Induced Mutations and Polyploidy Breeding

Abstract

Mutation is a sudden heritable change that occurs in the genetic information of an organism not caused by genetic segregation or genetic recombination but induced by chemical, physical or biological agents. Polyploids are organisms with multiple sets of chromosomes in excess of the diploid number. Polyploidy is a natural mechanism that provides adaptation and speciation.

1 Mutation Breeding

Mutation is a sudden heritable change that occurs in the genetic information of an organism not caused by genetic segregation or genetic recombination; but induced by chemical, physical or biological agents. Mutation breeding follows three strategies:

• (a) Induced mutagenesis: mutations occur because of irradiation (gamma rays, X-rays, ion beam, etc.) or treatment with chemical mutagens

• (b) Site-directed mutagenesis: mechanism of creating mutations at a defined site in a DNA molecule

• (c) Insertion mutagenesis: done through DNA insertions; by genetic transformation and insertion of T-DNA or activation of transposable elements

For crop breeding, multiple mutant alleles are the sources of genetic diversity. The vital issue in mutation breeding is the diligence to isolate and select individuals with target mutation. This process involves two major steps: mutant screening and mutant confirmation. In mutant screening, the breeder fixes certain traits to be selected. This involves selection of individuals that meet specific selection criteria like early flowering, disease resistance, etc. Mutant confirmation is done through reevaluating the putative mutants under controlled and replicated environments. By this process, false mutants can be revealed. In general, mutations vital for crop improvement usually involve single bases and may or may not affect protein synthesis.

1.1 History

Reports on mutant crops from China were available as early as 300 BC. Towards the late nineteenth century, Hugo de Vries was the first to identify mutations while “rediscovering” Mendelian laws. He could consider such variability as heritable that was distinctive from segregation and recombination. He coined the term “mutation”. Such variability was described as shock-like changes (leaps) in existing traits. After the discovery of mutagenic action of X-rays, radiation-induced mutations were used as tools for generating novel genetic variability. This was demonstrated in maize, barley and wheat by Stadler. The first commercial mutant variety was produced in tobacco in 1934. The number of commercially released varieties rose to 484 by 1995. This number sharply increased with time (Fig. 16.1). They include fruit trees, ornamentals and food crops. Agronomic traits like lodging resistance, early maturity, winter hardiness and product quality (e.g. protein and lysine content) were the most sought after traits in breeding. Mutagenesis became very popular from the 1950 as a breeding tool, and a range of crops and ornamentals were subjected to induced mutations to increase trait variation.

Fig. 16.1

Milestones in induced mutagenesis

1.2 Mutagenic Agents

Agents that induce artificial mutations are called mutagens. They are grouped as chemical and physical. Planting materials are exposed to physical and chemical mutagenic agents to induce mutations. Materials like whole plants, usually seedlings, and in vitro cultured cells can be used for mutation induction. Seed is the most commonly used plant material. Plant forms as bulbs, tubers, corms and rhizomes are also used. In vegetatively propagated crops, vegetative cuttings, scions or in vitro cultured tissues like leaf and stem explants, anthers, calli, cell cultures, microspores, ovules, protoplasts, etc. are used. Gametes can be mutated through immersion of spikes, tassels, etc. Whereas chemical mutagens are preferably used to induce point mutations, physical mutagens induce gross lesions, such as chromosomal abbreviation or rearrangements. Frequency and types of mutations are direct results of dosage and rate of exposure or rather than its type. The choice of a mutagen will be based on the safety of usage, ease of use, availability of the mutagens, effectiveness in inducing certain genetic alterations, suitable tissue, cost and available infrastructure among other factors.

1.3 Physical Mutagenesis

Physical mutagens, mostly ionizing radiations, have been used widely for developing more than 70% of mutant varieties for the last 80 years. Radiation is energy travelled through a distance in the form of waves or particles. Radiation is a high-energy level of electromagnetic (EM) spectrum that is capable of dislodging electrons from the nuclear orbits of the atoms. The impacted atoms, become ions, hence, the term ionizing radiation. These ionizing components of the EM include cosmic, gamma (γ) and X-rays. The most commonly used physical mutagens and their properties are shown in Tables 16.1a and 16.1b. X-rays were the first to be used to induce mutations. After this, various subatomic particles (neutrons, protons, beta particles and alpha particles) were used in nuclear generators to emit radiations. Gamma radiation from radioactive cobalt (⁶⁰Co) is widely used. Since it has high penetrating potential and is hazardous, gamma rays can be used for irradiating whole plants and delicate materials like pollen grains. In most cases, DNA double-strand breaks lead to mutation. Since gamma rays have shorter wavelength, they possess more energy than protons and X-rays, which gives them the strength to penetrate deeper into the tissue. Neutrons are used in dry seeds as they cause serious damage to the chromosomes. The mutagenic potential of UV rays had been confirmed in many organisms. Emission of UV light (250–290 nm) has a modest capacity to infiltrate tissues and goes deeper into the tissue and can cause a great number of variations in the chemical composition. The advantage of using physical mutagenesis over chemicals is the degree of accuracy and reproducibility. Among them, gamma rays are most sought after due to its uniform penetrating power. During the past two decades, ion beams have become more popular. They consist of particles travelling along a path that vary in mass from a simple proton to a uranium atom which is generated through particle accelerators. The positively charged ions are accelerated at a high speed (about 20%–80% of the speed of light) and form high-linear energy transfer (LET) radiation. LET radiation causes significant biological effects, such as chromosomal aberration, lethality, etc. Ion beams induce deletion of fragments of various sizes and are less repairable.

Table 16.1a Commonly used physical mutagens

Table 16.1b Types and properties of ionizing radiations used for plant-induced mutagenesis

For inducing mutations, doses that lead to 50% lethality (LD₅₀) have often been chosen. It is the amount of substance required (usually per body weight) to kill 50% of the test population. Very often it is argued that LD₅₀ is quite arbitrary and might lead to a high number of (mostly deleterious) mutations. LD₅₀ can lead to loss of desirable mutations due to plant mortality or due to poor agronomic performance. Therefore, in self-pollinated species, a mutation rate targeting a lower LD (e.g. LD20) with a survival rate of 80% appears to be more ideal. The isotope ⁶⁰Co has a half-life of 5.27 years and emits radiations of energies 1.33 MeV and 1.17 MeV (mega electron volt).

Ionizing radiations break chemical bonds in the DNA molecule, deleting a nucleotide or substituting it with a new one. Radiation being applied at a proper dose depends on radiation intensity and duration of exposure. Roentgen (r or R) is the unit to measure dosage of radiation. Rontgen is named after Wilhem Conrad Röntgen a German physicist, who during 1895 produced and detected electromagnetic radiation that earned him the first Nobel Prize in physics in 1901. The exposure may be chronic (continuous low dose administered for a long period) or acute (high dose over a short period). The dose rate is not necessarily positively correlated with the proportion of useful mutations. A high dose need not necessarily produce best results. The mutagen dose depends on the mutation load and the chance to find desirable mutations.

1.3.1 Ion Beams

Ion beams are usually generated by particle accelerators, e.g. cyclotrons, using ²⁰Ne, ¹⁴ N, ¹²C, ⁷Li, ⁴⁰Ar or ⁵⁶Fe as radioisotope sources. These ion beams are responsible for linear energy transfer (LET). Like physical mutagens, as LET increases, lethality, chromosomal aberration, etc., are also induced. The LET for gamma rays and X-rays accounts in the range of 0.2–2.0 keV/μm and hence is called low-LET radiations. However, the high-LET radiations from carbon (23 keV/μm) and iron (640 keV/μm) ion beams extend larger and wider ionization energy. High-LET ion beam radiations cause more localized, dense ionization within cells than those of low-LET radiations. The choice of ion beam depends on the characteristics of the ion with respect to electrical charge and velocity. Dose (in Gy=Gray units) is proportional to the LET (in keV/μm) and number of particles. An ideal irradiation dose provides highest mutation rate at any locus. Through applying different doses in a given time and screening the irradiated population, acceptable mutants can indicate the best dose. Scientists may consider traits like survival rate, growth rate, chlorophyll mutation, etc., as early indicators for mutation and this exercise requires sizeable investment. Advantages of ion beam mutagenesis include low dose with high survival rates, induction of high mutation rates with wide range of variation. Ion beam is an excellent tool for mutation breeding to improve horticultural and agricultural crops with high efficiency. In rice, salt-resistant lines were developed through ion beam irradiation. This was developed with 30–60 keV low-energy ion beam.

1.3.2 Aerospace Mutagenesis

In the recent past, plant materials were sent to aerospace to study probable mutation induction in space. The speculation is that cosmic radiation, microgravity, weak geomagnetic field, etc. contains the potential agents of mutation induction. However, much is not known on the genetics of aerospace mutagenesis. Gamma rays induce nucleotide substitutions and small deletions of 2–16 bp, and the mutation frequency is estimated to be one mutation/6.2 Mb. Fast neutrons are believed to result in kilobase-scale deletions.

More than 90% of the space radiation is composed of protons, neutrons, heavy particles, rays and microgravity. China had sent more than 400 varieties of 50 species to outer space by 8 recoverable satellites. From this exercise, more than 50 new varieties with high yield, high quality and drought tolerance have been commercialized. Though progress is made, mechanisms governing mutation induction is still under investigation.

1.4 Chemical Mutagenesis

Though the action is milder, the advantage with chemical mutagens is that they can be used without sophisticated machinery. However, undesirable changes are higher than in physical mutagenesis. Usually, the material is soaked in a solution of the mutagen to induce mutations. Extra care must be taken for health protection since chemical mutagens are carcinogenic. Thus, safety data sheets should be carefully read and the mutagenic agent should be appropriately inactivated before disposal. Although a large number of mutagens are available, only a small number is recognized by IAEA (International Atomic Energy Agency). Such mutagenic agents are responsible for over 80% of the registered new mutant plant varieties reported in the (IAEA) database. Of these, three compounds are significant: ethyl methanesulphonate (EMS), 1-methyl-1-nitrosourea and 1-ethyl-1-nitrosourea, which account for 64% of these varieties.

One of the most effective chemical mutagenic groups is the group of alkylating agents (these react with the DNA by alkylating the phosphate groups as well as the purine and pyrimidine). Another group is that of the base analogues (they are closely related to the DNA bases and can be wrongly incorporated during replication). Examples are 5-bromo-uracil and maleic hydrazide (Table 16.2). There is a clear advantage with the point mutations created by chemical mutagens. Point mutations have the potential to generate not only loss-of-function but also gain-of-function phenotypes. This happens when the mutation leads to a modified protein activity or affinity, like tolerance to the herbicide (glyphosate or sulphonylurea). Factors like concentration, the length of treatment and the temperature of the experiment influence the efficiency of mutagenesis. Since chemical mutagens are very reactive, it is advisable to use fresh batches of the chemical(s).

Table 16.2 Commonly used chemical mutagens

EMS reacts 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. Chemical mutagenesis induces a high frequency of nucleotide substitutions, and a majority of the changes (70–99%) in EMS-mutated populations are GC to AT base pair transitions. Sodium azide (Az) and methylnitrosourea (MNU) are also used in combination.

All chemical mutagens are strongly carcinogenic, and extreme care should be taken while handling and disposal. EMS is an IARC group 2B carcinogen. Working with MNU can be sometimes difficult as it is unstable above 20°C. EMS solutions can be deactivated in a solution of 4% (w/v) NaOH and 0.5% (v/v) thioglycolic acid. Chemical mutagens (EMS, DES, Az) have been applied for treating banana shoot tips to produce variants for tolerance to Fusarium wilt. EMS has also been successful in obtaining a wide range of variations in petal colour and in salt-tolerant lines in sweet potato.

1.5 Types of Mutations

Mutations can be broadly divided into (a) intragenic or point mutations (occurring within a gene in the DNA sequence); (b) intergenic or structural mutations within chromosomes (inversions, translocations, duplications and deletions) and (c) mutations leading to changes in the chromosome number (polyploidy, aneuploidy and haploidy). In addition, there are nuclear and extranuclear or plasmon (chloroplast and mitochondrial) mutations. Mutational changes at the molecular level are accomplished through substitution of one base by the other. This happens through mispairing of bases between pyrimidines and purines.

Basically, transitions (point mutations that changes purine to another purine A ↔ G or pyrimidine to another pyrimidine C ↔ T) and transversions (when a purine is changed to pyrimidine or vice versa) are the simplest kinds of base pair changes. However, they may result in phenotypically visible mutations (Fig. 16.2a). Another common error would be addition or deletion of a nucleotide base pair when one of the bases manages to pair with two bases or fails to pair at all. Such sequence changes in the reading frame of the gene’s DNA are known as frameshift mutations. Since they can change the message of the gene starting with the point of deletion/addition, they are more prominent (Fig. 16.2b). Base sequence may be inverted because of chromosome breakage. On the other hand, reunion of the broken ends can result in different DNA molecules in a reciprocal fashion. Duplication of a DNA sequence is yet another common mechanism changing the structure of gene leading to gene mutation.

Fig. 16.2

(a) Transition and transversion. Transitions are interchanges of pyrimidine (C T) or purine (A G) bases. Transversions are interchanges of pyrimidine for purine bases or vice versa (b) Frameshift mutation: This type of mutation occurs when the addition or loss of DNA bases changes a gene’s reading frame. A reading frame consists of groups of three bases that each codes for one amino acid. A frameshift mutation shifts the grouping of these bases and changes the code for amino acids. The resulting protein is usually non-functional. Insertions, deletions and duplications can all be frameshift mutations

1.6 Practical Considerations

The dose of a mutagen that ensures optimum mutation frequency with minimum unintended damage is regarded as the optimal dose. In case of physical mutagens, tests of radiosensitivity (from radiation sensitivity) give estimates. It gives an indication of the quantity of recognizable effects of radiation exposure. Since it is a predictive value, it gives guidance on the choice of optimal exposure dosage.

Important factors influencing the outcome of chemical mutagenesis are:

• (a) Inherent traits of tissue

• (b) Environment

• (c) Concentration of mutagen

• (d) Treatment volume

• (e) Treatment duration

• (f) Temperature

• (g) Presoaking of seeds

• (h) pH (7.0)

• (i) Catalytic agents (Cu²⁺ and Zn²⁺)

• (j) Post-treatment handling

Factors influencing the outcome of physical mutagenesis are:

• (a) Oxygen

• (b) Moisture content

• (c) Temperature

• (d) Physical ionizing agents (electromagnetic [EM] and ionizing radiation)

• (e) Dust and fibres (e.g. from asbestos)

• (f) Biological and infectious agents (both viral and bacterial)

In general, the steps differ for sexually and asexually propagated crops, but common principles also exist.

The common practical considerations are:

• (a) Thorough understanding of the genetic makeup of the traits to be improved. Polygenic traits have lesser chances of inducing mutations than monogenic traits.

• (b) Knowledge of reproduction – sexual or asexual. For asexually propagated species, it is to be either in vitro or in vivo. If it is sexually propagated, type of fertilization (self or cross) is to be used.

• (c) Determination of the material that is to be used for the propagation prior to treatment, i.e. gametes or seeds for sexually propagated crops; and stem cuttings, buds, nodal segments or twigs for asexually propagated ones.

• (d) Knowledge of the karyotype, especially when there are hybridization barriers.

• (e) Selection of an appropriate mutagen and dose. A pilot assay is advisable before large-scale treatment of propagules. Radiation dose is expressed in rads (radiation-absorbed dose) which is equivalent to absorption of 100 ergs/g (rad is a unit of absorbed radiation dose, defined as 1 rad = 0.01 Gy = 0.01 joule/kg). The unit kilorad (kR which is 1000 rads) which was in use earlier is replaced by gray units (Gy). These two can be interconverted as 1 kR is equivalent to 10 Gy. A concept of LD₅₀ (lethal dose 50%) is used to refer the optimum dose to be used in the experiment. By definition LD₅₀ is the dose which causes 50% lethality in the organism used for irradiation in definite time. Generally, irradiated populations are generated by using an LD₅₀ dose treatment and with a dose lower than LD₅₀. It can be determined by exposing different subsamples of the target plant material (seeds) to a range of doses (low to high) and monitoring survival of the plants in field (up to flowering or maturity). There are species sensitive to radiation. In such cases, doses lower than LD₅₀ are also appreciable to reduce mutation load. Therefore, it is preferred to work out radiosensitivity test between LD₂₅ or LD₃₀ and LD₅₀ to obtain desired mutation.

• (f) Appropriate infrastructure (irradiation house, laboratories, screen house, fields, etc.) desired mutant selection.

• (g) Isolation of chimaeras from stable mutants.

1.7 Mutation Breeding Strategy

The advantage of mutation breeding over other breeding strategies depends on efficient selection of superior variants in the second (M₂) or third (M₃) generation as summarized in Fig. 16.3. The generation nomenclature starts with M₀ for seed or pollen mutagenesis and M₀ V₀ for vegetative organs, where M stands for the meiotic and V for the vegetative generation. All materials are labelled with a “0” prior to mutagenesis and with a “1” after mutagenesis is performed. The first generation is unsuitable for evaluation as plants will be genotypically heterogeneous (chimaeric). The first generation suitable for selection in a seed-mutagenized material is M₂. Several cycles are needed to make a vegetatively propagated material genotypically homogeneous and to stabilize the inheritance of mutant alleles.

Fig. 16.3

Steps in mutation breeding. Traditional mutation breeding scheme. Each row describes the steps for a specific generation

The first step in mutation breeding is to reduce the number of potential variants among the mutagenized seeds or other propagules of the first (M₁) plant generation to a significant level to allow close evaluation and analysis. The population size needs to be effectively managed. Population size depends on the inheritance pattern of the target gene. Hence, it is advisable to select mutagens that yield high frequency of mutations in order to reduce the population size of M₁. Genetically, M₁ mutant plants are heterozygous because only one allele is affected by one mutation. Probability of mutating both the alleles is extremely low. In M₁, dominant mutations can be identified as recessive mutation where expression is impossible. Screening for mutations in subsequent generations among segregants is the advisable option. In this way, breeder generates homozygotes for dominant or recessive alleles. M₁ population must be self-pollinated as cross-pollination that will produce new variation. Screening and selection starts in M₂ generation. Three main types of screening/selection techniques are: physical/mechanical, visual/phenotypic and “other” methods. Physical or mechanical selection can be used efficiently to determine the shape, size, weight, density of seeds, etc. using appropriate sieving machinery. Visual screening is the most effective and efficient method for identifying mutant phenotypes. Visual/phenotypic selection is often used in selection for plant height, adaptation to soil, growing period, disease resistance, colour changes, earliness in maturity, climate adaptation, etc. In the category of “others”, physiological, biochemical, chemical and physicochemical procedures for screening may be used for selection of certain types of mutants. When a mutant line appears to possess a promising trait, the next stage is seed multiplication for extensive field trials. In this case, the mutant line, the mother cultivar and other varieties (local check) will be tested.

1.8 In Vitro Mutagenesis

In vitro mutagenesis is induction of mutations in explants or in vitro cultures (protoplasts, cells, tissues and organs). This is applicable to both seed-propagated and vegetatively propagated crops. In the latter, it is advantageous where a large number of uniformly growing in vitro cultures can be used. Cultured cells, organs and tissues have a developmental pattern; therefore those can be synchronized and separation of chimaeras can be done more efficiently. For seed crops, the use of haploid culture may provide additional benefits. In vitro mutagenesis involves the following steps:

• (a) Selection of proper target material (explants or cultures)

• (b) Mutagen selection, determination of proper dose and post-treatment handling and subcultures

• (c) Regeneration of plants for mutant selection

A variety of explants are available like apical meristems, axillary buds, roots and tubers. Subcultures will determine chimaeras. In the first vegetative generation (M₁V₁), mutations are not expressive. If superior mutants are detected early, these should be monitored for stability in further generations i.e. up to M₁V₄ or M₁V₆. In banana, using recurrent irradiation in vitro, increased in vitro shoot multiplication and morphological variations were observed. Resistant plants to black sigatoka were derived through carbon ion beam irradiation of in vitro plantlets of banana (cv. Williams and Cavendish Enano).

Chimaeras can be easily isolated in in vitro culture by repetitive subculturing, normally involving about four generations (M₁V₄). In seed crops, backcross to the original line can exclude unwanted mutant genes (see Table 16.3 for details). It is feasible to exercise selection of agronomically useful and genetically determined traits in in vitro culture. Usage of culture medium added with a certain amount of herbicide, salt or aluminium or exposure of cultures to physical stress such as cold or heat can be exercised. This is to select cells/tissues with required tolerance or resistance. Such cells/tissues can be isolated, multiplied through subcultures and regenerated into plants. In vitro cultured explants provide a wider choice of controlled selection where large populations can be screened as against lower number of individuals in the case of in vivo plants.

Table 16.3 In vitro mutagenesis in vegetatively propagated crops

1.9 Gamma Gardens or Atomic Gardens

This is a form of mutagenesis where plants are exposed to radioactive sources (cobalt-60) in order to generate mutations, some of which turned out to be useful. This resulted in the development of over 2000 new varieties of plants, most of which are now used in agricultural production. The “Todd’s Mitcham” peppermint variety, resistant to verticillium wilt, produced by Brookhaven National laboratory, USA, during 1950s, is one of the first examples of variety produced by a gamma garden. The Rio Star grapefruit, developed at the Texas A&M Citrus Center in the 1970s, now accounts for over three quarters of the grapefruit produced in Texas is yet another example. After World War II, there was a concerted effort to find peaceful uses of atomic energy. One of the ideas was to subject plants to irradiation to produce mutations in plenty, through which disease - or cold-resistant or unusual coloured varieties can be derived. Such experiments were conducted in giant gamma gardens of the USA, Europe and the former USSR. Though modern genetic engineering replaced the need for atomic gardening, still the legacy being continued by the Institute of Radiation Breeding in Japan that currently owns the largest and possibly the only surviving gamma garden in the world, at Hitachiōmiya in Ibaraki Prefecture (Fig. 16.4a). The circular garden measures 100 m in radius and enclosed by an 8-m high-shielding dike wall. Radiation (gamma rays) comes from a cobalt-60 source placed inside a central pole. The aim is to produce traits responsible for tolerance to fungus or consumer-friendly fruit colours. Overall development of new crop varieties with new traits is the purpose. In the words of nanotechnologist Paige Johnson of the University of Tulsa, Oklahoma, “if you think of genetic modification today as slicing the genome with a scalpel, in the 1960s they were hitting it with a hammer”.

Fig. 16.4

(a) Aerial view of the gamma garden at the Institute of Radiation Breeding, Hitachiōmiya, Ibaraki Prefecture, Japan. (b) Layout of a gamma garden

These gardens were designed to test effects of radiation on plant life. However, research gradually turned towards inducing beneficial mutations. They were typically five acres in size and were arranged in a circular pattern with a retractable radiation source in the middle (Fig. 16.4b). Plants were usually laid out like slices of a pie, stemming from the central radiation source. Radioactive bombardment will be usually for about 20 h, after which scientists wearing protective equipment would enter the garden to assess results. The plants nearest to the centre usually died, while the ones further out often featured tumours and other growth abnormalities. Plants beyond these were with a higher than usual range of mutations. These gamma gardens have continued to operate in the 1950s. Research into the potential benefits of atomic gardening has continued, most notably through a joint operation between the International Atomic Energy Agency (IAEA) and the UN’s Food and Agriculture Organization (FAO). Japan’s Institute of Radiation Breeding is well known for its modern-day usage of atomic gardening techniques.

2 Factors Affecting Radiation Effects

Ionizing radiation is energetic and penetrating, and its chemical effects in biological matter are due to initial physical energy deposition events, referred to as the track structure. Ionizing radiation exists in either particulate or electromagnetic types. The particulate radiation interacts with the biological tissue either by ionization or excitation. The ionizations and excitations tend to be localized, along the tracks of individual charged particles. While the photon penetrates the matter without interactions, it can be completely absorbed by depositing its energy or it can be scattered (deflected) from its original direction and deposit part of its energy as:

• (a) Photoelectric interaction: a photon transfers all its energy to an electron positioned usually in the outer shell of the atom. The electron ejects from the atom and begins to pass through surrounding matter.

• (b) Compton scattering: a portion of the photon energy is absorbed and the photon is scattered with reduced energy.

• (c) Pair production: the photon interacts with the nucleus and an electron and a positively charged positron is produced. This only occurs with photons with energies in excess of 1.02 MeV.

2.1 Direct and Indirect Effects

Radiation damage causes damage to DNA molecules either directly or indirectly. In the direct action, radiation disrupts the molecular structure. This structural change either damages or kills the cell. Later, surviving damaged cells may have abnormalities. This process becomes predominant with high-LET radiations such as particles and neutrons and high radiation doses. In the indirect action, the radiation hits the water molecules, the major constituent of the cell and other organic molecules in the cell, whereby free radicals such as hydroxyl (HO) and alkoxy (R-O) are produced. Exposure of cells to ionizing radiation induces high-energy radiolysis of H₂O molecules into H⁺ and OH⁻ radicals. Such radicals are chemically reactive and in turn recombine to produce superoxide (HO₂) and peroxide (H₂O₂) that incur oxidative damage to molecules of the cell.

Free radicals are characterized by an unpaired electron and causes molecular structural damage to the DNA. Hydrogen peroxide is also toxic to the DNA. The result of indirect action on the cell is impairment of function or death. Number of free radicals produced by ionizing radiation depends on the total dose. Majority of radiation-induced damage is by indirect action since water constitutes nearly 70% of the composition. In addition to the damages caused by water radiolysis products, cellular damage may also involve reactive nitrogen species (RNS) and other species. This can occur as a result of ionization of atoms on constitutive key molecules (e.g. DNA). Either direct or indirect, the ultimate effect is the biological and physiological alterations. This may be manifested seconds or decades later. In the evolution of these alterations, genetic and epigenetic changes may be involved (Fig. 16.5).

Fig. 16.5

Physical, biochemical and biological response mechanisms of radiation

2.2 Biological Effects

Biological effects are ionization of atoms of biomolecules that may cause chemical changes or eradicate its functions. The energy transmitted may act directly causing ionization of the biological molecule or indirectly act through ionization of the water molecules that surround the cell (Fig. 16.6). Due to this, proteins can lose the functionality of its amino groups and thus increasing its chemical responsiveness. Enzymes would be deactivated and lipids will suffer peroxidation. Carbohydrates will get dissociated and nucleic acid chains will have ruptures/modifications. By all means, DNA is the primary target of radiation as it contains genes with information of cell functioning and reproduction. The energy deposition is a random process. Even low doses can deposit enough energy to result in cellular changes or cell death. But cells can recuperate from this damage. If the repair of DNA damage is incomplete, signalling pathways leading to cell death through apoptosis (death of cells as a normal and controlled part of an organism’s growth or development) can happen. If mutation occurs, the cell will survive with modification in the DNA sequence. Mutated cells are capable of reproduction.

Fig. 16.6

Direct and indirect actions of radiation

3 Molecular Mutation Breeding

Cells with damaged DNA will survive only when these damages are repaired correctly or erroneously. The result of erroneous repairs will be fixed in the genome as induced mutations. The nature and extent of DNA damage determines the molecular feature of induced mutations. For example, EMS often leads to G/C to A/T transition, while ion beam could cause deletion of DNA fragment of various sizes. While nucleotide substitution may produce a dominant allele, DNA deletions will cause recessive mutations. So, when a recessive mutation is required, irradiation may be preferred. When we need herbicide resistance (dominant mutation), the use of chemical mutagen is preferred.

Mutagenesis research has been revolutionized by advances in genomics including methods to detect genetic variation and select mutant phenotypes like:

• (a) Transposon mutagenesis or transposition mutagenesis (a process that allows genes to be transferred to a host’s chromosome)

• (b) Insertional mutagenesis (creation of mutations of DNA by the addition of one or more base pairs. This can occur naturally or mediated by viruses or transposons)

• (c) TILLING (targeting induced local lesions in genomes), ecoTILLING (ecotype TILLING) and high-resolution melting (HRM)

• (d) Site-directed mutagenesis

  Of these, the last two will be dealt here in some detail, since the first two are largely done in microorganisms.

3.1 TILLING and EcoTILLING

TILLING is a method that allows directed identification of mutations in a specific gene. TILLING was first done in Arabidopsis thaliana and thereafter successfully used in corn, wheat, soybean, tomato and lettuce. TILLING relies on the ability of a special enzyme to detect mismatches in normal and mutant DNA strands when they are annealed. Seed is treated with either ethyl methanesulphonate (EMS) or sodium azide to generate a population of plants with random point mutations. By selectively pooling the DNA and amplifying with unlabelled primers, mismatched heteroduplexes are generated between wild-type and mutant DNA. Heteroduplexes are incubated with the plant endonuclease CEL I (that cleaves heteroduplex mismatched sites), and the resultant products are visualized on a Fragment Analyzer. Subsequent analysis of the individual plant DNA from the pool DNA identified the plant bearing the mutation. There are 10 steps in TILLING (Fig. 16.7). This is a high-throughput process to identify single-nucleotide mutations in a gene of interest. This is also a powerful detection method that can result from chemical-induced mutagenesis. TILLING was first used by Claire McCallum in the late 1990s in Arabidopsis.

Fig. 16.7

Steps in TILLING (figures are only representative)

Outline of the basic steps for typical TILLING and EcoTILLING assays:

• (a) Seeds are mutagenized with chemical mutagens. The resulting M₁ plants are self-fertilized.

• (b) DNA samples are prepared from M₂ individuals for mutational screening. DNA is collected from a mutagenized population (TILLING) or a natural population (EcoTILLING).

• (c) For TILLING, DNAs are pooled. Typical EcoTILLING assays do not use sample pooling, but pooling has been used to discover rare natural single-nucleotide changes.

• (d) After extraction and pooling, samples are typically arrayed into a 96-well format.

• (e) The target region is amplified by PCR with gene-specific primers that are end-labelled with fluorescent dyes.

• (f) Following PCR, samples are denatured and annealed to form heteroduplexes that become the substrate for enzymatic mismatch cleavage. Cleavage at mismatched site done by enzyme CEL I.

• (g) Cleaved bands representing mutations or polymorphisms are visualized using denaturing polyacrylamide gel electrophoresis.

EcoTILLING uses TILLING techniques to look for natural mutations. DEcoTILLING is an altered method of TILLING and EcoTILLING to identify fragments. After NGS sequencing technologies were discovered, TILLING by sequencing has been developed based on Illumina sequencing of target genes amplified from multidimensionality pooled templates to identify possible single-nucleotide changes (see Chap. 24 on Genomics for details).

3.2 Site-Directed Mutagenesis

Site-directed mutagenesis makes specific and intentional changes to the DNA. This is otherwise known as oligonucleotide-directed mutagenesis and is used for investigating the structure of DNA, RNA and protein molecules and for protein engineering. The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is also complementary to the template DNA around the mutation site, so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (point mutation), multiple base changes, deletion or insertion. DNA polymerase is used to extend the single-strand primer that copies the rest of the gene sequence. The gene thus copied contains the mutated site and is then introduced into a host cell as a vector and cloned. DNA sequencing is undertaken to select the desired mutation.

The aforesaid method using single-strand primer extension was inefficient due to a low yield of mutants.

Some of the modified methods for site-directed mutagenesis are:

• (a) Kunkel’s method: This was introduced by Thomas Kunkel in 1985. Here, the DNA fragment to be mutated is inserted into a phagemid (DNA-based cloning vector) and is then transformed into an E. coli strain deficient in two enzymes, dUTPase (dut) and uracil deglycosidase (udg). Both enzymes are part of a DNA repair pathway that protects the bacterial chromosome from mutations by the spontaneous deamination of dCTP to dUTP. The dUTPase deficiency prevents the breakdown of dUTP, resulting in a high level of dUTP in the cell. The uracil deglycosidase deficiency prevents the removal of uracil from newly synthesized DNA. As the double mutant E. coli replicates the phage DNA, its enzymatic machinery may, therefore, mis-incorporate dUTP instead of dTTP, resulting in single-strand DNA that contains some uracils (ssUDNA). The ssUDNA thus produced is extracted from the bacteriophage that is released into the medium and then used as template for mutagenesis. An oligonucleotide containing the desired mutation is used for primer extension. The heteroduplex DNA thus formed consists of one parental non-mutated strand containing dUTP and a mutated strand containing dTTP. The DNA is then transformed into an E. coli strain carrying the wild-type dut and udg genes. Here, the uracil-containing parental DNA strand is degraded, so that nearly all of the resulting DNA consists of the mutated strand.

• (b) Cassette mutagenesis: Cassette mutagenesis need not involve primer extension using DNA polymerase. Here, a fragment of DNA is synthesized and then inserted into a plasmid. It involves the cleavage by a restriction enzyme at a site in the plasmid and subsequent ligation of a pair of complementary oligonucleotides containing the mutation in the gene of interest to the plasmid. Usually, the restriction enzymes cut the plasmid permitting sticky ends of the plasmid and insert to ligate to one another. This method can generate mutants at close to 100% efficiency. The drawback with this method is that it will allow mutations only at sites that can be cleaved by the restriction enzymes.

• (c) PCR site-directed mutagenesis: Cassette mutagenesis mutates restriction sites only. This may be overcome by using polymerase chain reaction with oligonucleotide primers so that a larger fragment may be generated, covering two convenient restriction sites. The fragment containing the desired mutation can be separated from the original by gel electrophoresis. Variations employ three or four oligonucleotides, two of which may be non-mutagenic oligonucleotides that cover two convenient restriction sites and generate a fragment that can be digested and ligated into a plasmid, whereas the mutagenic oligonucleotide may be complementary to a location within that fragment well away from any convenient restriction site. These methods require multiple steps of PCR so that the final fragment to be ligated can contain the desired mutation. The design process for generating a fragment with the desired mutation and relevant restriction sites can be cumbersome. Software tools like SDM-Assist can simplify the process.

3.3 MutMap

MutMap is a method of rapid gene isolation using a cross of a mutant to wild-type parental line. The large F₂ population will be screened to isolate mutant through SNP (single-nucleotide polymorphism) analysis (Fig. 16.8). This technique applied in rice can be explained as follows:

Fig. 16.8

A scheme for MutMap in rice. A rice cultivar with a reference genome sequence is mutagenized by EMS. A semi-dwarf phenotype mutant is crossed to the wild-type plant of the same cultivar used for the mutagenesis. F₂ is raised from F₁ to have both mutant and wild-type phenotypes. Crossing of the mutant to the wild-type parental line ensures detection of phenotypic differences at the F₂ generation between the mutant and wild type. DNA of F₂ displaying the mutant phenotype are bulked and subjected to whole-genome sequencing followed by alignment to the reference sequence. SNPs with sequence reads composed only of mutant sequences (SNP index of 1) are closely linked to the causal SNP for the mutant phenotype (courtesy: Nature Biotechnology)

Use a mutagen (say EMS) to mutagenize a rice cultivar (X) that has a reference genome sequence. To make the mutated gene homozygous, plants of first mutant generation (M₁) are self-pollinated to raise M₂ and further generations. Phenotypes in the M₂ and advanced generations are screened to isolate recessive mutants with altered traits like plant height, tiller number and grain number per spike. This mutant is crossed with the cultivar used for inducing mutations (wild type). The resulting F₁ is self-pollinated, and the F₂ are grown in the field for scoring the phenotype. Since F₂ progeny are derived from a cross between the mutant and its parental wild-type plant, the number of segregating loci responsible for the phenotypic change is minimal (in most cases, one). But the segregation of phenotypes in F₂ shall be prominent even if the phenotypic differences are small. It is appropriate to use SNPs to identify nucleotide changes incorporated into the mutant. They are detected as insertion-deletions (indels) between mutant and wild type. In the F₂ progeny, the majority of SNPs will segregate in a 1:1 mutant/wild type ratio. However, the SNP responsible for the change of phenotype is homozygous in the progeny showing the mutant phenotype. When DNA samples are collected from recessive mutant of F₂ progeny, and bulk sequenced, 50% mutant and 50% wild-type sequence reads are expected. However, the causal SNP and closely linked SNPs should show 100% mutant and 0% wild-type reads. On the other hand, SNPs loosely linked to the causal mutation should have >50% mutant and <50% wild-type reads. If SNP index is defined as the ratio between the number of reads of a mutant SNP and the total number of reads corresponding to the SNP, this index would equal 1 near the causal gene and 0.5 for the unlinked loci.

4 The FAO/IAEA Joint Venture for Nuclear Agriculture

Over the last 45 years, the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture (headquartered in Vienna, Austria) supported worldwide countries’ efforts to attain food security. The Plant Breeding and Genetics Section of this programme assists countries in using radiation-induced mutations, facilitated by biotechnologies, to develop superior crop varieties. The mandate of Joint FAO/IAEA Programme is constitution of field projects in developing countries, coordination of collaborative research network and a research and development laboratory arm in Seibersdorf, outside Vienna, Austria. As of now, there are a total of 86 field projects relating to the development of mutants dealing with biotic, abiotic and nutritional aspects (Tables 16.4a, 16.4b and 16.4c) (The information provided is not exhaustive). Through Technical Cooperation Projects (TCP), the technology transfer is accomplished characterized through strengthening of human and infrastructural capabilities. The irradiation facilities (majority are with cobalt-60 sources) are provided through TCP.

Table 16.4a Applications of induced mutagenesis for biotic stress resistance in plant breeding

Table 16.4b Applications of induced mutagenesis for abiotic stress resistance in plant breeding

Table 16.4c Applications of induced mutagenesis in the improvement of crop quality and nutritional traits in plant breeding

As per FAO/IAEA Mutant Varieties Database, more than 3222 mutant varieties are released in different countries. China, India, the former USSR, the Netherlands, Japan and the USA are the leading countries having the highest number mutant varieties. Highest proportion of mutants (>50%) is with gamma rays compared to other mutagens (Table 16.5). Crop wise, cereals stand first followed by ornamentals and legumes (see Table 16.6). Rice stands first (700 mutant varieties) in among crops followed by barley, wheat, maize, durum wheat, oat, millet, sorghum and rye (Table 16.7). As per the FAO/IAEA database, 1825 mutants (accounting to 57%) have either better agronomic or botanical traits. Of these, 577 (18%) mutants are developed for increase in yield and related traits, 321 (10%) mutants for better quality and nutritional content, 200 (6%) mutants for biotic and 125 (4%) mutants for abiotic stress tolerance. These programmes have benefited the local economies through contributing millions of dollars annually.

Table 16.5 Number of officially released mutant varieties

Table 16.6 Number of released mutant varieties in cereals and legumes

Table 16.7 Leading rice varieties obtained by mutation breeding

4.1 Mutation Breeding in Different Countries

Continent wise, Asia stands first in terms of mutant varieties released (Fig. 16.9). China stands first in terms of development of new varieties through induced mutagenesis. It is well ahead of other countries in number of released varieties (Fig. 16.10). Crop wise, cereals own the maximum percentage of varieties released (48%) (Fig. 16.11).

Fig. 16.9

Number and proportion of mutant cultivars released, categorized by continents (source: IAEA mutant Database)

Fig. 16.10

Number of mutant cultivars released in different countries (source: FAO)

Fig. 16.11

Mutants released in various crops

Japan used irradiation, chemical mutagenesis and somaclonal variation to release 242 mutant varieties. Due to successful efforts of Institute of Radiation Breeding, 61% of these varieties were induced by gamma rays. Some mutant cultivars of Japanese pear exhibit resistance to diseases. In addition, 228 indirect use (hybrid) mutant varieties primarily generated in rice and soybean have found value as parental breeding germplasm resources in Japan. In 2005, the total cultivated area of mutant rice cultivars was 2,10,692 ha (12.4% of the total cultivated rice area). Income from mutant cultivars was estimated to be nearly 250 billion Yen (2.34 billion US dollars) in 2005.

India initiated sustained efforts to use induced mutations in the late 1950s. Between 1950 and 2009, India developed about 329 mutant varieties in rice, wheat, barley, pearl millet, jute, groundnut, soybean, chickpea, mung bean, cowpea, black gram, sugarcane, chrysanthemum, tobacco and dahlia. Indian Agricultural Research Institute (IARI), Bhabha Atomic Research Centre, Tamil Nadu Agricultural University and the National Botanical Research Institute were the prime institutions involved. Several gamma-irradiated rice mutants were released in India as high-yielding varieties under the series “PNR”. Two early ripening and aromatic rice varieties, “PNR 381” and “PNR 102”, are currently popular with farmers in the states of Haryana and Uttar Pradesh.

Wide use of high-yielding varieties made Vietnam the second largest exporter of rice, exporting 4.3 million tons per year. Currently, mutant varieties contribute to 15% of the annual rice production. Around 55 mutant varieties have been developed, most of which are rice. Mutant rice are planted in over 1.0 million ha, including Hatay, Bacgiang, Nghean, Vinhphuc, Hanam, Thaibinh and Hanoi of northern Vietnam, which led to poverty relief. Besides higher yield, varieties with aroma, protein and amylase content were also derived. Tolerance to salinity, cold, drought and lodging was given prime importance. Nearly 2,540,000 ha are cultivated with mutant varieties of crops with a return of 374.4 million USD.

In Thailand, the work on induced mutations in rice commenced in 1965 and was stimulated in cooperation with IAEA. Two aromatic indica-type varieties of rice, “RD6” and “RD15”, which were developed by gamma irradiation of a popular rice variety, “KhaoDawk Mali 105” (“KDML 105”) and were released in 1977 and 1978, respectively. Even after 40 years, these varieties are still popular. RD6 has glutinous endosperm and retains all of the grain characters, including the aroma of its parent variety. In contrast, RD15 is non-glutinous and aromatic, similar to the parent, but ripens 10 days earlier than the parent. According to the Bureau of Economic and Agricultural Statistics of Bangkok, during 1997–1998, RD6 was grown on 2,524,576 ha, covering 32.1% of the area under rice that produced 4,599,995 tons paddy.

In Bangladesh, more than 44 mutant varieties belonging to 12 different crop species have been released through mutation breeding. The Bangladesh Institute of Nuclear Agriculture in Mymensingh is the prime institution for mutation breeding that released up to eight mutant rice varieties. Rice mutants, including Binasail, Iratom-24 and Binadhan-6, were all planted in a cumulative area of 795,000 ha and contributed substantially towards food security.

USA produced a semi-dwarf gene allele (sd1) in rice through gamma ray mutagenesis. This triggered the American version of the “Green Revolution” in rice. Stadler, a high-yielding wheat mutant, is another success story. Stadler is resistant to leaf rust and loose smut with lodging resistance. Luther, a barley mutant, had 20% increased yield, shorter straw, higher tillering and better lodging resistance. Luther was grown in 120,000 acres with an estimated return of 1.1 million US dollars per year. It was used extensively in crossbreeding and several mutants were released. Pennrad is yet another high-yielding winter barley mutant with winter hardiness, early ripening and better lodging resistance grown in 100,000 ha in the USA. The grapefruit varieties, Star Ruby and Rio Red, developed through thermal neutron mutagenesis are sold under the trademark “Rio Star”.

In Pakistan, at the Nuclear Institute for Agriculture and Biology, crops selected for improvement include rice, chickpea, mungbean and cotton. Improvement has been sought in plant architecture, maturity period, disease resistance, etc. The primary triumph of the Nuclear Institute of Agriculture is the release of four improved varieties of rice that were obtained using induced mutagenesis (Table 16.7).

European countries have been active in mutation breeding programmes. Bulgaria released 76 new cultivars produced from induced mutagenesis of which maize has the largest number of varieties (26 varieties). Kneja 509, a maize hybrid, occupies up to 50% of the growing area. In other European countries, development of short height and high-yielding mutant cultivars of barley ‘Golden Promise’ and ‘Diamant’ have made a major impact on the brewing industry. These have also been used as parents for many leading barley cultivars across Europe, North America and Asia. Golden Promise (developed through gamma ray irradiation of malting cultivar ‘Maythorpe’) was released in Czechoslovakia in 1965 through gamma ray irradiation of ‘Valticky’. ‘Diamant’ has 12% increased yield, 15 cm shorter in height, occupying 43% of the barley area. Golden Promise is popular in Ireland, Scotland and the UK for brewing. These mutants are part of the commitment of the Joint FAO/IAEA programme for global food security. Mutation breeding-derived crop varieties around the world demonstrate the potential as a flexible and practicable approach to have desirable crop varieties. There are several host institutions all over the world to conserve mutant stocks (see Table 16.8). Few of the crop varieties released through classical mutagenesis since 2010 is available in Table 16.9.

Table 16.8 Some characterized mutant stocks of crops and the host institutions

Table 16.9 Few crop varieties released through classical mutagenesis since 2010

5 Polyploidy Breeding

Polyploids are organisms with multiple sets of chromosomes in excess of the diploid number. Polyploidy is a natural mechanism that provides adaptation and speciation. Among angiosperms, 50% to 70% of the species have undergone polyploidy during the course of evolution. Flowering plants form polyploids at a significantly high frequency of 1 in every 100,000 plants. To understand polyploidy, a few basic notations need be defined. The total number of chromosomes in a somatic cell is designated “2n”. The total number of chromosomes in a somatic cell is twice the haploid number (n) in the gametes (see Fig. 16.12). There may be more polyploid species in a given genera. The haploid chromosome number of diploid species of a polyploidy series is known as the basic chromosome number (x). For example, in wheat, we have tetraploid and hexaploid wheat (see Fig. 16.13). The ploidy of some of the major crops in the world is represented in Table 16.10.

Fig. 16.12

Different kinds of changes in chromosomes (x = basic chromosome number; 2n = somatic chromosome number)

Fig. 16.13

Derivation of bread wheat

Table 16.10 Examples of polyploid crops (somatic chromosome number is in brackets)

5.1 Types of Changes in Chromosome Number

Polyploids are classified as euploids or aneuploids based on their chromosomal composition. Euploids are in majority that are multiples of the complete set of chromosomes specific to a species. Based on composition of genome, euploids are either autopolyploids or allopolyploids. A common class of euploids are tetraploids (see Table 16.11).

Table 16.11 Common types of changes in chromosome number

Autopolyploidy

Autopolyploids are otherwise called autoploids. They are with multiple sets of basic set (x) of chromosomes of the same genome. In nature, autoploids result from union of unreduced gametes or can be induced artificially. Natural autoploids include tetraploid crops like alfafa, peanut, potato and coffee and triploid bananas. Such species occur spontaneously through chromosome doubling. In ornamentals and forages, chromosome doubling led to increased vigour. Induced autotetraploids in watermelon are utilized for producing seedless triploid hybrids. This is accomplished through treating diploids with mitotic inhibitors like dinitroanilines and colchicine. Apart from chromosome counts, ploidy status of induced polyploids can be determined through chloroplast count in guard cells; morphological features such as leaf, flower or pollen size (gigas effect) and flow cytometry.

Allopolyploidy

They are also called alloploids. Alloploids are a combination of genomes of different species. Hybridization of two or more genomes followed by chromosome doubling or fusion of unreduced gametes leads to such phenomena. This process occurs in nature as a key process of speciation in angiosperms and ferns. Economically important natural alloploids are strawberry, wheat, oat, upland cotton, oilseed rape, blueberry and mustard. Each genome is designated by a different letter to differentiate between the sources of the genomes in an alloploid. The cultivated mustards (Brassica spp.) can be explained in a triangle with each genome represented by a letter (Fig. 16.14a). The degree of homology between genomes differs with some being able to undergo chromosome pairing. The phenomenon becomes segmental alloploidy when only segments of chromosomes of the combining genomes differ. These chromosomes are not homologous but are homoeologous chromosomes. Homoeologous chromosomes indicate ancestral homology. Induced alloploidy is rare. Through hybridization and chromosome doubling, allotetraploid was induced in Cucumis sativus x Cucumis hystrix cross. This was done to explain the molecular mechanisms involved in diploidization (tendency of polyploids to act as diploids). Cytogenetic analysis carried out in advanced generations established molecular mechanisms involved in stabilization of newly formed allopolyploids.

Fig. 16.14

(a) Triangle showing origin of cultivated mustard. (b) Origin of amphidiploid (Raphanobrassica) formed from cabbage (Brassica) and radish (Raphanus). The fertile amphidiploid arose in this case from spontaneous doubling in the 2n = 18 sterile hybrid

A prototypic allopolyploid (allotetraploid) was synthesized by G. Karpechenko in 1928. He expected a fertile hybrid with leaves of cabbage (Brassica) and roots of radish (Raphanus). Both these species are with 18 chromosomes, and they allow intercrossing. Hybrid progeny was produced, but this hybrid was functionally sterile because chromosomes of cabbage and radish were not homologous. However, one part of the hybrid plant produced some seeds. On planting, these seeds produced fertile individuals with 36 chromosomes but were allopolyploids. They had apparently been derived from spontaneous, accidental chromosome doubling to 2n₁ + 2n₂ in one region of the sterile hybrid which underwent normal meiosis. Thus, in 2n₁ + 2n₂ tissue, there is a pairing partner for each chromosome, and balanced gametes of the type n₁ + n₂ are produced. These gametes fuse to give 2n₁ + 2n₂ allopolyploid progeny, which also are fertile. This kind of allopolyploid is sometimes called an amphidiploid. Unfortunately for Karpechenko, amphidiploid he made had roots of cabbage and the leaves of radish. He called this Raphanobrassica (Fig. 16.14b). Treating a sterile hybrid with colchicine doubles chromosomes thus make them fertile. Allopolyploidy is a major force of speciation.

Aneuploidy

Aneuploids contain either an addition or subtraction of one or more specific chromosome(s). Univalent and/or multivalent formation arises during meiosis. A range of 30–40% of the progeny derived from autotetraploid maize are aneuploids. Univalents arise because of unequal distribution of chromosomes during anaphase I. Similarly, multivalents are formed due to non-separation of homologous chromosomes during meiosis that leads to unequal migration of chromosomes to opposite poles. This process is called non-disjunction. Such aneuploids are with reduced vigour. Depending on the number of chromosomes gained or lost, aneuploids are classified as monosomy (2n-1), nullisomy (2n-2), trisomy (2n + 1), tetrasomy (2n + 2) and pentasomy (2n + 3).

5.2 Methods for Inducing Polyploidy

Colchicine first isolated in 1820 by the French chemists P. S. Pelletier and J. B. Caventou inhibits the formation of spindle fibres that temporarily arrests chromosomes at the anaphase stage. Colchicine is extracted from autumn crocus (Colchicum autumnale). Chromosomes have replicated during anaphase, but in the absence of cell division, polyploid cells are formed. Other mitotic inhibitors, namely, dinitroanilines, oryzalin, trifluralin, amiprophos-methyl and N₂O gas, have also been identified and used as chromosome doubling agents. Seedlings with actively growing meristems are seen to be the best material to induce polyploidy. Seedlings or apical meristems can be soaked in colchicine solution. Older shoots when treated lead to cytochimaeras. Chemical solutions can be applied to buds using cotton, agar or lanolin or by dipping branch tips into a solution for a few hours or days. The efficacy can be increased by using surfactants, wetting agents and other carriers (dimethyl sulphoxide). Polyploidy in low frequencies can be induced by the use of heat or cold treatment, X-ray or gamma ray irradiation. Exposure of maize plants or ears to high temperature (38–45 °C) at the time of first zygotic division produces 2–5% tetraploid progeny. Similar heat treatments are used in barley, wheat and rye to induce polyploidy.

Spontaneous induction of polyploidy in plants happens by several cytological means. Non-reduction of gametes during meiosis is one such way which is known as meiotic nuclear restitution. Such gametes are with 2n chromosomes like somatic cells. This could be due to aberrations related to spindle formation and abnormal cytokinesis. The union of non-reduced gametes form polyploids. This happens in open-pollinated diploid apples. In interspecific crosses between Digitalis ambigua and Digitalis purpurea, 90% of F₂ progenies show spontaneous allotetraploids. Autohexaploid Beta vulgaris (sugar beet) is another example. Alfalfa from cultivated autotetraploid varieties apparently are from the union of reduced (2x) and unreduced (4x) gametes. Polyspermy is another mechanism seen in orchids where one egg is fertilized by several male nuclei. The major pathways involved in polyploidy formation are represented in Fig. 16.15.

Fig. 16.15

Major pathways in the formation of polyploids

5.3 Molecular Consequences of Polyploidy

Polyploidy is widespread in flowering angiosperms and is one of the main causes behind the rapid diversification. It is a major route for the creation of new genes through gene duplication and diversification. This contention is still getting debated. Studies on molecular consequences of polyploidization commenced only recently.

Polyploids have a tendency to return to a diploidized state, a process known as diploidization. Diploidization experiences changes in chromosome organization, gene order, expression and epigenetic modification. This may involve abnormal chromosome segregation, rearrangement and breakage (Fig. 16.16a,b). In synthetic allotetraploids between doubled haploid Brassica oleracea (C genome) and Brassica rapa (A genome), abnormal chromosomal segregations led to aneuploidy in the first generation itself, with an aneuploidy rate of 24%. This aneuploidy rate rises to 95% in the 11th generation. This high rate of aneuploidy never reduces the homoelogs. The number of homeologs is maintained at four copies (i.e. the loss of chromosome 1 from the A genome is usually associated with gain of the same chromosome from the C genome, and vice versa). This is a compensating aneuploidy that indicates a dosage balance requirement. As such, the newly generated polyploids display higher rate of genome rearrangements leading to loss of chromosomal fragments (Fig. 16.16a).

Fig. 16.16

Genomic consequences of polyploidy. (a) Some possible scenarios with respect to genomic rearrangements, such as chromosome loss, chromosomal translocation and chromosome fragment loss, have been depicted in a simplified manner using only two chromosomes. P1, parent 1; P2, parent 2. (b) The process of gene loss in a parent-of-origin manner, termed fractionation. In the depicted scenario, the chromosomal copy from P2 loses most of the genes. (c) Proliferation of transposable elements over time. Such proliferation may lead to changes in gene order, gene function and gene expression

Polyploidization initially results in multiplication of gene content. Genome sequencing has thrown light on gene loss in species that were subjected to polyploidization during course of evolution over several million years (Ma). Only 17% of duplicate sequences were retained in A. thaliana after a paleopolyplodization (β) event that took place ~50 Ma. In Glycine max, two rounds of whole-genome duplications took place ~59 and ~13 Ma in the paleopolyploid phase. In the more recent duplication event, 56.6% of duplicates are no longer detectable, compared to 74.1% genes lost after the older Glycine polyploidization. Thus, for the younger and the older duplication events, the rates of gene loss are 4.4% and 1.3% per million years (Myr), respectively. This indicates that the greater rate of gene loss in the initial phases slowed down over time. The loss of polyploidy-derived genes is fractionation. This is a mechanism by which removal of duplicates derived from polyploidization happens (Fig. 16.16b). Also, at the expression level, this phenomenon is reflected. Genes located on one sub-genome show higher expression than indicating genome dominance. Fractionation of genes leads to preferential gene retention. A number of distinguishing characteristics are seen in retained duplicate sequences compared to those single copy sequences. They are biased gene function, higher gene complexity (number of exons and protein domains), increased gene expression and parental genome dominance. The elevated mutation rate in polyploids reflects over increased transposable element activities. The proliferation of transposons in polyploids is due to reduced population size, masked deleterious transposon insertion and/or conflict in transposition repressors due to genome merger (Fig. 16.16c).

5.4 Molecular tools for Exploring Polyploid Genomes

A combination of genetic mapping, molecular cytogenetics, sequence and comparative analysis can shed light on the nature of ploidy evolution, from the base of the plant kingdom to intra- and interspecific hybridization. Some of the techniques that can endeavour such analysis are as follows (see Chap. on Genomics for further details on these techniques):

• (a) In Situ Hybridization: In situ hybridization is a bridge between chromosomal and molecular level of genome investigations. This detects positions of unique sequences and repetitive DNAs along the chromosome(s). Fluorescent in situ hybridization (FISH) is a bit advanced, which detects fluorescent labels linked to DNA probes that can be visualized in a fluorescence microscope. Genomic in situ hybridization (GISH) is yet another advanced tool where total genomic DNA of species is hybridized as a probe on chromosomes. This leads to an analysis of whole genome discrimination rather than localization of specific sequences. There are several examples on the use of these techniques. In newly synthesized allotetraploid genotypes of Brassica napus, extensive genome remodelling due to homeologous pairing between the chromosomes of the A and C genomes were demonstrated. A combined GISH and FISH analysis demonstrated that in natural populations of Tragopogon miscellus, extensive chromosomal variation (mainly due to chromosome substitutions and homeologous rearrangements) was present up to the 40th generation following polyploidization.

• (b) Molecular Marker-Based Genetic Mapping: Genetic mapping in polyploids is complicated compared to diploid species. The need of large populations and use of complicated statistical methods make the process more difficult to obtain reliable genetic distance estimates. A simple way is to use only single-dose markers from each parent, i.e. those segregating 1:1 in the mapping population (e.g. a population obtained from the cross Mmmm × mmmm in a tetraploid species).

• (c) Methylation-Sensitive Molecular Markers: The use of an AFLP-like method using restriction enzymes sharing the same recognition site but having differential sensitivity to DNA methylation (isoschizomers – pairs of restriction enzymes specific to the same recognition sequence) is efficient for the determination of genome-wide DNA methylation patterns. This process otherwise known as methylation-sensitive amplified polymorphism (MSAP) is based on the use of the isoschizomers HpaII and MspI (both recognizing the 5’-CCGG sequence) but affected by the methylation state of the outer or inner cytosine residues. New and acceptable results were derived in newly synthesized polyploids by the use of this technique. In F₄ allotetraploids of Arabidopsis, frequent changes occurred when compared to the parents with increases and decreases in methylation. The change in methylation patterns equally affected both repetitive DNA sequences and low-copy DNAs.

• (d) Comparative Genome Analysis: Comparative genomics addresses several pertinent questions in genome evolution. Several phylogenetic and taxonomic studies revealed ancient polyploidy events and the evolution of novel genes that enabled adaptive processes. Recent genomic research revealed the relevance of polyploidy in angiosperm evolution and also suggested several ancient whole genome duplication (WGD) events. Transposable elements must have played a pivotal role in enhancing functional changes through genome reorganization following allopolyploidization.

• (e) High-Throughput DNA Sequencing: High-throughput DNA sequencing coupled with computational analysis provides answers for the genetic analysis of polyploids. In B. napus, the polyploidy issue was done by sequencing leaf transcriptome across a mapping population. The Wheat Genome Initiative (http://www.wheatgenome.org/) individual or groups of homeologous chromosomes were analysed by flow cytometry separation. While in cultivated wheat gene duplications were predominant, wild wheat was characterized by deletions. Exon capture helped in variant discovery in polyploids that played a crucial role in the origin of new adaptations. SNPs have been utilized in the detection of variation in plant polyploidy. Illumina GoldenGate assay identifies a high number of SNPs in tetraploid and hexaploid wheat. In elite maize inbred lines, more than one million SNPs have been identified in Illumina sequencing platform.