23

Mutagenesis in
plant breeding

Purpose and expected outcomes

It was previously pointed out that mutation is the ultimate source of variation. Without adequate variation, plant
breeding is impossible. To start a breeding program, the breeder must find the appropriate genotype (containing
the desired genes) from existing variation, or create the variation if it is not found in nature. Mutagenesis is the
process by which new alleles are created. The purpose of this chapter is to discuss mutagenesis as both a
technique and a breeding method. The newly created mutants may be used as parents in future breeding
programs, in which case mutagenesis is a breeding technique as a source of variation. However, an induced
mutant can be systematically processed through conventional breeding steps to be released as a cultivar, hence
making it a breeding method (mutation breeding). Mutations arise spontaneously in nature and are pivotal in
natural evolution. After com-pleting this chapter, the student should be able to:

1 Define mutation and mutagenesis.

2 Discuss mutagenic agents.
3 Discuss the steps in a mutation breeding program.
4 Discuss the limitations of mutation breeding.

23.1 Brief historical perspectives

The discovery of the mutagentic effects of X-rays on the fruit fly (Drosophila) by H. Muller in the 1920s paved the
way for researchers to experiment with its effects on various organisms. In 1928, H. Stubbe demonstrated the
use of mutagenesis in producing mutants in tomatoes, soybeans, and other crops. The
first commercial mutant was produced in tobacco in 1934. Reports by B. Sigurbjornsson and A. Micke
indicated 77 cultivars that were developed via muta-genesis prior to 1995. In 1995, the number was 484.
This number has since been significantly exceeded. They include food crops (e.g., corn, wheat, pea),
ornamentals (e.g., chrysanthemum, poinsettia, dahlia), and fruit trees (e.g., citrus, apple, peach).

Principles of Plant Genetics and Breeding, Second Edition. George Acquaah.

2012 John Wiley & Sons, Ltd. Published 2012 by John Wiley & Sons, Ltd.
 MUTAGENESIS IN PLANT BREEDING 437

Traits modified include agronomic ones, such as plant maturity, winter hardiness, lodging resistance, and
product quality (e.g., protein and lysine content), and numerous ornamental ones.
The role of mutations in plant breeding over the years has gone from skepticism, as was demonstrated by the
reaction of L.J. Stadler, who is said to have advised his students against using mutation breeding for commercial
crop improvement, to the over-opti-mism by protagonists who saw it as a revolutionary plant breeding method.
Currently, induced muta-tions are used more often in a supplementary role as a source of new alleles. However,
it is still important in breeding vegetatively propagated species, including field crops, ornamentals, and fruit and
forest species. It is especially useful in ornamental plant breeding where novelty is often advantageous and can
become commercially significant. Furthermore, with the advent of genetic engineering and its radical tools, which
allow targeted genetic alteration (versus the random genetic alteration produced by conventional mutagenesis), it
appears that breeders are gravitating towards this truly revolutionary technology for creat-ing new variability.
Nonetheless, every now and then, some breeders find good reason to use a technique or technology that has
been marginalized by advances in science and technology.
In conventional breeding of sexual plants, genetic variability is derived from recombination. Parents must not
be identical, or else there would be no seg-regation in the F2. Even when parents are dissimilar, they often have
similar “housekeeping genes”, which are common to both parents. Whereas segregation will not occur for these
common genes, mutagenesis can create variability by altering them.

23.2 Types of mutations

In terms of origin, mutations may be spontaneous (natural) or induced (artificial, with the aid of agents).
Spontaneous mutations arise at the very low rate of about 10 5 or 10 6 per generation for most loci in most
organisms. This translates to 1 in 100 000 or 1 in 1 000 000 gametes that may carry a newly mutated allele at
any locus. They are caused by mistakes in molecular processes associated with the replication of DNA,
recombination, and nuclear divi-sion. However, because mutagenic agents are com-mon in the general
environment, induced mutations,
as a result of these agents (natural radiations) are hard to distinguish from spontaneously induced
mutations due to cellular processes.
Mutations may also be classified according to the type of structural change produced as:

Ploidy variation. involving changes in chromo-some number (grain or loss in complete sets of chromosomes
or parts of a set).
Chromosome structure variation. involving changes in chromosome structure (e.g., duplica-tions of
segments, translocation of segments).
Gene mutation. changes in nucleotide constitution of DNA (by deletion or substitution).

Mutation may occur in the nuclear DNA or chro-mosomes, or in extra-nuclear (cytoplasmic) genetic
systems. A good example of the practical application of mutations in plant breeding is cytoplasmic – the
genetic male sterility gene, which occurs in chloroplasts.
Mutations that convert the wild type (the common phenotype) to the mutant form (the rare phenotype) are
called forward mutations, while those that change a mutant phenotype to wild phenotype are called reverse
mutations. Forward mutations are more common than reverse mutations. Recessive mutations are the most
common types of mutations. However, recessive alleles in a diploid are expressed only when they are in the
homozygous state. Conse-quently, an organism may accumulate a genetic load (the sum of deleterious genes
that are carried, mostly hidden, in the genome and may be transmitted to descendants) without any consequence
because of heterozygous advantage. As previously discussed, outcrossing species are susceptible to inbreeding
depression (loss of vigor) because of the opportunities for expression of deleterious recessive alleles.

23.2.1 Induced mutations versus spontaneous mutations

Spontaneous mutations produce novel alleles for the evolutionary process. Natural mutations have the benefit of
being processed through the evolutionary process, whereby viable mutants become recombined with existing
forms and become adapted under the guidance of natural selection. Mutagenesis can be used to create new
alleles that can be incorporated into existing cultivars through recombination
 438 CHAPTER 23

following hybridization and under the guidance of artificial selection. Modern crop production systems are capable
of providing supplemental care to enable a mutant that would not have survived natural selection to become
productive. As previously discussed, a sig-nificant number of commercial cultivars originated from mutation
breeding techniques. Furthermore, the rate of spontaneous mutation is low (10 –6 per locus). Artificial
mutagenesis aims at increasing muta-tion rates for desired traits.

23.2.2 Cell type: Gametic mutations versus somatic mutations

Mutations may originate in the gametic or somatic cells. Gametic mutations are heritable from one gen-eration to
the next and expressed in the entire plant. However, mutations in a somatic tissue will affect only that portion of
the plant resulting, in a condition called chimera. In species that produce tillers, it is possible to have a tiller
originate from a chimeric tis-sue, while others are normal. A chimera consists of two genetically distinct tissues
and may produce two distinct flowers on the same plant. However, the dual color is impossible to reproduce by
either sexual or asexual propagation. Commercial use of chimera is not attractive because the vegetative
propagules must, of necessity, comprise both kinds of tissues in order to reproduce the maternal features. It is
also well known that cells at the G2 (gap phase) and M (mitosis) stages are more sensitive to radiation than those
at the G1 and S (synthesis) stages.

23.2.3 Gene action: Dominant versus recessive mutations

In terms of gene action, a mutation may be a:

Recessive mutation. change of a dominant allele to a recessive allele (A ! a).

Dominant mutation. change of a recessive allele to a dominant allele (a ! A).

Open pollinated species may accumulate a large amount of recessive mutant alleles without any adverse
effects. However, upon selfing, the recessive alleles become homozygous and are expressed, lead-ing to the
phenomenon of inbreeding depression. Using recessive genes in breeding takes a longer time because it
requires an additional step of selfing in
order to identify and select the desired recombinants. On the other hand, dominant mutations manifest in the
current generation, needing no additional regen-eration to be observable.

23.2.4 Structural changes at the chromosomal level

Three types of structural changes in the chromosome can occur as a result of mutation.

1. Gene mutation

(a) Kind Gene mutations entail a change in the nucleotide constitution of the DNA sequence, adding or
deleting nucleotides.

Transitions and transversions. DNA consists of four bases – A, T, C, and G – that pair in a specific pattern,
G–C and A–T. The structure of the DNA may be modified in two ways – transition or trans-version of bases
(Figure 23.1). Mutation by transi-tion entails the conversion of one purine base to another purine (or a
pyrimidine to another pyrimi-dine). During replication, the second purine (differ-ent purine), having altered
base pairing properties, guides an incorrect base into position. Conse-quently, one normal base pair is
converted to another pair that is genetically incorrect. An agent of mutation (mutagen) such as nitrous acid
has been known to cause deamination of adinine to hypoxanthin, cytosine to uracil, and guanine to xan-
thine, the net effect being a replacement of A–T with G–C in the DNA structure. A transversion involves the
substitution of a purine by a pyrimidine and vice versa.
Tautomeric shifts. It is known that each of the bases of DNA can exist in rare states as a result of the
redistribution of electrons and protons in the molecule, events called tautomeric shifts. When

Transition A T A C
Transversion A T C G

T A GC

Figure 23.1 Mutations may occur by transition of transversion.

 MUTAGENESIS IN PLANT BREEDING 439

G A
Normal Normal
C T
G A
A G
Mutant Mutant
C A T G
T C

C G T A
Normal Normal
(a) C (b) T

Figure 23.2 Mutations may be caused by tautomeric shifts: (a) shift involving cytosine; (b) shift involving thymine.

this occurs, the base sometimes is unable to hydro-gen bond with its complementary base. Instead, some of these
altered bases succeed in bonding with the wrong bases, resulting in mutations when, dur-ing replication, one
purine (or pyrimidine) is substi-tuted for the other (Figure 23.2).
Effect of base analogues. Certain analogues of the naturally occurring bases in the DNA molecule have been
shown to have mutagenic effects. For example, the natural base thymine (T), a 5-methyluracil, has a
structural analog, 5-bromouracil (5-BU). The two bases are so similar that 5-BU can substitute for T during
replication, leading to base pair transition (Figure 23.3).
Single base deletions or additions. A variety of alkylating agents (e.g., sulfur and nitrogen mus-tards) can act
on the DNA molecule, reacting mainly with guanine (G) to alkylate and remove it from the DNA chain. The
missing spot may be occupied by any of the four bases to create muta-tions, usually by transition. Acridine is
also known
 to express its mutagenic effect through addition of deletion of bases.

(b) Effect at the protein level Four different effects are known to occur as a result of nucleotide substitution.

Silent mutation. Because the genetic code is degenerate (one amino acid can be coded by more than one
triplet), a change from ACG ! CGG has no effect as both triplets code for arginine.
Neutral mutation. This kind of mutation involves an altered triplet code that codes for a different but
chemically equivalent amino acid. For example, CAC may change to CGC, altering histidine to a chemically
equivalent amino acid, arginine. The change causes a change in the primary structure of the protein (amino
acid sequence) but the form of the resultant protein may be unchanged.
Missense mutation. Unlike neutral mutations, a missense mutation results when an altered triplet