This standard specifies the method for determining the biological activity of plant hormone secondary metabolites using the indicator plant method. This standard is applicable to the activity determination of plant hormone secondary metabolites such as auxin, cytokinin and gibberellin.

ICS07.080
TrrKAa-oJouaKAa
National Standard of the People's Republic of China
GB/T38577-—2020
Determination of the biological activity for plant hormone-related secondarymetabolites-Indicator plantmethod2020-03-31 Issued
State Administration for Market Regulation
National Standardization Administration
2020-03-31 Implementation
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. TrrKAa-cJouaKAa
GB/T38577—2020
Drafting units of this standard: China University of Metrology, China National Institute of Standardization, Zhejiang Institute of Inspection and Quarantine Science and Technology Main drafters of this standard: Zhang Yafen, Ye Zihong, Huang Chaoqun, Ma Aijin, Yu Xiaoping, Shen Juxuping, Xu Yipeng, Cui Haifeng, Li Yi, Chen Li, Wu Juan.
1 Scope
Determination of biological activity of secondary metabolites of plant hormones Indicator plant method
This standard specifies the method for determining the biological activity of secondary metabolites of plant hormones by indicator plant method TrrKAa-oJouaKAa
GB/T38577-—2020
This standard is applicable to the activity determination of secondary metabolites of plant hormones, such as auxin, cytokinin and gibberellin. Normative references
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, its latest version (including all amendments) applies to this document GB/T 6682 Specifications and test methods for water for analytical laboratories Terms, definitions and abbreviations
Terms and definitions
The following terms and definitions apply to this document. 3.1.1
Plant hormone secondary metabolites
Plant hormone-related secondary metabolites Active substances that regulate plant growth, development and dormancy, synthesized by plants themselves and obtained through microbial fermentation or chemical synthesis.
biological activity
Biological activity
The relative value of the ability of equal concentrations of plant hormone secondary metabolites to promote or inhibit plant growth, development and dormancy with their corresponding standards.
3.2 Abbreviations
The following abbreviations apply to this document.
GAa: gibberellic acid NAA: l-naphthylacetic acid ZT: zeatin
Within a certain concentration range, the concentration of plant hormone active substances is proportional to their ability to promote the growth of specific tissues or the synthesis of specific substances of the corresponding indicator plants. Taking the standard NAA as a reference, the standard curve is obtained according to the linear regression relationship between the relative elongation of wheat coleoptiles and the logarithm of NAA concentration. The ratio of NAA concentration to sample concentration that makes the relative elongation of coleoptiles equivalent within the linear range is calculated to represent the auxin activity of 1 mg/mL sample: Taking the standard ZT as a reference, the standard curve is obtained according to the linear regression relationship between the amount of amaranth synthesized in the cotyledons of the tail spike and the logarithm of ZT concentration. The ratio of ZT concentration to sample concentration that makes the amount of amaranth synthesized within the linear range is calculated. The ratio of the concentrations indicates the cytokinin activity of the 1 mg/mL sample: Taking the standard substance GA as a reference, the standard curve is obtained based on the linear regression relationship between the α-amylase activity in the aleurone layer of barley seeds and the logarithm of the GA concentration, and the GA concentration that makes the induced α-amylase activity equivalent within the linear range is calculated: The ratio of the sample concentration indicates the gibberellin activity of the 1 mg/mL sample 5 Reagents or materials
Unless otherwise specified, only analytical reagents are used, and the water is the first-grade water specified in GB/T6682. 5.1 Wheat seeds
Wheat (Triticum aestium L.). Long wheat 6135. Harvested in the same year, the grains are full, and the germination rate is ≥90%. 5.2
Amaranthus caudatus seeds
Amaranthus caudatus L., red variety. Harvested in the same year, full grains, germination rate>9o%5.3
Barley seeds
Barley (Hordeum vulgare L.), two-row barley. Harvested in the same year, full grains, germination rate ≥90%5.41% Sodium hypochlorite solution
Measure 100mL 10% sodium hypochlorite, dilute to 1000ml with water, prepare before use5.50.1% (W/V) starch solution
Weigh 1.00g soluble starch, add water to 50mL. Heat until completely dissolved, then add 8.16g potassium dihydrogen phosphate. After it dissolves, dilute to 1000ml with water. Prepare before use. I-KI solution
Weigh 0.60g potassium iodide and 0.06g iodine, dissolve with 0.05mol/L hydrochloric acid and dilute to 1000mL, prepare 5.71×10-8mol/L acetic acid buffer before use
Weigh 0.136g sodium acetate trihydrate, dissolve with water and dilute to 1000mL. Prepare 1×10-3mol/L sodium acetate solution: use a pipette to draw 57μL acetic acid solution. Dilute to 1000ml with water to prepare 1×10-mol/L acetic acid solution. Measure 590ml 1×10-mol/lL sodium acetate solution and 410ml 1×10-3mol/L acetic acid solution respectively, mix, add 1g streptomycin, and shake well.
3 Phosphate buffer containing L-tyrosine
Weigh 0.20g of l-tyrosine and dissolve it with 5.5ml of 0.5mol/l hydrochloric acid: weigh 2.39g of disodium hydrogen phosphate dodecahydrate and 0.91g of potassium dihydrogen phosphate. Dissolve it with water and dilute to 100ml to prepare 1/15mol/L phosphate buffer (pH7.0). Mix the two and dilute to 500ml and store at 4℃ for later use.
9 Phosphate-citric acid buffer·pH5.0
Weigh 1.80g of potassium dihydrogen phosphate, 1.02g of citric acid, 20g of decoction sugar, add 900ml of water to dissolve, measure with a pH meter and adjust the pH to 5.0. Dilute to 1000ml with water. Mix thoroughly and store at 4℃ for later use. 2
Purity 98%.
Purity ≥98%.
Purity ≥ 98%.
1mg/mL NAA standard preparation solution
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GB/T38577—2020
Weigh 10.0mg NAA standard, dissolve it with 0.5mL anhydrous ethanol, dilute it to 10mL with water, mix it thoroughly, and store it in a refrigerator at 4℃. The shelf life is 1 month.
5.141mg/mL GAs standard preparation solution
Weigh 10.0mg GA, standard, dissolve it with 0.5ml anhydrous ethanol, dilute it to 10mL with water, mix it thoroughly, and store it in a refrigerator at 4℃. The shelf life is 1 month.
1mg/mL ZT standard stock solution
Weigh 10.0mg ZT standard. Dissolve it with 0.5mL anhydrous ethanol and dilute it to 10mL with water. After fully mixing, refrigerate at 4℃ and keep it for 1 month.
5.16NAA standard working solution
Pipette 1mL 1mg/mlLNAA standard stock solution, add it to 9ml phosphate-citrate buffer and mix well. Get 1×10-mg/mL NAA solution. Dilute 10 times in sequence to get 1.00X10mg/ml, 1.00×10-mg/mL, 1.00×10-mg/ml.NAA standard working solution. Pipette 316μl.1mg/ml.NAA standard stock solution, 684l phosphate-citrate buffer and mix well to get 3.16×10-mg/ml NAA solution. Dilute 10 times in sequence to obtain 3.16×10-mg/ml3.16×10-mg/ml NAA standard working solution. Prepare before use. 5.17GA standard working solution
Pipette 1ml1mg/mlGA: standard stock solution and add to 9ml.Acetate buffer, mix well to obtain 1×10-mg/mL solution. Dilute 10 times in sequence to obtain 1.00X10-mg/ml, 1.00×10-mg/mL, and 1.00×10-mg/mlGA standard working solutions. Pipette 316μL 1mg/ml.GA, standard stock solution, 684μL acetate buffer, mix well to obtain 3.16×10-mg/mlGA solution. Dilute 10 times in sequence to obtain 3.16×10=mg/mL, 3.16×10-mg/mlL.3.16×10-mg/mLGA standard working solutions. Prepare before use.
ZT standard working solution
Pipette 1mL, 1mg/mLZT standard stock solution, add to 9mL. phosphate buffer containing L-tyrosine, mix well. Obtain 1×10-! mg/mL.ZT solution. Dilute 10 times in sequence to obtain 1.00×10-mg/mL, 1.00×10*mg/ml1.00×10-mg/mL ZT standard working solution. Pipette 316μlL1mg/ml.ZT standard stock solution, 684μl.phosphate buffer containing [-tyrosine, mix well. Get 3.16×10-1mg/mL.ZT solution. Dilute 10 times in sequence to get 3.16×10mg/ml, 3.16×10-smg/mL.ZT standard working solution. Prepare before use.
GB/T38577—2020
6 Instruments and equipment
Centrifuge: 10000×g.
Ultraviolet spectrophotometer.
pH meter: accuracy 0.01.
Electronic analytical balance: accuracy 1g. 0.01g. 0.0001g. 6.4
5Biochemical incubator: 25℃±1℃. Shading treatment. 6.5
6.6Shaker: 25℃±1℃, shading treatment6.7
Stereo microscope: with image processing system
Green light darkroom: wavelength 492nm~455nm.
Grinding beads: diameter 1mm~2mm.
Constant temperature water bath: 30℃±1℃.
High-speed oscillating grinder
7 Samples
7.1 Sample collection and preservation
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Weigh more than 10 mg of the pure substance to be tested extracted and purified from plant or microbial fermentation broth or chemically synthesized with an electronic analytical balance, dissolve it with 10 mL of ethanol, and record the concentration of the substance to be tested c (mg/mL). Then divide it into small portions and store it at 4°C in the dark. Divide it into at least 3 portions, and each portion after dividing it shall not be less than 1 ml. Weigh about 1000 mg of the solid product to be tested with an electronic analytical balance, dissolve it with 10 mL of the reagent specified in the product manual, or measure 10 mL of the liquid product with a measuring cylinder, and calculate the concentration of the substance to be tested according to the content of the substance to be tested described in the product manual, and record it as c. (mg/ml). Then divide it into small portions and store it according to the requirements of the product manual, and divide it into at least 3 portions, and each portion after dividing it shall not be less than 1 ml. 7.2
Sample preparation
During the test, select the corresponding buffer according to the different test objects (see Appendix A). Dilute the sample 5 times or 10 times in the range of 1×10-mg/mL~1×10-mg/ml, and record the concentration after dilution as c, (mg/mL). Prepare at least 3 concentration gradients for the sample, and repeat the measurement for each concentration sample at least 3 times.
8 Test steps
8.1 Auxin determination
8.1.1 Obtain wheat coleoptile segments
Wheat seeds are sterilized by soaking in a freshly prepared 1% sodium hypochlorite solution for 15 minutes, then washed with distilled water, and repeated once. Then, incubate in a 25°C biochemical incubator without light until the seedlings are about 25mm~35mm long. Select seedlings with a length of 25mm~30mm and remove the coleoptile from the base. Cut off the 3mm~5mm top in a green light dark room, cut the next 6mm segment, put it in phosphate-citrate buffer, shake it in a 25°C shaker for 1.5h (≤80r/min), and change the solution every 0.5h. 8.1.2 Measurement of coleoptile length
Place the obtained coleoptile segment under a stereo microscope and measure and record the length (L). After marking one by one, place it in equal amounts of phosphate-citrate buffer, NAA standard working solution of different concentrations, and sample solution of different concentrations, and culture it in a biochemical incubator at 25°C 4
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GB/T38577—2020
for 24h. Then place the treated coleoptile under a handheld microscope and measure and record the length (L). Calculate the actual elongation length △L of each coleoptile pin, and take 5 groups of data whose absolute difference of repeated test results does not exceed 20% of the arithmetic mean. Take the average value of the actual elongation length of the coleoptile after treatment with phosphate-citrate buffer △L. as the control. 8.1.3 Plotting the standard curve
Refer to Table A.1 in Appendix A for data measurement, recording and calculation. Calculate the relative elongation degree of the coleoptile after treatment with the standard working solution as △L/△L. Take the logarithm value x of the concentration of the standard working solution as the independent variable with a base of 10, and the relative elongation degree of the cut coleoptile after treatment as the dependent variable 3· draw the standard curve. 8.1.4 Measurement
Refer to Table A.1 to measure and calculate the relative elongation degree of the coleoptile after sample treatment (△L/△L)y ... Then, according to the effective y value, 2 is calculated from the standard curve, and the biological activity of the auxin-like substances in the sample is calculated according to formula (1). If there are multiple effective y values, the average value is calculated according to formula (1).
EAuin=CoX10
Wherein:
The biological activity of auxin, the unit is E;
The concentration of the sample to be tested, the unit is milligrams per milliliter (mg/mL); the logarithm of the concentration of the standard working solution;
The concentration of the diluted sample, the unit is milligrams per milliliter (mg/mL). The average value of the results of more than 3 independent experiments is used as the auxin activity of the sample, and the calculation result is retained to three significant figures. 8.24
Cytokinin determination
8.2.1 Obtaining Amaranthus caudatus seedlings
Soak about 2g of Amaranthus caudatus seeds in a freshly prepared 1% sodium hypochlorite solution for sterilization for 15 minutes, wash with distilled water, and repeat once. Then place it in a 25℃ biochemical incubator for imbibition for 5 hours. Then spread it flat on two layers of 18cm qualitative filter paper moistened with 9mlL distilled water in a culture dish, cover it with a film, and culture it in the dark at 25℃ for 72 hours. Then select the yellowed seedlings with uniform cotyledon size. 8.2.2 Measurement of erythrin concentration
Thirty etiolated seedlings were randomly selected as a test unit. They were induced and cultured in a biochemical incubator at 25℃ for 48 hours with equal amounts of phosphate buffer containing l-tyrosine, standard working solutions of different concentrations and sample solutions of different concentrations. Then 40 cotyledons with relatively uniform size and color were selected and transferred into a 2mL centrifuge tube containing 1mL distilled water. An appropriate amount of grinding beads were added and the cells were crushed by oscillation and grinding at a frequency of 50Hz for 5 minutes using a high-speed oscillating grinder. After centrifugation at 10000g for 5 minutes, 0.8mL of supernatant was taken and added into a centrifuge tube with 4.2ml distilled water added in advance. The tube was adjusted to zero with phosphate buffer containing L-tyrosine and measured with an ultraviolet spectrophotometer. The optical density values ​​ODs3 and ODo were read at wavelengths of 534nm and 650nm respectively. The difference (ODs34-OD50) was calculated and recorded as D, indicating the erythrin concentration. Calculate the average optical density value after treatment with phosphate buffer containing L-tyrosine as the control D. . 8.2.3 Standard curve drawing
Refer to Table A.2 to measure, record and calculate the data, with D.-D. indicating the increase in the concentration of lycopene after treatment with the standard working solution. Take the logarithm of the concentration of the standard working solution with a base of 10 as the independent variable, and the increase in the concentration of lycopene as the dependent variable y, and draw the standard curve.
GB/T38577-—2020
8.2.4 Measurement
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Refer to Table A.2 to measure and calculate the increase in the concentration of lycopene after the sample is treated. The test with a value within the linear range of the standard curve is considered a valid test, and the sample concentration c. is recorded. The data obtained from the invalid test is discarded. Then calculate from the standard curve based on the effective value, and calculate the biological activity of cytokinins in the sample according to formula (2). If there are multiple effective y values, calculate according to formula (2) and take the average value. EcTK=C×10
Where:
The biological activity of cytokinins, the unit is E: Co
The concentration of the sample to be tested, the unit is milligrams per milliliter (mg/ml); The logarithm of the concentration of the standard working solution:
The concentration of the diluted sample, the unit is milligrams per milliliter (mg/mL)...2)
Use the average of the results of more than three independent experiments as the cytokinin activity value of the sample, and retain three significant figures for the calculation result. 8.3
Gibberellin determination
8.3.1 Obtaining half-seeds of barley without embryos
Cut the barley seeds in half, select the half without embryos, soak and sterilize them in a freshly prepared 1% sodium hypochlorite solution for 15 minutes, wash with distilled water, and repeat once. Then swell on moistened sterilized filter paper at 25℃ for 48 hours. Then shake in acetic acid buffer at 50r/min~100r/min for 1.5 hours, change the water every 0.5 hours, take out and gently dry for use. 8.3.2 Measurement of α-amylase activity
Take 10 half-seeds of wheat without embryos that have been swollen as a test unit. Induce with 1mL of acetic acid buffer, different concentrations of GAs standard working solution and different concentrations of sample solution, shake and culture at 60r/min~100r/min in a 25℃ shaker for 24h. Grind the seeds and mix them well, centrifuge them at 1000r/min for 5min in a centrifuge to precipitate the seed debris. Then draw 0.4ml of the supernatant into a new 10ml centrifuge tube, add 1.6mL of 0.1% starch solution, mix well, and water bath at 30℃ for 10min. Add 2mL of α-Kl solution, make the volume to 5mL with distilled water, shake well until the solution turns blue, adjust to zero with acetic acid buffer, measure with a UV spectrophotometer, and read the OD value of the solution in each tube to indicate the α-amylase activity. Calculate the average optical density value D after acetic acid buffer treatment. As a control.
8.3.3 Standard curve drawing
Refer to Table A.3 to measure and record data and calculate, with D, minus D. Indicate the increase in α-amylase activity after treatment with the standard working solution. Take the logarithm of the concentration of the standard working solution as the independent variable with a base of 10, and the increase in α-amylase activity as the dependent variable y, and draw a standard curve.
8.3.4 Measurement
Measure and calculate the increase in α-amylase activity after sample treatment with reference to Table A.3 (D, -D.) y·Tests with y values ​​within the linear range of the standard curve are considered valid tests, and the sample concentration c is recorded.·The data obtained from invalid tests are discarded. Then calculate from the standard curve based on the valid y value, and calculate the biological activity of the hematin substances in the sample according to formula (3). If there are multiple valid values, calculate them according to formula (3) and take their average value.
Wherein:
EGA=CX10*
EGA—biological activity of gibberellin, unit is E; 6
Concentration of the diluted sample, unit is milligram per milliliter (mg/mL); logarithm of the concentration of the standard working solution;
Concentration of the sample to be tested, unit is milligram per milliliter (mg/mL). TrrKAa-cJouaKAa
GB/T38577—2020
The average value of the results of more than three independent experiments is used as the gibberellin activity value of the sample. The calculation result retains three significant figures. Repeatability
The absolute value difference of three independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. 7
GB/T38577—2020
A.1 The data records of auxin activity detection are shown in Table A.1. Table A.1
Measurement index
Lo1/μm
Len/um
Li/μm
AL/μm
Standard curve
Control group
Phosphate-citric acid solution
Appendix A
(Informative appendix)
Test data record table
Auxin activity test data record table
NAA standard solution concentration/(mg/mL)
1.00×10-
3.16×10-
Cytokinin activity test data record is shown in Table A.2.1.00×10-
3.16×10 -61
1.00×10-
Cytokinin activity detection data recording table Control group
Measurement index
ODanel
Phosphate
Buffer containing L-tyrosine
|ZT standard solution concentration/(mg/mL)
3.16×10-
1.00×10-
3.16×10-
1.00×10-
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Sample solution concentration/(mg/mL)
Sample solution concentration/(mg/mL)
And normalized standard
DD.
Standard activity
Phosphorus test
Buffer containing L-tyrosine
Activity A.2 (continued)
ZT standard solution indicator/(mg/mL)
1.00×10-5
3.16×10-
1.00×10-
The large amount is measured and the record is generated, see detail A.3. Activity A.3
Determine the activity of
and standardize the buffer solution
Standard activity
1.00×10-
3.16×10-
Activity of plant hormone production bzxz.net
1.00×10~
GA standard solution expression/(mg/mL)
1.00×10-5
3.16×10-
3.16×10-
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GB/T38577-—2020
Expression with long solution/(mg/mL)
100×10-
Expression with long solution,
(mg/mL)1D. ) The test with y·y value in the linear range of the standard curve is regarded as a valid test, and the sample concentration c is recorded. The data obtained from the invalid test is discarded. Then, according to the valid y value, the biological activity of the gibberellin in the sample is calculated according to formula (3). If there are multiple valid values, the average value is calculated according to formula (3).
Where:
EGA=CX10*
EGA—biological activity of gibberellin, unit is E; 6
concentration of the diluted sample, unit is milligram per milliliter (mg/mL); logarithm of the concentration of the standard working solution;
concentration of the sample to be tested, unit is milligram per milliliter (mg/mL). TrrKAa-cJouaKAa
GB/T38577—2020
The average value of the results of more than three independent experiments is used as the gibberellin activity value of the sample. The calculation result is retained to three significant figures. Repeatability
The absolute value difference of three independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. 7
GB/T38577—2020
A.1 The data records of auxin activity detection are shown in Table A.1. Table A.1
Measurement index
Lo1/μm
Len/um
Li/μm
AL/μm
Standard curve
Control group
Phosphate-citric acid solution
Appendix A
(Informative appendix)
Test data record table
Auxin activity test data record table
NAA standard solution concentration/(mg/mL)
1.00×10-
3.16×10-
Cytokinin activity test data record is shown in Table A.2.1.00×10-
3.16×10 -61
1.00×10-
Cytokinin activity detection data recording table Control group
Measurement index
ODanel
Phosphate
Buffer containing L-tyrosine
|ZT standard solution concentration/(mg/mL)
3.16×10-
1.00×10-
3.16×10-
1.00×10-
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Sample solution concentration/(mg/mL)
Sample solution concentration/(mg/mL)
And normalized standard
DD.
Standard activity
Phosphorus test
Buffer containing L-tyrosine
Activity A.2 (continued)
ZT standard solution indicator/(mg/mL)
1.00×10-5
3.16×10-
1.00×10-
The large amount is measured and the record is generated, see detail A.3. Activity A.3
Determine the activity of
and standardize the buffer solution
Standard activity
1.00×10-
3.16×10-
Activity of plant hormone production
1.00×10~
GA standard solution content/(mg/mL)
1.00×10-5
3.16×10-
3.16×10-
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GB/T38577-—2020
Use long solution to show the activity/(mg/mL)
100×10-
Use long solution to show the activity,
(mg/mL)1D.) The test with y·y value in the linear range of the standard curve is regarded as a valid test, and the sample concentration c is recorded. The data obtained from the invalid test is discarded. Then, according to the valid y value, the biological activity of the gibberellin in the sample is calculated according to formula (3). If there are multiple valid values, the average value is calculated according to formula (3).
Where:
EGA=CX10*
EGA—biological activity of gibberellin, unit is E; 6
concentration of the diluted sample, unit is milligrams per milliliter (mg/mL); logarithm of the concentration of the standard working solution;
concentration of the sample to be tested, unit is milligrams per milliliter (mg/mL). TrrKAa-cJouaKAa
GB/T38577—2020
The average value of the results of more than three independent experiments is used as the gibberellin activity value of the sample. The calculation result is retained to three significant figures. Repeatability
The absolute value difference of three independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. 7
GB/T38577—2020
A.1 The data records of auxin activity detection are shown in Table A.1. Table A.1
Measurement index
Lo1/μm
Len/um
Li/μm
AL/μm
Standard curve
Control group
Phosphate-citric acid solution
Appendix A
(Informative appendix)
Test data record table
Auxin activity test data record table
NAA standard solution concentration/(mg/mL)
1.00×10-
3.16×10-
Cytokinin activity test data record is shown in Table A.2.1.00×10-
3.16×10 -61
1.00×10-
Cytokinin activity detection data recording table Control group
Measurement index
ODanel
Phosphate
Buffer containing L-tyrosine
|ZT standard solution concentration/(mg/mL)
3.16×10-
1.00×10-
3.16×10-
1.00×10-
rrKAa-cJouaKAa
Sample solution concentration/(mg/mL)
Sample solution concentration/(mg/mL)
And normalized standard
DD.
Standard activity
Phosphorus test
Buffer containing L-tyrosine
Activity A.2 (continued)
ZT standard solution indicator/(mg/mL)
1.00×10-5
3.16×10-
1.00×10-
The large amount is measured and the record is generated, see detail A.3. Activity A.3
Determine the activity of
and standardize the buffer solution
Standard activity
1.00×10-
3.16×10-
Activity of plant hormone production
1.00×10~
GA standard solution content/(mg/mL)
1.00×10-5
3.16×10-
3.16×10-
rrKAa-cJouaKAa
GB/T38577-—2020
Use long solution to show the activity/(mg/mL)
100×10-
Use long solution to show the activity,
(mg/mL)
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