Some standard content:
ICS07.080
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National Standard of the People's Republic of China
GB/T38580—2020
Technical specification for mutagenesis breeding of microorganisms
Technical specification for mutagenesis breeding of microorganisms2020-03-31Issued
State Administration for Market Regulation
National Standardization Administration
Implementation on 2020-03-31
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/T38580—2020
Drafting units of this standard: Tsinghua University, Luoyang Huaqing Tianmu Biotechnology Co., Ltd., China National Institute of Standardization Main drafters of this standard: Zhang Du, Xing Xinhui, Li Mei, Jian Xingjin, Wang Liyan, Guo Xiaojie, Zhang Lele, Ma Aijin. 1
1 Scope
Technical specification for microbial mutagenesis breeding
This standard specifies the requirements and verification methods for microbial mutagenesis breeding. iriKAa~cJouakAa
GB/T38580—2020
This standard applies to ultraviolet, plasma and chemical mutagenesis breeding of microorganisms such as bacteria, actinomycetes, yeast and mold. 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 National Food Safety Standard
Methods for the treatment of mutagens, teratogens and carcinogens GB15193.19
Terms and Definitions
The following terms and definitions apply to this document. 3.1
Medium strains
Microorganisms that play a physiological metabolic role in the fermentation process for industrial, agricultural, food, medical and other purposes. 3.2
mutagenesis
Methods of inducing mutations in genetic material through artificial measures 3.3
mutagenesisbreedingofmicroorganisms
Breeding methods of inducing mutations in the genetic material of microorganisms through artificial mutagenesis and selecting the desired mutant strains from the mutant population to cultivate new varieties
4 Requirements
4.1 Personnel
Test personnel should be familiar with basic microbial operations, fully understand the risks and safety operation specifications of using microorganisms and mutagens before conducting relevant experiments, and take appropriate protection during the experiment. 2 Facilities and environment
Having a laboratory or workshop that can carry out basic microbial experiments, equipped with relevant water, electricity, gas and other infrastructure: The laboratory biosafety level must meet the requirements of the mutated microorganisms.
4.3 Mutagenic materials
4.3.1 General
Mutagenic materials should follow the following principles:
GB/T38580-2020
Single, dispersed cells, preferably in suspension; b)
The fewer nuclei, the better, preferably mononuclear; Select appropriate culture conditions according to the type of bacteria to ensure that the bacteria have strong vitality: e
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The preparation process of mutagenic materials should be carried out in a clean bench, and the added reagents and solutions should be sterilized or filtered in advance. 4.3.2 Bacterial suspension
Inoculate the bacteria into a suitable culture medium, culture to the logarithmic growth phase, collect the bacteria by centrifugation, wash twice with sterile saline, and then resuspend with an appropriate amount of saline to adjust the concentration of the bacteria to 1×10°CFU/ml~1X10*CFU/mL. 4.3.3 Spore suspension
Inoculate the strain onto a solid plate or slant and culture until a large number of spores are produced: wash with saline and collect by centrifugation. Wash twice with saline, resuspend with an appropriate amount of saline and fully disperse by mechanical oscillation: remove the mycelium by filtration. Finally, suspend with saline to obtain a uniform spore suspension, and adjust its concentration to 1×10*CFU/ml~1×10*CFU/mL. 4.3.4 Mycelium suspension
Inoculate the strain into a suitable culture medium and culture until a large number of vigorously growing mycelium are produced, take a certain amount of bacterial liquid and collect by centrifugation. Wash twice with saline, resuspend with an appropriate amount of saline and break the mycelium by mechanical oscillation, and filter out the unbroken mycelium. Finally, suspend with saline to obtain a uniform mycelium suspension, and adjust its concentration to 1X10*CFU/mL~1X10°CFU/mL. 4.3.5 Protoplast suspension
Inoculate the strain into a suitable culture medium, culture to obtain a large number of fresh mycelium or spores, and collect the mycelium or spores. Wash twice with an osmotic pressure stabilizer and resuspend, add a certain amount of wall-breaking enzyme to the resuspended liquid, perform enzymolysis under the optimal conditions, take samples at regular intervals and observe the release of protoplasts under a microscope. After the enzymolysis is completed, filter out the unenzymolyzed mycelium, collect the protoplasts by centrifugation, wash the protoplasts three times with an osmotic pressure stabilizer, and adjust the concentration to 1×10°CFU/mL~1X10°CFU/ml. 4.4 Mutagenesis breeding operation
4.4.1 Ultraviolet mutagenesis
The requirements for ultraviolet mutagenesis breeding operation are as follows:
a) Parameter setting: It is advisable to select the following parameters: ultraviolet wavelength 260nm, ultraviolet lamp power 10W~30W, irradiation distance 10cm~30cm. The irradiation time of bacteria, protoplasts, and Streptomyces spore suspensions should be 10s to 90s. The irradiation time of yeast and strawberry spore suspensions should be 1min to 10min. Appropriate parameters should be optimized for different microbial samples and mutagenesis requirements.
b) Turn on the UV lamp 20min to 30min in advance. e
The permeabilized material should be placed in a glass plate III with a sterile stirring bar and stirred while irradiating. d)
Each sample should be repeated three times, and the same mutagenic material should be used as a negative control without irradiating UV light. e
All steps after mutagenesis should be performed under red light in a dark room. The treated materials and negative controls should be kept in an ice bath for 1h to 2h away from light. g)
After mutagenesis, the sample materials and negative controls should be diluted in a gradient manner, and the bacterial solution of appropriate dilution should be applied to a solid plate and cultured away from light until a single colony is produced.
h) The number of colonies on the mutagenic and negative control plates should be counted, and the mutagenic lethality should be calculated. 4.4.2 Atmospheric pressure and room temperature plasma mutagenesis
The requirements for atmospheric pressure and room temperature plasma mutagenesis breeding are as follows: 2
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GB/T38580—2020
Parameter setting: The following parameters should be selected: irradiation power 100W~120W, irradiation distance 2mm, working gas flow 10L/min. a)
The irradiation time of bacteria, protoplasts, and streptomyces spore suspension should be selected from 30s~5min, and the irradiation time of yeast and mold spore suspension should be selected from 1min6min.
Each sample should be repeated three times, and the same mutagenic material should be taken as the negative control. The negative control is not irradiated. After the plasma irradiation is completed, the irradiated sample should be immediately placed in the prepared sterile centrifuge tube with culture medium added in advance and mixed evenly. The treated materials and negative controls should be diluted in a gradient, and the bacterial solution with appropriate dilution should be selected to coat the solid plate and cultured until a single colony is produced.
The number of colonies on the mutagenic and negative control plates should be counted, and the mutagenic lethality should be calculated. e)
Chemical mutagenesis
The requirements for chemical mutagenesis are as follows:
Choice of chemical mutagen: It is advisable to select appropriate chemical mutagens according to the mutagenic materials and mutagenic requirements. For commonly used chemical mutagens and their preparation methods, please refer to Appendix A and Appendix B. Chemical mutagen concentration: For commonly used mutagen concentrations and treatment times for different microorganisms, please refer to Appendix C. b)
Add mutagenic working solutions of different concentration gradients to the mutagenic materials, mix well and treat for different times. If necessary, add reaction termination solution to terminate the reaction, centrifuge, discard the supernatant, and wash the mutagenic bacteria with sterile deionized water. Three replicates should be set for each mutagenic dose group or each treatment time group. No chemical mutagen is added to the negative control group, and an equal amount of d)
sterile water is added instead.
The treated materials and negative controls should be diluted in a gradient manner, and the appropriate dilution of bacterial solution should be selected to coat the solid plate and cultured until a single colony is produced.
The number of colonies on the mutagenic and negative control plates should be counted, and the mutagenic lethality should be calculated. After using chemical mutagens, they should be treated according to GB15193.19 was treated with the method 5
Verification method
The result of mutagenesis was expressed as lethality, which was calculated according to formula (1): Ce-CT
Wherein:
Lethality;
Negative control plate colony count;
Test group plate colony count.
·(1)）
GB/T38580-—2020
(Informative Appendix)
Types, mechanisms of action of chemical mutagens and commonly used chemical mutagens Types, mechanisms of action of chemical mutagens and commonly used chemical mutagens are shown in Table A.1. Table A.1
Types of chemical mutagens
Base analogs
Alkylating agents
Frameshift mutagens
Oxidative mutagens
Metal ions
Mechanism of action
Commonly used mutagens
5-bromouracil (5-BU)
5-fluorouracil (5-FU)
Replaces the normal bases in DNA
Alkylating agents on the bases in the DNA molecule
Base modification
Frameshift mutation
Oxidative damage to DNA bases
The mechanism is still unclear
6-Azauracil (6-NU)
2-Aminopurine (2-AP)
6-Mercaptopurine (6-MP)
8-Aminoguanine (8-NG)
Nitrosoethylurea
Nitrosoguanidine
Diethyl sulfate
Sulfate (sulfonic acid) esters
Methyl methanesulfonate
Ethyl methanesulfonate
Acridine orange
Proflavin
Nitrite (and its salts)
Manganese ion
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B.1 Nitrosoguanidine (NTG)
The preparation method is as follows:
Appendix B
(Informative Appendix)
Preparation methods of commonly used chemical mutagens
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GB/T38580-—2020
pH6.0 phosphate buffer: prepare 0.2mol/L NaHPO·2H0 solution and 0.2mol/L NaHPO:2H0 solution respectively. Take out 12.3mL of the former and 87.7mL of the latter. Mix the two to get pH6.0 phosphate buffer 1mg/ml. Nitrosoguanidine solution: weigh 50mg nitrosoguanidine. Dissolve it with 5mL acetone. Store at 4℃. Add 45mL pH6.0 phosphate buffer to it before use, shake it to fully dissolve it, and filter to sterilize. Mutagenic terminator: 0.07mol/L pH8.6 disodium hydrogen phosphate buffer. Take 2.507g of sodium dihydrogen phosphate and dissolve it in 100ml sterile water, filter to sterilize.
Sodium nitrite (NIT)
The preparation method is as follows:
pH4.5 acetate buffer: weigh 18g sodium acetate and 9.8mL glacial acetic acid in a 1L beaker. Add a small amount of distilled water to dissolve, transfer to a 1.000mL volumetric flask to dissolve, mix thoroughly, filter and sterilize, and store at 4℃ for later use. -0.1mol/L sodium nitrite solution: take 0.69g sodium nitrite, dissolve in 100ml sterile water, filter and sterilize. When used, mix with pH4.5 acetate buffer in a volume ratio of 1:2 before use. Mutagenic terminator: 25% NaS0 solution. Weigh 4g NaSO·5H2O, dissolve in 6ml distilled water and place in a 4℃ refrigerator for later use, filter and sterilize when used. The volume ratio with the mutagenic solution is 1:1. B.3 Diethyl sulfate (DES)
The preparation method is as follows:
Diethyl sulfate is insoluble in water, but has a short half-life when dissolved in ethanol or acetaldehyde ether, which is 1 hour at 30°C, so it needs to be prepared before use. Dissolve 0.5 mL of DES in 4.5 mL of anhydrous ethanol to prepare a 10% stock solution, and filter and sterilize. B.4 Ethyl methanesulfonate (EMS)
Preparation method is as follows:
pH 7.0 phosphate buffer: weigh 9.39g KHPO,3HO and 3.5g KHPO, add distilled water to 1000mL, filter, sterilize at 121℃ for 15min or 115℃ for 30min; accurately weigh 5.408g EMS powder in a 10mL volumetric flask, first add 6mL pH 7.0 phosphate buffer to dissolve, then make up to 10mlL. Prepare 5.0mol/L mother solution, store at 4℃. Filter and sterilize before use. 5
GB/T38580-2020
Appendix C
(Informative Appendix)
The mutagenic conditions and lethality of commonly used chemical mutagens are shown in Table C.1. Table C.1
Mutagen
Sodium nitrite (NIT)
Nitrosoguanidine (NTG)
Diethyl sulfate
Working concentration
0.05mol/L
0.025mol/L
2mg/mL
0.025mol/L
0.025mol/L
0.05mol/L
0.025mol/L
2mg/mL
1mg/mL
1mg/mL
mutagenic strains
berberine-producing endophytes
fungal mycelium suspension
green wood spores
Maoyuan Streptomyces spores
sticky tape mold spores
white rot fungus spores
endophytic fungi of Fritillaria fasciata
mycelium suspension
Bacillus cereus
bacterial suspension
Maoyuan Streptomyces spores
Bacillus cereus
bacterial suspension
Bacillus subtilis
bacterial suspension||t t||Lactobacillus plantarum
Actinomycetes spore suspension
Neurosporium robusta
Spore suspension
Trichoderma viride spores
Streptomyces shandongensis
Mutagenesis time
10min~20min
Lethality
85%~95%
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Mutagenesis temperature
25℃~26℃
Mutagen
Diethyl sulfate
Ethyl methanesulfonate|| tt||Lithium chloride
Working concentration
Table C.1 (continued)
Mutagenic bacteria
Thermophilic anaerobic bacteria
Bacillus cereus
Actinomycetes spore suspension
Saccharomyces cerevisiae
Lactobacillus liquid
Actinomycetes spore suspension
Mutagenic time
Lethality
70%80%
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GB/T38580—2020
Mutagenic temperature1
Types of chemical mutagens
Base analogs
Alkylating agents
Frameshift mutagens
Oxidative mutagens
Metal ions
Mechanism of action
Common mutagens
5-bromouracil (5-BU)
5-fluorouracil (5-FU)
Replaces the normal bases in DNA
Alkylates the bases in the DNA molecule
Modification
Frameshift mutation
Oxidative damage to DNA bases
The mechanism is unclear
6-Azauracil (6-NU)
2-Aminopurine (2-AP)
6-Mercaptopurine (6-MP)
8-Aminoguanine (8-NG)
Nitrosoethylurea
Nitrosoguanidine
Diethyl sulfate
Sulfate (sulfonic acid) esters
Methyl Methyl methyl sulfonate
Ethyl methyl sulfonate
Acridine orange
Proflavin
Nitrite (and its salts)
Manganese ion
TrrKAa-cJouakAa
B.1 Nitrosoguanidine (NTG)
The preparation method is as follows:
Appendix B
(Informative Appendix)
Preparation methods of commonly used chemical mutagens
iriKAa~cJou akAa
GB/T38580-—2020
pH6.0 phosphate buffer: prepare 0.2mol/L NaHPO·2H0 solution and 0.2mol/L NaHPO:2H0 solution respectively. Take out 12.3mL of the former and 87.7mL of the latter. Mix the two to get pH6.0 phosphate buffer 1mg/ml. Nitrosoguanidine solution: weigh 50mg nitrosoguanidine. Dissolve it with 5mL acetone. Store at 4℃. Add 45mL pH6.0 phosphate buffer to it before use, shake it to fully dissolve it, and filter to sterilize. Mutagenic terminator: 0.07mol/L pH8.6 disodium hydrogen phosphate buffer. Take 2.507g of sodium dihydrogen phosphate and dissolve it in 100ml sterile water, filter to sterilize.
Sodium nitrite (NIT)
The preparation method is as follows:
pH4.5 acetate buffer: weigh 18g sodium acetate and 9.8mL glacial acetic acid in a 1L beaker. Add a small amount of distilled water to dissolve, transfer to a 1.000mL volumetric flask to dissolve, mix thoroughly, filter and sterilize, and store at 4℃ for later use. -0.1mol/L sodium nitrite solution: take 0.69g sodium nitrite, dissolve in 100ml sterile water, filter and sterilize. When used, mix with pH4.5 acetate buffer at a volume ratio of 1:2 before use. Mutagenic terminator: 25% NaS0 solution. Weigh 4g NaSO·5H2O, dissolve in 6ml distilled water and place in a 4℃ refrigerator for later use, filter and sterilize when used. The volume ratio with the mutagenic solution is 1:1. B.3 Diethyl sulfate (DES)
The preparation method is as follows:
Diethyl sulfate is insoluble in water, but has a short half-life when dissolved in ethanol or acetaldehyde ether, which is 1 hour at 30°C, so it needs to be prepared before use. Dissolve 0.5 mL of DES in 4.5 mL of anhydrous ethanol to prepare a 10% stock solution, and filter and sterilize. B.4 Ethyl methanesulfonate (EMS)
Preparation method is as follows:
pH 7.0 phosphate buffer: weigh 9.39g KHPO,3HO and 3.5g KHPO, add distilled water to 1000mL, filter, sterilize at 121℃ for 15min or 115℃ for 30min; accurately weigh 5.408g EMS powder in a 10mL volumetric flask, first add 6mL pH 7.0 phosphate buffer to dissolve, then make up to 10mlL. Prepare 5.0mol/L mother solution, store at 4℃. Filter and sterilize before use. 5
GB/T38580-2020
Appendix C
(Informative Appendix)
The mutagenic conditions and lethality of commonly used chemical mutagens are shown in Table C.1. Table C.1
Mutagen
Sodium nitrite (NIT)
Nitrosoguanidine (NTG)
Diethyl sulfate
Working concentration
0.05mol/L
0.025mol/L
2mg/mL
0.025mol/L
0.025mol/L
0.05mol/L
0.025mol/L
2mg/mL
1mg/mL
1mg/mL
mutagenic strains
berberine-producing endophytes
fungal mycelium suspension
green wood spores
Maoyuan Streptomyces spores
sticky tape mold spores
white rot fungus spores
endophytic fungi of Fritillaria balsamifera
mycelium suspension
Bacillus cereus
bacterial suspension
Maoyuan Streptomyces spores
Bacillus cereus
bacterial suspension
Bacillus subtilis
bacterial suspension||t t||Lactobacillus plantarum
Actinomycetes spore suspension
Neurosporium robusta
Spore suspension
Trichoderma viride spores
Streptomyces shandongensis
Mutagenesis time
10min~20min
Lethality
85%~95%
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Mutagenesis temperature
25℃~26℃
Mutagen
Diethyl sulfate
Ethyl methanesulfonate|| tt||Lithium chloride
Working concentration
Table C.1 (continued)
Mutagenic bacteria
Thermophilic anaerobic bacteria
Bacillus cereus
Actinomycetes spore suspension
Saccharomyces cerevisiae
Lactobacillus liquid
Actinomycetes spore suspension
Mutagenic time
Lethality
70%80%
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GB/T38580—2020
Mutagenic temperature1
Types of chemical mutagens
Base analogs
Alkylating agents
Frameshift mutagens
Oxidative mutagens
Metal ions
Mechanism of action
Common mutagens
5-bromouracil (5-BU)
5-fluorouracil (5-FU)
Replaces the normal bases in DNA
Alkylates the bases in the DNA molecule
Modification
Frameshift mutation
Oxidative damage to DNA bases
The mechanism is unclear
6-Azauracil (6-NU)
2-Aminopurine (2-AP)
6-Mercaptopurine (6-MP)
8-Aminoguanine (8-NG)
Nitrosoethylurea
Nitrosoguanidine
Diethyl sulfate
Sulfate (sulfonic acid) esters
Methyl Methyl methyl sulfonate
Ethyl methyl sulfonate
Acridine orange
Proflavin
Nitrite (and its salts)
Manganese ion
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B.1 Nitrosoguanidine (NTG)
The preparation method is as follows:
Appendix B
(Informative Appendix)
Preparation methods of commonly used chemical mutagens
iriKAa~cJou akAa
GB/T38580-—2020
pH6.0 phosphate buffer: prepare 0.2mol/L NaHPO·2H0 solution and 0.2mol/L NaHPO:2H0 solution respectively. Take out 12.3mL of the former and 87.7mL of the latter. Mix the two to get pH6.0 phosphate buffer 1mg/ml. Nitrosoguanidine solution: weigh 50mg nitrosoguanidine. Dissolve it with 5mL acetone. Store at 4℃. Add 45mL pH6.0 phosphate buffer to it before use, shake it to fully dissolve it, and filter to sterilize. Mutagenic terminator: 0.07mol/L pH8.6 disodium hydrogen phosphate buffer. Take 2.507g of sodium dihydrogen phosphate and dissolve it in 100ml sterile water, filter to sterilize.
Sodium nitrite (NIT)
The preparation method is as follows:
pH4.5 acetate buffer: weigh 18g sodium acetate and 9.8mL glacial acetic acid in a 1L beaker. Add a small amount of distilled water to dissolve, transfer to a 1.000mL volumetric flask to dissolve, mix thoroughly, filter and sterilize, and store at 4℃ for later use. -0.1mol/L sodium nitrite solution: take 0.69g sodium nitrite, dissolve in 100ml sterile water, filter and sterilize. When used, mix with pH4.5 acetate buffer in a volume ratio of 1:2 before use. Mutagenic terminator: 25% NaS0 solution. Weigh 4g NaSO·5H2O, dissolve in 6ml distilled water and place in a 4℃ refrigerator for later use, filter and sterilize when used. The volume ratio with the mutagenic solution is 1:1. B.3 Diethyl sulfate (DES)
The preparation method is as follows:
Diethyl sulfate is insoluble in water, but has a short half-life when dissolved in ethanol or acetaldehyde ether, which is 1 hour at 30°C, so it needs to be prepared before use. Dissolve 0.5 mL of DES in 4.5 mL of anhydrous ethanol to prepare a 10% stock solution, and filter and sterilize. B.4 Ethyl methanesulfonate (EMS)
Preparation method is as follows:
pH 7.0 phosphate buffer: weigh 9.39g KHPO,3HO and 3.5g KHPO, add distilled water to 1000mL, filter, sterilize at 121℃ for 15min or 115℃ for 30min; accurately weigh 5.408g EMS powder in a 10mL volumetric flask, first add 6mL pH 7.0 phosphate buffer to dissolve, then make up to 10mlL. Prepare 5.0mol/L mother solution, store at 4℃. Filter and sterilize before use. 5
GB/T38580-2020
Appendix C
(Informative Appendix)
The mutagenic conditions and lethality of commonly used chemical mutagens are shown in Table C.1. Table C.1
Mutagen
Sodium nitrite (NIT)
Nitrosoguanidine (NTG)
Diethyl sulfate
Working concentration
0.05mol/L
0.025mol/L
2mg/mL
0.025mol/L
0.025mol/L
0.05mol/L
0.025mol/L
2mg/mL
1mg/mL
1mg/mL
mutagenic strains
berberine-producing endophytes
fungal mycelium suspension
green wood spores
Maoyuan Streptomyces spores
sticky tape mold spores
white rot fungus spores
endophytic fungi of Fritillaria fasciata
mycelium suspension
Bacillus cereus
bacterial suspension
Maoyuan Streptomyces spores
Bacillus cereus
bacterial suspension
Bacillus subtilis
bacterial suspension||t t||Lactobacillus plantarum
Actinomycetes spore suspension
Neurosporium robusta
Spore suspension
Trichoderma viride spores
Streptomyces shandongensis
Mutagenesis time
10min~20min
Lethality
85%~95%
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Mutagenesis temperature
25℃~26℃
Mutagen
Diethyl sulfate
Ethyl methanesulfonate|| tt||Lithium chloride
Working concentration
Table C.1 (continued)
Mutagenic bacteria
Thermophilic anaerobic bacteria
Bacillus cereus
Actinomycetes spore suspension
Saccharomyces cerevisiae
Lactobacillus liquid
Actinomycetes spore suspension
Mutagenic time
Lethality
70%80%
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GB/T38580—2020
Mutagenic temperature39g and KHPO3.5g, add distilled water to 1000mL, filter, sterilize at 121℃ for 15min or 115℃ for 30min; accurately weigh 5.408g EMS powder into a 10mL volumetric flask, first add 6mL pH7.0 phosphate buffer to dissolve, then make up to 10mlL. Prepare a 5.0mol/L mother solution, store at 4℃. Filter and sterilize before use. 5
GB/T38580-2020
Appendix C
(Informative Appendix)
Common chemical mutagens mutagenic conditions and lethality Common chemical mutagens mutagenic conditions and lethality are shown in Table C.1. Table C.1
Mutagen
Sodium nitrite (NIT)
Nitrosoguanidine (NTG)
Diethyl sulfate
Working concentration
0.05mol/L
0.025mol/L
2mg/mL
0.025mol/L
0.025mol/L
0.05mol/L
0.025mol/L
2mg/mL
1mg/mL
1mg/mL
mutagenic strains
berberine-producing endophytes
fungal mycelium suspension
green wood spores
Maoyuan Streptomyces sporeswwW.bzxz.Net
sticky tape mold spores
white rot fungus spores
endophytic fungi of Fritillaria fasciata
mycelium suspension
Bacillus cereus
bacterial suspension
Maoyuan Streptomyces spores
Bacillus cereus
bacterial suspension
Bacillus subtilis
bacterial suspension||t t||Lactobacillus plantarum
Actinomycetes spore suspension
Neurosporium robusta
Spore suspension
Trichoderma viride spores
Streptomyces shandongensis
Mutagenesis time
10min~20min
Lethality
85%~95%
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Mutagenesis temperature
25℃~26℃
Mutagen
Diethyl sulfate
Ethyl methanesulfonate|| tt||Lithium chloride
Working concentration
Table C.1 (continued)
Mutagenic bacteria
Thermophilic anaerobic bacteria
Bacillus cereus
Actinomycetes spore suspension
Saccharomyces cerevisiae
Lactobacillus liquid
Actinomycetes spore suspension
Mutagenic time
Lethality
70%80%
TriKAa-cJouakAa
GB/T38580—2020
Mutagenic temperature39g and KHPO3.5g, add distilled water to 1000mL, filter, sterilize at 121℃ for 15min or 115℃ for 30min; accurately weigh 5.408g EMS powder into a 10mL volumetric flask, first add 6mL pH7.0 phosphate buffer to dissolve, then make up to 10mlL. Prepare a 5.0mol/L mother solution, store at 4℃. Filter and sterilize before use. 5
GB/T38580-2020
Appendix C
(Informative Appendix)
Common chemical mutagens mutagenic conditions and lethality Common chemical mutagens mutagenic conditions and lethality are shown in Table C.1. Table C.1
Mutagen
Sodium nitrite (NIT)
Nitrosoguanidine (NTG)
Diethyl sulfate
Working concentration
0.05mol/L
0.025mol/L
2mg/mL
0.025mol/L
0.025mol/L
0.05mol/L
0.025mol/L
2mg/mL
1mg/mL
1mg/mL
mutagenic strains
berberine-producing endophytes
fungal mycelium suspension
green wood spores
Maoyuan Streptomyces spores
sticky tape mold spores
white rot fungus spores
endophytic fungi of Fritillaria fasciata
mycelium suspension
Bacillus cereus
bacterial suspension
Maoyuan Streptomyces spores
Bacillus cereus
bacterial suspension
Bacillus subtilis
bacterial suspension||t t||Lactobacillus plantarum
Actinomycetes spore suspension
Neurosporium robusta
Spore suspension
Trichoderma viride spores
Streptomyces shandongensis
Mutagenesis time
10min~20min
Lethality
85%~95%
iiiKAa~cJouakAa
Mutagenesis temperature
25℃~26℃
Mutagen
Diethyl sulfate
Ethyl methanesulfonate|| tt||Lithium chloride
Working concentration
Table C.1 (continued)
Mutagenic bacteria
Thermophilic anaerobic bacteria
Bacillus cereus
Actinomycetes spore suspension
Saccharomyces cerevisiae
Lactobacillus liquid
Actinomycetes spore suspension
Mutagenic time
Lethality
70%80%
TriKAa-cJouakAa
GB/T38580—2020
Mutagenic temperature
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