20. Mutagenesis

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By Nino Brown; Institution of Plant Breeding, Genetics and Genomics, University of Georgia

Contents

Mutation Breeding

“A knowledge of the laws of mutation must sooner or later lead to the possibility of inducing mutations at will and so of originating perfectly new characters in animals and plants. And just as the process of selection has enabled us to produce improved races, greater in value and in beauty, so a control of the mutative process will, it is hoped, place in our hands the power of originating permanently improved species of animals and plants.” - De Vries, 1909

Importance of variation in plant breeding

Variation is the source from which plant breeders are able to produce new and important cultivars. Alleles of varying forms at given loci in a population can be selected and fixed within a new individual or line. We depend on recombination and independent assortment of favorable alleles to produce new and unique individuals from which to select and produce the lines that will serve as our cultivars. With tens of thousands of genes within each crop species genome, the possibilities seem limitless. This, however, is not so clearly the case.

Consider that variation within a population can be exploited by selecting individuals with new combinations of desirable traits or alleles. As discussed in other chapters on genetic diversity of crop plants, most crop species, which have been selectively bred for centuries, have large portions of their genome essentially fixed. This means then, that the portions of DNA, and therefore number of traits available for re-shuffling is reduced. This reduction, however ominous it may seem, has still allowed us to make significant gains in crop yield and quality in recent decades.

Importance of mutation in creating variation

So, while recombination of alleles provides offspring with presumably selectable variation for the spectrum of traits exhibited, it is only capable of creating new combinations of traits already existing. Recombination does not of itself produce novel traits. This ability is only attainable through the act of mutation, which can ultimately lead to new species. Harten gives his working definition of mutation in plants as “any heritable change in the idiotypic constitution of sporophytic or gametophytic plant tissue, not caused by normal genetic recombination or segregation” (Harten, 1998). These changes in our target plant can be passed on to progeny and used for human benefit through breeding. The occurrence of mutations within the genome of plants is rare, and in natural settings can be lethal. Through breeding and selection, beneficial mutants can be identified and used to improve target species. In this chapter, we will discuss the basic concepts surrounding mutation, as well as the application and exploitation of mutation in plant breeding programs.

Naturally occurring mutations

Mutations occur spontaneously in natural settings quite frequently. They can happen due to mistakes made during cell replication or exposure to mutagens such as radiation. It is estimated that a mutation occurs every 10-8 base pair per generation in eukaryotic genomes (Drake et al., 1998). In corn (Zea mays), mutations occur from 10-6 to 5 x 10-4 per base pair per generation (Stadler, 1930). Those that we can track easily in the offspring are mutations occurring in either the gametes or cells that give rise to the gametes. Mutations in somatic cells cannot be easily tracked, nor can they be passed on to future generations, and so are only important in vegetatively propagated species.

Mechanics of mutation

Before going any further in our discussion of mutation in plants, it is prudent to touch on the genetics behind mutations, the varying types of mutations, natural versus spontaneous, the mechanics of mutagenic agents, and the implications.

A mutation is any change within the genome of an organism that is not brought on by normal recombination and segregation. Causative agents are many, but include exposure to mutagenic agents such as radiation or certain chemicals, and mistakes made during normal cell division and replication. Most of these act upon the genome at random and are occurring all the time. These mutations are usually benign and go unnoticed in the organism due to the many cellular mechanisms that protect against these sorts of genetic mistakes. Mutations that are not caught by DNA repair mechanisms in the cell can go on to affect the organism and be present in future progeny. In Luria and Delbruck’s classic experiment on bacterial resistance, they demonstrated that mutations occurred within the population at random and go unnoticed until some sort of selective pressure is placed upon the population (Luria and Delbrück, 1943).

Spontaneous mutations are those that occur without human intervention. These types of mutation happen randomly and the cause of them therefore not easily traceable. We do know, however, several things that cause mutations or increase their frequency. This includes the activity of mutagens found in nature such as solar radiation or reactive chemicals such as depurinators or free radicals. Mistakes during the replication of DNA during mitosis or unequal crossing-over events during meiosis can go un-repaired in a cell, leading to mutant progeny cells. It is hard to tell at what rate these occur, because the cellular machinery typically catches these mistakes. Occasionally, however, some mistakes make it permanently into the organism’s genome.

Types of mutation

At the most basic level, there are only a handful of classifications of mutations regardless of causative event. Deletions and insertions involve the removal or addition of segments of DNA respectively. These segments can range from individual base-pairs to several thousand base-pairs long. Substitutions occur when a particular base is replaced with one of the other three nucleotide bases. Inversions are instances where a segment of chromosome is rotated and replaced in the opposite direction that the segment was facing. The last major classification is a reciprocal translocation. This involves the excision of segments from two non-homologous chromosomes. These excised portions are then inserted into the other chromosome. Chromosome A will gain the segment from Chromosome B and Chromosome B gains the segment from chromosome A.

Mutagens and implications of each

Several types of mutagenic agents exist and have been used extensively since their discovery to produce variation and answer genetic questions. HJ Muller first discovered and used the mutagenic properties of X-ray radiation to study the genetics of Drosophila flies and the mechanics of heredity (Muller, 1928). Following this discovery, the use of ionizing radiation and chemical mutagens has been used extensively in the study of genetics and contributed a great deal to our understanding of biology as a whole.

Ionizing Radiation

Ionizing radiation includes ultra-violet (UV) light, X-ray, Gamma rays, and neutrons. These high-energy forms of radiation cause double-strand breaks of the DNA double helix. Once pieces of the DNA are broken, cellular repair mechanisms stitch the pieces back together. These DNA repair systems can only handle low rates of radiation, however, and increases in the rate of exposure to ionizing radiation causes permanent mutations to occur and accumulate in an organism’s genome. Radiation causes deletions of nucleotides from the DNA sequence. These deletions can cause reading-frame shifts, inactive protein products, or faulty transcripts. This typically results in null mutations, which are those in which a particular gene is inactivated.

Chemical Mutagens

Chemical mutagens affect the DNA molecule through chemical reactions within the genome. Base analogs are chemicals with similar properties to the DNA bases. They can be incorporated by the cell into the genome, replacing the proper base. Alkylating agents such as ethyl methanesulfonate (EMS), react with guanine or thymine by adding an ethyl group which causes the DNA replication machinery to recognize the modified base as an adenine or cytosine, respectively. This base substitution typically does not result in reading frame shifts, but instead causes altered forms of a triplet sequence. Changing a single base within a coding region causes either a nonsense codon which stops transcription or an altered codon which changes the amino acid transcribed, which can de-activate, reduce efficiency of, or produce a new protein. Nitrous acid, a deaminating agent, removes the amine group from adenine or cytosine. When the cell replicates this altered area, it matches adenine to the deaminated cytosine, and cytosine to the deaminated adenine, resulting in similar effects to that of alkylating agents. The last type of chemical mutagen, intercalating agents, causes deletions, reading frame shifts, or random base insertions. These compounds insert themselves into the DNA between adjacent base pairs, thus disrupting replication and transcription machinery.

Transposable Elements

Transposable elements are a special class of mutagen. They are self-replicating segments of DNA that excise and/or insert themselves within the genome. Also known as transposons, these strange sequences were first proposed by the pioneering Barbara McCLintock working on maize (McClintock, 1948). Transposable elements, unlike other forms of mutagenesis, do not act upon the genome in a completely random fashion. Rather, they have certain “hot-spots” where they are more likely to insert or replicate themselves. By their insertion or deletion, they act upon the genes in which they are located or those adjacent to them. Transposable elements can cause gene disruptions, protein product alterations, or large-scale genome rearrangements. If inserted into the intron of a gene, they can cause transcriptional inefficiency (Hartwell et al., 2008).

Use of Mutation in Plant Breeding

Mutagenesis, the act of inducing mutations within an organism’s genome, has been used in plant breeding since Muller’s discovery of the mutagenic effects of X-rays on Drosophila flies (Muller, 1927). Table 1 shows a representative sample of the 3000 varieties that have been improved using mutagenesis (JointFAO/IAEA, 2011). The first crop species to be mutagenized was barley by LJ Stadler, who began using X-rays to induce mutations independently of Muller at around the same time (Stadler, 1928), although he published his first paper on the subject the following year. These early mutation experiments were designed mainly to discern genetic truths of inheritance and chromosomal theory. Recall that at this point Watson and Crick’s double-helix DNA model had still not been arrived at.

Dose, Rate, Species/Genotypes, Conditions of application, etc. Much of the early work done with ionizing radiation and chemical mutagens was an effort to determine efficient doses and exposures of the various agents to effect high percentages of mutations without causing lethality. The researchers noticed that the rates and doses varied tremendously for species, genotype, ploidy level, and the conditions in which treatment were conducted. Grays (Gy) are the measurement unit of radiation dose. For the sterilization of food products, processors typically use rates as high as 10 kGy. In the mutagenic treatment of plant material, doses can range from as low as 2 Gy for cell cultures or leaf tissues, to as high as 700 Gy for seed material (Ahloowalia and Maluszynski, 2001).

When determining the most efficient dose for one’s crop species, it is important to first consult the literature for any information on the mutagenic agent to be used and the crop it will be used on. Chances are good that someone has already used the particular mutagenic agent on your crop. If not, it is important to consider several factors. (1) The first of course is safety issues regarding the mutagen. A researcher needs to know what sort of certifications, licenses, and precautions must be taken while carrying out the experiment and handling the plant material after the experiment is complete. (2) Next is the mutagen’s type and mechanism. Knowing how the mutagen affects the experimental tissue will not only allow for greater safety, but will also help in making better decisions on how to carry-out the experiment. For instance, if using X-rays, distance from the source of radiation reduces dose. It would be unwise to place a bucket of seed in front of an X-ray source and assume all the seeds in the bucket received equivalent doses of radiation. Similarly, the mutagenic chemical, EMS, requires very stringent experimental conditions to effect mutations. (3) Tissue specific reactions. Seeds react differently than stem cuttings, meristem tissues or callous tissue to the same level of radiation. (4) Species and ploidy level affect mutational response to the mutagen a great deal. Some crop species are equipped to handle a greater mutational load than others. They can more efficiently repair damage to their DNA. Ploidy level of the target species also influences mutation response and will be discussed in a later section.

Screens and efficiency

Recall that most agents used to induce a mutagenic response act upon the target genome in a random fashion. There is no precise control of where mutations will occur within the genome, within particular cells, or to what degree or number of mutations will occur within the target genome. To limit or maximize the number of mutations occurring, it is possible to adjust the dosage of mutagen treatment to the desired effect. For instance, if the mutation response desired is a high level, the researcher will expose the experimental material to high doses, which will result in high lethality of plants, but a high mutational response. Lower doses will result in fewer mutations per genome, a higher survival rate, and possibly also a higher level of non-mutant recovery. The purposely ambiguous terms low and high are used here due to the variable nature of mutation experiments and the large number of factors involved. For example, Auld exposed germinating cotton seeds to 3 to 5 times the LD50 rate of EMS (3% v/v) to ensure recovery of mutants in his tetraploid Gossypium hirsutum subjects (Auld et al., 1998). In contrast, EMS treatment of germinating sugar beet seeds only required 0.5% v/v EMS to elicit a sufficiently mutagenic response (Hohmann et al., 2005).

Once the desired dose-response relationship has been established in the experimental materials, the next step is the implementation of the “screen.” The first level of the screen is simple, the M1 generation (M notation denotes generations after mutagenic treatment, so the parent is the M0 and the first generation after mutagenesis is the M1 generation) must be grown out. Most mutant alleles are recessive, so successful mutants would not be seen until the M2, at which point lethality and infertility will often significantly affect the plants in the population. So the first level of the screen is identifying viable mutants.

Next, a cheap, fast, high throughput method of phenotyping and screening for the trait of interest must be in place. This is the most important factor in any mutagenesis experiment. Due to the chimeric nature of mutation, it is likely that some of the viable plants seen in the M2 population will be non-mutagenized parent types. Identification of the desired mutants requires looking at and accurately identifying the mutants. Accurate measurements must be fast and affordable to process the necessary number of individual plants or lines to achieve success. Like all plant breeding methods, mutation breeding is also a numbers game.

In the sugar beet experiment noted above, of over 3200 M2 families derived from an early bolting line treated with EMS, only 9 families exhibited the desired non-bolting trait and eventually gave rise to 5 lines with the non-bolting phenotype (Hohmann et al., 2005). The success of this program was in its simple and efficient screen. The experimenter observed the M2 population for non-bolting visually. Many other traits are not so simply characterized, but can still be done fairly cheaply and quickly.

Let us use the cotton example from above. Auld treated 2 kg of germinating cotton seeds with EMS and the fiber from nearly 2000 individual M3 plants characterized using the High Volume Instrument (HVI). This machine measures cotton fiber quality but is considered less accurate than other methods. HVI measurement, however, is cheap, fast, and highly correlated to yarn and textile performance. Although not perfectly accurate, it is good enough for processing large numbers. These 2000 M3 individuals gave rise to 2 M4:5 lines with fiber lengths that exceeded the parent value by 10% (Auld et al., 1998). One out of every thousand plants was a desirable mutant.

Mutation breeding in Self Fertilizing Species

Breeding mutant traits is fairly straightforward in crops that are capable of self-fertilization. Because many mutations are recessive, after mutagenic treatment, the material should be self-fertilized and advanced to at least the M2 before phenotypic screening. At this point plants will be segregating for the recessive mutant trait. Positive mutant identifications should be kept for future selection. Because mutagens act randomly upon the genome, it is important to collect as many positive mutants as possible. This allows the breeder to have a series of lines from which to select for performance in addition to the presence of the mutant trait.

Mutation breeding in Cross Fertilizing Species

Cross-fertilizing species raise some difficulties. Because species which are predominantly cross-fertilizing typically exhibit significant inbreeding depression, the necessary self-fertilizations to identify mutants in the population result in reduced plant vigor due to the genetic background and not necessarily the mutations. This compounds the difficulty of successfully identifying mutations. Dominant mutations can be identified, but these occur very rarely. Crop species with self-infertility mechanisms are especially hard to use mutation breeding methods without elaborate crossing schemes. The numbers required to make this feasible, however, make this impractical if not essentially impossible.

Mutation breeding in vegetatively propagated species

When attempting to effect mutation in vegetatively propagated species such as sugarcane or banana, it is important to note the chimeric nature of mutagenic treatment. All cells exposed to the mutagen will not necessarily incur mutations, but those that do incur mutations, will give rise to cells exhibiting the mutation. For this reason it is important to treat parts of the plant that will give rise to either seed or vegetative propagules. Identification and propagation of the necessarily large numbers of plants to identify successful mutants is difficult for many vegetatively propagated plants, however, once one is identified, the mutation is fixed in the cloned progeny. Crop species where in vitro techniques exist and can be used to mutate plant material, allows for the regeneration of large numbers of plantlets. This system is highly amenable to both vegetatively and seed propagated species.

Mutation breeding in seed propagated species

Seeds treated with mutagenic agents give rise to chimeric plants. Chimeric plants produce both mutant and non-mutant seed. This can be problematic; however, one just needs to plant more seeds to find the desired mutants. As long as an efficient screening method is in place, this should produce no significant pitfalls. Mutagenic treatment of seed is by far the most popular method in mutation breeding programs.

Ploidy and how it affects mutation breeding

Mutagenesis of polyploid plant species is difficult. Because most mutations are recessive, plants must be homozygous to display the trait. Polyploid conditions can further complicate the process of reaching homozygosity for the mutation, so must be selfed for additional generations to ensure presence of the mutation.

The Future of mutation breeding

Recent advances in genomics technology have led to a radiation of genomic techniques into applied breeding in general and mutational breeding specifically. Technologies such as high throughput sequencing has allowed for the relatively cheap and fast genome sequencing of plants. Methods such as TILLING (Targeting Induced Local Lesions in Genomes), Zinc finger nuclease mediated mutagenesis, and the use of meganucleases, has allowed us to produce targeted mutations in crop plants to delineate gene function as well as improve cultivars. These new and more specific methods are very promising.

TILLING relies on high throughput sequencing to assemble an array of mutants for a particular target sequence. Plant materials are mutagenized, the DNA is extracted and the target sequence PCR amplified and sequenced to identify mutants and locate the polymorphisms (McCallum et al., 2000). Although the mutations are induced randomly across the plant genome, they are detected only in the gene of interest. This allows the researcher to keep only those plants with mutations in the desired region. A similar process, EcoTILLING, screens for the spontaneous mutations present due to natural variation within a population.

Zinc finger nucleases (ZFN) and Meganucleases (MN) present a more targeted approach to induced mutation. ZFNs can be tailored to bind to specific recognition sites associated with the desired sequence. Once dimerized, the target DNA is cleaved, and a donor sequence introduced (Bibikova et al., 2003). The donor sequence typically exhibits desired mutations or it can be used to introduce new transgenes altogether into the target genome. Meganucleases have a similarly specific mode of action, and a great deal of research is going into both of these promising techniques for targeted mutagenesis as well as plant transformation.

Conclusion

Mutation breeding has long been a beneficial tool in not only the plant breeder’s tool box, but also basic geneticist’s. In crops where diversity for a given trait is low or non-existent, induced mutagenesis provides an avenue of possibility. With a clear objective, efficient mutagenic protocol, and a high throughput and efficient phenotypic screening method, mutagenesis can be of great benefit for the improvement of crop plants.


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