CN107449858A - A kind of method for determining plant ammonium nitrogen utilization ratio - Google Patents
A kind of method for determining plant ammonium nitrogen utilization ratio Download PDFInfo
- Publication number
- CN107449858A CN107449858A CN201710397367.5A CN201710397367A CN107449858A CN 107449858 A CN107449858 A CN 107449858A CN 201710397367 A CN201710397367 A CN 201710397367A CN 107449858 A CN107449858 A CN 107449858A
- Authority
- CN
- China
- Prior art keywords
- nitrogen
- culture
- tissue
- nitrate
- ammonium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/0098—Plants or trees
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention discloses a kind of method for determining plant ammonium nitrogen utilization ratio.Clonal tissue culture seedling is cultivated in two kinds of different nitrate of stable nitrogen isotopes value as in the culture medium of only nitrogen source respectively；After tissue-cultured seedling after culture in 5 weeks, the stable nitrogen isotopes value of tissue culture seedling leaf and the carbon nitrogen mol ratio of blade under above two nitrate culture are determined；Equally, clonal tissue culture seedling is separately taken, is cultivated in ammonium salt respectively on the culture medium of the mixed nitrogen of different from two kinds of stable nitrogen isotopes values nitrate compositions；Determine tissue culture seedling leaf stable nitrogen isotopes value and the carbon nitrogen mol ratio of blade under two kinds of mixed nitrogen cultures；The share that tissue-cultured seedling utilizes nitrate is calculated, calculates the carbon nitrogen mol ratio that under mixed nitrogen culture tissue-cultured seedling utilizes ammonium nitrogen accordingly, also as tissue-cultured seedling ammonium nitrogen utilization ratio or treats measuring plants ammonium nitrogen utilization ratio.
Description
Technical field
The present invention relates to a kind of method for determining plant ammonium nitrogen utilization ratio, belong to crop production technology field.
Technical background
Nitrogen is necessary nutrient in plant growth and development process, is to form protein, nucleic acid, enzyme, chlorophyll etc.
Important composition composition.Therefore, soil nitrogen concentrations are considered as the important limiting factor of soil productivity.Plant inhales from soil
It is mainly nitrate nitrogen and ammonium nitrogen to receive the nitrogen utilized.And plant is when absorbing nitrate nitrogen, it is necessary to ability quilt after nitrate-nitrogen reduction
Plant assimilating, generally often reducing 1 molecule nitrate nitrogen needs to spend 15 molecule ATP, and ammonium nitrogen can be directly plant assimilating, together
Changing 1 molecule ammonium nitrogen only needs 5 molecule ATP.
The nitrogen assimilation and carbon assimilation of plant are synchronously carried out in plant, and the energy of nitrogen assimilation consumption comes from plant
Photosynthesis.Because plant assimilating ammonium nitrogen ratio assimilation nitrate nitrogen more saves energy, therefore, the amount of plant assimilating ammonium nitrogen is more
When will more save energy, so as to improve the assimilatory efficiency of carbon.However, when ammonium concentration is too high, the invalid of ammonium just occurs in plant
Circulation, will now occur the waste of energy.Therefore, when carrying out effective nitrogen management of plant, it is necessary in view of plant profit
With the efficiency of ammonium nitrogen.
Plant can be characterized as the organic carbon that plant often obtained using 1 mole of ammonium nitrogen using the efficiency of ammonium nitrogen and rub
That number.However, plant is typically to absorb nitrate nitrogen and ammonium nitrogen simultaneously, therefore, from ammonium nitrogen in plant leaf blade nitrogen content
Share is difficult to determine.Therefore, it is difficult to the utilization ratio of plant ammonium nitrogen is determined by conventional method.
The nitrogen content of plant leaf blade is the result of plant assimilating nitrate nitrogen and ammonium nitrogen, and blade carbon content is the knot of carbon assimilation
Fruit, because the carbon nitrogen assimilation of plant is synchronously carried out.Therefore, the nitrogen content of blade and the ratio of carbon content, namely assimilation are single
The carbon of position molal quantity needs the molal quantity of nitrogen, because Leaf nitrogen concentration is to come from nitrate nitrogen and ammonium nitrogen.Therefore, it is known that plant exists
Leaf N carbon mol ratio under Leaf N carbon mol ratio and nitrate nitrogen and ammonium nitrogen co-incubation under nitrate nitrogen culture, knows simultaneously
Road plant utilizes the share of nitrate nitrogen and ammonium nitrogen under nitrate nitrogen and ammonium nitrogen culture, it is possible to calculates plant and utilizes ammonium state
The nitrogen carbon mol ratio of nitrogen, plant utilize the carbon nitrogen mole that the inverse of the nitrogen carbon mol ratio of ammonium nitrogen is that plant utilizes ammonium nitrogen
Than that is, plant utilizes the carbon assimilation efficiency of ammonium nitrogen, and plant may be characterized as plant using the carbon assimilation efficiency of ammonium nitrogen and utilize
The efficiency of ammonium nitrogen.However, the nitrogen assimilation of the plant of plantation in the wild or water planting plantation, nitrate nitrogen and ammonium nitrogen had both occurred in leaf
Piece occurs in root again, therefore, it is difficult to judge the share from nitrate nitrogen and ammonium nitrogen in Leaf nitrogen concentration, cannot also calculate
Go out the carbon assimilation efficiency that plant utilizes ammonium nitrogen.
The content of the invention
The technical problem to be solved in the present invention is：A kind of method for determining plant ammonium nitrogen utilization ratio is provided, overcome existing
There is technology to draw average plant nitrogen utilization ratio and be difficult to differentiate between art wall of the plant using the efficiency of which kind of nitrogen source
Build.
Technical scheme：, can be by adjusting the basic element of cell division and life in the multiplicative stage of Plant Tissue Breeding
The concentration ratio of long element does not produce root in the whole multiplicative stage to ensure tissue-cultured seedling, and the assimilation of such nitrate nitrogen and ammonium nitrogen is just only
Occur in blade.Therefore, determine tissue-cultured seedling nitrate nitrogen as the blade carbon nitrogen content under independent nitrogen source culture (leaf dry weight
Mass percent), so that it may calculate nitrogen carbon mol ratio of the tissue-cultured seedling in nitrate nitrogen culture lower blade；Tissue-cultured seedling is determined in nitrate nitrogen
With the carbon nitrogen content (mass percent of leaf dry weight) of ammonium nitrogen culture lower blade, so that it may calculate nitrate nitrogen and ammonium nitrogen training
Support the nitrogen carbon mol ratio of lower tissue culture seedling leaf.
Stable nitrogen isotopes technology has been widely used the fields such as ecology at present, and stable nitrogen isotopes are used as nitrogen source
Indicator studies the nitrogen cycle in biosphere.Therefore nitre can be utilized to calculate tissue-cultured seedling by two-way stable nitrogen isotope techniques
The share of state nitrogen and ammonium nitrogen, then according to tissue-cultured seedling nitrate nitrogen culture lower blade nitrogen carbon mol ratio, in nitrate nitrogen and ammonium
Leaf N carbon mol ratio under state nitrogen co-incubation, it is possible to the nitrogen carbon mol ratio that tissue-cultured seedling utilizes ammonium nitrogen is calculated, from
And obtaining the carbon assimilation efficiency that tissue-cultured seedling utilizes ammonium nitrogen, i.e. tissue-cultured seedling utilizes the efficiency of ammonium nitrogen.According to tissue-cultured seedling in difference
Ammonium nitrogen utilization ratio under the conditions of nitrogen, you can effective nitrogen management is carried out to plant.
A kind of method for determining plant ammonium nitrogen utilization ratio, it is comprised the steps of：
First, the clonal tissue culture seedling of same gene type is obtained by plant tissue culture technique, by adjusting culture medium
Hormone ratio tissue-cultured seedling is in the multiplicative stage, form the clonal tissue culture seedling of unrooted；
Second, by isotope mass spectrometer determine for configure Plant Tissue Breeding culture medium in nitrate stabilization
Nitrogen isotope ratio, two kinds of nitrate that stable nitrogen isotopes value difference value is more than 10 ‰ are filtered out, its stable nitrogen isotopes value point
Wei not δ15N01And δ15N02；Respectively culture medium is configured using both nitrate as only nitrogen source；
3rd, above-mentioned clonal tissue culture seedling is cultivated respectively and made in two kinds of different nitrate of aforementioned stable nitrogen isotope value
For in the culture medium of only nitrogen source；
4th, after tissue-cultured seedling after culture in 5 weeks, culture medium of the above two nitrate as only nitrogen source is determined respectively
The stable nitrogen isotopes value of tissue culture seedling leaf under culture, its blade stable nitrogen isotopes value are designated as δ respectively15NN1And δ15NN2；Together
When determine carbon nitrogen content of any of which nitrate as the tissue culture seedling leaf under the medium culture of only nitrogen source, calculating
Its nitrogen carbon mol ratio, is designated as n；
5th, the culture medium of Plant Tissue Breeding is configured from ammonium salt, ensures its stable nitrogen isotopes value δ15NAWith δ15N01
Or δ15N02Difference be all higher than 10 ‰；Then by ammonium salt two kind nitrate groups different from aforementioned stable nitrogen isotope value respectively
Into mixed nitrogen, two kinds of different mixed nitrogen culture mediums are configured；
6th, equally, clonal tissue culture seedling is separately taken, it is cultivated respectively in the different mixed nitrogen culture medium of above two
On；
7th, equally, it is steady to determine tissue culture seedling leaf under above two mixed nitrogen culture after culture in 5 weeks for tissue-cultured seedling
Determine nitrogen isotope value, ammonium salt and stable nitrogen isotopes value are δ15N01Nitrate composition mixed nitrogen culture under tissue-cultured seedling
Blade stable nitrogen isotopes value is designated as δ15NN1mix, ammonium salt and stable nitrogen isotopes value are δ15N02Nitrate composition mixing nitrogen
Tissue culture seedling leaf stable nitrogen isotopes value under the culture of source is designated as δ15NN2mix, while determine any of which mixed nitrogen culture
The carbon nitrogen content of lower tissue culture seedling leaf, its nitrogen carbon mol ratio is calculated, is designated as m；
8th, by δ15NN1、δ15NN2、δ15NN1mixAnd δ15NN2mixSubstitute into equation：It can calculate
Go out the share F that tissue-cultured seedling utilizes nitratenit, namely treat that measuring plants utilize the share of nitrate；
9th, according to FnitCalculate the equipment usage F of the tissue-cultured seedling ammonium nitrogen under mixed nitrogen culturea, Fa=1-Fnit；
Tenth, according to Fnit, m and n calculate the carbon nitrogen mol ratio P that the tissue-cultured seedling under mixed nitrogen culture utilizes ammonium nitrogen, group
The carbon nitrogen mol ratio P that seedling utilizes ammonium nitrogen is trained, calculation formula is：Tissue-cultured seedling utilizes the carbon nitrogen mol ratio of ammonium nitrogen
P is also tissue-cultured seedling ammonium nitrogen utilization ratio, also as plant ammonium nitrogen utilization ratio AUE.
Advantage of the present invention
1) present invention can determine plant ammonium nitrogen utilization ratio measure, overcome prior art to draw average plant
Thing nitrogen utilization efficiency and be difficult to differentiate between plant using which kind of nitrogen source efficiency technology barriers.
2) present invention marks culture technique by two-way stable nitrogen isotope, in the same of measure plant ammonium nitrogen utilization ratio
When also determine plant nitrate nitrogen and the equipment usage by state nitrogen, according to plant ammonium nitrogen utilization ratio and ammonium salt equipment usage,
The ammonium nitrogen assimilation efficiency under different condition can be evaluated, so as to effectively manage the nitrogenous fertilizer of plant use, avoid nitrogen source waste and
Nitrogenous fertilizer excessively applies the harm to environment.
3) this method provides technical support for the Screening of Media of high-quality tissue-cultured seedling, that is, selects tissue-cultured seedling ammonium nitrogen to utilize
The culture medium of efficiency high.
4) this method carries out culture experiment using the clonal tissue culture seedling of same gene type, and the isotope error of measure is small,
Therefore the result of measure is more reliable.
5) the step of this method uses is few, calculates simple.
Inventive principle
Stable nitrogen isotopes technology has been studied the nitrogen cycle in biosphere with the indicator of nitrogen source extensively at present.It is natural
Two kinds of stable nitrogen isotopes of nitrogen are in boundary14N and15N, stable nitrogen isotopes value generally use δ15N (‰) expressions, nature
Middle δ15N change turns to -10 ‰~+20 ‰.The Leaf N isotope value for cultivating the plant under different nitrogen sources can react well
The feature of the stable nitrogen isotopes value of nitrogen source.Therefore, can quantitative identification by mass balance principle and isotopic dating method
It is different inorganic nitrogen-sourced in plant.
Tissue-cultured seedling can all have certain nitrogen isotope fractionation when assimilating nitrate nitrogen and ammonium nitrogen.Therefore, in mixing nitrogen
The stable nitrogen isotopes value for the tissue culture seedling leaf cultivated under the conditions of source is that nitrogen isotope fractionation occurs for assimilation nitrate nitrogen and ammonium nitrogen
The result of stable nitrogen isotopes value mixing afterwards.Therefore, the equipment usage of nitrate nitrogen and ammonium nitrogen is calculated, must accurately be known
The stable nitrogen isotopes value after nitrogen isotope fractionation occurs for tissue-cultured seedling assimilation nitrate nitrogen and ammonium nitrogen under road mixed nitrogen culture.By
When ammonium nitrogen is as independent nitrogen source, some tissue-cultured seedling are unable to normal growth, such as the tissue-cultured seedling of crucifer.Therefore,
From nitrate nitrogen of two kinds of stable nitrogen isotopes value difference values more than 10 ‰ tissue-cultured seedling is cultivated as independent nitrogen source simultaneously.Determine tissue culture
The seedling tissue culture seedling leaf stable nitrogen isotopes value under the different nitrate nitrogen culture of both stable nitrogen isotopes values respectively.So
Afterwards, by the ammonium nitrogen filtered out, the nitrate nitrogen different with two kinds of stable nitrogen isotopes values forms mixed nitrogen culture group simultaneously respectively
Seedling is trained, the difference of the stable nitrogen isotopes value of ammonium nitrogen and the stable nitrogen isotopes value of two kinds of nitrate nitrogens is all higher than 10 ‰.Respectively
Determine the stable nitrogen isotopes value of tissue culture seedling leaf under two kinds of mixed nitrogen cultures.Calculated finally by two end member mixed models
Go out the share that tissue-cultured seedling is utilized respectively nitrate nitrogen and ammonium nitrogen.
The isotopic dating method of two end members is expressed as：
δ15NN1mix=Fnitδ15NN1+Faδ15NA-T (1)
δ15NN2mix=Fnitδ15NN2+Faδ15NA-T (2)
Fa=1-Fnit (3)
Here δ15NN1And δ15NN2Respectively tissue-cultured seedling is more than 10 ‰ two kinds of nitrate in stable nitrogen isotopes value difference value
Blade stable nitrogen isotopes value under culture, δ15NA-TAssimilate ammonium salt for tissue-cultured seedling under mixed nitrogen culture and nitrogen isotope point occurs
Stable nitrogen isotopes value after evaporating.δ15NN1mixIt is that stable nitrogen isotopes value is δ15N01Nitrate and ammonium salt composition mixed nitrogen
The stable nitrogen isotopes value of tissue culture seedling leaf under culture.δ15NN2mixIt is that stable nitrogen isotopes value is δ15N02Nitrate and ammonium
The stable nitrogen isotopes value of tissue culture seedling leaf under salt composition mixed nitrogen culture.FnitPart is utilized for nitrate (nitrate nitrogen)
Volume, FaFor the equipment usage of ammonium salt (ammonium nitrogen).
Simultaneous equations (1) (2) and (3), solution obtains：
Know that tissue-cultured seedling, using after the share of nitrate nitrogen and ammonium nitrogen, will calculate tissue-cultured seedling utilization under mixed nitrogen culture
The carbon assimilation efficiency of ammonium nitrogen, the nitrogen carbon mol ratio of blade of the tissue-cultured seedling under mixed nitrogen culture must be calculated and in nitre state
Nitrogen carbon mol ratio of the nitrogen as the tissue culture seedling leaf under only nitrogen source culture.Any one nitrate is as only nitrogen source wherein
Medium culture under tissue-cultured seedling Leaf N carbon mol ratio be designated as n, tissue-cultured seedling under any one mixed nitrogen culture wherein
The nitrogen carbon mol ratio of blade is designated as m.The nitrogen carbon mol ratio of tissue culture seedling leaf can determine the nitrogen and carbon of blade by elemental analyser
Content (mass percent of leaf dry weight), then by being calculated.Under mixed nitrogen culture, the nitrogen carbon of tissue culture seedling leaf
Mol ratio is：
M=nFnit+y Fa (5)
Y is the nitrogen carbon mol ratio that tissue-cultured seedling utilizes ammonium nitrogen under mixed nitrogen.Under mixed nitrogen culture, tissue-cultured seedling utilizes
The carbon nitrogen mol ratio P of ammonium nitrogen is just y inverse, i.e. tissue-cultured seedling is using the carbon assimilation efficiency of ammonium nitrogen：
The carbon assimilation efficiency of ammonium nitrogen is utilized according to the tissue-cultured seedling being calculated, i.e., is also that tissue-cultured seedling ammonium nitrogen utilizes effect
Rate, also as plant ammonium nitrogen utilization ratio AUE.
Embodiment
Embodiments of the invention：
First step, the clonal tissue culture seedling of same gene type is obtained by plant tissue culture technique, trained by adjusting
Supporting the hormone ratio of base makes tissue-cultured seedling be in the multiplicative stage, forms the clonal tissue culture seedling of unrooted；
Second step：By isotope mass spectrometer determine for configure Plant Tissue Breeding culture medium in nitrate
Stable nitrogen isotopes ratio, filter out two kinds of nitrate that stable nitrogen isotopes value difference value is more than 10 ‰, its stable nitrogen isotopes
Value is respectively δ15N01And δ15N02；The culture medium of suitable concn is configured using both nitrate as only nitrogen source respectively；
Third step, above-mentioned clonal tissue culture seedling is cultivated in two kinds of different nitric acid of aforementioned stable nitrogen isotope value respectively
Salt is as in the culture medium of only nitrogen source；
Four steps, after tissue-cultured seedling after culture in 5 weeks, training of the above two nitrate as only nitrogen source is determined respectively
The stable nitrogen isotopes value of the tissue culture seedling leaf under base culture is supported, its blade stable nitrogen isotopes value is designated as δ respectively15NN1And δ15NN2；Simultaneously any of which nitrate is determined as the carbon nitrogen of the tissue culture seedling leaf under the medium culture of only nitrogen source to contain
Measure (mass percent of leaf dry weight), calculate its nitrogen carbon mol ratio, be designated as n；
5th step, the ammonium salt for configuring the culture medium of Plant Tissue Breeding to be suitably used for from stable nitrogen isotopes value,
Ensure its stable nitrogen isotopes value δ15NAWith δ15N01Or δ15N02Difference be all higher than 10 ‰；Then by ammonium salt respectively with it is above-mentioned steady
Determine the different two kinds of nitrate composition mixed nitrogen of nitrogen isotope value, configure two kinds of different mixed nitrogen culture mediums；
6th step, equally, separately takes clonal tissue culture seedling, it is cultivated respectively in the different mixed nitrogen training of above two
Support on base；
7th step, equally, tissue-cultured seedling determine tissue-cultured seedling leaf under above two mixed nitrogen culture after culture in 5 weeks
Piece stable nitrogen isotopes value, ammonium salt and stable nitrogen isotopes value are δ15N01Nitrate composition mixed nitrogen culture under group
Training seedling leaf stable nitrogen isotopes value is designated as δ15NN1mix, ammonium salt and stable nitrogen isotopes value are δ15N02Nitrate composition it is mixed
The tissue culture seedling leaf stable nitrogen isotopes value closed under nitrogen source culture is designated as δ15NN2mix, while determine any of which mixed nitrogen
The carbon nitrogen content (mass percent of leaf dry weight) of the lower tissue culture seedling leaf of culture, calculates its nitrogen carbon mol ratio, is designated as m；
8th step, by δ15NN1、δ15NN2、δ15NN1mixAnd δ15NN2mixSubstitute into equation： I.e.
The share F that tissue-cultured seedling utilizes nitrate can be calculatednit, namely treat that measuring plants utilize the share of nitrate；
9th step, according to FnitCalculate the equipment usage F of the tissue-cultured seedling ammonium nitrogen under mixed nitrogen culturea, Fa=1-
Fnit；
Tenth step, according to Fnit, m and n calculate the carbon nitrogen mol ratio that the tissue-cultured seedling under mixed nitrogen culture utilizes ammonium nitrogen
P, tissue-cultured seedling utilize the carbon nitrogen mol ratio P of ammonium nitrogen, and calculation formula is：Tissue-cultured seedling is rubbed using the carbon nitrogen of ammonium nitrogen
You are also tissue-cultured seedling ammonium nitrogen utilization ratio than P, also as plant ammonium nitrogen utilization ratio AUE.
Embodiment：The measure of Orychophragmus violaceus ammonium nitrogen utilization ratio
Culture materials：Clone Orychophragmus violaceus tissue-cultured seedling
Culture medium prescription is：MS+6-BA2.0mg/L+NAA0.1mg/L, 30g/L sucrose, agar：7.5g/L, pH value：
5.8, cultivate room temperature：25±2℃.Photoperiod：12h/d, intensity of illumination：50μmol·m-2·s-1, the stabilization of two kinds of nitrate
Nitrogen isotope value is respectively：δ15N01=22.67 ‰, δ15N02=8.08 ‰.The stable nitrogen isotopes value of ammonium salt is：δ15NA=-
2.64‰.δ is used respectively15N01And δ15N02Nitrate configuration concentration be 10mM, 20mM and 40mM culture medium, Orychophragmus violaceus tissue culture
After seedling cultivates 5 weeks in above-mentioned culture medium, the stable nitrogen isotopes value of its blade is determined respectively.Due to Orychophragmus violaceus tissue-cultured seedling with
δ15N01Nitrate configuration concentration be 10mM, 20mM and 40mM medium culture under blade stable nitrogen isotopes value difference it is different
Not significantly, and with δ15N02Nitrate configuration concentration be the stable nitrogen of blade under 10mM, 20mM and 40mM medium culture
Also difference is not notable for isotope value.Therefore, it is δ in stable nitrogen isotopes value15N01Nitrate culture under Orychophragmus violaceus tissue-cultured seedling
Blade stable nitrogen isotopes value δ15N1I.e. it is assumed that being the Orychophragmus violaceus tissue culture under 10mM, 20mM and 40mM are cultivated in nitrate concentration
The average value of the stable nitrogen isotopes value of seedling leaf, i.e. δ15N1=17.02 ‰ (n=9), similarly obtain δ15N2=5.71 ‰ (n=
9).It is δ in 10mM, 20mM and 40mM stable nitrogen isotopes value15N02Nitrate culture under Orychophragmus violaceus blade nitrogen carbon mol ratio
(n) it is respectively 0.053mol/mol, 0.050mol/mol and 0.063mol/mol.
It is constant to test 1 ammonium nitrogen concentration, the lower Orychophragmus violaceus tissue-cultured seedling ammonium nitrogen utilization ratio of nitrate processing
Implementation result is as follows：
It is respectively δ with stable nitrogen isotopes value by ammonium nitrogen according to the method for the present invention15N01And δ15N02Nitrate nitrogen group
Into mixed nitrogen, when configuring culture medium, the ammonium nitrogen concentration for ensureing culture medium is all 20mM, then sets the concentration of nitrate nitrogen
Respectively 5mM, 10mM, 20mM and 40mM.So the nitrogen concentration of culture medium is just 25mM, 30mM, 40mM and 60mM respectively.By nothing
Property system Orychophragmus violaceus tissue-cultured seedling cultivate respectively under above-mentioned four kinds of mixed nitrogen concentration gradients, after culture in 5 weeks, determine respectively
δ15N01The composition mixed nitrogen culture of nitrate nitrogen and ammonium nitrogen under Orychophragmus violaceus tissue culture seedling leaf stable nitrogen isotopes value δ15NN1mix
Be δ in stable nitrogen isotopes value15N02The composition mixed nitrogen culture of nitrate nitrogen and ammonium nitrogen under Orychophragmus violaceus tissue culture seedling leaf
Stable nitrogen isotopes value δ15NN2mix.It is δ to determine stable nitrogen isotopes value simultaneously15N02Nitrate nitrogen and ammonium nitrogen composition mix nitrogen
The carbon nitrogen content (mass percent of leaf dry weight) of Orychophragmus violaceus tissue culture seedling leaf under the culture of source, calculates its nitrogen carbon mol ratio,
M is designated as, the above results are as shown in table 1：
The stable nitrogen isotopes value and nitrogen carbon mol ratio of the lower Orychophragmus violaceus tissue culture seedling leaf of the nitrate nitrogen of table 1 processing
Note：Ammonium salt in culture medium is all 20mM.N=3.
As known from Table 1, in the case that ammonium nitrogen concentration is all 20mM in the medium, the concentration of nitrate nitrogen, Zhuge are increased
Dish tissue-cultured seedling is respectively in ammonium nitrogen and two kinds of stable nitrogen isotopes value differences away under larger nitrate nitrogen composition mixed nitrogen culture
The stable nitrogen isotopes value of blade all has maximum stable nitrogen isotope value all in gradual polarization under highest nitrate.
And the Leaf N carbon mol ratio of Orychophragmus violaceus tissue-cultured seedling is then fallen before as the increase of nitrate is presented, then tend to be constant
Trend.According to the δ of table 115NN1mixAnd δ15NN2mix, with reference to δ15N1And δ15N2, utilize equationAnd side
Journey Fa=1-FnitThe share F that the Orychophragmus violaceus tissue-cultured seedling under mixed nitrogen utilizes nitrate nitrogen is calculated in cannitWith utilize ammonium
The share F of state nitrogena, result of calculation is as shown in table 2：
Nitrate-N Use share (the F of the lower Orychophragmus violaceus tissue-cultured seedling of the nitrate nitrogen of table 2 processingnit) and ammonium nitrogen utilize part
Volume (Fa)
Note：Ammonium salt in culture medium is all 20mM.N=3.
As known from Table 2, in the case that ammonium salt concentration is all 20mM in the medium, the concentration of nitrate, Orychophragmus violaceus are increased
Tissue-cultured seedling using the share of ammonium nitrogen without significant change, but the equipment usage of ammonium nitrogen be all twice of Nitrate-N Use share with
On.Know that Orychophragmus violaceus tissue-cultured seedling, using after the share of nitrate nitrogen and ammonium nitrogen, exists under mixed nitrogen culture in conjunction with tissue-cultured seedling
Nitrate nitrogen is as Orychophragmus violaceus group under the nitrogen carbon mol ratio of Orychophragmus violaceus tissue culture seedling leaf under independent nitrogen source culture and mixed nitrogen culture
The nitrogen carbon mol ratio of seedling leaf is trained, passes through equation The utilization ratio of tissue-cultured seedling ammonium nitrogen can be tried to achieve, calculates knot
Fruit is as shown in table 3：
The lower Orychophragmus violaceus tissue-cultured seedling ammonium nitrogen utilization ratio (P) of the nitrate nitrogen of table 3 processing
Note：Ammonium salt in culture medium is all 20mM.N=3.
As known from Table 3, the utilization ratio of Orychophragmus violaceus tissue-cultured seedling ammonium nitrogen first gradually increases with the increase of nitrate concentration,
Then tend to constant.Therefore, when nitrate concentration is added to 20mM, tissue-cultured seedling ammonium nitrogen utilization ratio up to maximum, continues
Increase the concentration of nitrate to 40mM, do not increase tissue-cultured seedling ammonium nitrogen utilization ratio, increase the waste of nitrate on the contrary.
Test that 2 nitrate concentrations are constant, the nitrate and ammonium salt equipment usage of the lower Orychophragmus violaceus tissue-cultured seedling of ammonium salt concentration processing
Implementation result is as follows：
It is respectively δ with stable nitrogen isotopes value by ammonium salt according to the method for the present invention15N01And δ15N02Nitrate composition
Mixed nitrogen, when configuring culture medium, the nitrate concentration for ensureing culture medium is all 20mM, then sets the concentration difference of ammonium salt
For 0.5mM, 1mM, 2.5mM, 5mM, 10mM and 40mM.So the nitrogen concentration of culture medium be respectively just 20.5mM, 21mM,
22.5mM, 25mM, 30mM and 60mM.Clone Orychophragmus violaceus tissue-cultured seedling is cultivated in above-mentioned six kinds of mixed nitrogen concentration gradients respectively
Under, after culture in 5 weeks, Orychophragmus violaceus tissue-cultured seedling is determined respectively in δ15N01The composition mixed nitrogen culture of nitrate and ammonium salt under
Blade stable nitrogen isotopes value δ15NN1mixBe δ in stable nitrogen isotopes value15N02Nitrate and ammonium salt composition mixed nitrogen
Cultivate lower blade stable nitrogen isotopes value δ15NN2mix.It is δ to determine stable nitrogen isotopes value simultaneously15N02Nitrate nitrogen and ammonium nitrogen
Form the carbon nitrogen content (mass percent of leaf dry weight) of the Orychophragmus violaceus Orychophragmus violaceus tissue culture seedling leaf under mixed nitrogen culture, meter
Its nitrogen carbon mol ratio is calculated, is designated as m, the above results are as shown in table 4：
The stable nitrogen isotopes value and nitrogen carbon mol ratio of the lower Orychophragmus violaceus tissue culture seedling leaf of the ammonium salt of table 4 processing
Note：Nitrate in culture medium is all 20mM.N=3.
As known from Table 4, in the case that nitrate concentration is all 20mM in the medium, the concentration of ammonium salt, Orychophragmus violaceus are increased
Tissue-cultured seedling is respectively in ammonium salt and two kinds of stable nitrogen isotopes value differences away from the blade under larger nitrate composition mixed nitrogen culture
Stable nitrogen isotopes value is all being gradually reduced, and all has the stable nitrogen isotopes value of minimum under highest ammonium salt concentration.And Zhuge
The nitrogen carbon mol ratio of dish Orychophragmus violaceus then gradually increases with the increase of ammonium salt.Because the concentration of nitrate in culture medium is all
20mM, and Orychophragmus violaceus tissue-cultured seedling is δ in 20mM stable nitrogen isotopes value15N01Nitrate culture under the stable same position of nitrogen of blade
Plain value δ15N1=17.19 ‰ (n=3)；It is δ in 20mM stable nitrogen isotopes value15N02Nitrate culture under the stable nitrogen of blade
Isotope value δ15N2=5.81 ‰ (n=3).Therefore, according to the δ of table 415NN1mixAnd δ15NN2mix, with reference to δ15N1And δ15N2, utilize
Equation Can is calculated the Orychophragmus violaceus tissue-cultured seedling under mixed nitrogen and utilizes nitrate nitrogen and ammonium
The share of state nitrogen, result of calculation are as shown in table 5：
Nitrate-N Use share (the F of the lower Orychophragmus violaceus tissue-cultured seedling of the ammonium salt of table 5 processingnit) and ammonium nitrogen equipment usage (Fa)
Note：Nitrate in culture medium is all 20mM.N=3.
As known from Table 5, in the case where culture medium nitrate concentration is all 20mM, the concentration for increasing ammonium salt causes Orychophragmus violaceus
The equipment usage of tissue-cultured seedling ammonium salt gradually increases, and the equipment usage of nitrate then gradually reduces.The concentration of ammonium salt increases from 2.5mM
When being added to 5mM, there is obviously amplification in the ammonium salt equipment usage of Orychophragmus violaceus tissue-cultured seedling.Know that Orychophragmus violaceus tissue-cultured seedling is mixing
Under nitrogen source culture using the share of nitrate nitrogen and ammonium nitrogen after, in conjunction with tissue-cultured seedling in the case where nitrate nitrogen is as independent nitrogen source culture it is all
The nitrogen carbon mol ratio of Orychophragmus violaceus tissue culture seedling leaf, the side of passing through under the nitrogen carbon mol ratio of Pueraria lobota dish tissue culture seedling leaf and mixed nitrogen culture
JourneyThe utilization ratio of tissue-cultured seedling ammonium nitrogen can be tried to achieve, result of calculation is as shown in table 6：
The nitre state ammonium nitrogen utilization ratio (P) of the lower Orychophragmus violaceus tissue-cultured seedling of the ammonium salt of table 6 processing
Note：Nitrate in culture medium is all 20mM.N=3.
As known from Table 6, Orychophragmus violaceus tissue-cultured seedling ammonium nitrogen utilization ratio gradually increases with the increase of ammonium salt concentration, but most
Decline under high ammonium salt concentration.Therefore, it is not that the concentration of ammonium salt is higher under nitrate concentration is constant in the medium, group
It is higher to train seedling ammonium nitrogen utilization ratio.
In summary, when carrying out culture media nitrogen source optimization, it is necessary to consider tissue-cultured seedling ammonium nitrogen utilization ratio.Compare 2
Experiment, it has been found that under ammonium salt constant concentration, under the processing for increasing nitrate concentration, increase the concentration of nitrate to 20mM
When, Orychophragmus violaceus tissue-cultured seedling just obtains maximum ammonium nitrogen utilization ratio.And in the case where nitrate concentration is constant, increase ammonium salt concentration
Under processing, when increasing the concentration of ammonium salt to 10mM, Orychophragmus violaceus tissue-cultured seedling just obtains maximum ammonium nitrogen utilization ratio, and in highest
Under ammonium salt concentration, Orychophragmus violaceus tissue-cultured seedling ammonium nitrogen utilization ratio is relatively low.Therefore, when carrying out effective nitrogen management of plant,
The concentration of ammonium salt and the utilization ratio of plant ammonium nitrogen answer emphasis to consider.
Claims (6)
- A kind of 1. method for determining plant ammonium nitrogen utilization ratio, it is characterised in that：First, the clonal tissue culture seedling of same gene type is obtained by plant tissue culture technique, by adjusting swashing for culture medium Plain ratio makes tissue-cultured seedling be in the multiplicative stage, forms the clonal tissue culture seedling of unrooted；Second, by isotope mass spectrometer determine for configure Plant Tissue Breeding culture medium in nitrate stable nitrogen it is same Position element value, filters out two kinds of nitrate that stable nitrogen isotopes value difference value is more than 10 ‰, its stable nitrogen isotopes value is respectively δ15N01And δ15N02；Respectively culture medium is configured using both nitrate as only nitrogen source；3rd, above-mentioned clonal tissue culture seedling is cultivated in two kinds of different nitrate of aforementioned stable nitrogen isotope value as only respectively In the culture medium of one nitrogen source；4th, after tissue-cultured seedling after culture in 5 weeks, medium culture of the above two nitrate as only nitrogen source is determined respectively Under tissue culture seedling leaf stable nitrogen isotopes value, its blade stable nitrogen isotopes value is designated as δ respectively15NN1And δ15NN2；Survey simultaneously Determine carbon nitrogen content of any of which nitrate as the tissue culture seedling leaf under the medium culture of only nitrogen source, calculate its nitrogen Carbon mol ratio, is designated as n；5th, the culture medium of Plant Tissue Breeding is configured from ammonium salt, ensures its stable nitrogen isotopes value δ15NAWith δ15N01Or δ15N02Difference be all higher than 10 ‰；Then by ammonium salt respectively two kind nitrate different from aforementioned stable nitrogen isotope value form it is mixed Nitrogen source is closed, configures two kinds of different mixed nitrogen culture mediums；6th, equally, clonal tissue culture seedling is separately taken, it is cultivated respectively on the different mixed nitrogen culture medium of above two；7th, equally, tissue-cultured seedling determines the stable nitrogen of tissue culture seedling leaf under above two mixed nitrogen culture after culture in 5 weeks Isotope value, ammonium salt and stable nitrogen isotopes value are δ15N01Nitrate composition mixed nitrogen culture under tissue culture seedling leaf Stable nitrogen isotopes value is designated as δ15NN1mix, ammonium salt and stable nitrogen isotopes value are δ15N02Nitrate composition mixed nitrogen training Tissue culture seedling leaf stable nitrogen isotopes value under supporting is designated as δ15NN2mix, while determine any of which mixed nitrogen culture the following group The carbon nitrogen content of seedling leaf is trained, its nitrogen carbon mol ratio is calculated, is designated as m；8th, according to δ15NN1、δ15NN2、δ15NN1mixAnd δ15NN2mix, the share F that tissue-cultured seedling utilizes nitrate can be calculatednit, that is, treat Measuring plants utilize the share of nitrate；9th, according to FnitCalculate the equipment usage F of the tissue-cultured seedling ammonium nitrogen under mixed nitrogen culturea；Tenth, according to Fnit, m and n calculate the carbon nitrogen mol ratio P that the tissue-cultured seedling under mixed nitrogen culture utilizes ammonium nitrogen；Tissue-cultured seedling Using ammonium nitrogen carbon nitrogen mol ratio P also be tissue-cultured seedling ammonium nitrogen utilization ratio, as plant ammonium nitrogen utilization ratio AUE.
- A kind of 2. method for determining plant ammonium nitrogen utilization ratio according to claim 1, it is characterised in that：In the 4th step In rapid, side of any one nitrate as the tissue-cultured seedling Leaf N carbon mol ratio n under the medium culture of only nitrogen source is calculated Method is：Multiply any one nitrate as the nitrogen content of the tissue-cultured seedling under the medium culture of only nitrogen source with carbon content ratio Then it is any one nitrate as the tissue-cultured seedling Leaf N carbon mol ratio n under the medium culture of only nitrogen source with 0.857； Here nitrogen content and the mass percent that carbon content is leaf dry weight.
- A kind of 3. method for determining plant ammonium nitrogen utilization ratio according to claim 1, it is characterised in that：In the 7th step In rapid, the method for calculating tissue culture seedling leaf nitrogen carbon mol ratio m under mixed nitrogen culture is：Equally it is by mixed nitrogen culture the following group The nitrogen content and carbon content ratio for training seedling leaf are multiplied by 0.857, then are tissue culture seedling leaf nitrogen carbon mol ratio under mixed nitrogen culture m；Here nitrogen content and the mass percent that carbon content is leaf dry weight.
- A kind of 4. method for determining plant ammonium nitrogen utilization ratio according to claim 1, it is characterised in that：In the 8th step In rapid, the share F that tissue-cultured seedling utilizes nitrate is calculatednitMethod be：By δ15NN1、δ15NN2、δ15NN1mixAnd δ15NN2mixSubstitute into Equation：
- A kind of 5. method for determining plant ammonium nitrogen utilization ratio according to claim 1, it is characterised in that：In the 9th step In rapid, the equipment usage F of tissue-cultured seedling ammonium nitrogen under mixed nitrogen cultureaComputational methods be：Fa=1-Fnit。
- A kind of 6. method for determining plant ammonium nitrogen utilization ratio according to claim 1, it is characterised in that：In the tenth step In rapid, tissue-cultured seedling utilizes the carbon nitrogen mol ratio P of ammonium nitrogen, and calculation formula is：
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710397367.5A CN107449858B (en) | 2017-05-31 | 2017-05-31 | Method for measuring utilization efficiency of plant ammonium nitrogen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710397367.5A CN107449858B (en) | 2017-05-31 | 2017-05-31 | Method for measuring utilization efficiency of plant ammonium nitrogen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107449858A true CN107449858A (en) | 2017-12-08 |
| CN107449858B CN107449858B (en) | 2020-03-24 |
Family
ID=60487022
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710397367.5A Active CN107449858B (en) | 2017-05-31 | 2017-05-31 | Method for measuring utilization efficiency of plant ammonium nitrogen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107449858B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108801984A (en) * | 2018-05-21 | 2018-11-13 | 中国科学院地球化学研究所 | A kind of detection method of the plant inorganic nitrogen Assimilation rate based on Net Photosynthetic Rate |
| CN109169284A (en) * | 2018-10-10 | 2019-01-11 | 中国科学院地球化学研究所 | Under a kind of prediction mixed nitrogen in tissue-cultured seedling assimilation ammonium salt share and culture medium ammonium salt optimum concentration method |
| CN113567624A (en) * | 2021-07-21 | 2021-10-29 | 中国科学院地球化学研究所 | Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings |
| CN113884621A (en) * | 2021-08-16 | 2022-01-04 | 中国科学院地球化学研究所 | Method for quantitatively absorbing and utilizing bicarbonate by plants |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101953298A (en) * | 2010-08-26 | 2011-01-26 | 中国科学院地球化学研究所 | Method for dynamically measuring autotrophy capability of tissue culture seedling of plant |
| CN102053143A (en) * | 2009-11-05 | 2011-05-11 | 中国农业大学 | Method for marking soil nitrate nitrogen |
| CN102365925A (en) * | 2010-12-29 | 2012-03-07 | 中国科学院地球化学研究所 | Method for determining ability of plant on using nitrate |
| CN102792891A (en) * | 2012-08-09 | 2012-11-28 | 中国科学院地球化学研究所 | Method for determining tissue culture seedling autotrophic portion |
-
2017
- 2017-05-31 CN CN201710397367.5A patent/CN107449858B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102053143A (en) * | 2009-11-05 | 2011-05-11 | 中国农业大学 | Method for marking soil nitrate nitrogen |
| CN102053143B (en) * | 2009-11-05 | 2013-08-14 | 中国农业大学 | Method for marking soil nitrate nitrogen |
| CN101953298A (en) * | 2010-08-26 | 2011-01-26 | 中国科学院地球化学研究所 | Method for dynamically measuring autotrophy capability of tissue culture seedling of plant |
| CN102365925A (en) * | 2010-12-29 | 2012-03-07 | 中国科学院地球化学研究所 | Method for determining ability of plant on using nitrate |
| CN102792891A (en) * | 2012-08-09 | 2012-11-28 | 中国科学院地球化学研究所 | Method for determining tissue culture seedling autotrophic portion |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108801984A (en) * | 2018-05-21 | 2018-11-13 | 中国科学院地球化学研究所 | A kind of detection method of the plant inorganic nitrogen Assimilation rate based on Net Photosynthetic Rate |
| CN109169284A (en) * | 2018-10-10 | 2019-01-11 | 中国科学院地球化学研究所 | Under a kind of prediction mixed nitrogen in tissue-cultured seedling assimilation ammonium salt share and culture medium ammonium salt optimum concentration method |
| CN109169284B (en) * | 2018-10-10 | 2021-01-05 | 中国科学院地球化学研究所 | Method for predicting assimilation ammonium salt share of tissue culture seedlings under mixed nitrogen source and optimum ammonium salt concentration in culture medium |
| CN113567624A (en) * | 2021-07-21 | 2021-10-29 | 中国科学院地球化学研究所 | Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings |
| CN113884621A (en) * | 2021-08-16 | 2022-01-04 | 中国科学院地球化学研究所 | Method for quantitatively absorbing and utilizing bicarbonate by plants |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107449858B (en) | 2020-03-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101926267B (en) | Method for measuring bicarbonate ion utilizing capability of plant | |
| CN107449858A (en) | A kind of method for determining plant ammonium nitrogen utilization ratio | |
| CN102511362B (en) | Method for utilizing double markers to acquire share of inorganic carbon source utilized by plants | |
| CN102365925B (en) | Method for determining ability of plant on using nitrate | |
| Soussana et al. | Can we understand and predict the regulation of biological N 2 fixation in grassland ecosystems? | |
| CN105067772B (en) | A kind of method measuring plant gross photosynthesis carbon assimilation ability | |
| Zhan et al. | Chitin combined with selenium reduced nitrogen loss in soil and improved nitrogen uptake efficiency in Guanxi pomelo orchard | |
| Wang et al. | Evaluating the effects of nitrogen deposition on rice ecosystems across China | |
| de Melo Peixoto et al. | Silicon application increases biomass yield in sunflower by improving the photosynthesizing leaf area | |
| CN104904579A (en) | Method for applying trichoderma harzianum to vegetable hydroponics | |
| CN107153091B (en) | The measuring method of the inorganic nitrogen isotope fractionation value of plant assimilating ammonium salt | |
| CN105684899B (en) | The assay method of tissue-cultured seedling sucrose utilization rate | |
| Sesveren et al. | Response of Lactuva Sativa Var. Crispa to deficit irrigation and leonardite treatments | |
| Hayashi et al. | Potential ammonia emission from flag leaves of paddy rice (Oryza sativa L. cv. Koshihikari) | |
| Bialczyk et al. | Early vegetative growth of tomato plants in media containing nitrogen source as nitrate, ammonium, or various nitrate–ammonium mixtures with bicarbonate addition | |
| CN107027626B (en) | A method of quantitative tissue-cultured seedling nitrate utilization efficiency | |
| CN113567624B (en) | Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings | |
| CN103004352B (en) | Greenhouse crop fertigation method | |
| Mininni et al. | Restoration of soil fertility and management of mineral nutrition in a peach orchard under a sustainable farming system in semi-arid conditions | |
| CN109169284A (en) | Under a kind of prediction mixed nitrogen in tissue-cultured seedling assimilation ammonium salt share and culture medium ammonium salt optimum concentration method | |
| CN112501237B (en) | Method for predicting utilization capacity of microalgae bicarbonate | |
| CN107114238B (en) | A method of quantitative and prediction tissue-cultured seedling nitrate metabolic capability and nitrogen isotope fractionation value | |
| Yang et al. | Field Examinations on the Application of Novel Biochar-Based Microbial Fertilizer on Degraded Soils and Growth Response of Flue-Cured Tobacco (Nicotiana tabacum L.) | |
| Lim et al. | Ammonia Volatilization from Rice Paddy Soils Fertilized with 15 N-Urea Under Elevated CO2 and Temperature | |
| Liu et al. | Co-culture of rice and crayfish increases the global warming potential-An evidence from soil culture with different water content |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |