CN113567624B - Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings - Google Patents

Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings Download PDF

Info

Publication number
CN113567624B
CN113567624B CN202110823014.3A CN202110823014A CN113567624B CN 113567624 B CN113567624 B CN 113567624B CN 202110823014 A CN202110823014 A CN 202110823014A CN 113567624 B CN113567624 B CN 113567624B
Authority
CN
China
Prior art keywords
delta
tissue culture
nitrogen
nitrate
value
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.)
Active
Application number
CN202110823014.3A
Other languages
Chinese (zh)
Other versions
CN113567624A (en
Inventor
吴沿友
张开艳
吴沿胜
方蕾
邓智先
王世杰
刘丛强
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Geochemistry of CAS
Original Assignee
Institute of Geochemistry of CAS
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Institute of Geochemistry of CAS filed Critical Institute of Geochemistry of CAS
Priority to CN202110823014.3A priority Critical patent/CN113567624B/en
Publication of CN113567624A publication Critical patent/CN113567624A/en
Application granted granted Critical
Publication of CN113567624B publication Critical patent/CN113567624B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention discloses a method for quantitatively measuring the autotrophic and heterotrophic efficiency of different nitrogen sources of a tissue culture seedling. Respectively culturing the tissue culture seedlings on a sucrose culture medium of a mixed nitrogen source consisting of ammonium salt and two nitrates with different stable nitrogen isotope values; measuring the stable carbon and nitrogen isotope value of the leaves of the tissue culture seedlings; calculating the utilization portions of autotrophic and heterotrophic ammonium salt and nitrate of the tissue culture seedling, and acquiring the autotrophic and heterotrophic efficiency of different nitrogen sources of the tissue culture seedling according to the C/N ratio of the leaves of the tissue culture seedling and the utilization portions of the autotrophic and heterotrophic ammonium salt and nitrate. The invention can quantify the autotrophic efficiency and the heterotrophic efficiency of different nitrogen sources under different concentrations and different proportions under the condition of mixing the nitrogen sources, and provides a basis for the selection of the culture medium.

Description

Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings
Technical Field
The invention relates to a method for quantitatively measuring the autotrophic and heterotrophic efficiency of different nitrogen sources of a tissue culture seedling, belonging to the technical field of biology.
Technical Field
Tissue culture of plants is the most basic technology and means in current biotechnology and is now widely used in horticulture, agriculture and forestry production. It is a technique for quickly breeding plants under the condition of artificially providing a certain temp., light, humidity, nutrients and hormone, etc.
In the process of plant tissue culture, the growth modes of tissue culture seedlings are three: the first is autotrophic growth of plantlets by photosynthesis; the plantlets grow heterotrophically by virtue of an organic carbon source in a culture medium; thirdly, the plantlets are subjected to heterotrophic and autotrophic growth simultaneously by virtue of both organic carbon sources in the culture medium and artificial illumination. The conventional plant tissue culture rapid propagation technology is mostly carried out in a third way. Generally, the autotrophic ability of the tissue culture seedling determines the growth condition of the tissue culture seedling, and the tissue culture seedling with strong autotrophic ability is beneficial to improving the survival rate of the tissue culture seedling in the later domestication process.
Nitrogen is an essential element in the growth and development process of plants, and is an important component constituting proteins, nucleic acids, enzymes, chlorophyll and the like. Most plants absorb and utilize inorganic nitrogen mainly as nitrates and ammonium salts. In the coexistence of nitrate and ammonium salt, the utilization proportions of nitrate nitrogen and ammonium nitrogen are different under different concentrations and proportions of different nitrogen sources. Due to the different metabolic pathways and mechanisms of different nitrogen sources, the effects on the morphogenesis and other metabolism of plants, especially the autotrophic and heterotrophic metabolic efficiency of tissue culture seedlings, are different. The difference of the influence of different nitrogen sources on the efficiency of autotrophy and heterotrophy is known, the coupling process of carbon and nitrogen assimilation can be clearly known, the carbon and nitrogen coupling mechanism is clearer, and a decision is provided for nitrogen management (pollution reduction) of energy conservation (reduction of utilization of cane sugar) increase (increase) of (carbon) sink.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the method for quantitatively measuring the autotrophic efficiency and the heterotrophic efficiency of the tissue culture seedling under different nitrogen sources can simultaneously obtain the autotrophic and the heterotrophic share of the tissue culture seedling under the mixed nitrogen source and the utilization shares of different inorganic nitrogen sources, fills the blank that the autotrophic efficiency and the heterotrophic efficiency of the different nitrogen sources under the mixed nitrogen source cannot be obtained, and provides a decision for nitrogen management (pollution reduction) of increasing (adding) carbon for energy conservation (reducing the utilization of cane sugar).
It comprises the following steps:
firstly, all the cultures are selected to be cultured in the same culture chamber, and the used cane sugar is selected to be cane sugar with the same stable carbon isotope composition in the same manufacturer and the same batch;
secondly, regulating the hormone proportion of the culture medium to enable the tissue culture seedling to be detected to be in a proliferation stage by culturing the tissue culture seedling on the sugarcane and sucrose culture medium to form a rootless tissue culture seedling; simultaneously measuring delta of cane sugar in tissue culture medium13C value deltaCSAnd delta of leaf of plant to be tested grown in the tissue culture room only by using carbon dioxide13C value deltaC(ii) a Obtaining delta of leaves of a plant to be detected which only utilize carbon dioxide to grow in the tissue culture room13C value deltaCThe method comprises the following steps: during the experiment, the seeds corresponding to the tissue culture seedlings are sown in the corresponding culture medium, then cultured in the tissue culture room for 5 weeks, and finally the delta of the new leaves is determined13C value deltaC；
Third, by measuringPreparing stable nitrogen isotope ratio of nitrate in culture medium for plant tissue culture, and screening out two kinds of nitrate whose stable nitrogen isotope value difference is greater than 10 ‰, and its stable nitrogen isotope value is delta15N01And delta15N02(ii) a The two nitrates are respectively used as unique nitrogen sources to be prepared into a culture medium with the concentration of the nitrate to be investigated;
fourthly, respectively culturing the tissue culture seedlings to be detected with 1 single bud in a culture medium with two kinds of nitrates with different stable nitrogen isotope values as unique nitrogen sources;
fifthly, after the tissue culture seedling is cultured for 5 weeks, the stable nitrogen isotope values of the leaves of the tissue culture seedling cultured by the culture medium with the two nitrates as the unique nitrogen source are respectively measured, and the stable nitrogen isotope values of the leaves are respectively marked as delta15NN1And delta15NN2；
Sixth, by determining the stable nitrogen isotope ratio δ of ammonium salts in the medium used for preparing the plant tissue culture15NASelecting the stable nitrogen isotope ratio delta15NAAnd delta15N01And delta15N02Ammonium salts all greater than 10 per mill;
seventhly, the stable nitrogen isotope value is delta15N01And delta15N02The nitrate and the ammonium salt are respectively prepared into two groups of mixed nitrogen source culture media to be inspected;
eighthly, similarly, simultaneously culturing the tissue culture seedlings to be detected with 1 single bud on the two groups of mixed nitrogen source culture media respectively;
ninthly, similarly, after the tissue culture seedling is cultured for 5 weeks, measuring the stable nitrogen isotope value of the leaves of the tissue culture seedling cultured on the two groups of mixed nitrogen source culture media, wherein the stable nitrogen isotope value of the ammonium salt and the stable nitrogen isotope value are delta15N01The stable nitrogen isotope value of the leaf of the tissue culture seedling cultured by the mixed nitrogen source culture medium consisting of the nitrate is recorded as delta15NN1mixThe isotopic value of ammonium salt to stable nitrogen is delta15N02The stable nitrogen isotope value of the leaf of the tissue culture seedling cultured by the mixed nitrogen source medium consisting of the nitrate is recorded as delta15NN2mix；
Tenthly, simultaneously measuring the leaf stable carbon isotope value delta of the tissue culture seedling cultured on the two groups of mixed nitrogen source culture mediaCTAnd the C/N value R is determined by the following method: the carbon content C and the nitrogen content N of the leaf are simultaneously measured by an element analyzer, and the C/N value R is as follows: r is C/N;
eleventh, using δ15NN1、δ15NN2、δ15NN1mixAnd delta15NN2mixCalculating the nitrate utilization ratio f of the tissue culture seedlingsN(ii) a Further calculating the utilization portion f of ammonium saltA(ii) a Calculating the nitrate utilization ratio f of the tissue culture seedlingsNThe method comprises the following steps: will delta15NN1、δ15NN2And delta at the nitrate concentration15NN1mixAnd delta15NN2mixSubstituting the equation:
Figure BDA0003172522460000031
Figure BDA0003172522460000032
ammonium salt utilization fraction fAThe formula of (1) is: f. ofA＝1-fN。
Twelfth, using δCS、δCTAnd deltaC(ii) a Calculating the autotrophic portion f of the tissue culture seedlingsAT(ii) a Further calculating the heterotrophic part fHT(ii) a Calculating the autotrophic portion f of the tissue culture seedlingsATThe method comprises the following steps: will deltaCS、δCTAnd deltaCThe substitution equation:
Figure BDA0003172522460000033
Figure BDA0003172522460000034
heterotrophic fraction fHTThe formula of (1) is: f. ofHT＝1-fAT。
Thirteenth, R, f according to bladeA、fN、fATAnd fHTFurther obtaining the self-cultured tissue culture seedlings under different nitrogen source culture(ii) the efficiency of the heterotrophic and heterotrophic processes; the method for obtaining the autotrophic and heterotrophic efficiency of the tissue culture seedling under the culture of different nitrogen sources comprises the following steps: autotrophic efficiency of ammonium Nitrogen EAT-A，EAT-A＝R fAT/fA(ii) a Heterotrophic efficiency E of ammonium NitrogenHT-A，EHT-A＝R fHT/fA(ii) a Autotrophic efficiency of nitrate nitrogen EAT-N，EAT-N＝R fAT/fN(ii) a Heterotrophic efficiency E of nitrate nitrogenHT-N，EHT-N＝R fHT/fN。
The invention has the advantages of
1) The invention can simultaneously measure the carbon and nitrogen assimilation information of the tissue culture seedling under the mixed nitrogen source culture.
2) The method can obtain the contributions of different inorganic nitrogen sources to the autotrophic and heterotrophic ways of the tissue culture seedlings under different nitrogen source configurations, can evaluate the carbon-nitrogen coupling capacity of the tissue culture seedlings under different nitrogen source configurations, and provides technical support for the culture medium screening of high-quality tissue culture seedlings.
3) The method has the advantages that the acquisition of the autotrophic information and the heterotrophic information utilizes common experimental conditions, no additional experiment is added, the autotrophic information and the heterotrophic information are acquired simultaneously with the inorganic nitrogen information, and experimental errors are reduced, so that the acquired results are more reliable.
4) The method can rapidly acquire the information of assimilating different inorganic nitrogen sources of the tissue culture seedling by fewer experiments, and can predict the inorganic nitrogen assimilation effect of the tissue culture seedling under different nitrogen source configurations.
5) The method has the advantages of few steps and simple calculation.
Principle of the invention
The technique of stable nitrogen isotope is widely used for researching nitrogen circulation of biosphere by using indicator of nitrogen source. Two stable nitrogen isotopes of nitrogen element in nature are14N and15the value of N, stable nitrogen isotope is usually delta15N (‰) represents δ in nature15The change of N is-10 to +20 per mill. The leaf nitrogen isotope value of the plant cultured under different nitrogen sources can well reflect the characteristics of the stable nitrogen isotope value of the nitrogen source. Therefore, the difference in the plant body can be quantitatively distinguished by the mass balance principle and the isotope mixture modelInorganic nitrogen source (b).
The tissue culture seedling has certain nitrogen isotope fractionation when assimilating nitrate and ammonium salt. The stable nitrogen isotope value of the leaf blade of the tissue culture seedling cultured under the condition of mixed nitrogen source is the result of mixing the stable nitrogen isotope values after assimilating nitrate and ammonium salt and carrying out nitrogen isotope fractionation.
In the proliferation stage of plant tissue culture, the concentration ratio of cytokinin and auxin can be regulated to ensure that the tissue culture seedling does not produce root in the whole proliferation stage, so that the assimilation of nitrate and ammonium salt only occurs in the leaf. The invention determines the utilization ratio of nitrate and ammonium salt of tissue culture seedling by bidirectional nitrogen isotope labeling culture method. Firstly, two nitrates with the difference of stable nitrogen isotopes larger than 10 per thousand are used for simultaneously culturing tissue culture seedlings, then the two nitrates and the same ammonium salt are respectively used for simultaneously culturing the tissue culture seedlings, the difference of the stable nitrogen isotope value of the ammonium salt and the stable nitrogen isotope value of the two nitrates is ensured to be larger than 10 per thousand, and the stable nitrogen isotope values of the leaves of the tissue culture seedlings under the culture of two mixed nitrogen sources are respectively measured. And finally, calculating the proportions of the nitrate and the ammonium salt respectively used by the tissue culture seedlings through a two-end element mixed model.
The isotope mixture model for both end members is expressed as:
δ15NN1mix＝fNδ15NN1+fAδ15NA-T (1)
δ15NN2mix＝fNδ15NN2+fAδ15NA-T (2)
fA＝1-fN (3)
delta here15NN1And delta15NN2Respectively the leaf stable nitrogen isotope value, delta, of the tissue culture seedling under the culture of two nitrates with the stable nitrogen isotope value difference value more than 10 per mill15NA-TIs the stable nitrogen isotope value after the tissue culture seedling assimilates ammonium salt and is subjected to nitrogen isotope fractionation under the culture of a mixed nitrogen source. Delta15NN1mixIs stable nitrogen isotope value of delta15N01The nitrate and the ammonium salt form the stable nitrogen isotope value of the leaves of the tissue culture seedlings cultured by the mixed nitrogen source. Delta15NN2mixIs stable nitrogen isotope value of delta15N02The nitrate and the ammonium salt form the stable nitrogen isotope value of the leaves of the tissue culture seedlings cultured by the mixed nitrogen source. f. ofNIs the utilization fraction of nitrate, fAIs the utilization portion of ammonium salt.
Simultaneous equations (1), (2) and (3), solved to yield:
Figure BDA0003172522460000041
carbon element in nature also has two stable isotopes:12c and13c, their natural average abundances are 98.89% and 1.11%, respectively. The composition of the stable carbon isotope is usually delta13C (‰) represents delta in nature13The change of C is-90 to +20 per mill.
In the plant tissue culture process, the tissue culture seedling is usually subjected to hybrid growth, so that sugar is additionally provided as an organic carbon source. The tissue culture seedling can grow by using an organic carbon source and an inorganic carbon source in the growth process, so that the delta of a new leaf can be caused13The C value is derived from both organic carbon sources (heterotrophic sources) and inorganic carbon sources (autotrophic sources). Therefore, the isotope mixture model of the two end members can be used for obtaining the share of the tissue culture seedling using the organic carbon source. C3Plant delta13The variation range of C is-20 to-35 per mill, C4Plant delta13The variation range of C is-9 to-17 per mill. C3Representative of plant sucrose is beet sucrose, C4Representative sucrose of plants is sugarcane sucrose. The C can be obtained by selecting cane sugar as carbon source3Plant delta13C ratio utilizes CO in air2The carbon sources differ by about 10% per thousand, so that the respective proportions can be calculated using a model of isotopic mixing of the two end members.
The isotope mixture model of the two end-members can be expressed as:
δCT＝δH-fATδH+fATδC (5)
delta hereCTDelta of new leaves of tissue culture seedlings cultured by mixed nitrogen source13C value, δCComplete utilization of CO in air for tissue culture seedling2Delta as inorganic carbon source13C value, δHDelta for fully utilizing sucrose as organic carbon source for tissue culture seedlings13C value, fAHThe autotrophic portion when the inorganic carbon source is utilized for the study of the tissue culture seedling.
δCComplete utilization of CO in air for tissue culture seedling2Delta as inorganic carbon source13C value approximately equal to that of the corresponding seed of the tissue culture seedling is sown in the corresponding culture medium, then cultured in the tissue culture chamber for 5 weeks, and finally the delta of the newly grown leaf is measured13C value deltaC。δHDelta for fully utilizing sucrose as organic carbon source for tissue culture seedlings13C value, we know that the stable carbon isotope fractionation value of cane sugar under the tissue culture condition is 2.54 per mill, so that: deltaH＝δCS2.54% o, where deltaCSDelta of cane sugar13And C value. Thus, it is possible to obtain:
Figure BDA0003172522460000051
then:
fHT＝1-fAT (7)
where f isHTIs a heterotrophic share.
The C/N ratio R of a plant may characterize the nitrogen use efficiency of the plant. Similarly, the ratio of the utilization fraction of autotrophic carbon to ammonium nitrogen is indicative of the efficiency of ammonium nitrogen for autotrophic transport, i.e., EAT-A，EAT-A＝R fAT/fA(ii) a Heterotrophic efficiency E for obtaining ammonium nitrogen by the same methodHT-A，EHT-A＝R fHT/fA(ii) a Autotrophic efficiency of nitrate nitrogen EAT-N，EAT-N＝RfAT/fN(ii) a Heterotrophic efficiency E of nitrate nitrogenHT-N，EHT-N＝R fHT/fN。
Detailed Description
An embodiment of the invention, comprising the steps of:
firstly, all the cultures are selected to be cultured in the same culture chamber, and the used cane sugar is selected to be cane sugar with the same stable carbon isotope composition in the same manufacturer and the same batch;
secondly, regulating the hormone proportion of the culture medium to enable the tissue culture seedling to be detected to be in a proliferation stage by culturing the tissue culture seedling on the sugarcane and sucrose culture medium to form a rootless tissue culture seedling; simultaneous determination of the delta of sugarcane sucrose in the culture Medium13C value deltaCSAnd delta of leaf of plant to be tested grown in the tissue culture room only by using carbon dioxide13C value deltaC(ii) a Obtaining delta of leaves of a plant to be detected which only utilize carbon dioxide to grow in the tissue culture room13C value deltaCThe method comprises the following steps: during the experiment, the seeds corresponding to the tissue culture seedlings are sown in the corresponding culture medium, then cultured in the tissue culture room for 5 weeks, and finally the delta of the new leaves is determined13C value deltaC；
Thirdly, two nitrates with the stable nitrogen isotope value difference value of more than 10 per mill are screened out by measuring the stable nitrogen isotope ratio of the nitrate in the culture medium for configuring the plant tissue culture, and the stable nitrogen isotope values are delta and delta respectively15N01And delta15N02(ii) a The two nitrates are respectively used as unique nitrogen sources to be prepared into a culture medium with the concentration of the nitrate to be investigated;
fourthly, respectively culturing the tissue culture seedlings to be detected with 1 single bud in a culture medium with two kinds of nitrates with different stable nitrogen isotope values as unique nitrogen sources;
fifthly, after the tissue culture seedling is cultured for 5 weeks, the stable nitrogen isotope values of the leaves of the tissue culture seedling cultured by the culture medium with the two nitrates as the unique nitrogen source are respectively measured, and the stable nitrogen isotope values of the leaves are respectively marked as delta15NN1And delta15NN2；
Sixth, by determining the stable nitrogen isotope ratio δ of ammonium salts in the medium used for preparing the plant tissue culture15NASelecting the stable nitrogen isotope ratio delta15NAAnd delta15N01And delta15N02Ammonium salts all greater than 10 per mill;
seventhly, the stable nitrogen isotope value is delta15N01And delta15N02The nitrate and the ammonium salt are respectively prepared into two groups of mixed nitrogen source culture media to be inspected;
eighthly, similarly, simultaneously culturing the tissue culture seedlings to be detected with 1 single bud on the two groups of mixed nitrogen source culture media respectively;
ninthly, similarly, after the tissue culture seedling is cultured for 5 weeks, measuring the stable nitrogen isotope value of the leaves of the tissue culture seedling cultured on the two groups of mixed nitrogen source culture media, wherein the stable nitrogen isotope value of the ammonium salt and the stable nitrogen isotope value are delta15N01The stable nitrogen isotope value of the leaf of the tissue culture seedling cultured by the mixed nitrogen source culture medium consisting of the nitrate is recorded as delta15NN1mixThe isotopic value of ammonium salt to stable nitrogen is delta15N02The stable nitrogen isotope value of the leaf of the tissue culture seedling cultured by the mixed nitrogen source medium consisting of the nitrate is recorded as delta15NN2mix；
Tenthly, simultaneously measuring the leaf stable carbon isotope value delta of the tissue culture seedling cultured on the two groups of mixed nitrogen source culture mediaCTAnd the C/N value R is determined by the following method: the carbon content C and the nitrogen content N of the leaf are simultaneously measured by an element analyzer, and the C/N value R is as follows: r is C/N;
eleventh, using δ15NN1、δ15NN2、δ15NN1mixAnd delta15NN2mixCalculating the nitrate utilization ratio f of the tissue culture seedlingsN(ii) a Further calculating the utilization portion f of ammonium saltA(ii) a Calculating the nitrate utilization ratio f of the tissue culture seedlingsNThe method comprises the following steps: will delta15NN1、δ15NN2And delta at the nitrate concentration15NN1mixAnd delta15NN2mixSubstituting the equation:
Figure BDA0003172522460000071
Figure BDA0003172522460000072
ammonium salt utilization fraction fAThe formula of (1) is: f. ofA＝1-fN。
Twelfth, using δCS、δCTAnd deltaC(ii) a Calculating the autotrophic portion f of the tissue culture seedlingsAT(ii) a Further calculating the heterotrophic part fHT(ii) a Calculating the autotrophic portion f of the tissue culture seedlingsATThe method comprises the following steps: will deltaCS、δCTAnd deltaCThe substitution equation:
Figure BDA0003172522460000073
Figure BDA0003172522460000074
heterotrophic fraction fHTThe formula of (1) is: f. ofHT＝1-fAT。
Thirteenth, R, f according to bladeA、fN、fATAnd fHTFurther obtaining the autotrophic and heterotrophic efficiency of different nitrogen sources of the tissue culture seedling; the method for obtaining the autotrophic and heterotrophic efficiency of different nitrogen sources of the tissue culture seedling comprises the following steps: autotrophic efficiency of ammonium Nitrogen EAT-A，EAT-A＝R fAT/fA(ii) a Heterotrophic efficiency E of ammonium NitrogenHT-A，EHT-A＝R fHT/fA(ii) a Autotrophic efficiency of nitrate nitrogen EAT-N，EAT-N＝R fAT/fN(ii) a Heterotrophic efficiency E of nitrate nitrogenHT-N，EHT-N＝R fHT/fN。
Example 1: determination of autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings of brassica napus under different ammonium salt concentrations when nitrate concentration is 20mM
Culturing materials: clonal cabbage type rape tissue culture seedling
The formula of the culture medium is as follows: MS +6-BA 2.0 mg/L + NAA 0.2mg/L, 30g/L sugarcane sucrose, agar: 7.5g/L, pH: 5.8, culture room temperature: 25 +/-2 ℃. The photoperiod: 12h/d, illumination intensity: 50 μmol. m-2·s-1Delta. of cane sugar13C value deltaCSIs-11.64 per mill; the stable nitrogen isotope values of the two nitrates are respectively: delta15N01＝8.08‰，δ15N0222.67% o. The stable nitrogen isotope value of the ammonium salt is: delta15NA-2.64 ‰. According to the method of the invention, the ammonium salt is respectively mixed with stable nitrogen with the isotope value delta15N01And delta15N02The mixed nitrogen source was composed of nitrates to ensure that the nitrate concentration of the medium was 20mM each and then the ammonium salt concentration was set to 2.5mM, 5mM, 10mM and 40mM, respectively, when the medium was prepared. Thus, the inorganic nitrogen concentration of the medium was 22.5mM, 25mM, 30mM and 60mM, respectively. Culturing the tissue culture seedlings of the clonal cabbage type rape in the culture medium respectively, culturing for 5 weeks, and measuring C/N and delta of leaves of the tissue culture seedlings of the cabbage type rape respectively13C value deltaCTAnd at δ15N01The leaf stable nitrogen isotope value delta under the culture of the mixed nitrogen source consisting of nitrate and ammonium salt15NN1mixAnd a stable nitrogen isotope value of delta15N02The nitrate and the ammonium salt form a mixed nitrogen source to culture the stable nitrogen isotope value delta of the leaf15NN2mix. The measurement results are shown in table 1: meanwhile, seeds corresponding to the tissue culture seedlings are sown in corresponding culture media, then cultured in the tissue culture chamber for 5 weeks, and finally delta of the newly grown leaves is measured13C value deltaCDetermining delta of new leaf13C value deltaCIs-28.05 ‰ (n ═ 3).
TABLE 1 Stable carbon nitrogen isotope value and C: N of leaves of Brassica napus tissue culture seedlings treated with ammonium salt
Figure BDA0003172522460000081
Note: the nitrate in the medium was 20mM each. n is 3.
As can be seen from Table 1, when the nitrate concentration in the culture medium is 20mM, the concentration of the ammonium salt is increased, the leaf stable nitrogen isotope value of the tissue culture seedlings of Brassica napus is gradually reduced when the mixed nitrogen source consisting of the nitrate with the larger difference between the ammonium salt and the two stable nitrogen isotope values is cultured, and the leaf stable nitrogen isotope values are the smallest at the highest ammonium salt concentration; however, the leaf stable carbon isotope value of the cabbage type rape tissue culture seedling shows a trend of gradually decreasing and then gradually increasing.
Because the concentration of nitrate in the culture medium is 20mM, the stable nitrogen isotope value of the tissue culture seedlings of the cabbage type rape at 20mM is delta15N01Leaf stable nitrogen isotope value delta under nitrate culture15N13.30 ‰ (n ═ 3); stable nitrogen isotope value of delta at 20mM15N02Leaf stable nitrogen isotope value delta under nitrate culture15N215.53 ‰ (n ═ 3). Incorporating δ of Table 115NN1mixAnd delta15NN2mixUsing the equation
Figure BDA0003172522460000082
Figure BDA0003172522460000083
The nitrate and ammonium salt utilization ratio of the brassica napus tissue culture seedlings cultured by the mixed nitrogen source can be calculated, and the results are shown in table 2. Using delta from Table 1 simultaneouslyCTData, while at the same time deltaCS(-11.64‰)，δC(-28.05% o) substitution equation:
Figure BDA0003172522460000084
the autotrophic and heterotrophic share of the brassica napus tissue culture seedlings under the mixed nitrogen source culture can be calculated, and the results are shown in table 2.
TABLE 2 nitrate utilization fraction (f) of Brassica napus tissue culture seedlings under ammonium salt treatmentN) And the utilization ratio of ammonium salt (f)A＝1-fN) (ii) a Portion of autotrophy (f)AT) And heterotrophic fraction (f)HT)
Figure BDA0003172522460000085
Note: the nitrate in the medium was 20mM each. n is 3.
Vane R, f according to tables 1 and 2A、fN、fATAnd fHTFurther obtaining the autotrophic and heterotrophic efficiency of the tissue culture seedlings of the cabbage type rape under the ammonium salt treatment; see table 3.
TABLE 3 autotrophic and heterotrophic efficiency of cabbage type rape tissue culture seedlings under ammonium salt treatment by different nitrogen sources
Figure BDA0003172522460000086
Figure BDA0003172522460000091
Note: the nitrate in the medium was 20mM each. n is 3.
As can be seen from Table 3, in the case where the nitrate concentration in the medium was 20mM, the ammonium salt concentration of the tissue culture seedlings of Brassica napus was gradually decreased in both the autotrophic and heterotrophic efficiencies as the ammonium salt concentration was increased, and the autotrophic and heterotrophic efficiencies were minimized at the highest ammonium salt concentration. However, increasing the concentration of ammonium salt can significantly improve the heterotrophic efficiency of nitrate in tissue culture seedlings of Brassica napus, but there is an inflection point in improving the autotrophic efficiency of nitrate, and at 5mM ammonium salt, the nitrate autotrophic efficiency of tissue culture seedlings of Brassica napus is the greatest, and thereafter, the nitrate autotrophic efficiency gradually decreases with the increase of ammonium salt concentration. Maximum nitrate and ammonium heterotrophic efficiency is generally not sought during plant tissue culture, as maximum inorganic nitrogen heterotrophic efficiency often means lower autotrophic capacity, which is detrimental to the post-acclimation process of the cultured plantlets. However, the maximum autotrophic capacity of tissue culture seedlings is not obtained all the time during the tissue culture process. Considering that nitrogen is an essential element in the growth and development of plants, it is an important constituent constituting proteins, enzymes, chlorophyll, and the like. Therefore, it is very important to quantify the autotrophic efficiency of nitrate and ammonium salt of tissue culture seedlings. As can be seen from Table 3, when the concentration of nitrate in the culture medium is 20mM and the concentration of ammonium salt in the culture medium is 5mM, the sum of the autotrophic efficiency of nitrate and ammonium salt of the tissue culture seedlings of Brassica napus is the largest, which means that the inorganic carbon fixation efficiency of Brassica napus is the highest under the condition of the inorganic nitrogen mixture ratio. The continuous increase of the supply of inorganic nitrogen cannot improve the autotrophic efficiency of nitrate and ammonium salt of the tissue culture seedlings of the brassica napus, but causes the waste of the inorganic nitrogen.
Example 2: determination of autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings of brassica napus under different nitrate concentrations when ammonium salt concentration is 20mM
Culturing materials: clonal cabbage type rape tissue culture seedling
The formula of the culture medium is as follows: MS +6-BA 2.0 mg/L + NAA 0.2mg/L, 30g/L sugarcane sucrose, agar: 7.5g/L, pH: 5.8, culture room temperature: 25 +/-2 ℃. The photoperiod: 12h/d, illumination intensity: 50 μmol. m-2·s-1Delta. of cane sugar13C value deltaCSIs-11.64 per mill; the stable nitrogen isotope values of the two nitrates are respectively: delta15N01＝8.08‰，δ15N0222.67% o. The stable nitrogen isotope value of the ammonium salt is: delta15NA-2.64 ‰. According to the method of the invention, the ammonium salt is respectively mixed with stable nitrogen with the isotope value delta15N01And delta15N02The mixed nitrogen source was composed of nitrate salts, and when the medium was prepared, the ammonium salt concentration of the medium was ensured to be 20mM each, and then the nitrate salt concentrations were set to be 5mM, 10mM, 20mM, and 40mM, respectively. Thus, the nitrogen concentration of the medium was 25mM, 30mM, 40mM and 60mM, respectively. Culturing the tissue culture seedlings of the clonal cabbage type rape in the culture medium respectively, culturing for 5 weeks, and measuring C/N and delta of leaves of the tissue culture seedlings of the cabbage type rape respectively13C value deltaCTAnd at δ15N01The leaf stable nitrogen isotope value delta under the culture of the mixed nitrogen source consisting of nitrate and ammonium salt15NN1mixAnd a stable nitrogen isotope value of delta15N02The nitrate and the ammonium salt form a mixed nitrogen source to culture the stable nitrogen isotope value delta of the leaf15NN2mix. The measurement results are shown in table 4: sowing seeds corresponding to the tissue culture seedling in corresponding culture medium, culturing in the tissue culture chamber for 5 weeks, and measuring new leavesDelta of13C value deltaCDetermining delta of new leaf13C value deltaCIs-28.05 ‰ (n ═ 3).
TABLE 4 Stable carbon nitrogen isotope value and C: N of cabbage type rape tissue culture seedling leaf under nitrate treatment
Figure BDA0003172522460000101
Note: the ammonium salts in the medium were all 20 mM. n is 3.
From Table 4, it can be seen that, in the case that the concentrations of ammonium salts in the culture medium are both 20mM, the leaf stable nitrogen isotope values of the tissue culture seedlings of Brassica napus are gradually increased under the condition that the concentrations of the nitrates are increased, and the mixed nitrogen source composed of the nitrates with larger difference between the ammonium salts and the two stable nitrogen isotope values respectively is cultured, and the leaf stable nitrogen isotope values of the tissue culture seedlings of Brassica napus are the largest under the condition that the concentrations of the nitrates are the highest; the leaf stable carbon isotope value of the cabbage type rape tissue culture seedling shows a gradually decreasing trend.
The difference of the leaf stable nitrogen isotope values of the cabbage type rape tissue culture seedlings is not obvious under the three mononitrate concentrations (10mM, 20mM and 40mM) configured with the stable nitrogen isotope value of 8.08 per thousand nitrate, and the difference of the leaf stable nitrogen isotope values is also not obvious under the three mononitrate concentrations (10mM, 20mM and 40mM) configured with the stable nitrogen isotope value of 22.67 per thousand nitrate. Thus, delta of Brassica napus type tissue culture seedlings in this experiment15NN1Approximately equal to the average value of the stable nitrogen isotope value, delta, of the leaf blade under the culture of the three mononitrate (the stable nitrogen isotope value is 8.08 ‰)15NN1-3.17 ‰ (n ═ 9); delta of cabbage type tissue culture seedlings15NN2Also approximately equal to the average value of the stable nitrogen isotope value of the leaf blade under the culture of the three mononitrate (the stable nitrogen isotope value is 22.67 thousandths)15NN2-15.19 ‰ (n ═ 9). Incorporating delta from Table 415NN1mixAnd delta15NN2mixUsing the equation
Figure BDA0003172522460000102
The nitrate and ammonium salt utilization ratio of the brassica napus tissue culture seedlings cultured by the mixed nitrogen source can be calculated, and the results are shown in table 5. Using delta from Table 4 simultaneouslyCTData, while at the same time deltaCS(-11.64‰)，δC(-28.05% o) substitution equation:
Figure BDA0003172522460000103
the autotrophic and heterotrophic share of the brassica napus tissue culture seedlings under mixed nitrogen source culture can be calculated, and the results are shown in table 5.
TABLE 5 nitrate utilization fraction (f) of canola tissue culture seedlings under nitrate treatmentN) And the utilization ratio of ammonium salt (f)A＝1-fN) (ii) a Portion of autotrophy (f)AT) And heterotrophic fraction (f)HT)
Figure BDA0003172522460000111
Note: the ammonium salts in the medium were all 20 mM. n is 3.
Vanes R, f according to tables 4 and 5A、fN、fATAnd fHTFurther obtaining the autotrophic and heterotrophic efficiency of the tissue culture seedlings of the cabbage type rape under the nitrate treatment; see table 6.
TABLE 6 autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings of Brassica napus under nitrate treatment
Figure BDA0003172522460000112
Note: the ammonium salts in the medium were all 20 mM. n is 3.
As can be seen from Table 6, in the case that the ammonium salts in the medium were all 20mM, the ammonium salt autotrophic efficiency of the brassica napus tissue culture seedlings tended to increase gradually with the increase of the nitrate concentration, while the heterotrophic efficiency of the ammonium salts was once tended to decrease gradually, and increasing the nitrate supply was helpful to improve the ammonium nitrogen autotrophic efficiency of the brassica napus tissue culture seedlings. However, the heterotrophic efficiency of nitrate decreases with increasing nitrate, while the autotrophic efficiency of nitrate tends to increase and decrease. Considering that the maximum autotrophic efficiency of nitrate and ammonium salt helps the tissue culture seedlings to fix more inorganic carbon, the inorganic carbon fixation efficiency of the brassica napus tissue culture seedlings is highest when the concentration of ammonium salt is 20mM and the concentration of nitrate is 10 mM.
Example 3: determination of autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings of brassica napus under different inorganic nitrogen concentrations
Culturing materials: clonal cabbage type rape tissue culture seedling
The formula of the culture medium is as follows: MS +6-BA 2.0 mg/L + NAA 0.2mg/L, 30g/L sugarcane sucrose, agar: 7.5g/L, pH: 5.8, culture room temperature: 25 +/-2 ℃. The photoperiod: 12h/d, illumination intensity: 50 μmol. m-2·s-1Delta. of cane sugar13C value deltaCS-12.65% o; the stable nitrogen isotope values of the two nitrates are respectively: delta15N01＝8.08‰，δ15N0222.67% o. The stable nitrogen isotope value of the ammonium salt is: delta15NA-2.64 ‰. According to the method of the invention, the ammonium salt is respectively mixed with stable nitrogen with the isotope value delta15N01And delta15N02The nitrate forms a mixed nitrogen source, and when a culture medium is prepared, the concentration ratio of the nitrate to the ammonium salt in the culture medium is ensured to be 2: 1, and then setting the concentrations of inorganic nitrogen to 20mM, 40mM, 60mM, and 80mM, respectively. Culturing the tissue culture seedlings of the clonal cabbage type rape in the culture medium respectively, culturing for 5 weeks, and measuring C/N and delta of leaves of the tissue culture seedlings of the cabbage type rape respectively13C value deltaCTAnd at δ15N01The leaf stable nitrogen isotope value delta under the culture of the mixed nitrogen source consisting of nitrate and ammonium salt15NN1mixAnd a stable nitrogen isotope value of delta15N02The nitrate and the ammonium salt form a mixed nitrogen source to culture the stable nitrogen isotope value delta of the leaf15NN2mix. The measurement results are shown in table 7: at the same time, sowing the seeds corresponding to the tissue culture seedling in the corresponding culture medium, then culturing for 5 weeks in the tissue culture chamber, and finally measuring the new leavesDelta of13C value deltaCDetermining delta of new leaf13C value deltaCIs-28.05 ‰ (n ═ 3).
TABLE 7 Stable carbon and nitrogen isotope values and C: N of leaves of tissue culture seedlings of Brassica napus under treatment of different inorganic nitrogen concentrations
Figure BDA0003172522460000121
Note: the concentration ratio of nitrate to ammonium salt at each inorganic nitrogen concentration was 2: 1, n is 3.
As can be seen from Table 1, with the gradual increase of the inorganic nitrogen concentration, the leaf stable nitrogen isotope values of the tissue culture seedlings of the Brassica napus show a trend of gradually decreasing and then increasing under the culture of the mixed nitrogen source consisting of the nitrate with larger difference between the ammonium salt and the two stable nitrogen isotope values; the leaf stable carbon isotope value of the cabbage type rape tissue culture seedling shows a gradually decreasing trend.
The difference of the leaf stable nitrogen isotope values of the cabbage type rape tissue culture seedlings is not obvious under the three mononitrate concentrations (10mM, 20mM and 40mM) configured with the stable nitrogen isotope value of 8.08 per thousand nitrate, and the difference of the leaf stable nitrogen isotope values is also not obvious under the three mononitrate concentrations (10mM, 20mM and 40mM) configured with the stable nitrogen isotope value of 22.67 per thousand nitrate. Thus, delta of Brassica napus type tissue culture seedlings in this experiment15NN1Approximately equal to the average value of the stable nitrogen isotope value, delta, of the leaf blade under the culture of the three mononitrate (the stable nitrogen isotope value is 8.08 ‰)15NN1-3.17 ‰ (n ═ 9); delta of cabbage type tissue culture seedlings15NN2Also approximately equal to the average value of the stable nitrogen isotope value of the leaf blade under the culture of the three mononitrate (the stable nitrogen isotope value is 22.67 thousandths)15NN2-15.19 ‰ (n ═ 9). Delta from Table 715NN1mixAnd delta15NN2mixUsing the equation
Figure BDA0003172522460000122
Can be calculated to obtain the culture in the mixed nitrogen sourceThe tissue culture seedlings of Brassica napus were raised using the nitrate and ammonium salt content, and the results are shown in Table 8. Using delta from Table 7 simultaneouslyCTData, while at the same time deltaCS(-11.64‰)，δC(-28.05% o) substitution equation:
Figure BDA0003172522460000123
the autotrophic and heterotrophic share of brassica napus tissue culture seedlings cultured on mixed nitrogen sources can be calculated, and the results are shown in table 8.
TABLE 8 nitrate utilization fractions (f) of tissue culture seedlings of Brassica napus under treatment with different inorganic nitrogen concentrationsN) And the utilization ratio of ammonium salt (f)A＝1-fN) (ii) a Portion of autotrophy (f)AT) And heterotrophic fraction (f)HT)
Figure BDA0003172522460000131
Note: the concentration ratio of nitrate to ammonium salt at each inorganic nitrogen concentration was 2: 1. n is 3.
Vanes R, f according to tables 7 and 8A、fN、fATAnd fHTFurther obtaining the autotrophic and heterotrophic efficiency of the tissue culture seedlings of the cabbage type rape under the treatment of different inorganic nitrogen concentrations; see table 9.
TABLE 9 autotrophic and heterotrophic efficiency of tissue culture seedlings of Brassica napus under treatment of different inorganic nitrogen concentrations
Figure BDA0003172522460000132
Note: the concentration ratio of nitrate to ammonium salt at each inorganic nitrogen concentration was 2: 1, n is 3.
As can be seen from table 9, as the inorganic nitrogen concentration increases, the heterotrophic efficiency of both nitrate and ammonium salts gradually decreases, and the minimal heterotrophic efficiency of nitrate and ammonium salts is obtained at the highest inorganic nitrogen concentration. The autotrophic efficiency of the ammonium salt shows a gradually descending trend along with the increase of the inorganic nitrogen concentration, but a small-amplitude return rise occurs under the highest inorganic nitrogen concentration; with the increase of the inorganic nitrogen concentration, the autotrophic efficiency of the nitrate tends to increase and then gradually decrease. From the viewpoint of plant tissue culture, the obtaining of the maximum autotrophic efficiency of nitrate nitrogen and ammonium nitrogen is advantageous for improving the utilization efficiency of inorganic nitrogen, and therefore, when the concentration of inorganic nitrogen is 40mM (the concentration ratio of nitrate to ammonium salt is 2: 1), the autotrophic efficiency of inorganic nitrogen is the highest at this time.
In conclusion, the invention can obtain the autotrophic efficiency of nitrate nitrogen and ammonium nitrogen under different nitrogen source proportions. From the above results, it is understood that high nitrate and low ammonium contents are advantageous for the improvement of autotrophic efficiency, which is consistent with their different metabolic mechanisms and is also consistent with the reality.
The present invention is not limited to the above-described embodiments, and any obvious improvement, replacement or modification by those skilled in the art can be made without departing from the spirit of the present invention.

Claims (4)

1. A method for quantitatively measuring the autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings is characterized in that: comprises the following steps:
firstly, all the cultures are selected to be cultured in the same culture chamber, and the used cane sugar is selected to be cane sugar with the same stable carbon isotope composition in the same manufacturer and the same batch;
secondly, regulating the hormone proportion of the culture medium to enable the tissue culture seedling to be detected to be in a proliferation stage by culturing the tissue culture seedling on the sugarcane and sucrose culture medium to form a rootless tissue culture seedling; simultaneously measuring delta of cane sugar in tissue culture medium13C value deltaCSAnd delta of leaf of plant to be tested grown in the tissue culture room only by using carbon dioxide13C value deltaC；
Thirdly, two nitrates with the stable nitrogen isotope value difference value of more than 10 per mill are screened out by measuring the stable nitrogen isotope ratio of the nitrate in the culture medium for configuring the plant tissue culture, and the stable nitrogen isotope values are delta and delta respectively15N01And delta15N02(ii) a The two nitrates are respectively used as the only nitrogen sourceSetting the culture medium as the nitrate concentration to be inspected;
fourthly, respectively culturing the tissue culture seedlings to be detected with 1 single bud in a culture medium with two kinds of nitrates with different stable nitrogen isotope values as unique nitrogen sources;
fifthly, after the tissue culture seedling is cultured for 5 weeks, the stable nitrogen isotope values of the leaves of the tissue culture seedling cultured by the culture medium with the two nitrates as the unique nitrogen source are respectively measured, and the stable nitrogen isotope values of the leaves are respectively marked as delta15NN1And delta15NN2；
Sixth, by determining the stable nitrogen isotope ratio δ of ammonium salts in the medium used for preparing the plant tissue culture15NASelecting the stable nitrogen isotope ratio delta15NAAnd delta15N01And delta15N02Ammonium salts all greater than 10 per mill;
seventhly, the stable nitrogen isotope value is delta15N01And delta15N02The nitrate and the ammonium salt are respectively prepared into two groups of mixed nitrogen source culture media;
eighthly, similarly, simultaneously culturing the tissue culture seedlings to be detected with 1 single bud on the two groups of mixed nitrogen source culture media respectively;
ninthly, similarly, after the tissue culture seedling is cultured for 5 weeks, measuring the stable nitrogen isotope value of the lower leaf of the tissue culture seedling cultured on the two groups of mixed nitrogen source culture media, wherein the stable nitrogen isotope value of the ammonium salt and the stable nitrogen isotope value are delta15N01The stable nitrogen isotope value of the leaf of the tissue culture seedling cultured by the mixed nitrogen source culture medium consisting of the nitrate is recorded as delta15NN1mixThe isotopic value of ammonium salt to stable nitrogen is delta15N02The stable nitrogen isotope value of the leaf of the tissue culture seedling cultured by the mixed nitrogen source medium consisting of the nitrate is recorded as delta15NN2mix；
Tenthly, simultaneously measuring the leaf stable carbon isotope value delta of the tissue culture seedling cultured on the two groups of mixed nitrogen source culture mediaCTAnd carbon and nitrogen content, and recording the C/N ratio of the blade as R;
eleventh, using δ15NN1、δ15NN2And delta15NN1mixAnd delta15NN2mixCalculating the nitrate utilization ratio f of the tissue culture seedlingsN(ii) a Further calculating the utilization portion f of ammonium saltA；
Twelfth, using δCS、δCTAnd deltaC(ii) a Calculating the autotrophic portion f of the tissue culture seedlingsAT(ii) a Further calculating the heterotrophic part fHT(ii) a Calculating the autotrophic portion f of the tissue culture seedlingsATThe method comprises the following steps: will deltaCS、δCTAnd deltaCThe substitution equation:
Figure FDA0003514700080000021
heterotrophic fraction fHTThe formula of (1) is: f. ofHT＝1-fAT；
Thirteenth, R, f according to bladeA、fN、fATAnd fHTFurther obtaining the autotrophic and heterotrophic efficiency of different nitrogen sources of the tissue culture seedling; the method for obtaining the autotrophic and heterotrophic efficiency of different nitrogen sources of the tissue culture seedling comprises the following steps: autotrophic efficiency of ammonium Nitrogen EAT-A，EAT-A＝RfAT/fA(ii) a Heterotrophic efficiency E of ammonium NitrogenHT-A，EHT-A＝RfHT/fA(ii) a Autotrophic efficiency of nitrate nitrogen EAT-N，EAT-N＝RfAT/fN(ii) a Heterotrophic efficiency E of nitrate nitrogenHT-N，EHT-N＝RfHT/fN。
2. The method of claim 1, wherein the method comprises the steps of: in the second step, delta of leaf blade of plant to be tested grown only by carbon dioxide in the tissue culture room is obtained13C value deltaCThe method comprises the following steps: during the experiment, the seeds corresponding to the tissue culture seedlings are sown in the corresponding culture medium, then cultured in the tissue culture room for 5 weeks, and finally the delta of the new leaves is determined13C value deltaC。
3. The method of claim 1, wherein the method comprises the steps of: in the tenth step, the C/N value R of the blade is determined by: the carbon content C and the nitrogen content N of the leaf are simultaneously measured by an element analyzer, and the C/N value R is as follows: and R is C/N.
4. The method of claim 1, wherein the method comprises the steps of: in the eleventh step, the nitrate utilization part f of the tissue culture seedlings is calculatedNThe method comprises the following steps: will delta15NN1、δ15NN2And delta at the nitrate concentration15NN1mixAnd delta15NN2mixSubstituting the equation:
Figure FDA0003514700080000022
ammonium salt utilization fraction fAThe formula of (1) is: f. ofA＝1-fN。
CN202110823014.3A 2021-07-21 2021-07-21 Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings Active CN113567624B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110823014.3A CN113567624B (en) 2021-07-21 2021-07-21 Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110823014.3A CN113567624B (en) 2021-07-21 2021-07-21 Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings

Publications (2)

Publication Number Publication Date
CN113567624A CN113567624A (en) 2021-10-29
CN113567624B true CN113567624B (en) 2022-04-22

Family

ID=78165940

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110823014.3A Active CN113567624B (en) 2021-07-21 2021-07-21 Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings

Country Status (1)

Country Link
CN (1) CN113567624B (en)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102511362B (en) * 2011-10-27 2013-05-15 中国科学院地球化学研究所 Method for utilizing double markers to acquire share of inorganic carbon source utilized by plants
US8349587B2 (en) * 2011-10-31 2013-01-08 Ginkgo Bioworks, Inc. Methods and systems for chemoautotrophic production of organic compounds
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
CN105181820B (en) * 2015-09-07 2017-06-16 中国科学院地球化学研究所 A kind of determination method of the stable carbon isotope fractionation value during microalgae carton dioxide assimilation
CN105684899B (en) * 2016-01-21 2018-09-21 中国科学院地球化学研究所 The assay method of tissue-cultured seedling sucrose utilization rate
CN107449858B (en) * 2017-05-31 2020-03-24 中国科学院地球化学研究所 Method for measuring utilization efficiency of plant ammonium nitrogen

Also Published As

Publication number Publication date
CN113567624A (en) 2021-10-29

Similar Documents

Publication Publication Date Title
Hodgkinson Influence of partial defoliation on photosynthesis, photorespiration and transpiration by lucerne leaves of different ages
CN107449858B (en) Method for measuring utilization efficiency of plant ammonium nitrogen
Hajari et al. Nitrogen use efficiency of sugarcane (Saccharum spp.) varieties under in vitro conditions with varied N supply
Lötscher et al. Vertical leaf nitrogen distribution in relation to nitrogen status in grassland plants
Ullrich et al. Influence of light, temperature, and macronutrients on growth and scopolamine biosynthesis in Duboisia species
Jing et al. Regulatory effects of root pruning on leaf nutrients, photosynthesis, and growth of trees in a closed-canopy poplar plantation
CN113567624B (en) Method for quantitatively measuring autotrophic and heterotrophic efficiency of different nitrogen sources of tissue culture seedlings
Fei et al. Grain chalkiness is reduced by coordinating the biosynthesis of protein and starch in fragrant rice (Oryza sativa L.) grain under nitrogen fertilization
Cassells et al. Seasonal and physiological aspects of the isolation of tobacco protoplasts
CN116671395A (en) Cold resistance identification method for forage germplasm resources
CN105684899A (en) Determining method of tissue cultured seedling sucrose
CN107153091B (en) The measuring method of the inorganic nitrogen isotope fractionation value of plant assimilating ammonium salt
Zhang et al. The δ 15 N response and nitrate assimilation of Orychophragmus violaceus and Brassica napus plantlets in vitro during the multiplication stage cultured under different nitrate concentrations
Tian et al. Indica rice restorer lines with large sink potential exhibit improved nutrient transportation to the panicle, which enhances both yield and nitrogen-use efficiency
Li et al. Effects of benzyladenine and naphthalene acetic acid on growth and camptothecin accumulation in Camptotheca acuminata seedlings
Barbón et al. Influence of in vitro environment on somatic embryogenesis of Coffea arabica L. cv. Caturra rojo: the effects of carbon dioxide on embryogenic cell suspensions
Chabite et al. Mode of managing nutrient solution based on N use efficiency for lettuce (Lactuca sativa L.)
CN102511397A (en) Method for inducing populus calli and induction culture medium
CN109169284B (en) Method for predicting assimilation ammonium salt share of tissue culture seedlings under mixed nitrogen source and optimum ammonium salt concentration in culture medium
Bo et al. Biology organic fertilizer affects the quality and yield of flax under different irrigation regumes.
Timotiwu et al. The Effect of Silica And Manganese Aplication on Rice Growth and Yield
Daneshmand et al. Investigation of the effects of plant density and planting date on the quantitative and qualitative yields of two advanced soybean lines
CN108424208A (en) A kind of mill water culture nutrient solution and its preparation and application suitable for soybean nodulation
Vooková et al. Optimization of Algerian fir somatic embryos maturation
Sultonova Effect Of Mineral Elements On Potato Photosynthetic Productivity

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