Plants can be genetically modified by the use of chemical mutagens. Chemical mutagens interact with the DNA and cause mutations in the genome. Chemical mutagenesis is a powerful tool which can be applied for the development of plants with desired agronomic traits.

1. DEVELOPMENT OF NOVEL PLANT VARIETIES BY MUTAGENESIS
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2. Novel Varieties of Plants
Plant developed using chemical mutagens are not categorized as “Genetically Modified”
Can you discuss what are the advantages and disadvantages of Chemical Mutagenesis?
Will consumption of these plant products pose a greater danger to consumers as compared to
GMOs?
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3. Mutant Population Developed at IPB
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Two generations of mutants were developed from
an Isogenic F1 Hybrid Tomato line using the
chemical mutagen EMS. They were screened by
genotyping and phenotyping.

4. Lesson Objectives
1. To introduce the concept of chemical mutagenesis.
2. To discuss the experimental design for plant mutagenesis.
3. To develop a protocol for mutagenesis of an isogenic line of Triticum sp using Ethanemethane
sulfonate.
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5. Learning Outcomes
Upon completion of this module, the participants should demonstrate the ability to:
1. Design an experiment to mutate plants using a chemical mutagen.
2. Develop a procedure to isolate mutants on the basis of phenotypical screening and
genotyping.
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6. Developing a protocol
The development of a protocol for mutating plants involves several steps.
1. How will you determine the germination efficiency?
2. What are your criteria for the selection of the chemical mutagen?
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7. GERMINATION EFFICIENCY
ESTABLISHING THE DOSAGE OF MUTAGEN
SCREENING FOR MUTANTS (GENOTYPING / PHENOTYPICAL)
ESTABLISHING A MUTANT POPULATION
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8. STEP 1: GERMINATION EFFICIENCY
Prior to performing an experiment that involves mutagenesis, the
actual germination efficiency has to be determined.
Why does this have to be done?
Germination Efficiency = Total Number of Seeds Which Germinated
Total Number of Seeds Planted
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9. STEP 2: ESTABLISHING THE DOSAGE
Ethyl methanesulfonate (EMS) produces random mutations in genetic material by nucleotide
substitution; particularly by guanine alkylation.
Point mutations.
Rate of Mutation: 5x10−4 to 5x10−2 per gene.
The base analogue O6-ethylguanine.
G:C to A:T
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10. Modification of the base Guanine.
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11. The ideal dosage is the one which kills 50% of the seeds in the experimental trial. This can be
established by soaking the seeds in serially diluted solutions of the mutagen and determining
the concentration which kills 50% of the plants. Why?
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CONCENTRATION OF EMS PERCENTAGE OF
GERMINATION
SELECTION
1% 10 Which one?
0.75% 20
0.5% 30
0.25% 35
0.1% 40
0.01% 80

12. STEP 3: SCREENING FOR MUTANTS
Upon establishment of a mutant population, mutants have to be screened for specific desireable
traits, this can be done using the following protocols.
1. The application of a specific screen: ( e.g. salinity gradient, herbicide gradient, challenge test).
2. Phenotypical characterization (e.g. Brix index, Fatty Acid Content).
3. Genotypic screening. (e.g. PCR of specific genes, TILLING).
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13. SCREENING USING SELECTIVE AGENTS
Selective agents such as Herbicides can be utilized to screen for herbicide tolerance in
agronomic crops.
Another important trait is response to fertilizers.
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14. SCREENING BY TILLING
TILLING (Targeting Induced Local Lesions in Genomes) is a method which involves PCR of a
specific locus, followed by digestion with CEL Nuclease. This enzyme cleaves genes which
contain point mutations. The gel image below shows point mutations within a PCR product
which has been hybridized with the control followed by digestion with CEL Nuclease.
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M 1 2 3 4 5 6 7 8 9 10 11 12 N

15. STEP 4: ESTABLISHING A MUTANT POPULATION
The mutant population can be established by selfing of the selected mutants. Stable mutants
can be selected from the M2 population.
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M0 Plants obtained after treatment with EMS
M1 Plants obtained by selfing of M0
M2 Plants obtained by selfing of M1

16. Registration of novel varieties.
Novel plant varieties can be registered under the PLANT VARIETY PROTECTION ACT.
Malaysia registration portal: http://pvpbkkt.doa.gov.my/
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17. The Experiment: laboratory
1. We will be using Triticum aestivum seeds
2. Preparation of mutagen in buffer at concentrations of 0%, 0.01%, 0.1%, 0.2%, 0.5% and 1%.
3. Soaking of seeds overnight in the fume hood.
4. Washing and germination of seeds in petri plates.
5. Recording germination efficiency.
6. Phenotypical characterization.
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18. Record your observations.
EMS
Concentration
Germination Germination
Efficiency
50% Phenotypes
CONTROL 80 / 100 80% 40 /100
0.01%
0.1%,
0.2%,
0.5% 40 /100 32% 32 / 100
1%
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Estimate the germination efficiency and use this to compute the LD50 for the mutant population. For
example if the germination efficiency is 80% and the number of seeds germinating when treated with
0.5% EMS is 40 /100, the actual percentage of survivors is 32% which exceeds 50%.

19. Discussion Points
Does chemical mutagenesis for the development of agronomic crops
pose a greater risk as compared to Genetic Modification of specific
targets?
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