CN105165435A - Method for determining influence of acidity on selective absorption of plants to nitrogen source - Google Patents
Method for determining influence of acidity on selective absorption of plants to nitrogen source Download PDFInfo
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- CN105165435A CN105165435A CN201510664023.7A CN201510664023A CN105165435A CN 105165435 A CN105165435 A CN 105165435A CN 201510664023 A CN201510664023 A CN 201510664023A CN 105165435 A CN105165435 A CN 105165435A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention provides a method for determining the influence of acidity on selective absorption of plants to a nitrogen source. In the method, the processes of culture of aseptic seedlings, determination of the influence of long-term acidity on selective absorption of plants to the nitrogen source, determination of the influence of short-term specific acidity on absorption of plants to the nitrogen source, determination of the influence of short-term specific acidity on the absorption manner of plants to the nitrogen source, sample treatment, result computation and the like are carried out, so that the influence of acidity on absorption of plants to ammonium nitrogen, nitric nitrogen and amino acid is deeply analyzed. According to the method, the design is reasonable, the nutrition contribution of amino acid to plants can be deeply analyzed by adopting a completely aseptic cultivating environment, and the influence of acidity on absorption of plants to different nitrogen sources can be effectively and accurately distinguished by adopting an isotope labeling technology, so that the influence of environmental factors, especially the acidity, on absorption of plants to specific nitrogen sources can be accurately and deeply researched. The method provided by the invention provides a foundation to the research of the influence of future environmental change on plant growth, especially nitrogen absorption.
Description
Technical field
The present invention relates to Ecological Research Methods field, particularly relate to a kind of method measuring Effect of Acidity On Absorption plant selectable absorption nitrogenous source.
Background technology
Comprise multiple containing nitrogen compound in soil, as inorganic nitrogen, amino acid, polypeptide and protein, except inorganic nitrogen, amino acid also can as the important nitrogenous source of plant growth.The vascular plant of Chapin etc. (1993) Late Cambrian sterilized root is liked absorbing amino acid, sterilized root plant Aries hair beard grass in tundra, the arctic absorbs amino acid whose amount and at least accounts for 60% of total absorption of nitrogen, and with amino acid for the nitrogen of absorption when only nitrogen source is cultivated and the biomass of formation are all than many when taking inorganic nitrogen as nitrogenous source.Many researchers by root system short-term absorption test, laboratory organic nitrogen source sterile culture experiment, field plant to tagging organic substance (
13c,
15n double labelling) absorption and plant tissue
15the methods such as N natural abundance, show that many plants can absorb the organic nitrogen compound such as several amino acids, simple protein in surrounding medium, and just can be absorbed and used by plants without the need to organic nitrogen being converted into inorganic nitrogen.
Occurring in nature is soil types, temperature, humidity, acidity, CO such as
2the Eco Environmental Factors such as concentration and N level can affect the researchs such as the biological effectiveness .Warren of SOIL ORGANIC NITROGEN and find high mountain and area, arctic plant preferential absorption organic nitrogen (as Gly) under cryogenic, under the high temperature conditions preferential absorption inorganic nitrogen.Baligar etc. also report that acidity affects the accumulation of legume organic matter and biomass by photosynthesis, and then affect the absorption of fabaceous growth and nutrient.Xu etc. find that intensity of illumination is to Gly and NH
4 +absorption do not make significant difference, but by changing maize root system form and with the competitive relation of rhizospheric microorganism and then affect the absorption of plant to organic nitrogen.The crop such as paddy rice, tomato confirms high concentration CO
2tiller number, root/hat, photosynthesis, phytomass and plant nitrogen absorptive amount and microorganism amount of nitrogen fixation can be increased, also there is high yield and high quality effect simultaneously.Godlewski etc. try material with 15 kinds of farmlands and wild-type plant for supplying, aseptically study them to the absorption of organic nitrogen and environmental adaptation mechanism thereof, find that all root systems of plant can both secretory protein hydrolase, its secretory volume and Root morphology, rhizospheric environment are closely related.In addition, a lot of research finds under varying level external source organic nitrogen condition, and amino-acid nitrogen is suitable with inorganic nitrogen (ammonium nitrogen, nitrate nitrogen) to plant nitrogen nutrition contribution, is even greater than inorganic nitrogen.Different plant species can form the adaptation mechanism of oneself uniqueness, and then strengthen the Amino Acid Absorption in rhizospheric environment.
Acidity is one of most important envirment factor affecting plant growth, has important impact to the selective absorbing of different nitrogen sources.Along with a large amount of uses of chemical fertilizer, the arable land of China presents large-area acidifying.Acidity also exists huge difference with landing pit change, presents from north orientation south the trend reduced gradually.Research acidity has important ecological significance to nitrogen absorbed by plant.But amino acid can be decomposed rapidly by microorganism in its natural state, limit the research of amino acid nutrient to a certain extent.Therefore, long-term gnotobasis is the important foundation of research metabolism of amino acid physiology and such environmental effects nitrogenous source selective absorbing.
Summary of the invention
The object of this invention is to provide a kind of method measuring Effect of Acidity On Absorption plant selectable absorption nitrogenous source, be achieved through the following technical solutions:
(1) cultivation of aseptic seedling: choose full plant seed, soak 12h at normal temperatures, adopt 70% alcohol-pickled 1 minute successively afterwards, aseptic water washing 3 times, 10% hydrogen peroxide soak 5 minutes, aseptic water washing 3 times, 0.1molL
-1mercury chloride soaks 5 minutes, the sterilizing methods of aseptic water washing 5 times, seed after sterilizing is positioned in autoclaved culture dish the 3-5 days that germinates again, treat that plant root growth is to about 1cm, a seed is positioned over and sterilizedly fills in the 50ml centrifuge tube of 0.5% agar, be placed on aseptic culture platform and cultivate 1-3 days, the aperture that plant is 0.3cm by the diameter at centrifuge tube lid center grows, adopt the large 704 silicone rubber seal apertures in south, this silica gel is softer, the growth of plant can not be limited, after shaping silica gel, add containing 4mmolL
-1caCl
2, 2mmolL
-1k
2sO
4, 2mmolL
-1kH
2pO
4, 1.4mmolL
-1mgSO
47H
2o, 0.1 μm of olL
-1naMoO
42H
20,0.4 μm of olL
-1cuSO
45H
2o, 1 μm of olL
-1znSO
47H
2o, 8 μm of olL
-1h
3bO
3, 10 μm of olL
-1mnCl
2, 5 μm of olL
-1na
2eDTA and 18.3 μm olL
-1feSO
47H
2the nutrient solution of O, utensil used and all adopt autoclave sterilization without nitrogen nutrition liquid in test, adopted 0.22 μm of filter membrane to nitrogenous source sterilizing.
(2) long-term acidity absorbs the impact of nitrogenous source on plant selectable: supply examination nitrogenous source to be NO
3 -, NH
4 +, glycine, three kinds of nitrogenous source (NO
3 -, NH
4 +, glycine) etc. nitrogen quantity mixing, total concentration is 3mM, and each only mark wherein a kind of nitrogenous source (label Abundances is 50.22at.%
15nO
3 -, 50.17at.%
15nH
4 +, 50.16at.%
15n-glycine), acidity establishes pH=4.0,5.0,6.0,7.0,8.0 5 gradients, totally 15 process, adopts and adds 1molL
-1h
2sO
4or NaOH regulates acidity, often kind of acidity process arranges one blank, to detect natural abundance, within every three days, changes one time of nutrition liquid, cultivates after 25 days, the biomass of mensuration plant shoot and root system and
15n abundance.
(3) the specific acidity of short-term is on the impact of plant absorption nitrogenous source
With 3mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, adopt 1) method aseptic culture plant 25 days, under this nitrogenous source proportioning, plant root growth is vigorous, to reach the requirement detected sample quality, get the plant seedlings that growing way is basically identical, adopt sterile water centrifuge tube and root system of plant repeatedly to be rinsed, be placed on " hunger " in sterile water and cultivate a whole night (about 10h), plant is positioned over containing 3mM98.10at.%
15in the solution of N-glycine, if pH=4.2,6.2,8.2 3 acidity process, destructive sampling after absorbing 4h.
(4) the specific acidity of short-term is on the impact of nitrogen absorbed by plant source side formula
With 3mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, adopt 1) method aseptic culture plant 25 days, under this nitrogenous source proportioning, plant root growth is vigorous, to reach the requirement detected sample quality, get the plant seedlings that growing way is basically identical, adopt sterile water centrifuge tube and root system of plant repeatedly to be rinsed, be placed on " hunger " in sterile water and cultivate a whole night (about 10h), choose the plant seedlings that 6 strain growing ways are consistent, its root system is positioned in advance 50 μm of olL
-1cCCP(protonphorecarbonylcyanidem-chlorophenylhydrazone) in solution 1 hour, make its root system inactivation, then to contrast without the seedling of CCCP process, plant is positioned over containing 3mM98.10at.%
15in the solution of N-glycine, if pH=4.2,6.2,8.2 3 acidity process, carry out the short-term absorption pattern test marking nitrogenous source, destructive sampling after absorbing 4h.
(5) sample treatment
After the plant sample absorbed through Long-term absorption and short-term is gathered, by overground part and underground part separately, root system of plant first uses Ultrasonic Cleaning, then uses 0.5molL
-1caCl
2remove the nitrogen being adsorbed on root system surface, finally clean with aseptic water washing, and blot with blotting paper, root system of plant and overground part sample are with after freeze dryer freeze drying, weigh, pulverize with ball milling instrument, high-temperature sample disappears and boils oxidation, total nitrogen content semimicro kjeldahl apparatus distillation, finally uses 0.01molL
-1sulfuric acid titration, employing isotope mass spectrometer mensuration plant different parts
15n Abundances.
(6) nitrogenous source is measured
Be determine according to the 15N content in plant corpus according to the absorption of plant to a certain nitrogenous source, adopt following formulae discovery:
n uptake refer to the amount that root system of plant or overground part absorb a certain nitrogenous source;
a s root system of plant or overground part
15n Abundances;
a c refer to and do not add mark nitrogenous source
15the blank Abundances of N;
a f being the Abundances of a certain mark nitrogenous source in mixed nitrogen, in long-term acidity in the impact of plant absorption nitrogenous source, is 50.16% (glycine), 50.22% (NO
3 -), 50.17% (NH
4 +), in short-term absorbs, be 98.10% (glycine);
refer to the nitrogen pool of plant shoot or root system.
The present invention is the important foundation of research amino acid nutrient according to gnotobasis, the method of single nitrogenous source mark is the effective ways of specific forms of nitrogen in research environment factor pair plant absorption mixed nitrogen, effectively these two kinds of methods combining are got up, achieve the accurate and deep research environment factor especially acidity on the impact of the specific nitrogenous source of plant absorption.The present invention by cultivating aseptic seedling, measure long-term acidity the impact of nitrogenous source, the specific acidity of short-term absorbed on the impact of plant absorption nitrogenous source, the specific acidity of short-term to the process such as impact, sample treatment, result calculating of nitrogen absorbed by plant source side formula to plant selectable, analyse in depth acidity to plant absorption ammonium nitrogen, nitrate nitrogen, amino acid whose impact, for the change of research FUTURE ENVIRONMENT provides method basis to the impact of plant growth especially Nitrogen Absorption.The present invention adopts the culture environment of integral asepsis, analyses in depth amino acid and contributes the nutrition of plant, adopt isotope labelling techniques, can distinguish the impact of acidity on plant absorption different nitrogen sources effectively accurately.
Accompanying drawing explanation
Fig. 1 is acidity on the impact of a variety of Chinese cabbage overground part and root system fresh weight.
Fig. 2 is that acidity absorbs the impact of different nitrogen sources to a variety of Chinese cabbage overground part.
Fig. 3 is that acidity absorbs the impact of different nitrogen sources ratio to a variety of Chinese cabbage.
Fig. 4 is that acidity absorbs the impact of glycine mode to a variety of Chinese cabbage.
Fig. 5 is the impact that acidity absorbs a variety of Chinese cabbage overground part glycine.
Fig. 6 is the impact that acidity absorbs a variety of Chinese cabbage root system glycine.
Fig. 7 is the impact of acidity on a variety of Chinese cabbage root system glycine speed.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
embodiment one:
(1) cultivation of aseptic seedling: choose full plant seed, soak 12h at normal temperatures, adopt 70% alcohol-pickled 1 minute successively afterwards, aseptic water washing 3 times, 10% hydrogen peroxide soak 5 minutes, aseptic water washing 3 times, 0.1molL
-1mercury chloride soaks 5 minutes, the sterilizing methods of aseptic water washing 5 times, seed after sterilizing is positioned in autoclaved culture dish the 3-5 days that germinates again, treat that plant root growth is to about 1cm, a seed is positioned over and sterilizedly fills in the 50ml centrifuge tube of 0.5% agar, be placed on aseptic culture platform and cultivate 1-3 days, the aperture that plant is 0.3cm by the diameter at centrifuge tube lid center grows, adopt the large 704 silicone rubber seal apertures in south, this silica gel is softer, the growth of plant can not be limited, after shaping silica gel, add containing 4mmolL
-1caCl
2, 2mmolL
-1k
2sO
4, 2mmolL
-1kH
2pO
4, 1.4mmolL
-1mgSO
47H
2o, 0.1 μm of olL
-1naMoO
42H
20,0.4 μm of olL
-1cuSO
45H
2o, 1 μm of olL
-1znSO
47H
2o, 8 μm of olL
-1h
3bO
3, 10 μm of olL
-1mnCl
2, 5 μm of olL
-1na
2eDTA and 18.3 μm olL
-1feSO
47H
2the nutrient solution of O, utensil used and all adopt autoclave sterilization without nitrogen nutrition liquid in test, adopted 0.22 μm of filter membrane to nitrogenous source sterilizing.
(2) long-term specific acidity is on the impact of a variety of Chinese cabbage selective absorbing nitrogenous source: supply examination nitrogenous source to be NO
3 -, NH
4 +, glycine, three kinds of nitrogenous source (NO
3 -, NH
4 +, glycine) etc. nitrogen quantity mixing, total concentration is 3mM, and each only mark wherein a kind of nitrogenous source (label Abundances is 50.22at.%
15nO
3 -, 50.17at.%
15nH
4 +, 50.16at.%
15n-glycine), acidity is pH=4.0,5.0,6.0,7.0,8.0 5 gradients, totally 15 process ((T1:pH=4.0-50.22at.%
15nO
3 --NH
4 +-glycine, T2:pH=4.0-NO
3 --50.17at.%
15nH
4 +-glycine, T3:pH=4.0-NO
3 --NH
4 +-50.16at.%
15n-glycine, T4:pH=5.0-50.22at.%
15nO
3 --NH
4 +-glycine, T5:pH=5.0-NO
3 --50.17at.%
15nH
4 +-glycine, T6:pH=5.0-NO
3 --NH
4 +-50.16at.%
15n-glycine, T7:pH=6.0-50.22at.%
15nO
3 --NH
4 +-glycine, T8:pH=6.0-NO
3 --50.17at.%
15nH
4 +-glycine, T9:pH=6.0-NO
3 --NH
4 +-50.16at.%
15n-glycine, T10:pH=7.0-50.22at.%
15nO
3 --NH
4 +-glycine, T11:pH=7.0-NO
3 --50.17at.%
15nH
4 +-glycine, T12:pH=7.0-NO
3 --NH
4 +-50.16at.%
15n-glycine, T13:pH=8.0-50.22at.%
15nO
3 --NH
4 +-glycine, T14:pH=8.0-NO
3 --50.17at.%
15nH
4 +-glycine, T15:pH=8.0-NO
3 --NH
4 +-50.16at.%
15n-glycine, does not add at.% and shows that nitrogenous source is common
14n), interpolation 1molL is adopted
-1h
2sO
4or NaOH regulates acidity, often kind of acidity process arranges one blank, to detect natural abundance, within every three days, changes one time of nutrition liquid, cultivates after 25 days, the biomass of mensuration a variety of Chinese cabbage overground part and root system and
15n abundance.
(3) the specific acidity of short-term absorbs the impact of nitrogenous source on a variety of Chinese cabbage
With 3mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, adopt step (1) method aseptic culture a variety of Chinese cabbage 25 days, under this nitrogenous source proportioning, a variety of Chinese cabbage root growth is vigorous, to reach the requirement of instrument detection to sample quality, get a variety of Chinese cabbage seedling that growing way is basically identical, adopt sterile water centrifuge tube and a variety of Chinese cabbage root system repeatedly to be rinsed, be placed on " hunger " in sterile water and cultivate a whole night (about 10h), a variety of Chinese cabbage is positioned over containing 3mM98.10at.%
15in the solution of N-glycine, acidity establishes pH=4.2,6.2,8.2 3 gradients, adopts and adds 1molL
-1h
2sO
4or NaOH regulates acidity, carry out the short-term absorption test marking nitrogenous source afterwards, 6 repetitions are established in each process, and completely random arranges, and culture environment is consistent with the culture environment of preliminary seedling, destructive sampling after absorbing 4h.
(4) the specific acidity of short-term is on the impact of a variety of Chinese cabbage absorbed nitrogen source side formula
With 3mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, adopt 1) method aseptic culture a variety of Chinese cabbage 25 days, under this nitrogenous source proportioning, a variety of Chinese cabbage root growth is vigorous, to reach the requirement detected sample quality, get a variety of Chinese cabbage seedling that growing way is basically identical, adopt sterile water centrifuge tube and a variety of Chinese cabbage root system repeatedly to be rinsed, be placed on " hunger " in sterile water and cultivate a whole night (about 10h), choose a variety of Chinese cabbage seedling that 6 strain growing ways are consistent, its root system is positioned in advance 50 μm of olL
-1in CCCP solution 1 hour, make its root system inactivation, then to contrast without the seedling of CCCP process, a variety of Chinese cabbage is positioned over containing 3mM98.10at.%
15in the solution of N-glycine, acidity establishes pH=4.2,6.2,8.2 3 gradients, adopts and adds 1molL
-1h
2sO
4or NaOH regulates acidity, carry out the short-term absorption pattern test marking nitrogenous source, destructive sampling after absorbing 4h, after CCCP process root system, limit the active transport of root system, its nitrogenous source absorbed is Passive intake, is then the summation of active absorption and Passive intake without CCCP process.
(5) sample treatment
After a variety of Chinese cabbage sample absorbed through Long-term absorption and short-term is gathered, by overground part and underground part separately, a variety of Chinese cabbage root system first uses Ultrasonic Cleaning, then uses 0.5molL
-1caCl
2remove the nitrogen being adsorbed on root system surface, finally clean with aseptic water washing, and blot with blotting paper, a variety of Chinese cabbage root system and overground part sample are with after freeze dryer freeze drying, weigh, pulverize with ball milling instrument, high-temperature sample disappears and boils oxidation, total nitrogen content semimicro kjeldahl apparatus distillation, finally uses 0.01molL
-1sulfuric acid titration, employing isotope mass spectrometer mensuration plant different parts
15n Abundances.
(6) nitrogenous source is measured
A variety of Chinese cabbage to the absorption of a certain nitrogenous source according in a variety of Chinese cabbage body
15n content is determined, adopts following formulae discovery:
n uptake refer to the amount that root system of plant or overground part absorb a certain nitrogenous source;
a s root system of plant or overground part
15n Abundances;
a c refer to and do not add mark nitrogenous source
15the blank Abundances of N;
a f being the Abundances of a certain mark nitrogenous source in mixed nitrogen, in long-term acidity in the impact of plant absorption nitrogenous source, is 50.16% (glycine), 50.22% (NO
3 -), 50.17% (NH
4 +), in short-term absorbs, be 98.10% (glycine);
refer to the nitrogen pool of plant shoot or root system;
Adopt aseptic culture in conjunction with isotope-labeled method, the research acidity that can go deep into absorbs the impact of different nitrogen sources to a variety of Chinese cabbage, and the impact of the Nitrogen Absorption aspect that the change for following natural environment may bring provides theoretical foundation.
As can be seen from Figure 1, acidity has significant effect to Growth of Cabbage, and a variety of Chinese cabbage overground part and root system fresh weight present the trend first raising and reduce afterwards within the scope of pH=4.0-8.0, arrives maximum 7.0.And increase along with the continuation of acidity, there is obvious inhibitory action to Growth of Cabbage.The fit equation of nutrient solution pH and overground part fresh weight is: y=-0.17x
2+ 2.08x-3.73 (r=0.71
*), infer optimally upper grown pH be 6.23.
As can be seen from Figure 2, the selective absorbing of plant to different nitrogen sources constantly changes along with the change of acidity.From the absorption of acidity to different nitrogen sources, the absorption of glycine increases along with the increase of acidity, until pH=7, be significantly higher than pH=6,8 at pH=7 place, and the absorptive amount of glycine is a little more than nitrate nitrogen and ammonium nitrogen.Under the condition of pH=6, the absorptive amount of ammonium nitrogen and nitrate nitrogen is the highest, and this is consistent with the biomass rule of overground part.
Account for the ratio of total nitrogen from a certain nitrogenous source of Fig. 3, the nutrition contribution rate of nitrate nitrogen presents the trend reduced gradually, and ammonium nitrogen is then without visible trend, and this also meets forefathers and studies the ammonium nitrogen that shows to the insensitive conclusion of pH.
Fig. 4 shows, under low pH environment, a variety of Chinese cabbage Passive intake significantly increases, and active absorption slightly declines, and shows that acidity changes the mode of Root Absorption glycine.
As can be seen from Figure 5, acidity does not make significant difference to a variety of Chinese cabbage overground part glycine state nitrogen content, shows that acidity mainly acts on root system to the impact that a variety of Chinese cabbage absorbs nitrogenous source.
As can be seen from Figure 6, acidity significantly affects the mode of a variety of Chinese cabbage Root Absorption glycine, and under low pH condition, Passive intake increases, and active absorption reduces, and under high pH, active absorption and Passive intake all reduce.
As can be seen from Figure 7, high pH reduces the amount of active absorption, thus reduces the gross absorption of glycine, and under low pH, the absorption rate of glycine does not significantly reduce.
In sum, acidity has significant impact to glycine absorption and absorption pattern.Be under the condition of 6.2 at pH, under glycine gross absorption and active absorption amount are 4.2 and 8.2 conditions higher than pH, and Passive intake amount remarkable be 4.2 process lower than pH.The environmental influence of a variety of Chinese cabbage root growth root system active absorption and Passive intake, understand acidity in depth and absorb nitrogen rule and autogenous control to a variety of Chinese cabbage, be do not deal with the most important theories basis that soil continues acidifying.The a kind of of this Invention Announce measures the method that Effect of Acidity On Absorption plant selectable absorbs nitrogenous source, combines aseptic culture and isotope-labeled advantage, for the impact of research environment factor on plant absorption nitrogenous source provides method basis.
Claims (3)
1. measure the method that Effect of Acidity On Absorption plant selectable absorbs nitrogenous source, it is characterized in that, realized by following steps:
(1) cultivation of aseptic seedling: choose full plant seed, soak 12h at normal temperatures, adopt 70% alcohol-pickled 1 minute successively afterwards, aseptic water washing 3 times, 10% hydrogen peroxide soak 5 minutes, aseptic water washing 3 times, 0.1molL
-1mercury chloride soaks 5 minutes, the sterilizing methods of aseptic water washing 5 times, again the seed after sterilizing is positioned in culture dish the 3-5 days that germinates afterwards, treat that plant root growth is to about 1cm, a seed is positioned over and sterilizedly fills in the 50ml centrifuge tube of 0.5% agar, be placed on aseptic culture platform and cultivate 1-3 days, the aperture that plant is 0.3cm by the diameter at centrifuge tube lid center grows, and adopts the large 704 silicone rubber seal apertures in south, after shaping silica gel, add nutrient solution;
(2) long-term acidity absorbs the impact of nitrogenous source on plant selectable: supply examination nitrogenous source to be NO
3 -, NH
4 +, glycine, three kinds, etc. nitrogen quantity mixing, total concentration is 3mM, and each only mark wherein a kind of nitrogenous source, and label Abundances is 50.22at.%
15nO
3 -, 50.17at.%
15nH
4 +, 50.16at.%
15n-glycine, acidity is pH=4.0,5.0,6.0,7.0,8.0 5 gradients, totally 15 process, adopts and adds 1molL
-1h
2sO
4or NaOH regulates acidity, often kind of acidity process arranges one blank, to detect natural abundance, within every three days, changes one time of nutrition liquid, cultivates after 25 days, the biomass of mensuration plant shoot and root system and
15n abundance;
(3) short-term measures acidity to the impact of plant absorption nitrogenous source: with 3mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, adopt step (1) method aseptic culture plant 25 days, get the plant seedlings that growing way is basically identical, sterile water is adopted centrifuge tube and root system of plant repeatedly to be rinsed, be placed on " hunger " in sterile water and cultivate a whole night, plant is positioned over containing 3mM98.10at.%
15in the solution of N-glycine, with pH=4.2,6.2,8.2 3 acidity process, destructive sampling after absorbing 4h;
(4) the specific acidity of short-term is on the impact of nitrogen absorbed by plant source side formula: with 3mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, adopt step (1) method aseptic culture plant 25 days, get the plant seedlings that growing way is basically identical, sterile water is adopted centrifuge tube and root system of plant repeatedly to be rinsed, be placed on " hunger " in sterile water and cultivate a whole night, choose the plant seedlings that 6 strain growing ways are consistent, its root system is positioned in advance 50 μm of olL
-1in CCCP solution 1 hour, make its root system inactivation, then to contrast without the seedling of CCCP solution-treated, plant is positioned over containing 3mM98.10at.%
15in the solution of N-glycine, with pH=4.2,6.2,8.2 3 acidity process, carry out the short-term absorption pattern test marking nitrogenous source, destructive sampling after absorbing 4h;
(5) sample treatment: after the plant sample that the short-term through the Long-term absorption of step (2) and step (3), (4) absorbs is gathered, by overground part and underground part separately, root system of plant first uses Ultrasonic Cleaning, then uses 0.5molL
-1caCl
2remove the nitrogen being adsorbed on root system surface, finally clean with aseptic water washing, and blot with blotting paper, root system of plant and overground part sample are with after freeze dryer freeze drying, weigh, pulverize with ball milling instrument, high-temperature sample disappears and boils oxidation, total nitrogen content semimicro kjeldahl apparatus distillation, finally uses 0.01molL
-1sulfuric acid titration, employing isotope mass spectrometer mensuration plant different parts
15n Abundances;
(6) nitrogenous source is measured:
According to plant to the absorption of a certain nitrogenous source according in plant corpus
15n content is determined, adopts following formulae discovery:
n uptake refer to the amount that root system of plant or overground part absorb a certain nitrogenous source,
a s root system of plant or overground part
15n Abundances,
a c refer to and do not add mark nitrogenous source
15the blank Abundances of N,
a f being the Abundances of a certain mark nitrogenous source in mixed nitrogen, in long-term acidity in the impact of plant absorption nitrogenous source, is 50.16% glycine, 50.22%NO
3 -, 50.17%NH
4 +, in short-term absorbs, be 98.10% glycine,
refer to the nitrogen pool of plant shoot or root system.
2. a kind of method measuring Effect of Acidity On Absorption plant selectable absorption nitrogenous source according to claim 1, it is characterized in that, the nutrient solution of step (1) consists of: 4mmolL
-1caCl
2, 2mmolL
-1k
2sO
4, 2mmolL
-1kH
2pO
4, 1.4mmolL
-1mgSO
47H
2o, 0.1 μm of olL
-1naMoO
42H
20,0.4 μm of olL
-1cuSO
45H
2o, 1 μm of olL
-1znSO
47H
2o, 8 μm of olL
-1h
3bO
3, 10 μm of olL
-1mnCl
2, 5 μm of olL
-1na
2eDTA and 18.3 μm olL
-1feSO
47H
2o.
3. a kind of method measuring Effect of Acidity On Absorption plant selectable absorption nitrogenous source according to claim 1, it is characterized in that, in test, utensil used and nutrient solution all adopt autoclave sterilization, adopt 0.22 μm of filter membrane to nitrogenous source sterilizing.
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| CN114324048A (en) * | 2021-11-15 | 2022-04-12 | 广西壮族自治区中国科学院广西植物研究所 | Method for monitoring plant growth and nutritional characteristics of vine root traditional Chinese medicinal materials |
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