Principles of Plant Genetics and Breeding

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 mutagenesis). 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 oxygen 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
presoaked to prepare the cells to initiate metabolic
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.
Mutation breeding of seed-bearing plants
Objectives
The breeder should have clear objectives regarding the
trait to be induced. Induced mutations are neutral
mutants. They 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
genotype (secondary effect). Mutagenesis may cause
significant variation in quantitative traits.
Genotypes and source of material
Both self- and cross-pollinated species can be improved
with induced mutations. Seeds from self-pollinated
species are homozygous and homogeneous, making
it easier to identify mutants in the field. Outcrossed
species are heterozygous and heterogeneous, making it
more challenging to identify mutants. Dormant seeds
are easier to handle than vegetative material.
MUTAGENESIS IN PLANT BREEDING 205
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 F 1 in some
instances because it contains two genomes, which may
increase gene recombination and thereby produce a
greater amount of diversity in gene mutations.
Treatment
The goal in seed treatment is to treat enough seed to
eventually produce a large enough segregating population
in the second generation (M2 ). This means the
number of treated seed should generate sufficient first
generation plants (M1 ) that have sufficient fertility
to produce the size of M2 population needed. The
appropriate 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. The M2 population may consist of 20,000–50,000
plants.
The seed is multicellular and 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 vegetative phase
of development) and haplontic (competition between
mutated and normal pollen during fertilization) selection
in order to be included in the tissues forming seed
on the plants. Treating a seed with physical agents is
done by placing the seed in an appropriate container,
positioning it at a proper distance from the source of
radiation, and exposing it at a predetermined dose rate.
Gamma irradiation doses that have been used may be as
low as 0.5 krad (e.g., in corn) or as high as 25 krad (e.g.,
in dry seed of wheat), whereas the dose rate for X-rays
ranges between 10 and 25 krad. When using chemicals,
the proper concentration of mutagen is prepared at the
desirable pH and temperature. Seeds are soaked in the
mutagenic solution for the appropriate duration. EMS
concentrations that have been used range from less than
1% (e.g., in tomato) to about 4% (e.g., in wheat).
The common physiological injuries caused by the
mutagenic treatment are reduction in seedling height
(most frequently used identification of injury in the M1 ),