Mutation Breeding
Mutagenesis in Plant Breeding
The discovery of the mutagenic 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 tomato, soybean, and other crops. The first commercial mutant was produced in tobacco in 1934. Reports by B. Sigurbjornsson and A. Micke mentioned 77 cultivars that were developed via mutagenesis 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). 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 mutants.
The perceived role of mutation in plant breeding was initially treated with skepticism by some, as demonstrated by L. J. Stadler, who is said to have advised his students against using mutation breeding for commercial crop improvement as a reaction to the overoptimism by protagonists who saw it as a revolutionary plant breeding method. Currently, induced mutations 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 creating new variability. However, no approach should be written off as 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 segregation in the F2 generation. Even when parents are dissimilar, they often have similar “housekeeping genes” that are common to both parents. Whereas segregation will not occur for these common genes, mutagenesis can create variability by altering them.
Classification
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 one in 100,000 or one 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 division. However, because mutagenic agents are common 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:
· Genomic mutation: changes in chromosome number (gain or loss in complete sets of chromosomes or parts of a set).
· Structural mutation: changes in chromosome structure (e.g., duplications of segments, translocation of segments).
· Gene mutation: changes in the nucleotide constitution of DNA (by deletion or substitution).
Mutation may occur in the nuclear DNA or chromosomes, or in extranuclear (cytoplasmic) genetic systems. A good example of the practical application of mutations in plant breeding is the cytoplasmic-genetic malesterility gene, which occurs in chloroplasts.
In terms of gene action, a mutation may be recessive or dominant:
· 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).
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 a 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. Consequently, an organism may accumulate a genetic load 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.
Induced mutations versus spontaneous mutations
Spontaneous mutations produce novel alleles for the evolutionary process. Natural mutations have the benefit of being subjected to 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 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 significant number of commercial cultivars originated from mutation breeding techniques. Furthermore, the rate of spontaneous mutation is low (10−6 per locus). Artificial mutagenesis aims to increase mutation rates for desired traits.
Cell type: gametic versus somatic mutations
Mutations may originate in the gametic or somatic cells. Gametic mutations are heritable from one generation to the next and are 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 tissue, 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.
Gene action: dominant versus recessive mutations
As previously indicated, mutations may cause a dominant allele to be changed into a recessive allele (recessive mutation), or a recessive allele to be changed into a dominant allele (dominant mutation). 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, leading 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 regeneration to be observable.
Structural changes at the chromosomal level
Three types of structural changes in the chromosome can occur as a result of mutation.
Gene mutation
Kind of mutation Gene mutations entail a change in the nucleotide constitution of the DNA sequence, adding or deleting nucleotides.
· Transitions and transversions. As previously described, the DNA consists of four bases – A, T, C, and G – that pair in a specific pattern, G–C and A–T.
Figure 1.1 Mutations may occur by transition or transversion.
The structure of the DNA may be modified in two ways – transition or transversion of the bases (Figure 1.1). Mutation by transition entails the conversion of one purine base to another purine (or a pyrimidine to another pyrimidine). During replication, the second purine (a different purine), which has altered base-pairing properties, guides an incorrect base into position. Consequently, one normal base pair is converted to another pair that is genetically incorrect. An agent of mutation (a mutagen) such as nitrous acid has been known to cause deamination of adenine to hypoxanthin, cytosine to uracil, and guanine to xanthine, 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 this occurs, the base sometimes is unable to hydrogen bond with its complementary base. Instead, some of these altered bases succeed in bonding with the wrong bases, resulting in mutations when, during replication, one purine (or pyrimidine) is substituted for the other (Figure 1.2).
· Effect of base analogues. Certain analogues of the naturally occurring bases in the DNA molecule have
Figure 1.2 Mutations may be caused by tautomeric shifts: (a) shift involving cytosine, and (b) shift involving thymine.
Figure 1.3 Mutations may be caused by transition resulting from the substitution of base analogues for a natural base:
(a) mistake in incorporation, and (b) mistake in replication.
been shown to have mutagenic effects. For example, the natural base thymine (T), a 5-methyluracil, has a structural analogue, 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 1.3).
Single base deletions and additions. A variety of alkylating agents (e.g., sulfur and nitrogen mustards) 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 mutations, usually by transition. Acridine is also known to express its mutagenic effect through the addition of deletion of bases.
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 formation of the resultant protein may be unchanged.
· Missense mutation. Unlike neutral mutations, a missense mutation results when an altered triplet codes for a different amino acid that results in the protein being non-functional. For example, in hemoglobin of humans, a change of GAG (Glu) to GTG (Val) results in serious consequences.
· 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.
Frame shift mutation Insertion–deletion mutations (indels) may cause significant changes in the amino acid composition of a protein and hence its function. For example, GAG-CCG-CAA-CTT-C (corresponding to
Glu-Pro-Glu-Leu) may be altered by a 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 + A (at the beginning) → ACA-TCA-TCA, etc.
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 an 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). The subject is discussed in more detail in Chapter 13.
Structural chromosomal changes (aberrations)
Changes in chromosome structure begin with a physical break that may be caused by ionizing radiation (e.g., Xrays). After a break, several events may occur:
· The ends of the segment may be disunited.
· The break may be repaired to restore the chromosome to its original form (restitution).
· One or both ends of a break may join to the ends produced by a different break event (non-restitutional union). These events may result in one of four types of rearrangement – deletions, duplications, inversions, or translocations. The resulting consequences are variable.