I.

Definitions of Basic Terms

Mutagenesis is the process by which the genetic information of an organism is changed in

a stable manner. This happens in nature as a result of errors in DNA repair. Mutagenesis is the
process by which mutations are generated. The following terms are used frequently:
1. Mutation: This was originally defined in a series of articles by de Vries (1901, 1903 and 1905)
as a sudden heritable change in the genetic material not caused by recombination or
segregation. De Vries used the word “sudden” to differentiate between subtle changes that
could be explained by the normal processes of recombination. “Sudden” changes (mutations)
in plant forms (phenotypes) were obvious, apparent and unusual and therefore of interest.
Mutation however, especially at the gene sequence (genotype) level can lead to small and
subtle changes in phenotype which may not become immediately apparent and these can
now be detected using molecular techniques; thus the word “sudden” as applied to phenotype
can be deleted from the definition.
2. Mutants: individuals carrying a mutation that may be revealed using molecular means or
identified by phenotyping tools. Different types of mutant can be generated using
experimental mutagenesis.

Gambar 1. Distribution of mutant varieties (a) among continents (A&P: Australia and the Pacific);
(b) in Asia; (c) among different types of end-uses; and (d) within food crops
(FAO/IAEA, 2011)
 II. Type of Mutagenisis Technique

Gambar 2. Klasifikasi Mutagenesis berdasarkan Metode

2.1 Physical Mutagens
2.1.1 Description and Type of Technique
Physical mutagens comprise all nuclear radiations and sources of radio-activity including
ultraviolet light (a non-ionising radiation), several types of ionizing radiations, namely X- and
gamma-rays, alpha and beta particles, protons and neutrons. An overview of the main physical
mutagens used in plant mutation breeding is presented here, covering their physical
characteristics, their mode of action and all general principles and consideration on how they may
be applied for mutation induction in plants. Several types of ionizing radiation are available for
plant mutation induction. Each of these has the common feature of releasing ionizing energy.
However, there are several differences among ionizing radiations regarding the energy deployed
the penetrating capability and the level of hazard involved for operators (Table 1).
2.2.2 Target Plant Materials
1. Whole plants
Large plants have often been irradiated in a gamma field, a gamma greenhouse or a gamma
room. Seedlings or small plants, on the other hand, can easily be irradiated by most X-ray
machines or by gamma sources in a greenhouse or shielded rooms.
2. Seeds
Seeds are the favoured material for irradiation in many mutation induction experiments and
in practical mutation breeding. Seeds can be irradiated in many physical environments and they
can be desiccated, soaked, heated or frozen prior to the treatments. They can be stored for
extended periods of time in air-tight, vacuum and refrigerated conditions.
3. Pollen
A great advantage of pollen irradiation as opposed to the irradiation of seeds or growing
plants is the fact that the former rarely produces chimeras, i.e. the M 1 plants resulting from
fertilization by irradiated pollen of a non-irradiated egg cells will be fully homozygous for any
induced mutation. The disadvantages of pollen irradiation include the difficulty of obtaining
sufficient material from some species and the short viability of pollen grain in many plant species.
4. Meristems
Seed irradiation is essentially a treatment of the embryo meristems. The anatomy and
pattern of embryo meristems is important for mutagenic treatments of seeds (as well as other
plant material) since it determines whether a mutated cell will be lost during differentiation or
produce sufficient cell progenies to be found throughout much of the plant including germ cells.
5. Plant cells and in vitro tissue culture
The use of plant cells and tissues in culture offers exciting applications in crop mutation
breeding. The in vitro methods for plants micro-propagation launched in the early 60s, rapidly
became a powerful tool for scientists working on plant mutation induction, especially in VPCs.
Plant organ, tissue and cell culture provided ways for:
1) Rapid and mass propagation of target M 0 plants;
2) Rapid and mass propagation of any mutant populations;
3) Thorough and easy methods for analysing mutations morphogenetic and physiologic
changes associated with mutation induction;
4) Easy and rapid ways of dissociating chimeras;
5) In vitro mutagenesis; and
6) in vitro screening.
2.1.3 Faktor yang Mempengaruhi Aktivitas Mutagenesis
1. Environmental factors
1) Oxygen
Oxygen is one of the best-known modifiers of radiation sensitivity and the biological effects
of irradiation are usually greater in the presence of oxygen. Other factors, such as water content,
temperature and post-irradiation storage conditions appear to be secondary. Environmental
factors are less important with densely ionizing radiations such as fast neutrons.
2) Water content
Even minor differences in water content can have a very pronounced influence on the end
biological effect. Seeds stored under normal laboratory conditions are often in the range of 10.0
and 11.5 percent water and a difference of only 0.2 to 0.3 percent might greatly alter radio-
sensitivity of some species. This should be weighed in, when choosing an effective mutagenic
dose; a dose too low might not induce any mutations, and a dose too high might result in excess
sterility or no surviving plants.
3) Temperature
The temperature of plant cells before, during and after irradiation may affect the total amount
of genetic damage induced by X- or gamma-rays. However, the effect of temperature as a
modifying factor of radiation damage is not clearly understood and appears unimportant to the
plant breeder. A combination of heat shock and oxygen-free hydration was found to be most
protective against the post-irradiation oxygen-dependent damage (scored as seedling injury and
chromosomal aberrations).
2. Biological factors
1) Nuclear and interphase chromosome volume
the higher the chromosome number, the higher the resistance to radiation, resulting from
the fact that other chromosomes or parts of chromosomes might compensate for the mutations,
this is particularly true for polyploid species (Datta, 2014). Chromosomal organisation, number
and position of centromeres and chromosome size are also associated with radio-sensitivity (large
chromosomes are generally more radio-sensitive than small chromosomes).
2) Cell cycle
The sensitivity of the cell or more precisely of the nucleus is dependant of the length of the
cell cycle, and the stage of the cell division: mitosis or meiosis. A thorough and very informative
review of the effects of radiation on cell division and plant growth could be found in Lagoda et al.,
(2012).
3) Genetics
Differences in mutagen sensitivity among genotypes are common. Differences in radio-
sensitivity among genotypes within a species are usually much less than between species.
Therefore, for plant breeders wishing to induce mutations, the genotype factors can be ignored,
and a routine radio-sensitivity test will determine an appropriate dose rate for mutation induction.
2.2 Chemical Mutagens
2.2.1 Description
Chemical mutagenesis in reverse genetics has enjoyed a renaissance since the early 2000s
due to technological innovations including Targeting Induced Local Lesions in Genomes
(TILLING) and, more recently, Next Generation DNA Sequencing (NGS) technologies. These
advances have yielded important new insights into the mechanism and spectrum of chemically-
induced mutations. Example spectra for key mutagens and crops are included to help guide plant
breeders and/or researchers in designing plant mutagenesis experiments. Likewise, advances in
in vitro plant tissue culture created new opportunities in expanding chemical mutagenesis to in
vitro tissues. This is particularly important for the vegetatively propagated crops (VPCs) which
have lagged behind annual seed propagated crops for mutation breeding.
Gambar 3. Chemical mutagens most frequently applied in generating mutant varieties. Among
top agents are EMS (ethyl methanesulphonate), with 106 officially registered mutant
varieties, NEU (nitrosoethyl urea) (57), MNU (N-methyl N-nitrosourea) (53), colchicine
(46), and EI (ethylenimine) (36). Figure 2.1b. Officially released mutant crop varieties
registered in the MVD produced via chemical mutagenesis
2.2.2 Main Chemical Mutagens
1. Alkylating agents
Alkylating agents are electron deficient (i.e. electrophilic) compounds with one or more alkyl
groups, which can be transferred to biological molecules such as DNA that contain nucleophilic
groups. Most alkylating agents are pro-mutagens, i.e. they undergo transformation to produce
reactive intermediates. These intermediates can react with DNA by alkylating the phosphate
groups in the phosphodiester backbone as well as the various imino- or carbonyl- groups present
on the purine (adenine, guanine) or pyrimidine (cytosine, thymine) bases.
Alkylation is defined as the transfer of an alkyl group from one molecule to another. The
alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbene
(or their equivalents). The dialkyl nitroso amines (e.g. diethyl nitroso amine) are stable
compounds, which apparently act on DNA only after enzymatic activation (removal of one alkyl
group)
2. Sodium azide
Sodium azide is also considered a pro-mutagen as it is metabolized in vivo to a powerful
chemical mutagen through an organic intermediate, identified in barley as L-azido-alanine. It
appears that L-azido-alanine itself does not directly interact with DNA, but that mutagenesis is
mediated by the host-plant cellular processes involved in DNA excision-repair (Owais and
Kleinhofs, 1988; Sadiq and Owais, 2000).
Sodium azide induces chromosome aberrations at a very low rate. SA is a powerful
mutagen for inducing point mutations. Both in barley and rice, GC to AT transitions were the
predominant mutation type. It appears that sodium azide-induced mutagenesis has a different
local sequence context bias (GGR) compared to EMS (Tai et al., 2016). Therefore, combining
different mutagenic compounds may expand the spectrum of induced mutations and the resulting
mutant phenotypes.
3. Other chemical mutagens
The following groups of chemical mutagens: (i) base analogues; (ii) antibiotics; (iii)
acridines; (iv) nitrous acid; and, (v) hydroxylamine. Leitão, (2012) described the acridines under
a more general category of intercalating agents together with topoisomerase inhibitors and
poisons. The exploitation of these chemical mutagens for plant genetic improvement is much
more restricted compared to the alkylating agents and azide, because they are either less
effective, less well studied or more challenging to handle from a health or safety perspective.
Colchicine is considered a chemical mutagen senso latu, because its main effect is on ploidy and
not on gene.
Colchicine is widely used in plant breeding work to produce changes in ploidy. The
increased number of chromosomes usually brings about changes in plant morphology and
functions. Colchicine treatments of meristem-containing propagules or tissues can be performed
in many ways using concentrations ranging from 0.005 percent to 1.5 percent (van Harten, 1998).
Common methods for chromosome doubling involve soaking the seeds in a colchicine solution,
applying colchicine using a brush on growing shoot apices, or culturing (in vitro) plantlets in
colchicine-containing medium.
2.2.3 Target Plant Materials
1. Seeds
Seeds are the most commonly used target tissue for chemical mutagenesis. For example,
seeds of cereals or legumes can be easily stored and shipped and treated in large quantities.
Soaking the seeds in the mutagen solution is the most convenient and most widely used method,
thus small grain cereals and other seeds that imbibe rapidly are easy targets. Plant species and
varieties may respond differently to a certain chemical mutagenic treatment.
2. Explants Tissue
In vitro explants are currently becoming a useful target for chemical mutagenesis. In vitro
systems may offer several advantages such as the availability of more standardized conditions
and the possibility to prevent or restrict the formation of chimeras. A wide range of plants can be
regenerated from single cells via in vitro tissue culture. This provides an excellent opportunity for
combining tissue culture protocols with mutagenesis techniques. As is the case with radiation
treatment of VPCs, appropriate tissue culture techniques will greatly facilitate the regeneration of
a whole homohistont plant from a single cell to avoid the development of chimeras.
2.2.3 Advantages and limitations of chemical mutagenesis
The advantages and limitations of chemical mutagenesis for experimental plant
mutagenesis or plant breeding have been previously summarised (van Harten, 1998). Taking into
consideration the more recent findings pertaining to plant chemical mutagenesis, these can be
updated as stated below.
1. Advantages
1) Well characterized mutation spectrum producing mainly point mutations.
2) Less chromosomal damage when compared to physical mutagens.
3) High mutation frequency allows to create allelic variation at any target gene.
4) Mutations appear to be evenly spread across the entire genome.
5) Standardized protocols for seed treatment of the major seed propagated food crops.
6) Can be equally applied to in vitro tissues or explants.
7) EMS mutagenesis can be applied in a standard laboratory setting.
2. Limitations
1) Dense mutation rate, this may require several rounds of backcrossing to remove
undesirable mutations.
2) Penetration in multi-cellular or woody plant tissues is often difficult or has low
reproducibility.
3) Materials or seeds that are dormant or have long germination times, e.g. nuts, may
require special pre-treatments or manipulations.
4) Limited repertoire of well-characterized chemical mutagens for plant mutagenesis.
5) May not be effective to induce large chromosomal variations that are heritable.
6) Health and safety concerns due to toxic or carcinogenic properties.
 III. Mutagenesis and Experimental Mutagenesis

Mutagenesis can be exploited experimentally (Experimental Mutagenesis) by physical,

chemical and biological means.

1. Induced mutagenesis. This type of mutagenesis is induced by the use of radiation or

chemical mutagens and is a random process. Note that target-selected mutagenesis,
including the random mutagenesis and selection of mutants at a selected locus, belongs to
this category. The commonly used term “mutation induction” has the same meaning as
“induced mutagenesis”. Mutant plants and organisms produced by induced mutagenesis are
referred to as induced mutants.

Gambar 4. Ilustrasi Induksi Mutagenesis

2. Insertion mutagenesis. This type of mutagenesis results from DNA insertions, either
through genetic transformation and insertion of T-DNA (T-DNA insertion mutagenesis) or
activation of transposon elements (transposon mutagenesis or transposition mutagenesis).
The consequent mutants are known as insertion mutants or transposon mutants.
 Gambar 5. Mutagenesis Penyisipan
3. Site-directed mutagenesis. This type of mutagenesis is the process of creating a mutation
at a defined site in a DNA molecule. In plants, this is achieved by genetic transformation
followed by homologous recombination between the T-DNA fragment and indigenous DNA
molecules. The mutants thus generated are characterised by the replacement of an
indigenous DNA fragment by a foreign molecule, which can be as limited as one nucleotide.
 VI. Mutation Genetics and Breeding

The term mutation breeding (‘Mutationszüchtung’) was first coined by Freisleben and Lein
(1944) to refer to the deliberate induction and development of mutant lines for crop improvement.
The term has also been used in a wider sense to include the exploitation of natural as well as
spontaneous mutants, and in the development of any variety possessing a known mutation from
whatever source. The argument is semantic as all genetic variation is ipso facto mutation, in the
broad sense mutation breeding can be regarded simply as breeding. However, the term ‘mutation
breeding’ has become popular as it draws attention to deliberate efforts of breeders and the
specific techniques they have used in creating and harnessing desired variation in developing
elite breeding lines and cultivated varieties.
Similarly the term mutant variety is simply a variety (var.), but draws attention to the fact
that it carries an important trait controlled by a known mutant gene or it has been developed using
mutation techniques. In some parts of the world the term cultivar (cv.) is used to describe a
cultivated variety, for consistency, this book uses the word ‘variety’. However it should be noted
that the word ‘variety’ is also used as a botanical taxonomic descriptor. The following terms are
relevant to mutation genetics and breeding:
1. Mutant selection. The process of identifying individuals with a target mutant phenotype; this
includes two major steps: mutant screening and mutant confirmation (or mutant verification).
Mutant screening is a process of selecting out individuals from a large mutated population
that meet the selection criterion. For example, M 2 plants flowering three days earlier than
their wild type parent(s) are screened as potential early flowering mutants and plants without
disease symptom might be screened as potential disease resistant mutants. Since flowering
is dependent on both genotypic and environmental factors they can only be regarded as
“putative mutants”, which means they are not necessary “true mutants”. This is the case for
many traits including disease resistance as here non-infection may simply be the result of the
absence of the pathogen.
2. Mutant confirmation is the process of re-evaluating the putative mutants under replicated
and stringent conditions, using larger sample sizes (usually the progenies of selected putative
individuals, e.g. M 3 lines of selected M 2 plants). Many putative mutants of quantitative traits,
e.g. growth duration, yield, quality, disease resistance, might be proven to be false mutants.
V. Experimental Mutagenesis in Crop Genomics

Gene identification and the functional analysis of allelic variation for a particular trait are two
major fields in plant genomics research. The use of T-DNA insertion mutants and transposon
mutants are considered to be the most direct means for filling the genotype - phenotype gap and
directly connect the (disrupted) gene with the mutated traits. This is commonly referred to as
reverse genetics, as it begins with an altered gene and works towards the effects on traits.
However, these tools are not always applicable to all plant species, and they have inherent
limitations, for example, most of the mutants produced by T-DNA insertion are either knockout
mutants or over-expression mutants (when the T-DNA with a strong promoter is inserted into the
flanking sequence of a gene) and hence it is very difficult to study the effects of altered sequence
variation (different alleles of a gene). In addition many crop plants are recalcitrant to
transformation technologies and are excluded from such analyses.