Most mutations are recessive and, hence, are observed only when the homozygous genotype occurs.

This
446 CHAPTER 23
 MUTAGENESIS IN PLANT BREEDING 447

heredity whereby scientists start with a gene or an unknown genetic sequence and attempt to find the
phenotype(s) associated with it. A common approach to reverse genetics is to alter or disrupt the DNA sequence
or gene and look for the effects of such an action. Several techniques are used for disrupting the DNA element;
the common ones include:

Site-directed mutagenesis. Scientists can change either the regulatory regions in the promoter or a gene, or make
minor changes in the codon in the open reading frame. These changes comprise of deletions and point mutations.
Such changes may result in null allele, in which the gene is no longer functional. This deletion mutational event is
referred to as gene knockout. Rather than delete genes right away, scientists may devise a technique to include
“conditional alleles” that have normal functions until they are activated. These are called gene knock-in (e.g., Cre
or Flp recombinases that are activated by chemical, heat shock, etc.).
Gene silencing. In contrast to gene knockout, which permanently inactivates a gene, genes may be temporarily
inactivated (gene knockdown). This is achieved by the technique of gene silencing involv-ing double stranded RNA
(called RNA interfer-ence or RNAi) or Morpholino oligosaccharides. RNAi interferes with gene expression without
actu-ally mutating the DNA of interest. It relies on cellu-lar components (Dicer proteins, RISC complex) for efficacy.
The Morpholino antisense oligosaccharides bind and block access to the target mRNA without any helper
molecules or accelerating the degrada-tion of mRNA.
Interference using transgenes. Interference of gene expressions may be accomplished through the use of
trangenes to overexpress a normal gene of interest or overexpress mutant forms of a gene that is known to
interfere with the normal gene function.

23.12.2 Targeted induced local lesions in genomes (TILLING)

Modern advances in genome research have resulted in the availability of whole genome sequences for many
model organisms. Coupled with advances in geno-mics technologies and availability condition is readily
satisfied in species that are natu-rally inbreeding. The situation practically excludes species that are
polyploid, propagated clonally, or have a fertility regulating mechanism (e.g., self-incompatibility) from being
amenable to mutation breeding (except when dominant mutations are targeted).

23.10.4 Limited pre-existing genome

The researcher cannot change what does not exist. Consequently, mutations can only be induced in
existing genes.

23.10.5 Mutations are generally random events

A modern biotechnology technique, site-directed mutagenesis, has been used to induce targeted muta-
tions. However, conventional mutagenesis remains unpredictable, not being able to be directed to spe-cific
genes.

23.11 Selected significant successes of mutation breeding

Mutation breeding has been used to improve a num-ber of important crops (Table 23.3). The first com-mercial
cultivar derived from mutagenesis was “Chlorina”, a tobacco cultivar. It was developed with X-ray radiation in
1930. Since then, several hundreds of commercial cultivars or ornamentals, field crops, fruit crops, and other
plant kinds have been released for production. These include barley cultivars “Pallas” and “Mari” that were
developed by Swedish scientists. These genotypes and other cultivars developed
through hybridizations involving the two mutants, significantly impacted barley production in Denmark and
Sweden. In the United States, “Pemrad” and “Luther” cultivars of barley were significant in the production of
the crop in the 1970s. Dwarf cultivars of cereals have been developed by mutagenesis. Clas-sic examples
are “Norin 10” of the Green Revolution fame and “Calrose 76” rice, which strongly impacted California rice
production.

23.12 Molecular techniques for enhancing efficiency of induced mutagenesis

Conventional mutation breeding requires the rapid generation of large mutant populations of desirable genetic
backgrounds (homozygous for the mutation events and without chimeras) within which selection of desirable
mutants is pursued. The need to plant large field trials or conduct laboratory assays on the large numbers of
plants in order to discover muta-tional events is laborious and expensive. Molecular techniques are able to
directly query target genes for changes (mutations), thus reducing or eliminating the need for fields trials involving
large populations. The polymerase chain reaction (PCR) technique may be used to amplify target regions of the
genome by using enzymatic mismatch cleavages.

23.12.1 Reverse genetics

Classical genetics is an approach to understanding heredity by finding out the gene(s) responsible for a
particular trait of phenotype in an organism. This is also called forward genetics. Reverse genetics, as the
name implies, is an approach to understanding
Table 23.3 Selected general areas of achievement in mutation breeding.

1. Disease resistance – e.g., Verticilium wilt resistance in perpermint, Victorial blight resistance in barley,
downy mildew resistance in pearl millet.
2. Modification of plant structure – e.g., bush habit in dry bean, dwarf mutants in wheat and other cereals.
3. Nutritional quality augmentation – e.g., opaque and floury endosperm mutants in maize.
4. Chemical composition alteration – e.g., low-euricic acid mutants of rape seed.
5. Male sterility – for use in hybrid breeding in various crops.
6. Horticultural variants – development of various floral mutants.
7. Breeding of asexually propagated species – numerous species and traits.
8. Development of genetic stock – various lines for breeding and research.
9. Development of earliness in many species.
of cost effect and high throughput DNA genotyping and chemical mutagenesis protocols, researchers can
now identify mutations in nearly every gene of interest. Mutational
analysis has been a major tool for researchers in understanding the basic mechanisms of disease, devel-
opment, cell biology and metabolism. Earlier efforts employed the forward genetics approach that
involved systematic screening for mutations that pro-duced visible phenotypes as a result of defective
devel-opmental processes. Though effective, this approach has severe limitations. Many mutations can
remain undetected in phenotypic screening because of sheer genetic complexity. Further, practical
challenges occur because it is not feasible to screen the enormous number of genomes that will be
needed in order for rare mutations or phenotypes to be identified.
To overcome some of the limitations of the forward genetics approach, researchers resort to the
genera-tion or targeted discovery of a mutation within a gene of known sequence, an approach called
reverse genet-ics. The key limitation of this approach is the fact that it tends to be organism specific. Well
established reverse genetic systems include those for Drosophila, Xenopus, Caenorhabditis elegans, and
zebrafish. A new approach to mutational analysis developed by Henikoff and colleagues, dubbed
TILLING (tar-geted induced local lesions in genomes), and first applied to Arabidopsis thaliana, provides
a more gen-eralized approach to identifying mutations in genes that are only known by their sequence.
An advantage of this approach is its ability to generate an allelic series of mutations that is not biased by
phenotypic selection.
TILLING starts with first assembling chemically induced mutation-carrying gametes or living indi-
viduals (usually thousands, but this depends on the size of the gene and frequency of induced
changes) (Figure 23.5). Chemical mutagens that are best suited to this approach are those that
generate point mutations (e.g., EMS and ENU). Different mutagens may be used to diversify an
allelic series. A sample of DNA is taken from each individual for high throughput analysis of
heterozygosity. Geno-typing involves gene-specific PCR with labeled primers, melting and re-
annealing, followed by treatment with Cel 1 endonuclease and analysis on automated sequencing
machines (verification of identified mutants). The differential double-end labeling technique
employed allows mutants to be detected on complementary strands. Upon identify-ing a mutant in a
pool, genotyping is repeated for the DNA samples in that pool to identify the indi-vidual mutants.