Missense mutation.

Unlike neutral mutations, a missense mutation results when an altered triplet

C
Normal
C C
A
Bu
Mutant
C A T

C Bu A
Normal
C
Bu
Normal
C

(a) Mistake in incorporation

 A

A T
G
Bu
Mutant
A G C

T Bu A
Normal
A
Bu
T

(b) Mistake in replication

Figure 23.3 Mutations may be caused by transition resulting from the substitution of base analogues for a
natural base: (a) mistake in incorporation; (b) mistake in replication.
 440 CHAPTER 23

codes for a different amino acid. Not all missense mutations lead to appreciable protein changes. For example, an
amino acid may be replaced by another of very similar chemical properties, thereby altering the structure without
impacting its normal function (a neutral mutation). Also, the amino acid substitu-tion could occur in a region of the
protein which does not significantly affect the protein secondary structure or function. On the other hand, some
mis-sense mutations have devastating consequences. For example, in hemoglobin of humans, a change of GAG
(Glu) to GTG (Val) results in serious conse-quences (sickle cell anemia).
Nonsense mutation. A nonsense mutation causes an existing amino acid to be changed to a stop codon
(e.g., TAA, TAG), resulting in premature termination of protein synthesis.

(c) Frame shift mutation Insertion–deletion mutations (indels) may cause significant changes in the amino acid
composition of a protein, and hence in its function. For example, GAG–CCG–CAA–CTT–C (corresponding to
Glu–Pro–Glu–Leu) may be altered by deletion of G that shifts the reading frame to the right by one nucleotide to
produce AGC–CGC– AAC–TTC (corresponding to Ser–Arg–Asi–Phe). Very simply: CAT–CAT–CAT–CAT þ A (at
the beginning)
------! ACA–TCA–TCA, and so on.

2. Genomic mutation
Errors in cell division resulting from disorders in the spindle mechanism may result in improper distribution of
chromosomes to daughter cells. Such errors may cause some cell division products to inherit more or less of the
normal chromosome number. These errors, called chromosomal mutations, are of two main
kinds: euploidy (cells inherit additional complete set of the basic chromosome set – n) and aneuploidy (certain
chromosomes are missing from the basic set or added to the set in some cell division products).

3. Structural chromosomal changes (aberrations)

Changes in chromosome structure begin with a physi-cal break that may be caused by ionizing radiation (e.g., X-
rays). After a break, several events may occur:

The ends of the segment may be disunited.

The break may be repaired to restore the chromo-some to its original form (restitution).
Or, one or both ends of a break may join to the ends produced by a different break event (non-restitu-tional
union). These events may result in one of four types of rearrangement – deletions, duplica-tions, inversions,
or translocations. The resulting consequences are variable.

23.3 Mutagenic agents

Agents of artificial mutations are called mutagens. They may be grouped into two broad categories – physical
mutagens and chemical mutagens. The specific agents vary in ease of use, safety issues, and effectiveness in
inducing certain genetic alterations, suitable tissue, and cost, among other factors.

23.3.1 Physical mutagens

The principal physical mutagens are ionizing radia-tions (Table 23.1). X-rays were first to be used to induce
mutations. Since then, various subatomic parti-cles (neutrons, protons, beta particles, alpha particles)

Table 23.1 Examples of commonly used physical mutagens.

Mutagen Characteristics

X-rays Electromagnetic radiation; penetrates tissues from a few millimeters to many centimeters.
Gamma rays Electromagnetic radiation produced by radioisotopes and nuclear reactors; very penetrating into tissues;
sources are 60Co and 137Ce.
Neutrons A variety exists (fast, slow, thermal); produced in nuclear reactors; uncharged particles and penetrates
tissues to many centimeters. Source 235U.
Beta particles Produced in particle accelerators or from radioisotopes; are electrons; ionize; shallowly penetrating;
sources include 32P and 14C.
Alpha particles Derived from radioisotopes; a helium nucleus capable of heavy ionization; very shallowly penetrating.
Protons Produced in nuclear reactors and accelerators; derived from hydrogen nucleus; penetrates tissues up to
several centimeters.
 MUTAGENESIS IN PLANT BREEDING 441

have been generated using nuclear reactors. Gamma radiation from radioactive cobalt ( 60Co) is widely used. It is
very penetrating and dangerous. Neutrons are hazardous and less penetrating, but they are known to seriously
damage chromosomes. They are best used for materials such as dry seed, whereas the gentler gamma radiation
is suited also for irradiating whole plants and delicate materials such as pollen grains. The relative biological
effectiveness (RBE) of fast neutrons is higher than for gamma rays and X-rays. The breeder is interested in
identifying and using treatments with high RBE for maximizing the number of mutants produced. Treatments with
high RBE have high ionization density. Modifying the treat-ment environment (e.g., oxygen, moisture content of
tissue) can increase the effectiveness of the treatment.
Ionizing radiations cause mutations by breaking chemical bonds in the DNA molecule, deleting a nucleotide or
substituting it with a new one. The radi-ation should be applied at the proper dose, a factor that depends on
radiation intensity and duration of exposure. The dosage of radiation is commonly meas-ured in Roentgen (r or R)
units. The exposure may be chronic (continuous low dose administered for a long period) or acute (high dose
over a short period). The quality of mutation (proportion of useful mutations) is not necessarily positively
correlated with dose rate. A high dose does not necessarily yield the best results. A key limitation of use of
physical mutagens is the source. Special equipment or facilities are required for X-rays and nuclear-based
radiations.
About 70% of the world’s successful cultivars derived from mutagenesis were products of gamma-ray
radiation.

23.3.2 Chemical mutagens

Chemical mutagens are generally milder in their effect on plant material (Table 23.2). They can be applied
without complicated equipment or facilities. The ratio of mutational to undesirable modifications is generally
higher for chemical mutagens than for phys-ical mutagens. However, practical success with chemi-cal mutagens
lags behind achievements with physical mutagens. Usually, the material is soaked in a solution of the mutagen to
induce mutations. Chemical muta-gens are generally carcinogenic and must be used with great caution. One of
the most effective chemical mutagenic groups is that of alkylating agents (they react with the DNA by alkylating
the phosphate
Table 23.2 Examples of commonly used chemical mutagens.
Mutagen group Examples

Base analogs 5-bromo-uracil, 5-bromo-

deoxyuridine
Related compounds Maleic hydrazide, 8-ethoxy
caffeine
Antibiotics Actinomycin D, mitomycin C,
streptonigrin
Alkylating agents
– Sulfur mustards Ethyl-2-chloroethyl sulfide
– Nitrogen mustards 2-chloroethyl-dimethyl amine
– Epoxides Ethylene oxide
– Ethyleneimines Ethyleneimine
– Sulfonates, etc. Ethyl methanesulfonate (EMS)
– Diazoalanes Diazomethane
– Nitroso compounds N-ethyl-N-nitroso urea
Azide Sodium azide
Hydroxylamine Hydroxylamine
Nitrous acid Nitrous acid
Acridines Acridine orange

groups as well as the purines and pyrimidines). Another group is the base analogues (they are closely related to
the DNA bases and can be wrongly incor-porated during replication); examples are 5-bromo-uracil and maleic
hydrazide. Other chemical mutagens commonly used are ethyl methane sulfo-nate (EMS) and diethylsulfonate
(DES). The half-life (time taken for degradation of half the initial amount of alkylating agent) for EMS in water is
about 93 hours at 20 8C but only 10 hours at 37 8C. Conse-quently, chemical mutagens are best freshly
prepared for each occasion. Plant breeders who undertake wide crosses often need to manipulate the
chromosome number to have fertility. To do this, the most com-mon chemical mutagen used is colchicine.
Chemical mutagens produce more gene mutations and less chromosome damage.

23.4 Types of tissues used for mutagenesis

Whole plants and plant parts may be used for muta-genesis, depending on the mutagen to be used, the species,
and the research objective, among other fac-tors. Seeds are the most commonly used material for mutagenesis
involving physical or chemical mutagens. Whole plants are suited to chronic exposure to physi-cal mutagens. A
major drawback to seeds and whole
 442 CHAPTER 23

plants is that chimeras are commonly produced. Pollen grains can be used to avoid chimeras. The resulting
plants from pollen mutagenesis are hetero-zygous for any genetic change. However, sufficient quantities of
pollen are difficult to obtain; also, they do not stay viable for long.
The plant breeder may also use cuttings and cell or tissue culture explants for mutagenesis. In tissue cul-ture,
cells or tissue are treated with the appropriate mutagens and regenerated into new whole plants.

23.5 Factors affecting the success

of mutagenesis

Mutations are random events, even when scientists induce them. The plant breeder using the technique can
increase the success rate by observing the following:

Clear objective. A program established to select one specific trait is more focused and easier to con-duct with
a higher chance of success than a program designed to select more than one trait.
Efficient screening method. Mutation breeding programs examine large segregating populations to increase
the chance of finding the typically rare desirable mutational events. An efficient method of screening should
be developed for a mutation breeding program.
Proper choice of mutation and method of treat-ment. Mutagens, as previously discussed, vary in vari-ous
properties including source, ease of use, penetration of tissue, and safety. Some are suitable for soft tissues,
whereas others are suited to hard tissue.
Dose and rate. The breeder should decide on the appropriate and effective dose rate (dose and dura-tion of
application). The proper dose rate is deter-mined by experimentation for each species and genotype. Plant
materials differ in sensitivity to mutagenic treatment. It is difficult to find the pre-cise dose (intensity), but careful
experimentation can identify an optimum dose rate. Mutagenic treat-ments invariably kill some cells. Overdose kills
too many cells and often produces crippled plants, whereas underdose tends to produce too few mutants. Not only
are dose–response relationships rarely known, but they are influenced by the experi-mental conditions. It is
recommended that three dose levels be used in a project – the optimal rate, plus one dose above and below this
rate.
 Proper experimental conditions. It is known that the oxygen level in the plant material affects the amount of
damage caused by the mutagen, the higher the oxygen level, the greater the injury to the material tends to be. The
change in the effect of a mutagen with oxygen supply is called the oxygen enhancement ratio. In studies in which
enhanced mutagen frequency is desired, the experimental conditions may be supplemented with oxygen (e.g.,
bubbling oxygen through the mutagen solution in the case of chemical muta-genesis). Where such enhancement is
undesirable, an oxygen-free environment should be used. The effect of oxygen on mutagenesis is dependent upon
the moisture content of the tissue. The higher the tissue moisture, the lower the tissue oxygen supply. Mutagens
vary in the importance of moisture in their effectiveness in inducing mutation, X-rays being more affected than
gamma rays, for example. Dry seeds are better to use when enhanced mutation frequency due to oxy-gen status
of the research environment is not desired. In chemical mutagenesis, temperature has an effect on the half-life of
the mutagen, high temperature accelerating the reaction.

Another experimental factor affecting the success of mutagenesis is the pH of the environment in chemical
mutagenesis. For example, EMS is most effective at pH 7.0, whereas sodium azide is most effective at pH 3.0.
Sometimes, dry seeds need to be pre-soaked to prepare the cells to initiate meta-bolic activity. It is important to
mention that it is easier to modify the experimental condition when seeds are being used for the mutation program
than when other materials are used.

23.6 Mutation breeding of seed-bearing plants

23.6.1 Objectives
The breeder should have clear objectives regarding the trait desired to be induced. Induced mutations are
equivalent to natural mutations and hence, once observed, the normal procedure for plant breeding is applicable.
It should be borne in mind that mutagenic treatments have both primary and secondary effects. In addition to the
specific desired alteration (primary effect), mutagens tend to alter the background geno-type (secondary effect).
Mutagenesis may cause signi-ficant variation in quantitative traits.
MUTAGENESIS IN PLANT BREEDING 443
23.6.2 Genotypes and source of material
Both self- pollinated and cross-pollinated species can be improved with induced mutations. Seeds
from self-pollinated species are homozygous and homoge-neous, making it easier to identify
mutants in the field. Outcrossed species are heterozygous and heter-ogeneous, making it more
challenging to identify mutants. Dormant seeds are easier to handle than vegetative material.
Mutation breeding is often used to correct a specific deficiency in an adapted and high yielding
genotype. This otherwise desirable genotype may be susceptible to a disease or may need modification
in plant architecture. A mutant is easier to spot if the parent (source genotype) is genetically pure (as
opposed to being a mixture). Also, mutants are easier to identify if the mutant trait is distinctly different
from the parental trait (e.g., it is easier to spot a true dwarf mutant if a tall parent is used for the project).
Some suggest using an F1 in some instances because it contains two genomes, which may increase
gene recombination and thereby produce a greater amount of diversity in gene mutations.

23.6.3 Treatment
The goal in seed treatment is to treat enough seed to eventually produce a large enough segregating
popula-tion in the second generation (M2). This means the number of treated seed should generate
sufficient first generation plants (M 1) that have sufficient fertility to produce the size of M 2 population
needed. The appro-priate number is determined through preliminary experimentation. One of the side
effects of mutagen treatment is sterility, which should be factored into the decision process to determine
the number of seeds to treat. M2 population may be 20 000–50 000 plants.
The seed is multicellular. Hence, a mutation in a single cell will give rise to a chimeric plant. Thus, an
M1 plant is subject to both diplontic (competition between mutated and normal tissue during the vege-
tative phase of development) and haplontic (compe-tition between mutated and normal pollen during
fertilization) selection in order to be included in the tissues forming seed on the plants. Treating seed with
physical agents is done by placing the seed in an appropriate container, positioning it at a proper dis-
tance from the source of radiation, and exposing it at a predetermined dose rate. Gamma irradiation
doses as low as 0.5 krad (e.g., in corn) and as high as