Mutagenesis techniques like chemical mutagenesis using ethyl methane sulfonate (EMS) and physical mutagenesis using gamma irradiation and X-rays can be used to induce mutations in Jatropha curcas, an economically important plant. The document describes methods for mutagenesis in different tissues of J. curcas, including seeds, under in vitro and in vivo conditions. It also discusses approaches for detecting mutations, such as TILLING, and analyzing mutant populations to select plants with desirable traits for further breeding.

1. seminar
Mutagenesis in the laboratory
Supervisor
Dr. Niazi
Provisioner
Nazila gharibi
2021

2. ➢ Genetic variation is a source of
phenotypic diversity
▪ It is estimated that food production should be at least doubled by the year 2050 in order
to meet the needs of a continually growing population
➢ Primarily, simple selection of desirable
offspring was the first method of
breeding and this utilized the
occurrence of spontaneous
mutations
Introduction
➢ mutation induction has been an
important tool for crop breeding
since the release of the first mutant
variety of tobacco in the 1930s

3. Mutation
induction
Introgression,
Recombination
Tissue
Culture
Alter By
Design
REVERSE
GENETICS
FORWARD
GENETICS
GENETIC VARIATION
SELECTION
Crop improvement strategies based on genetic variation
- Transgenics
- genome editing
- somaclonal
variation
❖ Where sufficient variation does not exist naturally, it can be created through
either Random or Targeted processes
Out - crossing
starts with a phenotype and
moves towards identifying
the gene(s) responsible
starts with a known gene
and assays the effect of its
disruption by analyzing the
resultant phenotypes

4. Figure . Distribution of mutant crop varieties by continents
(Accessed on 15th July, 2015).
➢ The first commercial mutant
variety was produced in tobacco
in 1934
➢ Prior to 1995, reported 77
cultivars that were developed via
mutagenesis.
➢ In 1995, the number of
commercially released varieties
increased to 484.
▪ Some of the plants include fruit trees (e.g., apple, citrus, peach), ornamentals (e.g.,
chrysanthemum, dahlia, poinsettia), food crops (e.g., rice, barley, wheat, corn, pea), etc.

5. MUTATIONS
GENE MUTATION CHROMOSOMAL MUTATION GENOME MUTATION
At DNA level At protein level
- Transition
- Transversion
- Frame shift
- Silent
- Missense
- Non sense
- Neutral
Break in
Homologous
chromosome
Break in non
Homologous
chromosome
- Deletion
- Inversion
- Duplication
- Translocation
Aneuploidy
- Monosomic
- Trisomic
- Nullisomic
- Disomic

6. Mutagenesis is the process whereby sudden heritable changes occur in the
genetic information of an organism not caused by genetic segregation or genetic
recombination, but induced by chemical, physical or biological agents
MUTATIONS
Induced
mutations
Mutagens are those agents, with induces the mutations in a DNA molecules
MUTATIONS
Spontaneous
mutations
Random Targeted
About 35% of the 1440 commercial
varieties of roses, 25% of apple varieties
and 45% of potato seeds in the United
States

7. Mutagens
Biological
Chemical
Physical
IONIZING Non-IONIZING Transposon
Bacteria
Virus
Metals
De-amination
agents
Intercalating
agents
Alkylating
agents
Base
Analogues
Type of Mutagens

8. • H.j.Mullar first of all artificially induces the mutations
in flies
• L.j.stadler developed mutations in Barley and maize
Classified to:
(A). Ionizing Radiation
Particulate
Non-Particulate
(B). Non- Ionizing Radiation
Physical Mutagens
α - Ray
β – Ray
Fast neutrons
𝑪𝒐𝟔𝟎
𝑪𝒔𝟏𝟑𝟕
γ – Ray
X - Ray
Uv -Rays

9. ➢ The main Ionizing Radiations are
X-ray, α- ray, β-ray, γ-ray
➢ When these radiations react with
water, they produced highly active
free radicals (OH)
➢ These free radicals reacts with
DNA and the phosphodiester bond
of DNA resulted into mutagenic
effects
Physical Mutagens
X-ray
Wavelengths = ./01 - 10 nm
Penetration power = varies in
tissues from a few mm in the
long wavelength state to a few
cm in the short wavelength
state.
γ-ray
Release by decomposition
of isotopes such as 𝐶𝑜60
and 𝐶𝑠137
(half-life 3.5
years - 30 years)
Neutrons
produced in nuclear
reactors. The emission of
neutrons leads to the
release of high energy
and mutations.
Ionizing Radiation

10. ▪ UV rays don’t have such energy that they
cause ionization so its a non-ionizing radiation
▪ Mercury lamps have a wavelength of 250 to
290 nm and act as a source of ultraviolet light
in radiation.
▪ Nitrogenous bases absorbs UV lights and the
absorption is maximum at 260 nm.
▪ It causes the formation of Thymine dimer
(Pyrimedine dimer). If two thymine occur
together in one strand of DNA, UV light
causes fusion to form thymine dimer.
▪ At the site of thymine dimer confirmation of
DNA is changed, so rate of error during DNA
replication and transcription is high.
Physical Mutagens
Non- Ionizing Radiation

11. Typical symptoms of exposure to UV-B radiation on control leaves of cotton plants:
(a) No symptoms, (b) Initial chlorotic patches, and (c) Necrotic patches after
prolonged exposure. Adapted with permission from Prasad et al. (2003b)
Due to the long wavelength of ultraviolet light, its penetration into the tissue is limited
and it is used to induce mutations in cell cultures or protoplasts.
To do this, protoplasts are placed a few centimeters apart in unopened petri dishes under
a UV lamp. This reduces colony formation by 10 to 50 percent compared to non-mutant
protoplast culture.
Physical Mutagens

12. ❑ Rate of mutation a Amount of Radiation
The damage maybe
(a) Ss break
(b) Ds break
(c) Alteration in N sequence
✓ The ss damage may be
repaired in a cell but
others causes DELETION,
DUPLICATION, INVERSION
and TRANSLOCATION
✓ These radiations are used
for the treatment of
CANCER by giving the dose
of radiations at regular
interval
Physical Mutagens

13. Physical Mutagens

14. Factors influencing the outcome of mutagenesis using physical mutagens
Oxygen >> The interplay between oxygen and ionising radiation continue from
irradiation to post-irradiation storage
Moisture content >> Seed moisture content is important.
Temperature >> preheating of cell lines has been shown to increase the incidence of
mutation events.
Other physical ionizing agents >> The presence of other unintended agents
(electromagnetic and ionizing radiation) increase mutation frequencies.
Dust and fibers >> Particles in the environment including dust and fibers (e.g. from
asbestos) increase significantly the incidence of mutagenicity of irradiation
Biological and infectious agents >> hormonal concentrations and Infectious agents
(both viral and bacterial) have been shown to elevate radiosensitivity.
Troubleshooting

16. Chemical Mutagens
BASE ANALOGUES
- Maleic hydrazide
- Uridine derivatives
CHEMICALS
- Nitrous acid (HN𝐎𝟐)
- Hydroxyl amine (𝐇𝟐NO)
- Sodium azide (Na𝐍𝟑)
INTERCALATORS
- Ethidium bromide
- Proflavine
- Acridine orange
- daunorubicin
ALKYLATING AGENT
- Ethyl methyl sulphate
(EMS)
- Nitrogen mustards
- Mitomycin
- Methyl methane
sulfonate (MMS)
- Diethylsulfate (DES)
- Nitrosoguanidine (NG)
C . Auerbach – used the chemicals to induce the mutations

17. ❖ Base Analogues
➢ some chemicals are similar to N bases,
and these are called as base analogues.
➢ 5-Bermodioxyuridine (BrdU) and 5-
Bermoyuracil, are Analogs of thymine
and enter the DNA structure instead of
thymine.
➢ The frequency of mutations by these in
plants is low and therefore their use is
very limited.
In this way, 5-Bromo-Uracil can promote a change
of an AT base pair into a GC base pair
Chemical Mutagens

18. ➢ Some chemicals like Nitrogen,
Sulphur , mustard gas, MMS, EMS,
Nitrosoguanidine, NMU, NEV
➢ Due to these agents Methyl and
Ethyl groups transferred to N-
bases
➢ It resulted into changes in base
coupling potentials
➢ Transition and transversion type
mutation developed due to these
➢ Alkylating agents are compounds
with high reactivity
➢ EMS is commonly used for
mutations in plants ( ./1 – 3%)
➢ Urea nitroso is not stable in
alkaline solutions so it should be
PH= 5/6
Chemical Mutagens
❖ Alkylating Agents

19. ❖ Deamination Agents
➢ By the action of Nitrous Acid, amino
group is eliminated from The N base.
➢ Adenin…….. Hypoxanthine ( KETO)
Hypoxanthine ( KETO) binds with cytosin
Chemical Mutagens

20. Chemical Mutagens
❖ Deamination Agents

21. ➢ Certain dyes like proflavine,
Acridine orange , ICR-170, ICR-191,
are strong mutagens
➢ These dyes inserted in between the
N bases and configuration of DNA
changed
➢ The interval between two purine
raised from 3.4A to 6.8A
Chemical Mutagens
❖ INTERCALATORS

22. ➢ The other important chemical
is Hydroxyl Amine
➢ Adding OH group to amino
group of cytosine
➢ It is responsible for GC…..AT
transition
Chemical Mutagens

23. Troubleshooting
▪ Factors that are critical to induced mutagenesis assays include the condition
of the mutagenic solution, the inherent characteristics of the target
tissue and the environment.
❖ Factors influencing the outcome of mutagenesis using chemical mutagens
Concentration of mutagen >>This is the most critical factor with the results of assays
depending to a great extent on the use of optimal concentrations of the mutagen.
Treatment volume >> The samples should be immersed completely in the mutagen
solution the volume of which must be large enough to prevent the existence of
concentration gradients during treatment. As a guide, a minimum of 0.5–1.0 ml of mutagen
solution per seed is suitable for most cereals
Treatment duration >>
The treatment should be long enough to permit hydration and infusion of the mutagen to
target tissue. The relevant seed characteristics that impact on this include seed size,
permeability of the seed coat and cell constituents. For EMS, this is 93 h at 20 ◦ C or 26 h at
30 ◦ C.

24. Troubleshooting
Temperature >> Related to hydrolysis is the temperature of the environment in which
the plant material is treated.
Presoaking of seeds >> This enhances the total uptake, the rate of uptake and the
distribution of mutagen within the target tissue. The duration of pre-soaking depends
primarily on the anatomy of the seed.For barley, a pre-soaking period of 16–20 h is
sufficient
pH >> The hydrogen ion concentration of the solution influences the hydrolysis of EMS.
by maintaining the pH of the EMS solution at the optimal vale of 7.0, injury to seeds and
explants is minimized.
Catalytic agents >> Certain metallic ions such as Cu2+ and Zn2+ have been implicated
in the enhancement of chromosomal aberrations induced by EMS. It is for this reason
that it is recommended to use deionized water to prepare the EMS emulsion.
Post-treatment handling The by-products of the incubation process (resulting from
hydrolysis) and residual active ingredients should be promptly washed off the incubated
target tissues after treatment.

25. Virus Transposon
Biological Agents
Bacteria
o Insertional Mutagenesis
➢ Identification of mutated genes
➢ Markers for chromosomal localization of mutated genes as
well as cloning of the original gene
➢ Selection of mutant plants based on their altered
phenotype from transgenic plants

26. • Whole plants, usually Seedlings, and in vitro Cultured cells. Nevertheless, the
most commonly used plant material is Seed.
The starting materials for the induction of mutations are vegetative cuttings, scions, or in
vitro cultured tissues like leaf and stem explants, anthers, callus, cell cultures,
microspores, ovules, protoplasts, etc.
➢ Gametes, usually inside the inflorescences, are also targeted for mutagenic
treatments through immersion of spikes, tassels, etc.
➢ it is noteworthy that the frequency and types of mutations are direct results of the
dosage and rate of exposure or administration of the mutagen rather than its type
Multiple forms of plant propagules, such as bulbs, tubers, corms and rhizomes and
more recently, the induction of mutations in vegetatively propagated plants is
becoming more efficient as scientists take advantage of totipotency using single cells
and other forms of in vitro cultured plant tissues.
Types of planting materials for performing Mutagenesis

27. Methods for generating mutant varieties

29. Abstract
▪ Jatropha curcas is a semi-wild
▪ economically important shrub useful as a
source of biofuel or in soil reclamation
▪ it requires genetic improvement in order to
select the best genotypes for these
purposes.
the general methods for mutation induction
(chemical and physical mutagenesis) :
➢ ethyl methane sulfonate (EMS)
➢ gamma irradiation
➢ X-rays
induced mutants in different tissues of J.
curcas under in vitro and in vivo conditions.

30. ❖ Jatropha curcas is one of the most valuable
crops for its ability to produce seeds, which
contain 60–63 % of protein and 30–45 % of
toxic oil that renders the seedcake and oil
unsuitable for animal or human
consumption.
❖ The narrow genetic base in J. curcas hinders
efficient genetic improvement.
❖ In fact, the ultimate breeding objectives of
the J. curcas accessions are to reduce toxicity
and improve productivity under adverse
climatic conditions.
❖ To increase genetic diversity, mutagenesis
can be applied for plant improvement.
Introduction

31. Introduction
Ethyl methane sulfonate (EMS)
CH3SO2OC2H5
One of the most effective and
commonly used chemical mutagens
a monofunctional alkylating agent
base changes, breakage of the DNA
backbone, and mispairing
gamma rays, X-rays
Physical mutagens
low LET and produce energy in the form
of electromagnetic waves
most commonly used for mutation
breeding
deletions, point mutations, single- and
double-stranded brakes, and even
chromosome deletions

32. The Mutated populations (M1) are
generated
to reduce chimerism M2 or higher
populations are produced.
Entire mutant populations are
screened by either phenotypic
evaluation for selection of
phenotype of interest (forward
genetics) or by genotypic evaluation
for detection of novel allele in gene
of interest as well as study of gene
function (reverse genetics)
Schematic diagram of the basic
steps in physical and chemical
mutagenesis on different J.
curcas tissues
Methods

33. Methods
Schematic representation
of EMS mutagenesis of in
vitro material

34. Methods
Mutagenesis by chemical Agents
EMS Mutagenesis of In Vitro Material
Mutagenesis by physical Agents Agents

35. X-ray RS-2400 source Gamma cell irradiator cobalt-60 source

36. Further Analyses
❖ The second generation (and
higher) after chimera
dissolution of in vivo and in
vitro plants can be screened
for the selection of candidate
genes based on phenotypes
or genotypes.

37. ❖ Mutations can be detected with various direct and indirect methods such as:
➢ denaturing high-performance liquid chromatography (DHPLC),
➢ denaturing gradient gel electrophoresis (DGGE)
➢ temperature gradient capillary electrophoresis (TGCE)
➢ heteroduplex analysis (HD)
➢ single-stranded DNA conformation polymorphism (SSCP)
➢ chemical or enzymatic cleavage of mismatches (CECMs)
➢ Targeting Induced Local Lesions in Genome (TILLING)
➢ whole genome sequencing
➢ exome capture sequencing
➢ restriction-site-associated DNA (RAD) sequencing
➢ genotyping by sequencing (GBS)
Further Analyses

38. TILLING strategy using endonuclease digestion

39. TILLING strategy using two non-enzymatic methods

40. Conclusions
❖ For half a century, induced mutations have played an important role in plant breeding,
contributing to increased food production in both developed and developing economies.
Classical mutation breeding continues to be used for the benefit of communities in
parallel with application of modern genomic tools for mutation induction and discovery
in advanced laboratories.
❖ With 3211 registered mutant varieties in more than 170 different plant species, mutation
breeding has proven flexible, workable and ready to use on any crop if objectives and
selection methods are clearly identified. A range of mutagens are at our disposal to
induce mutations from the single nucleotide level to the genome level. Induced
mutations have not only played an unprecedented role in developing new crop cultivars
and novel products from existing crops, but also increasingly contribute to our
understanding of gene function and biochemical pathways. Along with newly emerged
‘omics’ techniques, induced mutations are contributing to the development of newly
emerging subject of systems biology.