CN111386987B - Planting method for improving selenium content of pakchoi without adding exogenous selenium - Google Patents
Planting method for improving selenium content of pakchoi without adding exogenous selenium Download PDFInfo
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- 239000011669 selenium Substances 0.000 title claims abstract description 98
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 97
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 97
- 238000000034 method Methods 0.000 title claims abstract description 42
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- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 title claims abstract description 41
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 claims abstract description 92
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims abstract description 46
- 229960004889 salicylic acid Drugs 0.000 claims abstract description 46
- 239000002689 soil Substances 0.000 claims abstract description 28
- 238000005507 spraying Methods 0.000 claims abstract description 11
- 238000007796 conventional method Methods 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000009331 sowing Methods 0.000 claims abstract description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
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- 238000003756 stirring Methods 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 2
- 235000020679 tap water Nutrition 0.000 claims description 2
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- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 3
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 3
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- 230000003213 activating effect Effects 0.000 description 3
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- 241001301148 Brassica rapa subsp. oleifera Species 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 108010060806 Photosystem II Protein Complex Proteins 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
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- 230000029553 photosynthesis Effects 0.000 description 2
- 238000010672 photosynthesis Methods 0.000 description 2
- 239000003375 plant hormone Substances 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 235000019738 Limestone Nutrition 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 235000010582 Pisum sativum Nutrition 0.000 description 1
- 102000008114 Selenoproteins Human genes 0.000 description 1
- 108010074686 Selenoproteins Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
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- 235000018823 dietary intake Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
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- 230000036737 immune function Effects 0.000 description 1
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- 229910052745 lead Inorganic materials 0.000 description 1
- 239000006028 limestone Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- 229910052748 manganese Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 238000005502 peroxidation Methods 0.000 description 1
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- 230000006461 physiological response Effects 0.000 description 1
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- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 229940082569 selenite Drugs 0.000 description 1
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
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Classifications
-
- 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
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/15—Leaf crops, e.g. lettuce or spinach
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C21/00—Methods of fertilising, sowing or planting
- A01C21/005—Following a specific plan, e.g. pattern
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Soil Sciences (AREA)
- Botany (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention belongs to the technical field of planting of pakchoi, and particularly relates to a planting method for improving selenium content of pakchoi without adding exogenous selenium. A planting method for improving selenium content of pakchoi without adding exogenous selenium comprises the following steps: (1) preparing a salicylic acid solution; (2) selecting a field for planting the pakchoi: firstly, detecting the pH value and the total selenium content of soil, and planting pakchoi in the selected land; turning over, preparing land, sowing, fertilizing and watering according to a conventional method; (3) in the 4-5 leaf stage of the pakchoi, the salicylic acid solution obtained in the step (1) is diluted by 100 times by adding water and is uniformly sprayed on the front and back surfaces of the pakchoi leaves; (4) spraying salicylic acid solution for 1 time in 6-7 leaf stage of pakchoi; (5) other daily management is carried out according to a conventional method. According to the method, on the basis of certain amount of soil selenium, the selenium content of overground parts and roots of the pakchoi can be obviously improved only by spraying the salicylic acid solution.
Description
Technical Field
The invention belongs to the technical field of planting of pakchoi, and particularly relates to a planting method for improving selenium content of pakchoi without adding exogenous selenium.
Background
Selenium (Se) is one of essential trace elements of human beings, animals and certain microorganisms, participates in the synthesis of various selenase and selenoprotein in human bodies, has important effects on improving human and animal immunity, eliminating oxygen free radicals in vivo and maintaining body health, and selenium deficiency not only causes the reduction of human immune function, but also can cause diseases. Research shows that the phenomenon of selenium deficiency of human bodies is relatively common in the world, about 10 hundred million people have insufficient daily selenium intake, and the development of human health is seriously threatened by the selenium deficiency of human bodies.
Inorganic selenium is not suitable for direct consumption due to its toxicity. Crops are the main source of organic selenium in food chain because of the capability of absorbing and transforming inorganic selenium in soil, and most of selenium in human and animal bodies is directly or indirectly obtained from crops. For most people with selenium deficiency, dietary intake is the most important method for supplementing selenium. Therefore, the key to solving the problem of selenium deficiency of the crowd is to improve the selenium content of crops.
In order to better utilize selenium resources in the environment of physical absorption and serve as food sources to relieve the situation of selenium intake shortage of people and animals, a great deal of crop selenium bioaugmentation research is carried out at home and abroad, wherein the application of exogenous selenium through leaf surfaces or soil is a common practice mode for producing selenium-containing foods. The applied exogenous selenium is various, including selenate, selenite, nano-selenium, amino acid chelated selenium and various selenium-containing fertilizers. Meanwhile, in a part of areas with high selenium content in soil, the purpose of promoting the accumulation of crop selenium can be achieved by improving the biological effectiveness of the soil selenium, such as adding limestone powder, pig manure, EDTA (ethylene diamine tetraacetic acid), microbial agents and the like to the soil. In addition, a method for breeding high-efficiency selenium-rich varieties by using a traditional breeding mode or improving the selenium absorption and tolerance capacity of crops by using a transgenic technology and the like is also provided.
On the whole, the existing methods for increasing the selenium content of crops can be summarized into three types: respectively adding exogenous selenium, activating soil selenium and breeding (creating) high-efficiency selenium-rich varieties, wherein the method for adding exogenous selenium is most commonly and widely applied in the practical process. It is obvious that the method of adding exogenous selenium and activating soil selenium aims at providing crops with sufficient active selenium, physiological response and tolerance capability of the crops to selenium are not considered, and thus, a plurality of negative effects are easily caused. The technology of applying the exogenous selenium has the problems of low utilization rate of the exogenous selenium (the utilization rate of selenium fertilizer applied on soil is only 5-30 percent, the utilization rate of sodium selenite applied on soil is 0.89-3.25 percent), seedling burning, residue (41.82-82.64 percent), environmental pollution and the like; the method for activating the selenium in the soil changes the basic chemical properties of the original soil to a certain extent, has the risks of high operation difficulty, high cost, potential damage to the properties of the soil and the like, and can seriously cause the loss of beneficial elements, thereby weakening the soil strength and polluting downstream water bodies. In addition, the method for breeding (transforming) the crop varieties has long research period and high cost, has great potential but is difficult to realize production and application in a large range, and the safety of the transgenic agricultural products still has great controversy. Therefore, there is a need to search for a more energy-saving, environment-friendly, simple and effective selenium bioaugmentation method.
Research finds that improving the selenium tolerance of crops is one of the important bottlenecks in increasing the selenium content of crops. Particularly, for conventional cultivated crops with low selenium enrichment capacity, it is important to enhance the stress resistance to selenium stress. In most crops, the absorbed selenium is combined with protein by replacing the position of sulfur in the crops, the structure and the function of the original protein are disturbed, excessive active oxygen is induced to be generated, lipid peroxidation is caused, the plant tolerance is reduced, and finally, the toxic effect is generated, so that excessive selenium cannot be accumulated. The plant hormone exists in all higher plants, is an important active substance for the plants to respond to the external environment change and regulate the growth and development of the plants, and has the growth and development regulating effect similar to that of natural hormone.
Salicylic acid is one of plant hormones, can be used as a signal molecule required by plant stress resistance reaction to activate a plant defense protection mechanism, not only relates to the resistance to external biotic stress, but also participates in various abiotic stresses (high temperature, low temperature, salinization, drought, heavy metal, ultraviolet radiation, ozone and other adversities), and plays a key role in plant signal conduction and stress resistance reaction. Has been widely applied to relieve the stress of various heavy metal elements such as Cd, Hg, Ni, Pb, Mn and the like.
Research shows that, besides salicylic acid generated by plants, exogenous salicylic acid plays the same important role in plant resistance, and can effectively relieve cadmium toxicity in peas, copper toxicity in cotton and the like. Also has obvious promoting effect on promoting/reducing the accumulation of beneficial/harmful elements of the soil by plants.
Disclosure of Invention
The invention provides a planting method for improving selenium content of pakchoi without adding exogenous selenium, which takes crops as a main body and salicylic acid as a core applied substance, and promotes the selenium tolerance of the crops by the characteristics of regulating the growth of the crops and improving the stress resistance of the crops through the salicylic acid, thereby realizing the obvious improvement of the selenium accumulation amount of the crops.
The technical scheme provided by the invention is as follows:
a planting method for improving selenium content of pakchoi without adding exogenous selenium comprises the following steps:
(1) preparing a salicylic acid solution: dissolving salicylic acid in absolute ethyl alcohol in a beaker, adding tap water and Tween-20, uniformly stirring, transferring to a volumetric flask, fixing the volume, and uniformly shaking to obtain a salicylic acid solution;
(2) selecting a field for planting the pakchoi: firstly, detecting the pH value and the total selenium content of soil, and planting pakchoi in the selected land; turning over, preparing land, sowing, fertilizing and watering according to a conventional method;
(3) diluting with salicylic acid solution in water at 4-5 leaf stage of caulis et folium Brassicae Capitatae to obtain salicylic acid solution of 100 μmol/L, and uniformly spraying on the front and back surfaces of caulis et folium Brassicae Capitatae;
(4) diluting with salicylic acid solution in water at 6-7 leaf stage of caulis et folium Brassicae Capitatae to obtain salicylic acid solution of 50 μmol/L, and uniformly spraying on the front and back sides of caulis et folium Brassicae Capitatae leaf;
(5) other daily management is carried out according to a conventional method.
Preferably, the concentration of the salicylic acid solution in the step (1) is 10 mmol/L.
Preferably, the amount of Tween-20 added in step (1) is 0.5%.
Preferably, the soil pH of the land selected in the step (2) is more than or equal to 7.2, and the total selenium content is more than or equal to 0.27 mg/kg.
Preferably, the amount of the salicylic acid solution of 100. mu. mol/L used in the step (3) is 40 kg/mu.
Preferably, the amount of the salicylic acid solution of 50. mu. mol/L used in the step (4) is 40 kg/mu.
Compared with the prior art, the invention has the beneficial effects that:
compared with the existing method, the planting method of the invention is obviously different from the existing method, and the planting method of the invention does not need to additionally add various exogenous selenium and does not need to change the soil for crop growth, so the problems of environmental pollution and the like caused by applying exogenous selenium in large quantity or changing the soil property do not exist. In addition, there is no long time, high cost of breeding input and risk of transgenic crops. The method has the advantages of simple operation, low cost, environmental protection, high efficiency and the like, and has important significance for fully exerting the advantages of the selenium-rich soil resources in the selenium-rich areas.
Detailed Description
The present invention will be more readily understood by reference to the following examples, which are given without limiting the scope of the invention.
Example 1：
A planting method for improving selenium content of pakchoi without adding exogenous selenium comprises the following steps:
(1) preparing 10mmol/L salicylic acid solution: dissolving 13.812g of salicylic acid in a beaker by using 20mL of absolute ethanol, adding 400-600mL of pure water and simultaneously adding 5mL of Tween-20 (Tween-20), uniformly stirring, transferring to a 10L volumetric flask, and carrying out constant volume and shaking uniformly to obtain a 10mmol/L salicylic acid solution.
(2) Selecting a field suitable for planting the pakchoi, firstly detecting soil, wherein the soil property is as follows: the pH value is 7.20, the total nitrogen is 2.26g/kg, the total phosphorus is 0.95g/kg, the total potassium is 3.0g/kg, the organic matter is 36.2g/kg, and the total selenium content is 0.27 mg/kg. Turning over, preparing land, sowing, fertilizing and watering according to a conventional method.
(3) Diluting 100 times with 10mmol/L salicylic acid solution in 4-5 leaf stage of Chinese cabbage to obtain 100 μmol/L salicylic acid solution, and uniformly spraying onto the front and back sides of leaf of Chinese cabbage with 100 μmol/L salicylic acid solution 40kg per mu.
(4) Diluting with 10mmol/L salicylic acid solution with water 200 times at 6-7 leaf stage of Brassica campestris to obtain 50 μmol/L salicylic acid solution, and uniformly spraying on the front and back surfaces of Brassica campestris leaf with 40kg of 50 μmol/L salicylic acid solution per mu;
(5) other daily management is carried out according to a conventional method.
Planting pakchoi in a field with the soil pH value of 7.20, the total nitrogen content of 2.26g/kg, the total phosphorus content of 0.95g/kg, the total potassium content of 3.0g/kg, the organic matter content of 36.2g/kg and the total selenium content of 0.27 mg/kg; meanwhile, pakchoi planted without spraying salicylic acid solution in the planting process is used as a control group, and the selenium content determination results are shown in table 1.
TABLE 1 Effect of the planting method of the present invention on the selenium content of pakchoi
Note: the values in the table are mean. + -. standard deviation. Different lower case letters indicate significant differences (P <0.05) between the groups performing the technique and the control.
As can be seen from Table 1, the total selenium content of the roots and the overground parts of the pakchoi obtained by the planting method of the invention is obviously increased. The total selenium content of the above-ground part sample (drying sample) of the pakchoi in the control group is 0.234 +/-0.042 mg/kg, and the selenium content of the root part sample (drying sample) is 0.056 +/-0.022 mg/kg; after the technology is implemented, the total selenium content of the overground part sample (drying sample) of the pakchoi is 0.404 plus or minus 0.048mg/kg, and the selenium content of the root part sample (drying sample) is 0.193 plus or minus 0.025 mg/kg. After the planting method is adopted, the selenium content of the overground part of the pakchoi is 1.73 times of that of the control, and the selenium content of the root part of the pakchoi is 3.45 times of that of the control. Meanwhile, T test on the average selenium content of the planting method and the control group shows that the selenium content of samples (dried samples) of the overground part and the root part of the pakchoi obtained by the planting method is obviously different from that of the control group (P is less than 0.05). Therefore, the method can obviously improve the selenium content of the pakchoi by spraying 100 times of diluent (100 mu mol/L salicylic acid) and 200 times of diluent (50 mu mol/L salicylic acid) of 10mmol/L salicylic acid in sequence under the conditions of not applying exogenous selenium and not changing the property structure of soil.
In addition, after the planting method is adopted, the main growth indexes and resistance indexes of the pakchoi are obviously changed.
TABLE 2 Effect of the planting method of the present invention on the growth index of pakchoi
Note: the values in the table are mean. + -. standard deviation. Different lower case letters indicate significant differences (P <0.05) between the groups performing the technique and the control.
As can be seen from Table 2, the total biomass (dry root weight), the aboveground biomass (dry overground part weight), the root biomass (dry root weight) and the maximum chemical quantum yield Fv/Fm of the pakchoi are all obviously increased in growth indexes after the planting method disclosed by the invention is adopted, wherein the root biomass and the maximum chemical quantum yield are obviously increased (P < 0.05). The increase of the maximum photochemical quantum yield reflects the increase of the maximum light energy conversion efficiency of the reaction center of the photosynthesis photosystem II, which indicates that the photosynthesis capability is enhanced. The electron transfer rate and photochemical quenching coefficient of photosystem II of the examples and the control group are not significantly different (P > 0.05).
See table 3 for changes in the crop resistance index.
TABLE 3 Effect of the planting method of the present invention on the Chinese cabbage resistance index
Note: the values in the table are mean. + -. standard deviation. Different lower case letters indicate significant differences (P <0.05) between the groups performing the technique and the control.
As can be seen from table 3, the content of Malondialdehyde (MDA) in example 1 is significantly reduced (P <0.05), and the reduced content of malondialdehyde indicates that the lipid peroxidation degree of the crop cell membrane is low, and the degree of stress to the crop cell membrane is weak. In addition, the example 1 obviously improves the oxidation resistance of the leaves of the pakchoi, the superoxide dismutase (SOD) and the Peroxidase (POD) of the leaves are obviously improved (P is less than 0.05), especially the SOD enzyme activity of the technical group is 2.57 times of that of a control, and the SOD and the POD are important antioxidant enzymes of plants, and play an important role in helping the plants to respond to stress and eliminate active oxygen in the plants. The CAT enzyme activity of the examples was not significantly different from the control (P > 0.05).
In conclusion, the salicylic acid can effectively promote the growth of crops, improve the antioxidant level of the crops, reduce the peroxidation degree of cell membrane lipid, enhance the selenium tolerance of the crops by adjusting the growth physiological state of the salicylic acid, and improve the selenium content of the crops.
The planting method provides a new idea and method for promoting selenium accumulation of crops, and the original technology which mainly provides sufficient active selenium is changed into the technology for regulating the growth of crops and improving the stress resistance, so that the selenium accumulation of the crops is promoted. The key point of the method is that on the basis of certain storage of the selenium in the soil, the selenium content of overground parts and roots of the pakchoi can be obviously improved only by spraying the salicylic acid solution, so that the salicylic acid can be used as an important substance for improving the selenium content of the pakchoi, and a new method is provided for improving the selenium content of crops.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (1)
1. A planting method for improving selenium content of pakchoi without adding exogenous selenium is characterized by comprising the following steps:
(1) preparing a salicylic acid solution: dissolving salicylic acid in absolute ethyl alcohol in a beaker, adding tap water and Tween-20, uniformly stirring, transferring to a volumetric flask, fixing the volume, and uniformly shaking to obtain a salicylic acid solution;
(2) selecting a field for planting the pakchoi: firstly, detecting the pH value and the total selenium content of soil, and planting pakchoi in the selected land; turning over, preparing land, sowing, fertilizing and watering according to a conventional method;
(3) diluting with salicylic acid solution in water at 4-5 leaf stage of caulis et folium Brassicae Capitatae to obtain salicylic acid solution of 100 μmol/L, and uniformly spraying on the front and back surfaces of caulis et folium Brassicae Capitatae;
(4) diluting with salicylic acid solution in water at 6-7 leaf stage of caulis et folium Brassicae Capitatae to obtain salicylic acid solution of 50 μmol/L, and uniformly spraying on the front and back sides of caulis et folium Brassicae Capitatae leaf;
(5) other daily management is carried out according to a conventional method;
wherein the concentration of the salicylic acid solution in the step (1) is 10mmol/L, and the addition amount of Tween-20 is 0.5%;
the soil pH of the selected land parcel in the step (2) is more than or equal to 7.2, and the total selenium content is more than or equal to 0.27 mg/kg;
the dosage of the 100 mu mol/L salicylic acid solution in the step (3) is 40 kg/mu;
the amount of the 50 mu mol/L salicylic acid solution used in the step (4) is 40 kg/mu.
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