of C and
15the urea mixed solution of N carries out carbon nitrogen double-tagging.Compared with additive method, application such an approach achieves vegetable material marked more uniformly and
15n and
13the high-recovery of C.
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
Gas labelling method, Split-Root Nutrient Solution Method and overground part labelling method can be divided at the isotope labelling method that rhizodeposition research field is main at present, hereafter put up with the maximally related overground part labelling method with the present invention and describe in detail, and analyze its relative merits.
Plant shoot labeling method uses abundant usually
15n-urea,
15n-NO
3 -or
15n-NH
4 +solution carries out single or multiple pulse labeling, can be divided into leaf marking method, petiole labelling method and stem stalk labelling method according to the difference at mark position.
Leaf marking method is mainly sprayed by blade, smear or the mode such as blade tip end immersion is carried out isotope and fed.During blade profile elongate (as wheat, oat etc.), usually from most advanced and sophisticated or centre, blade is cut off, immerse in the solution containing label isotope, by naturally absorbing of crop, solution is sucked in plant body; When blade is comparatively roomy (as soybean, mung bean etc.), the method for smearing or spraying usually is adopted to mark.For preventing blade from sustaining damage, the general label solution using low concentration, the isotope solution concentration of use often lower than 1.0%, as 0.25% and 0.40%, 0.5%.
Advantage: blade picked-up solution speed is fast, can easily for multiple labelling; Method of operating is easier, can carry out relatively large leaf marking in field; Not by the impact of crop growth stage, can mark at any growth time.
Shortcoming: mark uneven, 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; Adopt the part of label solution process to contain higher isotopic abundance than other parts, cause 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 absorbed by plant can not Accurate Determining.
Petiole labelling method and leaf marking similar, choose a slice blade and cut blade-section, by petiole insert containing label isotope solution in, utilize nature absorb carry out isotope labeling.The tagged element amount that in petiole labelling method, plant absorbs comparatively leaf marking method will be lacked, if the petiole containing high concentration tagged element comes off, then and can contaminated soil.Compared with feeding 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 the environmental baseline etc. of surrounding all affect the absorption of plant to solution.Solution absorption and labeling effciency depend on the growth phase of weather conditions and plant, this is because the absorption of solution is promoted by the transhipment of transpiration pull and nitrogen.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.
Namely label solution is expelled in plants stems stalk with micro syringe by microinjection.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 usually 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
15the distribution of N between Different Organs and utilization ratio, but due to the tagged element amount used in research 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 causes 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 the limitation of original labeling method in N, C simultaneously tracking, comparatively large to plant disturbance, affect naturally secreting of root system of plant deposited material, the isotope recovery is low, and the problem such as result of study is unstable.
A kind of isotope stem stalk double-tagging tracing method, the method step is as follows:
(1) preparation of material;
(2) mark;
(3) sampling and mensuration;
(4) calculate.
Detailed operation process of the present invention is as follows:
(1) preparation of material:
The material needed has: the gas chromatography bottle (1.5mL) of pre-opening, degreasing cotton thread, sewing-needle (medium size), needle-threader, thin copper wire, plastic tube (diameter 5mm), abundance are 99%
15n-urea, abundance are 99%
13c-glucose, volume fraction is alcohol water blend, cotton ball soaked in alcohol, tweezers, little watering can, the little U-shaped hard wire of 75%.
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, the segment being cut into about 12cm long is for subsequent use.
2. thin copper wire:
Thin copper wire is cut into the segment that about 5cm is long, for subsequent use.
3. plastic tube:
Plastic tube is cut into the segment that about 8cm is long, and centre position is cut U-shaped gap, enable fine rule wear from one end as, pass in the middle of pipe, then penetrate, then pass from the other end.
4. isotope solution:
Take a certain amount of by mark demand
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 for subsequent use.Dose of an isotope can regulate according to research purpose, as longer in mark and sampling interval time, should suitably increase label isotope consumption.
After above-mentioned thing is ready, all apparatus (except isotope solution) is carried out steam sterilizing 20min at 121 DEG C, is then placed in sterile hood for subsequent use.
(2) mark:
1. labeling process general introduction:
Mark in the latter stage of nourishing and growing of plant, in plant position to be marked (about 3 centimeters in plant distance ground) boring (diameter is 0.25mm), with degreasing cotton thread through this hole, the two ends of degreasing cotton thread are inserted and is equipped with in the reagent bottle of label solution; Described degreasing cotton thread overcoat has plastic tube, to prevent the loss of label solution; Fix degreasing cotton thread with thin copper wire, do not misplace between plastic tube and plant, and fixation, prevent gap from causing too greatly solution transpiration loss; To be the abundance prepared be described label solution 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, is all absorbed by plants to make solution residual on degreasing cotton thread; After whole labeling process completes, the material of mark and device are taken off from plant gently, Collection and conservation.
2. mark detailed step:
1. by removings such as plant peripheral obstacles, potted plant in this way, then by basin along cleaning, should prevent from making troubles in operating process, and polluting;
2. with the article that the alcohol water blend that volume fraction is 75% sprays hand, tweezers and around may touch, carry out disinfection, plant of protecting from infection;
3. clip cotton balls with tweezers and dip the alcohol water blend that volume fraction is 75%, at plant position to be marked (apart from 3cm place, ground) surface smear, carry out disinfection;
4. with needle-threader, degreasing cotton thread is penetrated syringe needle, by pin through plastic flexible pipe, pull out from middle osculum, action wants slow, prevents degreasing cotton thread from skidding off;
5. pin is passed plant (pin instills one or two deionized water in pinprick porch immediately through after plant, prevents from marking overlong time, plant injury), in the middle of plastic tube, the osculum other end enters, and pulls out gently from exit end;
6. with thin copper wire two parts of flexible pipe closed up and be fixed on labeled plant, reducing the space between plant and flexible pipe as much as possible, reducing rising loss;
7. insert the two ends of bottom hose with a little U-shaped hard wire, drive flexible pipe through the gas chromatography bottle cap of pre-opening, then take off bottle, take out U-shaped hard wire, cotton thread pin is chosen, bottle of then screwing on.(cotton thread is also unsuitable oversize, but end must arrive bottle bottom);
8. be that the syringe of 1mL adds index liquid in bottle with volume, add 1mL ~ 2mL deionized water again after having absorbed, the process do not marked adds the deionized water of same volume;
9. after solution is completely absorbed, by labelling apparatus, slowly take off from plant, reduce the injury to plant as far as possible;
10. after having marked, on plant ground watch glasses, the silk screen in 1mm aperture, collected fallen leaves every several days, prevented the isotope in falling 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. aboveground vegetation part flush with ground is cut, merotomize by the Different Organs such as cauline leaf of plant, load in paper bag, carry out mark, at 60 DEG C, dry 72h, weigh, use bowl mill grind away, to be measured;
2. the soil in earth pillar is taken out, be poured on one piece of clean Polypropylence Sheet, and scrape with the soil of little scoop by earth pillar inwall, get all soil as much as possible;
3. smashed to pieces by soil, whole 2mm that crosses sieves, and obtains axial root system, cleans, dry 72h, weigh, grind at 60 DEG C with distilled water, to be measured.
4. weigh the quality of whole wet soil, soil is fully mixed and gets about 100g, dry and measure water cut;
5. about 100g wet soil is separately got, sieved and washed by wet screening 200 mesh sieve, obtain the residue radicula in soil, radicula is cleaned and dry 72h at 60 DEG C, weigh (not for isotope assay, for estimating the amount of residual radicula in soil, thus comprehensively calculate root biomass in soil).
6. get sub-fraction soil sample, at 60 DEG C, dry 72h, bowl mill grinds, to be measured;
C. measure:
Mass spectrometer is used to measure in Plants and Soils sample
13c with
15the abundance of N, uses the total carbon of carbon blood urea/nitrogen analyzer working sample, full nitrogen.
(4) calculate:
The method proposed according to JanzenandBruisma (1989) calculates.
When adding tracer element, the SI standard unit atomic percent usually used surpasses atom%excess (atompercentexcess) and represents isotopic relative content.Atomic percent is super to be referred in certain test specimen, the difference of the abundance of stable nuclide and its natural abundance.The computing formula that nitrogen and carbon isotope atomic percent surpass is as follows:
15atom N percentage surpasses (atom%
15nexcess) in=sample
15abundance-the natural abundance (1) of N
13c atomic percent surpasses (atom%
13cexcess) in=sample
13abundance-the natural abundance (2) of C
In actual computation process, plant part or soil
15n (
13c) atomic percent super usual 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 NdfR (NitrogenderivedfromRhizodeposition) and CdfR (CarbonderivedfromRhizodeposition) respectively, 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 is respectively the content of full nitrogen and total carbon in soil.
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 estimate plant rhizodeposition nitrogen more accurately than only planting a strain.Crucially must plant unmarked contrast, be used as the natural abundance that atomic percent surpasses calculating, and should not use in air
15natural abundance (the 0.3663atom% of N
15n).
Beneficial effect of the present invention is:
The inventive method can carry out labeled in situ under soil cultivation condition, does not need to upset the space distribution of root system, keeps contacting of soil and root, and can with containing
13the glucose of C and
15the urea mixed solution of N carries out carbon nitrogen double-tagging.Compared with additive method, application such an approach achieves vegetable material marked more uniformly 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. experimental field overview:
Test and carried out at academy of agricultural sciences of Baicheng City in Jilin Province rail mounted net canopy (45 ° of 37'N, 122 ° of 48'E) in 2012.Local climate belongs to the continental monsoon climate in temperate zone, average annual sunshine time 2919.4h, average annual temperature 4.9 DEG C, frostless season 157d, average annual precipitation 407.9mm.Potted plant soil takes from academy of agricultural sciences experimental field, and the organic 12.4g/kg of topsoil soils, full nitrogen 1.17g/kg, alkali-hydrolyzable nitrogen 88.6mg/kg, available phosphorus 12.3mg/kg, effective potassium 71.8mg/kg, pH are 6.86.Preceding crop is white swallow No. 2 oats.Weather data is provided by Baicheng City weather bureau.
2. earth pillar prepares to process with test:
Height is used to be 50cm, internal diameter is that the bucket of 37cm is as Potted-plant container, get topsoil soil in above-mentioned plot, air-dry, crossing aperture is 3mm sieve, and every barreled enters 20kg, the soil watered to whole bucket all soaks, after showing native water cut and dropping to suitable humidity, sow, crop moisture content in the time of infertility remains on about 40% of maximum water holding capacity (quality).
Supply examination soybean (GlycinemaxL) to be No. 10, white agriculture, academy of agricultural sciences of Baicheng City in Jilin Province provides; Sow after vernalization, 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, pass through
13c with
15n stem stalk is fed double-tagging isotope tracer technique, the total amount of research rhizodeposition carbon, nitrogen and distribution transfer case.
3. labeling method:
Within after soybean planting the 5th week, bring into use original position cotton core feeding method to marking, a Zhou Houzai once marks.Labeling method: the hole of boring a diameter 0.25mm in about three centimeters in plant distance ground, with a cotton thread through this eye, connects plants stems and a bottle being filled with label solution.The plastic tube that the outer surface cover one of cotton thread is soft, to prevent the loss of label solution.Fix cotton thread with a thin copper wire, do not misplace between plastic tube and plant.Label solution is use quality volume fraction 4% (g/mL, refers to the quality volume fraction in mixed solution, lower same)
13glucose solution and the quality volume fraction of C mark (atomic percent surpasses 99.9%) are 0.2% (g/mL's)
15the mixed aqueous solution of the urea liquid of N mark (atomic percent surpasses 99.9%).Mark 2 times, each consumption is 1mL.Used tool is at 121 DEG C of steam sterilizing 20min.After solution is all absorbed, then add 1mL ~ 2mL deionized water, be all absorbed by plants to make solution residual on cotton core.At later stages, in soil upper cover, aperture is the silk screen of 1mm, and periodic collection is fallen leaves, and prevents the isotope in falling leaves from affecting soil.
4. sampling and assay method:
Along earth's surface, plant above ground portion is cut during sampling.Soybean is divided into stem, leaf, pod, seed by organ, is dug out by earth pillar, and all soil excessively aperture is the sieve of 2mm and collects all visible root systems in soil layer by hand, 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 at water and wash wet screening, pick fine root with tweezers, as supplementing the manual root system that can not all sort out; In the soil having crossed 2mm sieve, finally get a part cross 1mm sieve, air-dry weigh native as rhizosphere.Weigh after all plant samples and soil sample 60 DEG C of low temperature dryings at least 72h, sample uses in mass spectrometer working sample after pulverizing with bowl mill
13c with
15the abundance of N, uses carbon blood urea/nitrogen analyzer working sample organic carbon, total nitrogen content.
5. computing formula
When adding tracer element, the SI standard unit usually used is that atomic percent surpasses a%e (atompercentexcess).
Abundance-the natural abundance of stable nuclide in super (the atom%excess)=test specimen of atomic percent
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 NdfR (NitrogenderivedfromRhizodeposition) and CdfR (CarbonderivedfromRhizodeposition) respectively, 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 is respectively the content of full nitrogen and total carbon in soil.
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 between 0.44atom% and 1.21atom%, isotopic abundance relatively uniform (table 1).
15the N recovery reaches 85.1%, far above other labeling method.Reclaim
15n concentrates in seed and beanpod, meets the general Distribution dynamics of nitrogen.In sum, this kind of labeling method is soybean
15application comparison success in N isotope labeling.
Table 1 soybean plant strain different parts
15atom N percentage is super, the recovery and distributed data table
Plant uses stem stalk double label method once to mark weekly (midfeather 1 week), and totally twice, mark and start 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:
Shown by table 2 result, in soybean, plant haulm
13c abundance is the highest, follows by blade, foot end, beanpod and seed, plant
13c abundance between 0.04atom% and 0.41atom%, isotopic abundance relatively uniform (table 2).
13the C recovery reaches 70.8%, far above other labeling method, reclaims
13c concentrates in stem stalk.In sum, this kind of labeling method is soybean
13application comparison success in C isotope labeling.
Table 2 soybean plant strain different parts
13c atomic percent is super, the recovery and distributed data table
Plant uses stem stalk double label method once to mark weekly (midfeather 1 week), and totally twice, mark and start 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. experimental field overview:
With experimental example 1.
2. earth pillar prepares to process with test:
Supply examination naked oats (AvenanudaL) to be white swallow No. 2, academy of agricultural sciences of 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 marking, and a Zhou Houzai once marks.Labeling method is with experimental example 1.
4. sampling and assay method:
Along earth's surface, plant above ground portion is cut during sampling.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 between 0.55atom% and 2.03atom%, isotopic abundance relatively uniform (table 3).
15the N recovery reaches 83.3%, far above other labeling method.Reclaim
15n concentrates in seed, meets the general Distribution dynamics of nitrogen.In sum, this kind of labeling method is oat
15application comparison success in N isotope labeling.
Table 3 oat plant different parts
15atom N percentage is super, the recovery and distributed data table
Plant uses stem stalk double label method once to mark weekly (midfeather 1 week), and totally twice, mark and start from after planting the 5th week (July 4).In table, data are mean value ± standard error (n=6).
The 6.2 mark carbon recovery:
Shown by table 2 result, in soybean, plant haulm
13c abundance is the highest, follows by seed, foot end and blade, plant
13c abundance between 0.09atom% and 0.20atom%, isotopic abundance relatively uniform (table 4).
13the C recovery reaches 50.9%, slightly higher or close than other labeling method recovery, reclaims
13c concentrates in stem stalk.In sum, this kind of labeling method is oat
13application comparison success in C isotope labeling.
Table 4 oat plant different parts
13c atomic percent is super, the recovery and distributed data table
Plant uses stem stalk double label method once to mark weekly (midfeather 1 week), and totally twice, mark and start 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. experimental field overview:
With experimental example 1.
2. earth pillar prepares to process with test:
Supplying examination naked oats (AvenanudaL.) to be white swallow No. 2 (Baiyan2), is that being academy of agricultural sciences of Baicheng City in Jilin Province provides in vain green No. 11 (Bailv11) for examination mung bean (Vignaradiate); On June 29 2011 mung bean (sowing after vernalization) sowing time, the oat sowing time is July 17; Research method is under the soil using column simulation land for growing field crops and root growth environment, passes through
13c with
15n stem stalk is fed double-tagging isotope tracer technique, the total amount of research rhizodeposition carbon, nitrogen and distribution transfer case.
By 24 long be 55cm, diameter is that the pvc pipe of 20cm is imbedded in the dark pond of about 40cm, each earth pillar bottom surface pad diameter is about the plastic pallet of 25cm, load 19kg and cross the dry ground that aperture is 3mm sieve, the soil watered to whole pvc pipe all soaks, after showing native water cut and dropping to suitable humidity, sow.White swallow No. 2 is planted as protection row around pond.
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 process mung bean all marks, and oat is unmarked.CK1, CK2 are unmarked in contrast, and for measuring mung bean, oat natural abundance, 4 repetitions are established in test.Nonoculture oat every post 4 cave, nonoculture mung bean every post 2 cave, intercropping every post 2 cave oat, 2 cave mung beans.4, every cave seed during sowing, after emerging, thinning is to every cave 1 strain.
Process setting data table tested by table 5
3. labeling method:
During mung bean branching stage, with original position cotton core feeding method, mung bean is marked.Labeling method is with experimental example 1.
4. sampling and assay method:
Along earth's surface, plant above ground portion is cut during sampling.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 nitrogen Bulk elemental recovery is higher, loses less, and loss cause may be solution vapour loss, maturity stage fallen leaves etc.
During Mung Bean Blooming fruiting period, each position intercropping overall recovery is all higher than nonoculture (blade intercropping comparatively nonoculture slightly declines), 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
15atom N percentage is super, the recovery and distributed data table
In table, alphabetical implication is as follows: S
p: mung bean nonoculture, fruiting period of blooming, samples; S
m: mung bean and oat intercropping, Mung Bean Blooming fruiting period, samples; I
p: mung bean nonoculture, the maturity stage samples; I
m: mung bean and oat intercropping, the mung bean maturity stage samples.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%, and during intercropping, the abundance of oat aerial part is minimum is 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%, and during intercropping, the abundance of oat aerial part is minimum is 0.001atom%, and 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 existed in soil utilize the soil organism as carbon source, and carbon is consumed, and nitrogen element to be only broken down into gaseous state that loss just can occur is relevant depositing to after in soil.
The nonoculture of table 7 mung bean, mung bean and oat Intercropping System plant different parts
13c atomic percent is super, the recovery and distributed data table
In table, alphabetical implication is as follows: S
p: mung bean nonoculture, fruiting period of blooming, samples; S
m: mung bean and oat intercropping, Mung Bean Blooming fruiting period, samples; I
p: mung bean nonoculture, the maturity stage samples; I
m: mung bean and oat intercropping, the mung bean maturity stage samples.In table, data are mean value ± standard error (n=4).
Test findings shows, blooms in fruiting period, 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%.
Mung bean each position overall recovery during fruiting period of blooming, no matter separate room is done, in beanstalk
13the recovery of C element is the highest, is respectively 60.91%, 55.27%; During the mung bean maturity stage, each position intercropping overall recovery is all higher than nonoculture (except root system), and no matter separate room is done, in beanstalk
13the recovery of C element is all the highest, is respectively 45.38%, 48.47%;
The mung bean maturity stage, overall recovery was compared with fruiting period of blooming, and intercopping declines all to some extent, and beanstalk, beans leaf decline the most obvious, and beanpod, Mai Gen, wheat cauline leaf, overall recovery all raises in soil.
Comprehensive above three experimental example, we are known,
15n-
13c stem stalk double-tagging tracer technique successfully can 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 plant according to formula; In addition, adopting said method can mung bean in pass flag mung bean and oat Intercropping System, and tracks the transfer of isotope to intercropping oat, goes out nitrogen and carbon transfer amount by formula to calculating.
Claims (6)
1. an isotope stem stalk double-tagging tracing method, is characterized in that, the method step is as follows:
(1) preparation of material;
(2) mark;
(3) sampling and mensuration;
(4) calculate;
The material prepared 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, volume fraction is alcohol water blend, cotton balls, tweezers, watering can, the U-shaped iron wire of 75%;
The method carrying out marking in described step (2) is as follows: mark in the latter stage of nourishing and growing of plant, in plant position to be marked boring, with degreasing cotton thread through this hole, is inserted at the two ends of degreasing cotton thread and is equipped with in the reagent bottle of label solution; Described degreasing cotton thread overcoat has plastic tube, to prevent the loss of label solution; Fix degreasing cotton thread with copper wire, do not misplace between plastic tube and plant, and fixation, prevent gap from causing too greatly solution transpiration loss; To be the abundance prepared be described label solution 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, is all absorbed by plants to make solution residual on degreasing cotton thread; After whole labeling process completes, the material of mark and device are taken off from plant, Collection and conservation.
2. a kind of isotope stem stalk double-tagging tracing method according to claim 1, is characterized in that, described in the concrete steps carrying out marking as follows:
1. the barrier around plant is removed, prevent from making troubles in operating process, and pollute;
2. with the article that the alcohol water blend that volume fraction is 75% sprays hand, tweezers and can touch, carry out disinfection, plant of protecting from infection;
3. clip cotton balls with tweezers and dip the alcohol water blend that volume fraction is 75%, smear at plant portion faces to be marked, carry out disinfection;
4. with needle-threader, degreasing cotton thread is penetrated syringe needle, by pin through plastic tube, from the pull-out of plastic tube centre exit, prevent degreasing cotton thread from skidding off;
5. pin is passed plant, enter from the other end of plastic tube centre exit, from one end outlet pull-out of the non-threading of plastic tube;
6. with copper wire two parts of plastic tube closed up and be fixed on labeled plant;
7. drive flexible pipe through the gas chromatography bottle cap of pre-opening with U-shaped iron wire, then take off bottle, take off U-shaped iron wire, bottle of then screwing on, described degreasing cotton thread end will arrive gas chromatography bottle bottom;
8. be that the syringe of 1mL adds label solution in bottle with volume, add 1mL ~ 2mL deionized water again after having absorbed, the space management do not marked adds the deionized water of volume same with label solution;
9., after label solution is completely absorbed, the material of mark and device are taken off from plant;
10. after having marked, on marked plant ground watch glasses, aperture is the silk screen of 1mm, and periodic collection is fallen leaves, and prevents the isotope in falling leaves from affecting soil.
3. 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.
4. a kind of isotope stem stalk double-tagging tracing method according to claim 3, it is characterized in that, the material prepared in described step a is as follows: valve bag, marking pen, tweezers, scoop, sieve, sample grinding machine, paper bag, Polypropylence Sheet, balance, paper bag.
5. a kind of isotope stem stalk double-tagging tracing method according to claim 3, it is characterized in that, in described step b, sampling procedure is as follows:
1. marked aboveground vegetation part flush with ground is cut, classify by the Different Organs of plant, load in paper bag, at 60 DEG C, dry 72h respectively, weigh, use bowl mill grind away, to be measured;
2. soil used for marked plant is taken out;
3. smashed to pieces by soil, whole aperture is excessively the sieve of 2mm, obtains axial root system, cleans, dry 72h, weigh, grind through gained axial root system at 60 DEG C with distilled water, to be measured;
4. weigh the quality of whole soil, after fully being mixed by soil, get 100g, dry and measure water cut;
5. separately get 100g soil, sieved and washed by wet screening 200 mesh sieve, obtain the residue radicula in soil, gained radicula cleaned and dry 72h at 60 DEG C, weighing;
6. get appropriate soil sample, at 60 DEG C, dry 72h, bowl mill grinds, to be measured.
6. a kind of isotope stem stalk double-tagging tracing method according to claim 3, it is characterized in that, in described step c, assay method is as follows: use mass spectrometer to measure respectively in marked plant sample and pedotheque
13c with
15the abundance of N, uses total carbon respectively in the plant sample that marks of working sample and pedotheque of carbon blood urea/nitrogen analyzer and total nitrogen content.
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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 |
| CN107047265B (en) * | 2016-12-31 | 2022-06-21 | 中国农业科学院农业资源与农业区划研究所 | Circulating system for culturing high-abundance isotope carbon and nitrogen double-labeled plant sample |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101926267A (en) * | 2010-08-09 | 2010-12-29 | 中国科学院地球化学研究所 | Method for measuring bicarbonate ion utilizing capability of plant |
| CN102511362A (en) * | 2011-10-27 | 2012-06-27 | 中国科学院地球化学研究所 | Method by utilizing double markers to acquire share of inorganic carbon source utilized by plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101926267A (en) * | 2010-08-09 | 2010-12-29 | 中国科学院地球化学研究所 | Method for measuring bicarbonate ion utilizing capability of plant |
| CN102511362A (en) * | 2011-10-27 | 2012-06-27 | 中国科学院地球化学研究所 | Method by utilizing double markers to acquire share of inorganic carbon source utilized by plants |
Non-Patent Citations (2)
| Title |
|---|
| Evaluation of the wick method for in situ C－13 and N-15 labelling of annual plants using sugar-urea mixtures;Florian Wichern;《Plant Soil 》;20101231;105－115 * |
| 荒漠草地植物稳定性氮同位素对水分变化的响应;禹朴家;《干旱区研究》;20120331;第29卷(第2期);347－351 * |
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