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GB/T 38484-2020 English PDF (GBT38484-2020)

GB/T 38484-2020 English PDF (GBT38484-2020)

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GB/T 38484-2020: Determination of the biological activity for plant hormone-related secondary metabolites - Cytological method

This Standard specifies the method for determining the biological activity for plant hormone-related secondary metabolites by cytological evaluation. This Standard is applicable to the determination of the activity of auxin, cytokinin and gibberellin, which are the plant hormone-related secondary metabolites.
GB/T 38484-2020
GB
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
ICS 07.080
A 21
Determination of the Biological Activity for Plant
Hormone-Related Secondary Metabolites - Cytological
Method
ISSUED ON: NOVEMBER 19, 2020
IMPLEMENTED ON: NOVEMBER 19, 2020
Issued by: State Administration for Market Regulation;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Terms, Definitions and Abbreviations ... 4
4 Principle ... 5
5 Reagents or Materials ... 6
6 Apparatus ... 8
7 Measurement Procedures ... 8
8 Repeatability ... 11
Appendix A (Informative) Record Sheet of the Test Data ... 12
Determination of the Biological Activity for Plant
Hormone-Related Secondary Metabolites - Cytological
Method
1 Scope
This Standard specifies the method for determining the biological activity for plant hormone-related secondary metabolites by cytological evaluation.
This Standard is applicable to the determination of the activity of auxin, cytokinin and gibberellin, which are the plant hormone-related secondary metabolites. 2 Normative References
The following documents are essential to the application of this document. For the dated documents, only the versions with the dates indicated are applicable to this document; for the undated documents, only the latest version (including all the amendments) is applicable to this document.
GB/T 6682 Water for Analytical Laboratory Use ?€? Specification and Test Methods 3 Terms, Definitions and Abbreviations
3.1 Terms and definitions
For the purpose of this document, the following terms and definitions apply. 3.1.1 Plant hormone-related secondary metabolites
Active substances synthesized by plants and obtained through microbial fermentation or chemical synthesis that have the ability to regulate plant growth, development and dormancy.
3.1.2 Biology activity
The relative value of a unit concentration of plant hormone-related secondary metabolites and its corresponding standard to promote or inhibit plant growth, development and dormancy.
5 Reagents or Materials
Unless otherwise specified, only use analytical reagent.
5.1 Water. Class-I specified in GB/T 6682.
5.2 AuxRE::GUS cell line. Arabidopsis thaliana Columbia (Col-0) ecotype, contains auxin response elements AuxREs, carries a single copy of gus gene; and the number of viable cells is ???1??106 cells/mL.
5.3 GARE::GUS cell line. Arabidopsis thaliana Columbia (Col-0) ecotype, contains gibberellin response element TATC-boxs, carries a single copy of gus gene; and the number of viable cells is ???1??106 cells/mL.
5.4 CTKRE::GUS cell line. Arabidopsis thaliana Columbia (Col-0) ecotype, contains the cytokinin response element ARR1AT, carries a single copy of the gus gene; and the number of viable cells is ???1??106 cells/mL.
5.5 Cell line culture solution. Take 4.74g of MS culture medium without sucrose and agar; add 950mL of water to dissolve; add 30.00g of sucrose, 0.75g of calcium chloride, 0.35g of potassium nitrate; mix well. Then adjust the pH to 5.60 with sodium hydroxide; add water to make constant volume to 1000mL; use the 0.22??m filter membrane for filtering and sterilization. It may be prepared immediately before use. 5.6 DPBS, pH 7.4. Take 8.00g of sodium chloride, 0.20g of potassium chloride, 2.90g of disodium hydrogen phosphate dodecahydrate, and 0.24g of potassium dihydrogen phosphate; add water and make constant volume to 1000mL; after mixing well, store at room temperature for later-use. Similar commercial products may be used. 5.7 Extraction solution. Take 3.72g of disodium ethylenediaminetetraacetic acid dihydrate; add 700??L of ??-mercaptoethanol, 1mL of polyethylene glycol octylphenol ether; dissolve with DPBS and make constant volume to 1000mL; mix well and store at room temperature for later-use.
5.8 Extraction solution containing 1mmol/L 4-MUG. Take 105.6mg of 4-MUG; dissolve it with the extraction solution and make constant volume to 300mL; and prepare it immediately before use.
5.9 0.2mg/mL bovine serum albumin solution. Take 20.0mg of bovine serum protein standard substance; dissolve it with DPBS and make constant volume to 100 mL; it shall be prepared immediately before use.
5.10 Reaction terminating reagent. Take 21.20g of Na2CO3; dissolve in water and make constant volume to 1000mL.
5.21 GA3 standard working solution. Pipette 200??L of 1mg/mL GA3 standard stock solution, 800??L of cell line culture solution; and mix well to obtain 2??10-1 mg/mL GA3 solution. Dilute 10 times successively to obtain 2.00??10-4 mg/mL, 2.00??10-5 mg/mL, 2.00??10-6 mg/mL GA3 standard working solutions. Pipette 632??L of 1mg/mL GA3 standard stock solution, 368??L of cell line culture solution; and mix well to obtain 6.32??10-1 mg/mL GA3 solution. Dilute 10 times successively to obtain 6.32??10-5 mg/mL, 6.32??10-6 mg/mL GA3 standard working solutions. It shall be prepared immediately before use.
5.22 ZT standard working solution. Pipette 200??L of 1mg/mL ZT standard stock solution, 800??L of cell line culture solution; and mix well to obtain 2??10-1 mg/mL ZT solution. Dilute 10 times successively to obtain 2.00??10-3 mg/mL, 2.00??10-4 mg/mL, 2.00??10-5 mg/mL ZT standard working solutions. Pipette 632??L of 1mg/mL ZT standard stock solution, 368??L of cell line culture solution; and mix well to obtain 6.32??10-1 mg/mL ZT solution. Dilute 10 times sequentially to obtain 6.32??10-4 mg/mL, 6.32??10-5 mg/mL ZT standard working solutions. It shall be prepared immediately before use. 6 Apparatus
6.1 pH meter: accuracy of 0.01.
6.2 Electronic analytical balance: accuracy of 0.1mg, 0.01g, 1g.
6.3 Light incubator: 25??C??1??C.
6.4 Constant temperature water bath: 37??C.
6.5 Cell culture plate.
6.6 Microplate reader: can detect absorbance and fluorescence intensity at the same time.
6.7 Microplate: black.
7 Measurement Procedures
7.1 Preparation of the specimen
Convert according to the mass of the effective substance (or substance to be tested) of the test sample; take a sufficient amount of solid sample or pipette a sufficient amount of liquid sample; and select the appropriate reagent or water to dissolve and prepare the effective substance (or substance to be tested) solution as the specimen to be tested according to the product instruction manual or the characteristics of the substance to be tested; and the concentration is recorded as ??0 (mg/mL). When testing, 7.2.3 Determination of GUS protease activity in sample
Take an appropriate amount of sample solution (make the total protein content 20??g~200??g); record the volume V2. Add 1mL of the extraction solution containing 1mmol/L 4-MUG; put it into 37??C water bath; then after 5min, 25min, 45min, 65min and 85min in the water bath, take out 200??L of the reaction solution. Add 800??L of the reaction terminating reagent and mix well. Then pipette 200??L into the sample well of the microplate. Pipette 200??L of 4-MU working solution (4-MU content is 200nmol, 100nmol, 50nmol, 25nmol, 12.5nmol, 6.25nmol, respectively); and add it to the sample well of the microplate. Note that each sample detection microplate is required to bring standard substance; and each standard substance or sample solution is repeatedly added to 3 sample wells. And the reaction terminating reagent is used as a blank control to adjust the zero. Measure the fluorescence intensity under the excitation wavelength of 365nm and the absorption wavelength of 455nm. Draw a standard curve with the concentration of the 4-MU standard substance as the abscissa and the fluorescence intensity as the ordinate. And use the standard curve to convert the fluorescence intensity after the sample reaction into the amount of 4-MU substance actually produced after the sample reaction. Finally, draw a standard curve with the reaction time of the sample the abscissa, and the amount of 4-MU produced by the sample reaction as the ordinate. And calculate the amount, n, of 4-MU substance produced through hydrolysing 4-MUG per minute by the GUS protein; and according to Formula (2) calculate the GUS enzyme activity, y, in the sample.
Where:
y - GUS enzyme activity in the sample, in [pmol/(min?€???g)];
n - the amount of 4-MU substance produced through hydrolysing 4-MUG per minute by the GUS protein in the sample, in pmol/min;
??total - total protein concentration in the sample, in ??g/??L;
V2 ?€? sampling loading volume when measuring the enzyme activity; in ??L. The calculation results shall be retained to three digits after the decimal point. 7.3 Drawing of the standard curve
Determine the induced expression level of GUS protein after treatment with the standard working solution as described in 7.2; and record the data with reference to Appendix A. Draw a standard curve by using 10 as the base to take the logarithmic value x of the standard substance working solution concentration as the independent ??total

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