Method for reducing exogenous cyanide injury of rice by enriching co2 and regulating amino acids
By cultivating rice seedlings in a CO2-enriched environment and under controlled conditions, the problem of toxic damage to rice caused by high concentrations of CO2 and cyanide was solved, the growth rate and CN- treatment efficiency of rice were improved, and the amino acid content was regulated, thus optimizing the nitrogen cycle.
Patent Information
- Application Number
- CN202410737661.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-07
- Publication Date
- 2025-12-12
- Estimated Expiration
- 2044-06-07
AI Technical Summary
High concentrations of CO2 and cyanide cause toxic damage to rice and affect the balance of amino acid content. There is an urgent need for a method to reduce the toxic damage of exogenous CN- and regulate amino acid content.
By enriching the CO2 environment during rice seedling cultivation and adding cyanide to the standard nutrient solution to remove Fe ions, the CO2 concentration and stress period were controlled. The absorption rate and degradation rate of CN- in rice were measured, and the amino acid content was analyzed.
It significantly improved the relative growth rate, CN- absorption rate and degradation rate of rice, while increasing the valine and arginine content in leaves and roots, thus improving carbon and nitrogen utilization efficiency.
Smart Images
Figure CN118716041B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of plant toxicology, and particularly relates to a method for reducing exogenous cyanide damage and regulating amino acids of rice by enriching CO2. BACKGROUND
[0002] Amino acids (AA) are one of the important intermediate metabolites of plant nitrogen metabolism, which not only participates in protein synthesis, but also participates in and regulates many physiological metabolic processes to cope with various stresses. Under normal circumstances, the content of various amino acids in the plant body is kept at a certain constant level to ensure the balance of various related metabolic reactions. Under stress conditions, the content of various amino acids in the plant body will fluctuate to some extent to regulate the disorder and damage caused by the stress environment to the plant function, and therefore, the change of the content of amino acids is considered as one of the biological markers of the plant to the environmental stress. Nowadays, plants not only suffer from various pollutants in the soil and groundwater, but also need to face the increasing atmospheric carbon dioxide (CO2). In the past fifty years, the content of CO2 in the atmosphere has been increasing at an average rate of 2.5 ppm per year, and it is expected to reach 700 ppm by the end of this century. High concentration of CO2 not only causes climate change, but also changes the biological composition and agricultural cultivation mode in the earth's ecosystem.
[0003] Many industrial and agricultural activities use cyanide (CN - ) in large doses, causing a large amount of CN - -containing wastewater and waste residues to enter the environment through different ways, resulting in different degrees of pollution of the soil and water in many areas (especially in economically underdeveloped areas).
[0004] Studies have shown that many plants can absorb and assimilate exogenous CN - , and the ultimate metabolites are aspartic acid (Asp) / asparagine (Asn) and ammonium nitrogen (NH4 + ), which all participate in the nitrogen nutrient cycle in the plant body. Studies have also found that the increase of [CO2] gas fertilizer can enhance the photosynthetic capacity, carbon assimilation capacity and nitrogen demand of plants, thereby improving the production potential of crops.
[0005] Therefore, there is an urgent need for a simple and effective method for promoting the degradation of CN - in the plant body to reduce the toxic damage of exogenous CN - to plants, while promoting the nitrogen cycle. SUMMARY
[0006] The purpose of the present application is to provide a method for reducing exogenous cyanide damage and regulating amino acids of rice by enriching CO2, to reduce the toxic damage of exogenous CN - to plants, while being able to regulate the content of amino acids in the rice body.
[0007] The technical scheme adopted by the present application is a method for reducing cyanide exogenous damage to rice and regulating amino acids by enriching CO2, comprising the following steps:
[0008] S1: rice seedlings are divided into an experimental group and a control group and placed in conical bottles; a cyanide mixed solution prepared by adding a nutrient solution is added to the conical bottles, and the CN - concentration of the nutrient solution before stress is controlled and the volume of the nutrient solution is measured;
[0009] S2: the conical bottles are placed in a CO2 incubator, the CO2 concentration of the experimental group is increased from the environmental concentration to the enriched concentration, and the CO2 concentration of the control group is the environmental concentration;
[0010] S3: the number of days of the stress period is controlled, the nutrient solution containing KCN in the conical bottles is replaced regularly, and rice seedlings and a nutrient solution after the stress are obtained; the CN - content in the rice seedlings and the nutrient solution is measured, and the volume of the nutrient solution is measured;
[0011] S4: the CN - absorption rate and the CN - degradation rate of the rice seedlings in the experimental group and the control group are calculated;
[0012] S5: the amino acid content in the rice seedlings in the experimental group and the control group is analyzed.
[0013] Further, the nutrient solution in S1 is a standard nutrient solution without Fe ions; and the cyanide is KCN.
[0014] Further, the concentration of the cyanide in the nutrient solution in S1 is 1.0-3.0 mg CN / L.
[0015] Further, the enriched CO2 concentration in S2 is 695-705 ppm.
[0016] Further, the number of days of the stress period in S3 is 2-6 days, and the frequency of replacing the nutrient solution containing KCN in the conical bottles is 2 days / once.
[0017] Further, the rice before stress in S1 needs to be weighed, and the rice seedlings after stress in S3 need to be weighed.
[0018] Further, S4 further comprises calculating the relative growth rate of the rice seedlings in the experimental group and the control group, and the calculation formula of the relative growth rate RGR is:
[0019]
[0020] wherein, M (i) is the initial fresh weight of the rice seedlings, and M(f) The fresh weight of rice seedlings after stress.
[0021] Furthermore, in S4, CN - The formula for calculating the absorption rate R is:
[0022]
[0023] In the formula, C f Indicates CN in the nutrient solution after stress - Concentration, V f C represents the volume of the nutrient solution after stress. i Indicates CN in the nutrient solution before stress - Concentration, V i This indicates the volume of the nutrient solution before stress.
[0024] Furthermore, in S4, CN - The formula for calculating the degradation rate v is:
[0025]
[0026] In the formula, m (i) Indicates CN in the nutrient solution before stress - mass, m (f) Indicates CN in the nutrient solution after stress - mass, m (root) This indicates the amount of CN remaining in the rice root system after stress. - mass, m (shoot) This indicates the amount of CN remaining in rice leaves after stress. - Mass, t represents the duration of stress, M (f) The fresh weight of rice seedlings after stress.
[0027] The beneficial effects of this invention are
[0028] 1. This invention significantly improves the relative growth rate of rice seedlings under KCN stress by increasing the CO2 content in the environment during the cultivation of KCN-stressed rice seedlings, thereby reducing the resistance to KCN. - Absorption rate, for CN - The degradation rate.
[0029] 2. By comparing the amino acid content in rice seedlings under different conditions, it was found that under the same stress period, the method of the present invention can simultaneously increase the content of valine and arginine in plant leaves and roots; at the same time, for the nine essential amino acids synthesized by plants, the method of the present invention can simultaneously increase the content of five essential amino acids in the roots: phenylalanine, leucine, valine, threonine, and methionine.
[0030] 3, The method improves the utilization efficiency of carbon and nitrogen, and has very important practical significance for energy saving and emission reduction and resource utilization of toxicants. BRIEF DESCRIPTION OF DRAWINGS
[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.
[0032] Figure 1 is a relative growth rate comparison chart of rice seedlings, wherein a is a relative growth rate comparison chart of rice seedlings after stress treatment for 2 days, b is a relative growth rate comparison chart of rice seedlings after stress treatment for 4 days, and c is a relative growth rate comparison chart of rice seedlings after stress treatment for 6 days.
[0033] Figure 2 is a CN - absorption rate and degradation rate comparison chart of rice seedlings, wherein a is the CN - absorption rate of rice seedlings after stress treatment for 2 days, b is the CN - degradation rate of rice seedlings after stress treatment for 2 days, c is the CN - absorption rate of rice seedlings after stress treatment for 4 days, d is the CN - degradation rate of rice seedlings after stress treatment for 4 days, e is the CN - absorption rate of rice seedlings after stress treatment for 6 days, and f is the CN - degradation rate of rice seedlings after stress treatment for 2 days. DETAILED DESCRIPTION
[0034] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.
[0035] The complete steps of the method of the present application are as follows:
[0036] Culture steps of the used rice seedlings: After the rice seeds (Xiangzaoxian No. 45) are soaked in deionized water for 24 h, sand culture experiments are carried out in an artificial climate chamber, and the culture conditions are as follows: constant temperature (25±0.5 ℃), constant humidity (60±2%), constant light (20000 lux), and constant irrigation of modified ISO8629 nutrient solution (NH4Cl as nitrogen source); after the seedlings are cultured for 16 d, 10 seedlings with uniform growth are selected as a group, and the seedlings are temporarily cultured in the nutrient solution for 12 h to adapt to the water culture environment.
[0037] Table 1 Composition of modified ISO8692 nutrient solution
[0038] No. Reagent Concentration No. Reagent Concentration 1 NH4Cl 282 3.9 μmol / L 7 H3BO3 299 2.1 nmol / L 2 MgCl2.6H2O 59.0 μmol / L 8 MnCl2.4H2O 209 7.0 nmol / L 3 CaCl2·2H2O 122.4 μmol / L 9 Na2MoO4.2H2O 28.9 nmol / L 4 MgSO4.7H2O 60.9 μmol / L 10 [CuSO4·2H2O] 0.1 nmol / L 5 KH2PO4 246.0 μmol / L 11 ZnSO4 22.0 nmol / L 6 NaHCO3 1785.5 μmol / L 12 CoCl2.6H2O 6.3 nmol / L
[0039] Here, the standard nutrient solution without Fe ion component is used, and Hoagland's nutrient solution without Fe ion component can also be used. Because Fe ion is easily complexed with CN - to form ferricyanide, thereby changing the CN - existing form in the aqueous solution, the Fe ion component in the nutrient solution must be removed.
[0040] S1: The selected rice seedlings are divided into a test group and a control group, and are respectively placed in 50 mL conical bottles, and cyanide mixed solutions prepared by using the modified ISO8692 nutrient solution are respectively added to the conical bottles to control the concentration of CN - in the nutrient solution. Each conical bottle is wrapped with tin foil paper for light shielding treatment, so as to minimize water loss and inhibit the growth of algae.
[0041] S2: The conical bottles are placed in a CO2 incubator, and the CO2 concentration of the test group is increased from the environmental concentration to the enriched concentration, and the CO2 concentration of the control group is the environmental concentration.
[0042] S3: The number of stress periods is controlled, and the nutrient solution containing KCN in the conical bottle is replaced regularly.
[0043] S4: The test group and the control group are the same in all conditions except the CO2 concentration; the relative growth rate, CN - absorption rate, and CN - degradation rate of the rice seedlings in the test group and the control group are calculated.
[0044] S5: The amino acid content in the rice seedlings in the test group and the control group is analyzed.
[0045] The cyanide prepared by using the nutrient solution in S1 is KCN or NaCN.
[0046] The concentration of the cyanide in the nutrient solution in S1 is 1.0-3.0 mg CN / L.
[0047] The CO2 enrichment concentration in S2 is 695-705 ppm.
[0048] The number of days of stress period in S3 is 2-6 days; the frequency of replacing the nutrient solution containing KCN in the conical flask is 2 days / time.
[0049] The reason for replacing the nutrient solution containing KCN in the conical flask at a frequency of 2 days / time: this depends on the absorption rate of the plant to the nutrient components in the nutrient solution within 2 days, to avoid the plant from other nutrient shortages.
[0050] Experimental group
[0051] Example 1
[0052] S1: The selected rice seedlings were placed in 50 mL conical flasks respectively. KCN mixed solution prepared with modified ISO8692 nutrient solution was added respectively, the concentration of KCN in the nutrient solution: 3.0 mg CN / L. Each conical flask was wrapped with tin foil for light shielding treatment, to minimize water loss and inhibit the growth of algae.
[0053] S2: The conical flasks were placed in a CO2 incubator, with a CO2 concentration of 695 ppm.
[0054] S3: The stress period is 2 days.
[0055] S4: Calculate the relative growth rate, CN - absorption rate, CN - degradation rate of rice seedlings.
[0056] S5: Analysis of the amino acid content in the rice seedlings.
[0057] Example 2
[0058] S1: The selected rice seedlings were placed in 50 mL conical flasks respectively. KCN mixed solution prepared with modified ISO8692 nutrient solution was added respectively, the concentration of KCN in the nutrient solution: 3.0 mg CN / L. Each conical flask was wrapped with tin foil for light shielding treatment, to minimize water loss and inhibit the growth of algae.
[0059] S2: The conical flasks were placed in a CO2 incubator, with a CO2 concentration of 700 ppm.
[0060] S3: The number of days of stress period is 4 days, and the nutrient solution containing KCN in the conical flask is replaced every 2 days.
[0061] S4: Calculate the relative growth rate, CN - absorption rate, CN - degradation rate of rice seedlings.
[0062] S5: Analysis of the amino acid content in the rice seedlings.
[0063] Example 3
[0064] S1 : The selected rice seedlings were placed in 50 mL conical flasks. KCN mixed solution prepared with modified ISO8692 nutrient solution was added, with a KCN concentration of 3.0 mg CN / L in the nutrient solution. Each conical flask was wrapped with tin foil for light protection, to minimize water loss and inhibit the growth of algae.
[0065] S2: The conical flasks were placed in a CO2 incubator with a CO2 concentration of 705 ppm.
[0066] S3: The stress period was 6 days, with the KCN-containing nutrient solution in the conical flasks being replaced every 2 days.
[0067] S4: The relative growth rate, CN - uptake rate, and CN - degradation rate of the rice seedlings were calculated.
[0068] S5: Analysis of the amino acid content in the rice seedlings.
[0069] Example 4
[0070] S1 : The selected rice seedlings were placed in 50 mL conical flasks. KCN mixed solution prepared with modified ISO8692 nutrient solution was added, with a KCN concentration of 2.0 mg CN / L in the nutrient solution. Each conical flask was wrapped with tin foil for light protection, to minimize water loss and inhibit the growth of algae.
[0071] S2: The conical flasks were placed in a CO2 incubator with a CO2 concentration of 700 ppm.
[0072] S3: The stress period was 2 days.
[0073] S4: The relative growth rate, CN - uptake rate, and CN - degradation rate of the rice seedlings were calculated.
[0074] S5: Analysis of the amino acid content in the rice seedlings.
[0075] Example 5
[0076] S1 : The selected rice seedlings were placed in 50 mL conical bottles, respectively, and KCN mixed solution prepared with modified ISO8692 nutrient solution was added, the concentration of KCN in the nutrient solution was 2.0 mg CN / L. Each conical bottle was wrapped with tin foil for light shielding treatment, which minimized water loss and inhibited the growth of algae.
[0077] S2: The conical bottles were placed in a CO2 incubator with a CO2 concentration of 700 ppm.
[0078] S3: The stress period was 4 days, and the nutrient solution containing KCN in the conical bottle was replaced every 2 days.
[0079] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0080] S5: The amino acid content in the rice seedlings was analyzed.
[0081] Example 6
[0082] S1 : The selected rice seedlings were placed in 50 mL conical bottles, respectively, and KCN mixed solution prepared with modified ISO8692 nutrient solution was added, the concentration of KCN in the nutrient solution was 2.0 mg CN / L. Each conical bottle was wrapped with tin foil for light shielding treatment, which minimized water loss and inhibited the growth of algae.
[0083] S2: The conical bottles were placed in a CO2 incubator with a CO2 concentration of 700 ppm.
[0084] S3: The stress period was 6 days, and the nutrient solution containing KCN in the conical bottle was replaced every 2 days.
[0085] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0086] S5: The amino acid content in the rice seedlings was analyzed.
[0087] Example 7
[0088] S1 : The selected rice seedlings were placed in 50 mL conical bottles, respectively, and KCN mixed solution prepared with modified ISO8692 nutrient solution was added, the concentration of KCN in the nutrient solution was 2.0 mg CN / L. Each conical bottle was wrapped with tin foil for light shielding treatment, which minimized water loss and inhibited the growth of algae.
[0089] S2: Place the conical flask in a CO2 incubator with a CO2 concentration of 700 ppm.
[0090] S3: Stress cycle of 2 days.
[0091] S4: Calculate the relative growth rate, CN - uptake, CN - degradation rate of the rice seedlings.
[0092] S5: Analyze the amino acid content in the rice seedlings.
[0093] Example 8
[0094] S1 : Place the selected rice seedlings in 50 mL conical flasks, respectively, and add KCN mixed solution prepared with the modified ISO8692 nutrient solution, with a KCN concentration of 1.0 mg CN / L in the nutrient solution. Wrap each conical flask with tin foil for light protection, minimize water loss, and inhibit the growth of algae.
[0095] S2: Place the conical flask in a CO2 incubator with a CO2 concentration of 700 ppm.
[0096] S3: Stress cycle of 4 days, with the KCN-containing nutrient solution in the conical flask being replaced every 2 days.
[0097] S4: Calculate the relative growth rate, CN - uptake, CN - degradation rate of the rice seedlings.
[0098] S5: Analyze the amino acid content in the rice seedlings.
[0099] Example 9
[0100] S1 : Place the selected rice seedlings in 50 mL conical flasks, respectively, and add KCN mixed solution prepared with the modified ISO8692 nutrient solution, with a KCN concentration of 1.0 mg CN / L in the nutrient solution. Wrap each conical flask with tin foil for light protection, minimize water loss, and inhibit the growth of algae.
[0101] S2: Place the conical flask in a CO2 incubator with a CO2 concentration of 700 ppm.
[0102] S3: Stress cycle of 6 days, with the KCN-containing nutrient solution in the conical flask being replaced every 2 days.
[0103] S4: Calculate the relative growth rate, CN - uptake, CN - degradation rate of the rice seedlings.
[0104] S5: Analysis of the amino acid content in the rice seedlings.
[0105] Control group
[0106] Comparative Example 1
[0107] S1 : The selected rice seedlings were placed in 50 mL conical flasks, 50 mL of modified ISO8692 nutrient solution was added. Each conical flask was wrapped with tin foil for light shielding treatment, which minimized water loss and inhibited the growth of algae.
[0108] S2: The conical flasks were placed in a CO2 incubator with a CO2 concentration of 700 ppm.
[0109] S3: The stress period was 2 days.
[0110] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0111] S5: Analysis of the amino acid content in the rice seedlings.
[0112] Comparative Example 2
[0113] S1 : The selected rice seedlings were placed in 50 mL conical flasks, 50 mL of modified ISO8692 nutrient solution was added. Each conical flask was wrapped with tin foil for light shielding treatment, which minimized water loss and inhibited the growth of algae.
[0114] S2: The conical flasks were placed in a CO2 incubator with a CO2 concentration of 700 ppm.
[0115] S3: The stress period was 4 days, and the ISO8692 nutrient solution in the conical flasks was replaced every 2 days.
[0116] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0117] S5: Analysis of the amino acid content in the rice seedlings.
[0118] Comparative Example 3
[0119] S1 : The selected rice seedlings were placed in 50 mL conical flasks, 50 mL of modified ISO8692 nutrient solution was added. Each conical flask was wrapped with tin foil for light shielding treatment, which minimized water loss and inhibited the growth of algae.
[0120] S2: The conical flask was placed in a CO2 incubator with a CO2 concentration of 700 ppm.
[0121] S3: The stress period was 6 days, and the ISO 8692 nutrient solution in the conical flask was replaced every 2 days.
[0122] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0123] S5: The amino acid content in the rice seedlings was analyzed.
[0124] Comparative Example 4
[0125] S1: The selected rice seedlings were placed in 50 mL conical flasks, and a KCN mixed solution prepared using the modified ISO 8692 nutrient solution was added, with a KCN concentration of 3.0 mg CN / L in the nutrient solution. Each conical flask was wrapped with tin foil for light shielding treatment, to minimize water loss and inhibit the growth of algae.
[0126] S2: The conical flask was placed in a CO2 incubator with a CO2 concentration of 350 ppm.
[0127] S3: The stress period was 2 days.
[0128] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0129] S5: The amino acid content in the rice seedlings was analyzed.
[0130] Comparative Example 5
[0131] S1: The selected rice seedlings were placed in 50 mL conical flasks, and a KCN mixed solution prepared using the modified ISO 8692 nutrient solution was added, with a KCN concentration of 3.0 mg CN / L in the nutrient solution. Each conical flask was wrapped with tin foil for light shielding treatment, to minimize water loss and inhibit the growth of algae.
[0132] S2: The conical flask was placed in a CO2 incubator with a CO2 concentration of 350 ppm.
[0133] S3: The stress period was 4 days, and the nutrient solution containing KCN in the conical flask was replaced every 2 days.
[0134] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0135] S5: Analysis of the amino acid content in the rice seedlings.
[0136] Comparative Example 6
[0137] S1 : The selected rice seedlings were placed in 50 mL conical flasks, to which a KCN mixed solution prepared using a modified version of ISO8692 nutrient solution was added, the concentration of KCN in the nutrient solution being 3.0 mg CN / L. Each conical flask was wrapped with tin foil for light shielding treatment, to minimize water loss and inhibit the growth of algae.
[0138] S2: The conical flasks were placed in a CO2 incubator, the CO2 concentration being 350 ppm.
[0139] S3: The stress period was 6 days, during which the nutrient solution containing KCN in the conical flasks was replaced every 2 days.
[0140] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0141] S5: Analysis of the amino acid content in the rice seedlings.
[0142] Comparative Example 7
[0143] S1 : The selected rice seedlings were placed in 50 mL conical flasks, to which a KCN mixed solution prepared using a modified version of ISO8692 nutrient solution was added, the concentration of KCN in the nutrient solution being 2.0 mg CN / L. Each conical flask was wrapped with tin foil for light shielding treatment, to minimize water loss and inhibit the growth of algae.
[0144] S2: The conical flasks were placed in a CO2 incubator, the CO2 concentration being 350 ppm.
[0145] S3: The stress period was 2 days.
[0146] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0147] S5: Analysis of the amino acid content in the rice seedlings.
[0148] Comparative Example 8
[0149] S1 : The selected rice seedlings were placed in 50 mL conical bottles, and a KCN mixed solution prepared using a modified version of ISO8692 nutrient solution was added. The concentration of KCN in the nutrient solution was 2.0 mg CN / L. Each conical bottle was wrapped with tin foil for light shielding treatment to minimize water loss and inhibit the growth of algae.
[0150] S2: The conical bottles were placed in a CO2 incubator with a CO2 concentration of 350 ppm.
[0151] S3: The stress period was 4 days, and the nutrient solution containing KCN in the conical bottles was replaced every 2 days.
[0152] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0153] S5: The amino acid content in the rice seedlings was analyzed.
[0154] Comparative Example 9
[0155] S1 : The selected rice seedlings were placed in 50 mL conical bottles, and a KCN mixed solution prepared using a modified version of ISO8692 nutrient solution was added. The concentration of KCN in the nutrient solution was 2.0 mg CN / L. Each conical bottle was wrapped with tin foil for light shielding treatment to minimize water loss and inhibit the growth of algae.
[0156] S2: The conical bottles were placed in a CO2 incubator with a CO2 concentration of 350 ppm.
[0157] S3: The stress period was 6 days, and the nutrient solution containing KCN in the conical bottles was replaced every 2 days.
[0158] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0159] S5: The amino acid content in the rice seedlings was analyzed.
[0160] Comparative Example 10
[0161] S1 : The selected rice seedlings were placed in 50 mL conical bottles, and a KCN mixed solution prepared using a modified version of ISO8692 nutrient solution was added. The concentration of KCN in the nutrient solution was 1.0 mg CN / L. Each conical bottle was wrapped with tin foil for light shielding treatment to minimize water loss and inhibit the growth of algae.
[0162] S2: The conical flask was placed in a CO2 incubator with a CO2 concentration of 350 ppm.
[0163] S3: The stress period was 2 days.
[0164] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0165] S5: The amino acid content in the rice seedlings was analyzed.
[0166] Comparative Example 11
[0167] S1 : The selected rice seedlings were placed in 50 mL conical flasks, and a KCN mixed solution prepared using a modified version of ISO8692 nutrient solution was added, with the concentration of KCN in the nutrient solution being 1.0 mg CN / L. Each conical flask was wrapped with tin foil for light shielding treatment, to minimize water loss and inhibit the growth of algae.
[0168] S2: The conical flask was placed in a CO2 incubator with a CO2 concentration of 350 ppm.
[0169] S3: The stress period was 4 days, with the KCN-containing nutrient solution in the conical flask being replaced every 2 days.
[0170] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0171] S5: The amino acid content in the rice seedlings was analyzed.
[0172] Comparative Example 12
[0173] S1 : The selected rice seedlings were placed in 50 mL conical flasks, and a KCN mixed solution prepared using a modified version of ISO8692 nutrient solution was added, with the concentration of KCN in the nutrient solution being 1.0 mg CN / L. Each conical flask was wrapped with tin foil for light shielding treatment, to minimize water loss and inhibit the growth of algae.
[0174] S2: The conical flask was placed in a CO2 incubator with a CO2 concentration of 350 ppm.
[0175] S3: The stress period was 6 days, with the KCN-containing nutrient solution in the conical flask being replaced every 2 days.
[0176] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0177] S5: Analysis of the amino acid content in the rice seedlings.
[0178] Comparative Example 13
[0179] S1 : The selected rice seedlings were placed in 50 mL conical flasks, each of which was added with 50 mL of the modified ISO8692 nutrient solution. Each conical flask was wrapped with tin foil for light shielding treatment, so as to minimize water loss and inhibit the growth of algae.
[0180] S2: The conical flasks were placed in a CO2 incubator, in which the CO2 concentration was 350 ppm.
[0181] S3: The stress period was 2 days.
[0182] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0183] S5: Analysis of the amino acid content in the rice seedlings.
[0184] Comparative Example 14
[0185] S1 : The selected rice seedlings were placed in 50 mL conical flasks, each of which was added with 50 mL of the modified ISO8692 nutrient solution. Each conical flask was wrapped with tin foil for light shielding treatment, so as to minimize water loss and inhibit the growth of algae.
[0186] S2: The conical flasks were placed in a CO2 incubator, in which the CO2 concentration was 350 ppm.
[0187] S3: The stress period was 4 days, in which the ISO8692 nutrient solution in the conical flasks was replaced every 2 days.
[0188] S4: The relative growth rate, CN - absorption rate, CN - degradation rate of the rice seedlings were calculated.
[0189] S5: Analysis of the amino acid content in the rice seedlings.
[0190] Comparative Example 15
[0191] S1 : The selected rice seedlings were placed in 50 mL conical flasks, each of which was added with 50 mL of the modified ISO8692 nutrient solution. Each conical flask was wrapped with tin foil for light shielding treatment, so as to minimize water loss and inhibit the growth of algae.
[0192] S2: The conical flasks were placed in a CO2 incubator, in which the CO2 concentration was 350 ppm.
[0193] S3: Stress cycle is 6 days, wherein, the ISO8692 nutrient solution in the conical flask is replaced every 2 days.
[0194] S4: Calculate the relative growth rate of rice seedlings, CN - absorption rate, CN - degradation rate.
[0195] S5: Analysis of amino acid content in rice seedlings.
[0196] Each example, comparative example has 3 biological repeats.
[0197] Degradation experiment of exogenous CN - by rice seedlings:
[0198] Herein Examples 1-9 and Comparative Examples 1-15, the relative growth rate of rice seedlings under different CO2 concentrations, CN - removal rate, CN - degradation rate under different concentrations of KCN solution stress can be calculated.
[0199] (1) Calculation of the relative growth rate of rice seedlings:
[0200] See Figure 1 , for all examples, comparative examples, weigh the weight of rice seedlings before and after stress, and calculate the relative growth rate of rice seedlings according to the formula, the calculation formula of relative growth rate RGR(%) is as follows:
[0201]
[0202] Wherein, M (i) is the initial fresh weight of rice seedlings in this experiment (g), M (f) is the fresh weight of rice seedlings after stress (g).
[0203] (2) Analysis of CN - content in nutrient solution and total CN - content in rice seedlings.
[0204] CN - content analysis in nutrient solution: spectrophotometry was used. A certain volume (1-5 mL) of nutrient solution before stress, nutrient solution after 2 days of stress, and nutrient solution after 4 days of stress were collected in a colorimetric tube, respectively; add NaOH (0.1%) to 10 mL scale line;
[0205] Add 5 mL of buffer solution containing KH₂PO₄ and Na₂HPO₄, then quickly add 0.2 mL of 1% [m / v] chloramine-T solution, shake well and let stand for 3–5 min. Next, add 5 mL of a mixture of isonicotinic acid and pyrazolone, and dilute to 25 mL with ultrapure water. Mix well and place the colorimetric tube in a 32°C water bath for 40 minutes. Finally, measure the absorbance at 638 nm and calculate the CN content in the nutrient solution. - Content; among which, the isonicotinic acid solution in the mixture of isonicotinic acid and pyrazolone is obtained by dissolving 1.5g of isonicotinic acid in 24mL of 2% NaOH solution and diluting it with water to 100mL; the pyrazolone solution is obtained by dissolving 0.25g of pyrazolone in 20mL of N,N-dimethylformamide solution.
[0206] Total CN in rice seedlings - Content analysis: Total CN in rice seedlings was determined by distillation. - Content. Add 10 mL of 1% NaOH solution to the collection bottle at the end of the distillation system. Accurately weigh and chop the rice seedling root or leaf sample into a 500 mL round-bottom flask, add 200 mL of ultrapure water, then add 10 mL of 10% EDTA solution and 10 mL of 85% phosphoric acid. Start the distillation process. When the solution in the collection bottle is close to 100 mL, turn off the distillation system and transfer the liquid in the collection bottle to a 100 mL volumetric flask for final volume determination. The CN content in the collected solution is then analyzed. - Content determination and analysis of CN in nutrient solution - The content analysis methods are the same.
[0207] Rice seedlings react with CN in nutrient solution - Calculation of removal rate:
[0208] CN in the nutrient solution before and after KCN solution stress was obtained. - The changes in CN concentration and nutrient solution volume were analyzed, and the concentration of CN in the nutrient solution was calculated using the following formula. - Absorption rate R (%):
[0209]
[0210] In the formula, C f Indicates CN in the nutrient solution after stress - Concentration, V f C represents the volume of the nutrient solution after stress. i Indicates CN in the nutrient solution before stress - Concentration, V i This indicates the volume of the nutrient solution before stress. The response of rice seedlings to CN in the nutrient solution... - The absorption rates are shown in Table 1.
[0211] (3) The degradation rate of CN by rice seedlings in the nutrient solution - Calculation of degradation rate:
[0212] According to CN - The degradation amount of CN by rice seedlings in the nutrient solution was calculated according to the material balance in the rice reactor, and the degradation rate of CN by rice seedlings was calculated according to the following formula: - The degradation amount of CN by rice seedlings in the nutrient solution was calculated according to the material balance in the rice reactor, and the degradation rate of CN by rice seedlings was calculated according to the following formula: - The degradation amount of CN by rice seedlings in the nutrient solution was calculated according to the material balance in the rice reactor, and the degradation rate of CN by rice seedlings was calculated according to the following formula: - The degradation amount of CN by rice seedlings in the nutrient solution was calculated according to the material balance in the rice reactor, and the degradation rate of CN by rice seedlings was calculated according to the following formula: - The degradation amount of CN by rice seedlings in the nutrient solution was calculated according to the material balance in the rice reactor, and the degradation rate of CN by rice seedlings was calculated according to the following formula: - The degradation amount of CN by rice seedlings in the nutrient solution was calculated according to the material balance in the rice reactor, and the degradation rate of CN by rice seedlings was calculated according to the following formula:
[0213]
[0214] In the formula, m (i) represents the mass of CN in the nutrient solution before stress (μg), m - represents the mass of CN in the nutrient solution after stress (μg), m (f) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (root) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (shoot) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (f) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg).
[0215] In the formula, m (i) represents the mass of CN in the nutrient solution before stress (μg), m - represents the mass of CN in the nutrient solution after stress (μg), m (f) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (root) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (shoot) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (f) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg).
[0216] Table 1: Relative growth rate of rice seedlings, removal rate and degradation rate of exogenous CN in examples and comparative examples -
[0217]
[0218] Experimental data analysis: Compared with Comparative Example 5, it can be seen from Example 2 that the relative growth rate of rice seedlings under KCN stress is increased by 2.0 times, the CN absorption rate is increased by 1.79 times, and the CN degradation rate is increased by 1.53 times. - -
[0219] In the formula, m (i) represents the mass of CN in the nutrient solution before stress (μg), m - represents the mass of CN in the nutrient solution after stress (μg), m (f) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (root) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (shoot) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). (f) represents the mass of CN remaining in the rice roots after stress (μg), and m - represents the mass of CN remaining in the rice leaves after stress (μg). Figure 1In the figures, a[CO2] represents a CO2 concentration of 350 ppm (environmental concentration), and e[CO2] represents a CO2 enrichment concentration. Referring to Figure a, compared to Comparative Example 10, Example 4, Example 1, and Comparative Example 13, the relative growth rate of Example 7 increased by approximately 1.5 times. Referring to Figure b, compared to Comparative Example 11, Example 5, and Comparative Example 8, Example 2, and Comparative Example 5, the relative growth rate of Example 8 increased by approximately 2 times. Referring to Figure c, compared to Comparative Example 12, Example 6, Example 3, and Comparative Example 6, Example 3, and Comparative Example 15, the relative growth rate of Example 9 increased by approximately 2 times.
[0220] exist Figure 2 In the figure, a[CO2] represents a CO2 concentration of 350 ppm (environmental concentration), and e[CO2] represents a CO2 concentration enriched at a higher concentration. See Figure a for comparisons of Example 7 and Comparative Example 10, Example 4 and Comparative Example 7, and Example 1 and Comparative Example 4. - The absorption rate increased by approximately 1.5 times; see Figure b, Example 7 compared to Comparative Example 10, Example 4 compared to Comparative Example 7, Example 1 compared to Comparative Example 4, CN - The degradation rate increased by approximately 1.5 times; see Figure c, Example 8 compared to Comparative Example 11, Example 5 compared to Comparative Example 8, Example 2 compared to Comparative Example 5, CN - The absorption rate increased by approximately 1.7 times; see Figure d, Example 8 compared to Comparative Example 11, Example 5 compared to Comparative Example 8, Example 2 compared to Comparative Example 5, CN - The degradation rate increased by approximately 1.5 times; see Figure e, Example 9 compared to Comparative Example 12, Example 6 compared to Comparative Example 9, Example 3 compared to Comparative Example 6, CN - The absorption rate increased by approximately 1.7 times (see Figure f). This is in comparison to Example 9 and Comparative Example 12, Example 6 and Comparative Example 9, and Example 3 and Comparative Example 6. - The degradation rate increased by approximately 1.5 times.
[0221] Depend on Figure 1-2 The experimental data show that, under the same KCN concentration stress, increasing the CO2 content from 350 ppm to 700 ppm can significantly improve the relative growth rate and KCN content of rice seedlings. - Absorption rate, CN - The degradation rate can reduce exogenous CN. - Toxic damage to plants. Figure 1 , Figure 2 The p-value < 0.05 indicates a significant difference between the experimental group and the control group.
[0222] Determination of the content of various amino acids in different tissues of rice:
[0223] For all examples, after the end of the stress, the root and leaf tissues of rice seedlings were collected in liquid nitrogen and frozen, and 100 mg of ground plant tissue powder was mixed with 1.0 mL of a methanol / acetonitrile / water solution (v:v:v = 2:2:1). After homogenization and ultrasonic treatment for 30 min, the mixture was centrifuged at 14000g at 4°C for 20 min. The supernatant was analyzed for the content of 21 amino acids in the plant tissue using ultra-high liquid chromatography (1290 Infinity LC, Agilent Technologies) coupled with mass spectrometry (QTRAP, AB Sciex 5500).
[0224] For example, examples 2, comparative examples 2, 5, and 14, the specific results are shown in Tables 2 and 3.
[0225] Table 2: Content of various amino acids in the leaves of rice seedlings in examples 2, comparative examples 2, 5, and 14 (unit: nmol / g FW)
[0226]
[0227]
[0228] *NA indicates that the concentration in the sample is below the detection limit.
[0229] Table 3: Content of various amino acids in the roots of rice seedlings in examples 2, comparative examples 2, 5, and 14 (unit: nmol / g FW)
[0230]
[0231] *NA indicates that the concentration in the sample is below the detection limit.
[0232] Based on the data results in Tables 2 and 3, the effect of the synergistic regulation of enriched CO2 and KCN on the content of amino acids in the tissues of rice seedlings was evaluated:
[0233] The amino acids that meet the following conditions at the same time were screened out:
[0234] (1) The content of amino acids in the tissues of rice seedlings in comparative example 2 (enriched CO2) must be significantly greater than that in comparative example 14 (environmental CO2);
[0235] (2) The content of amino acids in the tissues of rice seedlings in comparative example 5 (environmental CO2 + KCN stress) must be significantly greater than that in comparative example 14 (environmental CO2);
[0236] (3) Example 2 (rich CO2+KCN stress) The content of amino acids in the rice seedling tissue must be significantly greater than Comparative Example 14 (ambient CO2), Comparative Example 2 (rich CO2), Comparative Example 5 (ambient CO2+KCN stress), and the screening results are as follows:
[0237] In the leaves: valine, asparagine, aspartate, arginine.
[0238] In the roots: serine, phenylalanine, tyrosine, leucine, valine, threonine, methionine, glutamate, proline, citrulline, arginine.
[0239] Among them, rich CO2 and KCN have a synergistic regulatory effect on the content of valine and arginine in the roots and leaves of rice seedlings, and the rest of the amino acid species are limited to the leaves or roots, showing tissue specificity of the synergistic regulation of rich CO2 and KCN.
[0240] The invention point of the present application is:
[0241] 1. The present application can significantly improve the relative growth rate, CN - absorption rate, CN - degradation rate of plants by increasing the CO2 content in the environment during the cultivation of KCN stressed rice seedlings, thereby reducing the toxic damage of exogenous CN - to plants.
[0242] 2. The method of the present application reduces the toxic damage of exogenous CN - to plants while increasing the content of five essential amino acids in the plant roots.
[0243] Each embodiment in the specification is described in a related manner, and the same or similar parts between each embodiment can be referred to each other, and each embodiment mainly explains the difference from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the related parts can be referred to the part of the method embodiment.
[0244] The above only describes the preferred embodiments of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application is included in the protection scope of the present application.
Claims
1. A method for reducing cyanide exogenous damage and regulating amino acids in rice by enriching CO2, characterized in that, The method comprises the following steps: S1: the rice seedlings are divided into an experimental group and a control group, and placed in conical bottles respectively; a cyanide mixed solution prepared by adding a nutrient solution is added in the conical bottles, and the CN - concentration of the nutrient solution before stress is controlled, and the volume of the nutrient solution is measured; S2: placing the conical flask in a CO2 incubator, increasing the CO2 concentration of the experimental group from the ambient concentration to the enriched concentration, and keeping the CO2 concentration of the control group at the ambient concentration; S3: control the number of days of stress cycle, replace the nutrient solution containing KCN in the conical flask regularly; obtain the rice seedlings after stress ends, the nutrient solution; measure the CN content in the rice seedlings, the nutrient solution, and measure the volume of the nutrient solution - content and measure the volume of the nutrient solution S4: Calculate CN of rice seedlings in experimental group and control group - absorption rate, CN - degradation rate; S5: analyzing the amino acid content in the rice seedlings of the experimental group and the control group.
2. The method for reducing cyanide exogenous injury and regulating amino acids of rice by enriched CO2 according to claim 1, characterized in that, The nutrient solution in S1 is a standard nutrient solution without Fe ions; and the cyanide is KCN.
3. The method for reducing exogenous cyanide injury and regulating amino acids in rice by enriched CO2 according to claim 1, characterized in that, The concentration of the cyanide in the nutrient solution in S1 is 1.0-3.0 mg CN / L.
4. The method for reducing cyanide exogenous injury and regulating amino acids of rice by enriched CO2 according to claim 1, characterized in that, The enriched CO2 concentration in S2 is 695-705 ppm.
5. The method for reducing cyanide exogenous injury and regulating amino acids of rice by enriched CO2 according to claim 1, characterized in that, The stress period in S3 is 2-6 days, and the frequency of replacing the nutrient solution containing KCN in the conical flask is 2 days / time.
6. The method for reducing cyanide exogenous injury and regulating amino acids of rice by enriched CO2 according to claim 1, characterized in that, The rice before stress in S1 needs to be weighed, and the rice seedlings after stress in S3 need to be weighed.
7. The method for reducing cyanide exogenous injury and regulating amino acids of rice by enriched CO2 according to claim 6, characterized in that, In S4, the relative growth rates of the rice seedlings of the experimental group and the control group are calculated, and the calculation formula of the relative growth rate RGR is: wherein M (i) is the initial fresh weight of the rice seedling, M (f) is the fresh weight of the rice seedling after stress.
8. The method for reducing cyanide exogenous injury and regulating amino acids of rice by enriched CO2 according to claim 1, characterized in that, The CN in S4 - The formula for calculating the absorption rate R of CN in S4 is: wherein C f represents the volume of the nutrient solution after the stress, C - represents the concentration of CN f represents the volume of the nutrient solution after the stress, C i represents the concentration of CN - represents the concentration of CN i represents the volume of the nutrient solution before the stress.
9. The method for reducing cyanide exogenous injury and regulating amino acids of rice by enriched CO2 according to any one of claims 1-8, characterized in that, The CN in S4 - The degradation rate v is calculated by the formula: wherein m (i) CN in the nutrient solution before stress - mass, m (f) CN in the nutrient solution after stress - mass, m (root) CN remaining in the rice roots after stress - mass, m (shoot) CN remaining in the rice leaves after stress - mass, t indicates the length of stress, M (f) fresh weight of the rice seedlings after stress
Citation Information
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Method for treating cyanide pollution by adding cysteine and application thereof
CN115259392A