Definition of Key Terms

Why Mutation Breeding?

• Mutation induction rarely produces new alleles; it produces alleles which are already known to occur spontaneously or may be discovered if an extensive search were made.
• Induced mutations are comparable to spontaneous mutations in their effects and in the variability they produce.
• But the induced mutations have a great advantage over the spontaneous ones; they occur at a relatively higher frequency so that it is practical to work with them.'

Mutation breeding is especially valuable in the following situations:

• When a specific useful trait is absent from the entire available germplasm — no natural source exists anywhere in the gene pool.
• When a variety is nearly perfect but needs improvement in a single character — and no donor variety with that character is available for conventional hybridization.
• In clonal (vegetatively propagated) crops — hybridization changes the entire genotype; mutagenesis allows improvement of specific characters within a clone without altering its overall genetic identity.
• When disease resistance sources are unavailable in cultivated species or accessible wild relatives — mutagenesis may generate new resistance alleles.

CLASSIFICATION OF MUTATIONS

Mutations are classified on three separate bases. These classifications are important for understanding which mutations are useful in which types of breeding programmes.

2.1  Classification Based on Genetic Basis — Three Types

Type 1: Gene Mutations (Point Mutations)

• Produced by changes in the base sequence of a gene. The change may involve:
• Base substitution (transitions and transversions): Replacement of one nucleotide base with another.
  • Transitions: purine for purine (A-G) or pyrimidine for pyrimidine (C-T).
  • Transversions: purine for pyrimidine or vice versa.
• Deletion: One or more nucleotide base pairs deleted from the gene. Creates a frameshift if not in multiples of 3.
• Addition (insertion): One or more nucleotide base pairs inserted into the gene. Also creates a frameshift.
• Gene mutations may be:
  • Missense mutations (codon changes to specify a different amino acid);
  • Nonsense mutations (codon changes to a stop codon — premature termination);
  • Frameshift mutations (insertion or deletion shifts the reading frame — usually highly disruptive);
  • Silent mutations (synonymous — codon change codes for the same amino acid due to degeneracy of genetic code).

Type 2: Chromosomal Mutations

• Arise due to changes in chromosome structure or chromosome number.
• Two categories:
  • Structural: Deletions, duplications, inversions, translocations — rearrangements of chromosome segments.
  • Numerical: Aneuploidy (loss or gain of individual chromosomes) and euploidy/polyploidy (change in complete genome sets).

Type 3: Cytoplasmic (Plasmagene) Mutations

• Produced by changes in the base sequence of cytoplasmic genes — genes located in the mitochondria or chloroplasts rather than in the nucleus.
• Location: Mitochondria and chloroplasts both contain their own circular DNA genomes and can undergo mutations independently of the nuclear genome.
• Inheritance: Cytoplasmic mutations are inherited maternally — through the egg cytoplasm, not through pollen. They do not follow Mendelian ratios.
• Expression: The mutant character occurs in buds or somatic tissues which are used for propagation in clonal crops. This means cytoplasmic mutations are most easily recovered in vegetatively propagated plants where the mutant bud or cutting can be multiplied directly.
  • Examples: Cytoplasmic male sterility (CMS) — the primary economically important cytoplasmic mutation; associated with mitochondrial DNA rearrangements. Variegation in certain plant species due to plastid (chloroplast genome) mutations. Streptomycin resistance in Chlamydomonas — a chloroplast mutation.

Classification Based on Origin

A. Spontaneous Mutations

• Spontaneous mutations occur in natural populations without any known external cause.
• They arise from:
  • Errors in DNA replication — polymerase errors, tautomeric shifts in bases
  • Spontaneous depurination or deamination of bases
  • Activity of transposable elements (jumping genes) — Barbara McClintock's discovery in maize
  • Background ionising radiation from natural sources (cosmic rays, radioactive elements in soil)

The general spontaneous mutation rate is approximately 1 in 10^6 (1 in one million) per gene per generation 

B. Induced Mutations

• Induced mutations are produced artificially by treating biological material with mutagenic agents (mutagens).
• Mutation induction rarely produces new alleles; it produces alleles which are already known to occur spontaneously or may be discovered if an extensive search were made.'This means induced mutagenesis does not create entirely unprecedented genetic changes — it accelerates the occurrence of changes that could theoretically occur spontaneously.

Classification Based on Magnitude of Phenotypic Effect

Macromutations (Oligogenic Mutations)

• Large phenotypic effects recognisable on an individual plant basis. Can be seen easily in M2 generation. Selection begins in M2.
• Genetic basis: Single major gene (oligogene) mutated.
• Examples
  • Ancon sheep breed (short-legged mutant in merino sheep, Pod maize (before cob development) to cob maize; colour mutations; dwarfism mutations.

Micromutations (Polygenic Mutations)

• Small phenotypic effects that cannot be recognised on individual plant basis. Can only be detected by comparing groups of plants statistically. Selection must be delayed to M3 or later generations.
• Genetic basis: Multiple minor genes (polygenes) mutated; each contributes a small change.
• Examples: Small yield improvements, small changes in grain quality, minor changes in maturity, altered disease resistance levels.
• Detection: Statistical analysis required; progeny rows compared;  Intensive and careful evaluation of a large number of M3 progeny rows allows identification of mutants with altered quantitative traits.

General Characteristics of Mutations

• Mutations are generally recessive: Most induced mutations produce recessive alleles. This is why mutations are hidden in M1 (heterozygous) and only appear in M2 (where homozygous recessive plants segregate out). However, dominant mutations also occur and can be selected in M1 plants directly.
• Mutations are generally harmful: Most mutations have deleterious effects on the organism. Only a small proportion — approximately 0.1% are beneficial and suitable for crop improvement. This is why large populations (thousands of M2 rows) must be screened to find useful mutants.
• Mutations are random: Mutations may occur in any gene. The breeder cannot direct mutation to a specific gene. However, some genes have higher mutation rates than others — gene-specific differences in mutation frequency exist.
• Mutations are recurrent: The same mutation may occur again and again — identical alleles can arise independently multiple times. This is evidence that mutations are not entirely random at the molecular level — certain sites within genes are more susceptible to mutation ('hotspots').
• Induced mutations commonly show pleiotropy: A mutagen often affects not one but several linked or nearby genes simultaneously. A useful mutation is frequently accompanied by unwanted changes in other characters, requiring backcrossing to remove the undesirable side effects.

CSE 2018 (Q3, 20M) — Describe mutation — types with genetic basis. Explain the role of mutagens. IFoS 2020 (Q3c, 10M) — Describe the role of mutation in development of disease resistant varieties. IFoS 2022 (Q3c, 10M) — What is mutagenesis? Describe the procedure for mutation breeding.

PHYSICAL MUTAGENS 

• All physical mutagens are forms of radiation.
• Physical mutagens are classified based on whether they ionise atoms (ionising radiation) or not (non-ionising radiation), and based on whether they consist of particles or electromagnetic waves:

Classification of Physical Mutagens  Ionising Radiation: Particulate radiation: Alpha-rays (DI = Densely Ionising) Beta-rays (SI = Sparsely Ionising) Fast neutrons (DI) *commonly used Thermal (slow) neutrons (DI) Non-particulate (electromagnetic) radiation: X-rays (SI) *commonly used Gamma-rays (SI) *most commonly used in crop breeding Non-Ionising Radiation: UV radiation (100-3900 Angstrom; 10-390 nm) = commonly used in mutation breeding.

Beta-Rays

• Nature: Beta-rays are high-energy electrons produced by the decay of radioactive isotopes such as tritium (3H), phosphorus-32 (32P), sulphur-35 (35S), etc.
• Mechanism of ionisation: High-energy electrons are slowed down by positively charged molecules in their path. They transfer energy to electrons of atoms — knocking them out of their orbits (ionisation) or pushing them to higher energy orbits (excitation). Electrons move in a zig-zag path as they are deflected by atoms. After losing energy, they attach to an atom making it negatively charged.
• Penetrating power: Very low penetrating power compared to X-rays. Beta-rays deposit most of their energy within a short distance from their source. This limits their usefulness for treating bulky plant material but makes them useful for surface treatments.
• Use in mutation breeding: Limited use in crop plants due to poor penetration. More useful for studies with microorganisms or when applied as radioisotope incorporations into plant tissue.

Alpha-Rays

• Alpha particles consist of two protons and two neutrons each — they are essentially helium nuclei. Produced by fission of radioactive isotopes of heavier elements.
• Movement: Because they are heavy particles, alpha-particles move in a straight line. They have a strong attraction for electrons and pull them away from the nuclei of atoms in their path.
• Penetrating power: Alpha particles are much less penetrating than neutrons or even beta-rays. They deposit their energy in a very short distance (dense ionisation near the source). After losing energy, each alpha particle captures two electrons and becomes an atom of helium.
• Use in mutation breeding: Very limited use in crop plants due to extremely poor penetration through plant tissues.

Fast Neutrons and Thermal Neutrons

• Fast neutrons: Produced in cyclotrons or atomic reactors as a result of radioactive decay of heavier elements. The velocity of fast neutrons is reduced by graphite or heavy water to generate thermal (slow) neutrons.
• Nature: Neutrons are uncharged particles — they are not repelled by the positively charged nuclei of atoms and can move through biological tissues in a straight line. They are highly penetrating.
• Mechanism: Neutrons do not cause ionisation directly. Ionisation is produced by two mechanisms:
  • Elastic scattering — nuclei of atoms are 'kicked away' by the neutron; these displaced nuclei then cause ionisation.
  • Capture-and-decay — thermal neutrons are captured by atomic nuclei, which become unstable and emit gamma-rays; the gamma-rays then cause ionisation.
• Ionisation type: Both fast and thermal neutrons are densely ionising (DI) — they produce dense tracks of ionised atoms along their paths, similar to alpha particles.
• Use in mutation breeding: Fast neutrons are commonly used in mutation breeding'. They are particularly effective in inducing chromosome breaks and deletions. Notable Indian example: Castor variety 'Aruna' (NPH1) was developed by fast neutron treatment of parent variety HC 6 at TNAU. 

X-Rays

• Nature: X-rays are non-particulate (electromagnetic) radiation with a wavelength of approximately 10^-11 to 10^-7 cm. They consist of photons (small packets of energy). X-rays are produced by X-ray tubes — when high-energy electrons hit a metal target.
• Hard vs Soft X-rays: X-rays are classified as hard (0.1-0.001 Angstrom wavelength) or soft (10-1 Angstrom wavelength) depending on their wavelength. Hard X-rays have more energy and penetrate further.
• Ionisation mechanisms:
  • Photoelectric effect: Low-energy photons transfer all energy to individual electrons, ejecting them from their orbits (primary ionisation). These high-energy electrons produce secondary ionisations — which are of greater significance than primary ones.
  • Compton scattering: A high-energy photon transfers part of its energy to eject an electron, while the photon continues at longer wavelength with less energy. Repeated interactions produce multiple ionisations.
  • Pair production: A very high-energy photon passing near an atomic nucleus is completely absorbed, producing a high-energy electron and a high-energy positron, both of which produce further ionisations.
• Penetrating power: X-rays and gamma-rays are highly penetrating and sparsely ionising (SI) — they can penetrate through entire seeds or plant parts, producing ionisations throughout.
• Historical significance: Stadler (1928) used X-rays to demonstrate induced mutations in barley and maize — the first demonstration of induced mutagenesis in crop plants. Muller (1927) used X-rays in Drosophila.

Gamma-Rays

• Nature: Gamma-rays are produced by radioactive decay of certain elements such as radium, carbon-14 (14C), and cobalt-60 (60Co). Cobalt-60 (60Co) is the most commonly used source of gamma-rays for mutation breeding and biological studies.
• Properties: X-rays and gamma-rays are similar in their physical properties and biological effects — both are non-particulate electromagnetic radiation of similar wavelengths and energy ranges. The main difference is their source: X-rays from machines; gamma-rays from radioactive decay.
• Gamma Garden (Gamma Field): A specialised facility for large-scale mutation breeding using gamma radiation. A cobalt-60 source is placed at the centre of a circular field. Plants are grown at various distances from the source — those closest receive higher doses. Gamma gardens exist at the Bhabha Atomic Research Centre (BARC), Trombay, Mumbai. Internationally, the gamma field at IAEA, Vienna was historically important.
• Use in mutation breeding: Gamma-rays are the most widely used physical mutagen in crop improvement worldwide. Most important Indian mutation-bred varieties were produced using gamma rays: Sharbati Sonora wheat (M.S. Swaminathan, from Sonora 64), Jagannath rice (from T141), Co 8152 sugarcane (from Co 527).

Ultraviolet (UV) Radiation

• Nature: UV rays have a wavelength of 100 to 3,900 Angstroms (10 to 390 nm). UV is present in solar radiation and can also be produced by mercury vapour lamps or tubes.
• Energy level: UV is a low-energy radiation — it does NOT cause ionisation. It is non-ionising radiation. It has a very limited penetrating capacity (usually limited to one or two cell layers).
• Molecular mechanisms of mutagenesis
  • UV rays generally produce dimers of thymine, uracil, and sometimes cytosine present in the same strand of DNA — these are pyrimidine dimers. The covalent bond between adjacent pyrimidines distorts the DNA helix and prevents normal replication.
  • UV also produces addition of a molecule of water to the 5,6 double bond of uracil and cytosine, promoting deamination of cytosine — converting cytosine to uracil, which is then misread as thymine during replication.
  • The most effective UV wavelength for mutagenesis is 2,540 Angstroms (254 nm) — this is because DNA bases show maximum absorption at this wavelength (close to the absorption maximum of nucleotide bases). 
• Limitations in crop plants: Poor penetration means UV is limited to irradiation of pollen grains and very small eggs. In plants, pollen grains may be irradiated and used for pollination.
• Practical difficulties that have prevented wider use of UV in crop breeding:
  • The difficulty in collecting large quantities of pollen grains in most crop species (except maize and similar crops), and
  • The limited duration of pollen viability after collection.

Where UV is used: Commonly used for mutagenesis in microorganisms (bacteria, fungi, yeast) where penetration is not a problem — a single-cell layer receives full UV dose. In higher plants, primarily limited to pollen irradiation in maize.

Comparison of Physical Mutagens

Mutagen	Type	DI or SI	Penetrating Power	Main Use in Crop Breeding
Alpha-rays	Particulate	DI	Very low	Rarely used; poor penetration
Beta-rays	Particulate	SI	Low	Limited; surface treatments, radioisotopes
Fast neutrons	Particulate	DI	High	Chromosome breaks/deletions; Castor Aruna (TNAU)
Thermal neutrons	Particulate	DI	High	Research; limited crop use
X-rays	EM radiation (SI)	SI	High	Jagannath rice, historical wheat varieties
Gamma-rays	EM radiation (SI)	SI	Very high	MOST WIDELY USED; Sharbati Sonora wheat; Co 8152 sugarcane; Gamma Field at BARC
UV (non-ionising)	EM radiation	Non-ionising	Very low	Microorganisms; maize pollen only in crops

DI = Densely Ionising; SI = Sparsely Ionising; EM = Electromagnetic.

IFoS 2018 (Q3a, 15M) — Describe the physical mutagens — their types, properties, and use in crop improvement. CSE 2018 (Q3, 20M) — Describe chemical and physical mutagens and their mechanisms of action.

CHEMICAL MUTAGENS 

• Chemical mutagenesis was first demonstrated by Charlotte Auerbach and J.M. Robson
• Chemical mutagens generally produce point mutations (gene mutations) more specifically than physical mutagens, which tend to produce chromosome breaks and large-scale rearrangements in addition to point mutations.

Classification of Chemical Mutagens

GROUP 1: Alkylating Agents Examples: sulphur mustards, nitrogen mustards, epoxides, imines (e.g., ethylene imine/EI), sulphates and sulphonates, diazoalkanes, nitroso compounds (e.g., MNNG — N-methyl-N-nitro-N-nitroso-guanidine; also EMS — ethyl methane sulphonate*; DES — diethyl sulphate) GROUP 2: Acridine Dyes Examples: acriflavine, proflavine, acridine orange, acridine yellow, ethidium bromide GROUP 3: Base Analogues Examples: 5-bromouracil (5-BU), 5-chlorouracil GROUP 4: Others Examples: nitrous acid, hydroxyl amine, sodium azide

Alkylating Agents — Mechanism of Action

Alkylating agents are the most important group for plant mutation breeding. They work by transferring alkyl groups (methyl, ethyl, etc.) to the bases in DNA, particularly to the N7 position of guanine (most common target) and the O6 position of guanine.

EMS — Ethyl Methane Sulphonate

• EMS is the most widely used chemical mutagen in modern plant breeding, particularly for TILLING.
• Mechanism: EMS adds an ethyl group to the O6 position of guanine (O6-ethylguanine), which then mispairs with thymine instead of cytosine during DNA replication. This results predominantly in G:C to A:T transitions — the most common type of base substitution produced by EMS. Because EMS primarily produces G to A transitions, it creates a specific, predictable spectrum of mutations useful for functional genomics.
• Advantages: Very high mutation frequency; produces point mutations rather than chromosome breaks; dissolved in water for seed soaking; relatively easy to use.
• Indian varieties from EMS: TAU-1 blackgram.

DES — Diethyl Sulphate

• Similar mechanism to EMS — a sulphonate that alkylates guanine. Used as an alternative to EMS in some mutation breeding programmes. Similar mutation spectrum.

MNNG — N-methyl-N-nitro-N-nitroso-guanidine

• An extremely potent alkylating agent. Methylates O6-guanine very efficiently.
• Used in research-scale mutation breeding.
• Requires stringent safety precautions due to high mutagenicity.
• More commonly used in bacterial and microorganism genetics than in crop breeding.

Ethylene Imine (EI)

• A cyclic imine that acts as an alkylating agent.Used for seed treatment in some mutation breeding programmes.

Acridine Dyes — Mechanism

• Acridine dyes (acriflavine, proflavine, acridine orange, acridine yellow, ethidium bromide) are flat, planar molecules that intercalate (insert) between adjacent base pairs in the DNA double helix. This physical insertion:
• Causes the DNA to become locally unwound and extended.
• When the DNA is replicated, the intercalating molecule can cause 'slippage' — the replication machinery slips and inserts an extra base (resulting in a +1 frameshift insertion) or skips a base (resulting in a -1 frameshift deletion).
• These frameshift mutations are usually more severe than base substitutions because they alter the reading frame of the gene for all codons downstream of the mutation.
• Ethidium bromide: Widely used as a DNA stain in gel electrophoresis (the orange/red fluorescent dye). It is also mutagenic due to its intercalating property — this is why laboratory workers are cautioned to wear gloves when handling ethidium bromide gels.

Base Analogues — Mechanism

• Base analogues are chemicals that structurally resemble normal DNA bases and are incorporated into DNA in place of the normal base during replication.
• 5-Bromouracil (5-BU): Structurally resembles thymine (5-methyluracil) and is incorporated in place of thymine. However, 5-BU can occasionally adopt a different tautomeric form that resembles cytosine — when in this form, it pairs with guanine instead of adenine. Over successive replications, this leads to A:T → G:C transitions.
• 5-Chlorouracil: Similar to 5-bromouracil; also a thymine analogue. Less commonly used.

Other Chemical Mutagens

• Nitrous acid (HNO2): A deaminating agent — removes amino groups from cytosine (converting it to uracil) and from adenine (converting it to hypoxanthine). Deaminated cytosine (uracil) pairs with adenine instead of guanine — producing C:G to T:A transitions. Most effective at low pH. Used in in vitro mutagenesis of viruses and phages rather than crop breeding.
• Hydroxylamine (NH2OH): Reacts specifically with cytosine — hydroxylates it to produce 4-hydroxylaminocytosine, which pairs with adenine instead of guanine. Produces C:G to T:A transitions specifically. Highly specific; used in research rather than large-scale crop breeding.
• Sodium azide (NaN3): Sodium azide inhibits the enzyme catalase, leading to accumulation of hydrogen peroxide, which can damage DNA. More precisely, sodium azide is metabolically converted to an azidoalanine adduct which is the actual mutagen.

Comparison Table — Key Chemical Mutagens

Mutagen	Group / Type	Molecular Mechanism	Best Known Use
EMS (Ethyl Methane Sulphonate)	Alkylating agent	Alkylates O6-guanine → G:C to A:T transitions (mispairing)	TILLING populations in rice, wheat, maize, Arabidopsis; TAU-1 blackgram
DES (Diethyl Sulphate)	Alkylating agent	Same as EMS — alkylates guanine	Alternative to EMS in some crop programmes
MNNG	Alkylating agent (nitrosamide)	Methylates O6-guanine; extremely potent	Research-scale; not widely used in field crops
Ethylene Imine (EI)	Alkylating agent (imine)	Alkylating; crosses-links DNA	Seed treatment in some crop programmes
Acridine orange / Ethidium bromide	Acridine / intercalating dye	Intercalates between bases → frameshift insertions/deletions	Research (Ethidium bromide as stain — also mutagenic)
5-Bromouracil	Base analogue	Replaces thymine; tautomeric shift → A:T to G:C transitions	In vitro mutagenesis; less used in crops
Sodium azide	Other (oxidative)	Metabolic conversion to mutagenic adduct; oxidative DNA damage	Barley mutation breeding; widely used in Scandinavian barley; rice
Nitrous acid	Deaminating agent	Deaminates cytosine to uracil → C:G to T:A transitions	Viral/phage genetics; not widely used in crop breeding
Colchicine	Spindle inhibitor	NOT a DNA mutagen; blocks spindle formation → chromosome doubling (polyploidy)

Important: Colchicine is listed by some students as a chemical mutagen — this is INCORRECT. Colchicine does not damage DNA. It is a spindle inhibitor that causes chromosome doubling. It produces chromosomal numerical changes (polyploidy) but is NOT a mutagen in the classic sense of altering gene sequence. This distinction may be tested directly.

IFoS 2021 (Q3c, 10M) — Describe chemical mutagens and their mechanism of action. CSE 2021 (Q3b, 10M) — Write on chemical mutagens — EMS, MMS, NMU — their mechanisms.

PROCEDURE OF MUTATION BREEDING

Prerequisites for a Mutation Breeding Programme

• A mutation breeding programme should have well-defined and clear-cut objectives.
• If the experimenter starts a mutagenesis programme just with the hope that he will discover something useful, he is most likely wasting his time and resources.
•  This is because the ratio of beneficial to useless mutations is very small (approximately 1 in 800 mutations, i.e., about 0.1%), and identifying desirable mutations from among the undesirable ones is a very difficult task indeed.

Key prerequisites:

• Clear objectives: What specific character is to be improved? Oligogenic or polygenic? This determines the handling procedure.
• Selection of variety for mutagenesis: The variety selected for mutagenesis should be the best variety available in the crop.There is no benefit in isolating useful mutants in an inferior variety — the mutant lines will also be inferior in background performance. Exception: if an extensive search for new alleles is needed (e.g., novel dwarfing genes in wheat), mutants may be sought in tall varieties that are not the best.
• Large enough facilities: The programme must be 'large enough with sufficient facilities to permit effective screening of large populations. Screening thousands of M2 rows requires substantial land, labour, and technical capacity.

Choice of Plant Part for Mutagenesis

What plant part should be treated depends on whether the crop is sexually or asexually propagated, and on the mutagen to be used. 

Seeds (Most Commonly Used)

• In sexually propagated crops, seeds are the most commonly used plant part for mutagenesis.
• Dry dormant seeds are biologically almost inert — they can withstand a range of extreme conditions (soaking, desiccation, heating, freezing, oxic or anoxic regimes) making mutagen treatment practical.
• Mutagenic treatment of seeds is essentially treatment of embryo meristems.

Pollen Grains

• Pollen grains may be used but are infrequently treated because:
• It is difficult to collect large quantities of pollen grains in most crop species (except maize and similar wind-pollinated crops)
• Hand pollination with treated pollen is laborious
• Pollen viability is relatively short — limiting the window for treatment and use
• Pollen grains are the ONLY plant part that can be successfully treated with UV radiation, because UV has such poor penetration that a pollen monolayer on a surface can receive effective UV dosage. A pollen monolayer is exposed to UV rays of 250 to 290 nm wavelength.

Vegetative Propagules (Buds and Cuttings)

• Used in clonal crops where seeds are either not produced or are genetically variable (heterozygous).
• In case of fruit trees when they are propagated by clones — the desirable cuttings are exposed to irradiation.

Whole Plants

• Whole plants are generally irradiated during the flowering stage so that it is equivalent to irradiation of developing pollen grains and egg cells.
• However, treatment of whole plants requires special facilities — a Gamma Garden (gamma field) — and is practical in only a few places.
• The BARC Trombay gamma field is India's primary facility for this purpose. 

Dose of the Mutagen

The choice of dose is critical.

• Mutagen treatments reduce germination, growth rate, vigour, and fertility. The damage generally increases with mutagen dose, but may not be proportional.
• An optimum dose produces maximum mutation frequency and causes minimum killing. Too high a dose kills too many treated individuals; too low a dose produces too few mutations.
• LD50: Most workers consider a dose close to LD50 to be approximately optimal. LD50 is the dose that kills 50 per cent of the treated individuals. LD50 varies with crop species and with the specific mutagen used. A preliminary experiment to determine LD50 is always required before starting a large-scale mutation breeding programme.

Varying the dose:

• For radiation: dose can be varied by changing the radiation intensity (distance from source) or the duration of treatment
• For chemical mutagens: dose is varied by changing concentration of the chemical solution or the duration of soaking

The M1 Generation — Chimaeras

The M1 generation is produced directly from the mutagen-treated plant parts. 

Critical concept: 'Since mutation is a single cell event, the M1 plants will carry an induced mutation only in PARTS of the shoot — they will be chimaeras.' A chimera is an individual with one genotype in some of its parts and another genotype in the others.

Types of Chimaeras

• L1 layer: Gives rise to epidermis only.
• L2 layer: Produces a part of leaf mesophyll AND the gametes (egg cells and pollen). This is the critical layer for mutation breeding in sexually reproducing crops.
• L3 layer: Yields the rest of the plant body.

Based on which layers are affected, two types of chimaeras are recognised:

• Periclinal chimaera: The whole of L1, L2, or L3 layer is affected. This is the more stable type.
• Sectorial chimaera: Only a sector (part) of L1, L2, or L3 is affected. This type is unstable in clonal crops — it must be made periclinal through successive clonal propagation and selection.

Which chimaera can be transmitted to the next generation (M2)?

• In sexually reproducing species: ONLY L2 chimaeras (periclinal or sectorial) are transmitted to M2 through gametes. L1 and L3 mutations are NOT transmitted because these layers do not contribute to gamete formation.
• In clonal crops: ALL chimaeras can potentially be recovered — either as periclinal chimaeras directly, or by inducing adventitious buds from wound tissue, or through tissue culture.

Practical Consequence of Chimaeras in M1

• Because M1 plants are chimaeras, they are NOT selected. Selection begins in M2.
• However:
  • Dominant mutations may appear directly in M1 plants and can be selected there.
  • M1 plants should be protected from outcrossing (by isolation or bagging) to prevent contamination of M2 progenies with foreign pollen.
• About 20-25 seeds from each M1 spike are harvested separately to raise M2 progeny rows.' This ensures each M2 row traces to a specific M1 plant spike, maintaining identifiability of mutations.

5.5  Handling Generations — Oligogenic Traits

M1 Generation

• Several hundred seeds (enough to give approximately 500 fertile M1 plants at harvest) are treated with the mutagen and planted.
• M1 plants are protected from outcrossing by isolation or inflorescence bagging.
• M1 plants will be chimaeras — no selection for target trait in M1.
• About 20-25 seeds from each M1 spike are harvested separately — each spike gives one M2 progeny row.

M2 Generation — SELECTION STARTS HERE

• About 2,000 progeny rows are grown. Each row traces to one M1 plant spike.
• Careful regular observations are made for distinct mutations. Only macromutations are detectable in individual M2 plants.
• All plants in M2 rows suspected of containing new mutations are harvested separately to raise individual plant progenies in M3.
• If the mutant is distinct and clearly identifiable, it is immediately selected for multiplication and testing.
• Expected frequency: Only 1-3% of M2 rows may be expected to have beneficial mutations. Most mutations will be useless for crop improvement — this requires screening thousands of rows.

Alternative M2 approach:

• M2 may be grown as a BULK — one or more equal-number seeds from each M1 spike mixed together. Individual plants are then selected within the bulk M2. This simplifies the programme but reduces traceability.

M3 Generation

• Progeny rows from individually selected M2 plants are grown.
• Poor and inferior mutant rows are eliminated.
• If mutant progenies are homogeneous (all plants showing the mutant phenotype), two or more M3 progenies containing the same mutation may be bulked.
• Mutant M3 rows are harvested in bulk for a preliminary yield trial in M4.

M4 Generation

• A preliminary yield trial is conducted with a suitable check variety.
• Promising mutant lines are selected for replicated multilocation trials.

M5 to M7 Generations

• Replicated multilocation yield trials are conducted. Outstanding lines may be released as new varieties.
• Low-yielding mutant lines are retained for use in hybridization programmes — even if the mutant yield is low, its unique gene (e.g., disease resistance, quality character) can be transferred to a better background by crossing.

Handling Generations — Polygenic Traits

M1 and M2

• Grown the same way as for oligogenic traits.
• In M2, vigorous, fertile, and normal-looking plants that do NOT exhibit a mutant phenotype are selected (unlike oligogenic where mutant-looking plants are selected). Their seeds are harvested separately to raise individual plant progeny rows in M3.

M3 — Main Selection Generation for Polygenic Traits

• Progeny rows from individual selected M2 plants are grown.
• Careful observations for SMALL deviations in phenotype from the parent variety.
• Inferior rows are discarded. Few homogeneous rows may be harvested in bulk.
• Selection is done in M3 rows showing segregation.
• Intensive and careful evaluation of a large number of M3 progeny rows allows identification of mutants with altered quantitative traits, e.g., partial or horizontal disease resistance. Such mutants occur at frequencies approaching 1% or even higher, so that their isolation becomes quite cost effective.' This explains why mutation breeding for horizontal disease resistance is practical.

M4 and M5-M8

• M4: Preliminary yield trials.
• M5-M8: Replicated multilocation trials depending on when progenies become homogeneous. Outstanding progenies released as varieties.

Handling Mutations in Clonal Crops

• Mutation breeding in clonal (vegetatively propagated) crops has special features.
• Clonal crops are generally highly heterozygous. Hybridization would change the entire genotype of the clone. Mutagenesis is the ONLY practical method to improve specific characters of a clone without changing its genetic makeup.
• Dominant mutations: Can be recovered directly in M1 from treated buds/cuttings. Most practically useful mutations in clonal crops contain dominant mutations — visible in the heterozygous state.
• Recessive mutations: Can only be utilised if the clone used for mutagenesis was ALREADY heterozygous for the gene in question (Aa). When treated, mutation from A to a in the already-Aa clone gives aa in some cells — which can be exposed if the clone is induced to produce shoots or seeds homozygous for aa. Such situations are rare.
• Periclinal chimaeras in clonal crops: May be commercially valuable as stable periclinal chimaeras. A periclinal chimaera has the epidermis (L1) or subepidermis (L2) of one genotype and the internal tissue (L3) of another. Some commercially valuable ornamental plants and fruit trees exist as stable periclinal chimaeras.
• Examples of clonal mutation breeding: Banana and chrysanthemum mutation breeding programmes 

IFoS 2022 (Q3c, 10M) — What is mutagenesis? Describe the procedure for mutation breeding. IFoS 2020 (Q3c, 10M) — Describe the role of mutation in development of disease resistant varieties.

Applications of Mutation Breeding

• Induction of desirable mutant alleles not available in germplasm: When no natural source of the desired allele exists anywhere — not in cultivated varieties, wild relatives, or gene bank — mutagenesis may create it. This is the most fundamental justification for mutation breeding.
• Improving specific characteristics of an adapted variety: Particularly valuable for clonal crops where hybridization would disrupt the entire genotype. For self-pollinated crops, mutagenesis may not be simpler or quicker than the backcross procedure when the character is available in another variety — but when no donor exists, mutation breeding is the only option.
• Improvement of quantitative characters including yield: Mutagenesis produces genetic variation in polygenic traits this variation is 'usually as much as 50% of that generated in F2 generation' from hybridization, and sometimes equal to or greater. This makes mutation a valid source of quantitative genetic variation.
• Treatment of F1 hybrids to increase variability and break linkages: F1 hybrids from intervarietal crosses may be treated with mutagens to increase genetic variability and facilitate recombination among linked genes. However, this method has not been widely used in practice.
• Irradiation of interspecific hybrids to produce translocations: Radiation of interspecific hybrid F1 plants can produce translocations, facilitating transfer of chromosome segments carrying useful genes from wild species to cultivated species chromosomes. 

Merits of Mutation Breeding

• Creates inexhaustible variation: Mutations can theoretically be induced at any gene in any crop — providing access to variation not limited by what exists naturally in the germplasm.
• More effective for oligogenic traits: Mutation breeding is more effective for improving oligogenic characters such as disease resistance than polygenic traits — because a single major gene mutation can confer complete resistance, detectable easily in M2.
• Best for clonal crops: Mutation breeding is the simplest, quickest, and best way to introduce a new character into vegetatively propagated crops without disrupting their existing superior genotype.
• Indispensable when no other method works: When no improvement is possible through hybridization (no donor exists, no successful cross possible), mutagenesis is the last resort.

Limitations of Mutation Breeding

• Very low frequency of useful mutations: Only approximately 0.1% of induced mutations are beneficial. Therefore, large M2 populations (thousands of rows) must be grown and carefully studied, requiring considerable time, labour and resources.
• Large-scale screening required: Efficient, quick, and inexpensive screening techniques are needed to handle large populations. Mutation breeding works best for characters with easily detectable, distinct phenotypes. For characters requiring elaborate testing (e.g., nutritional quality, end-use quality), screening thousands of M2 plants is 'virtually impractical'.
• Undesirable side effects: Desirable mutations are commonly associated with undesirable mutations in linked genes — chromosomal aberrations, sterility, quality defects. The mutant lines often have to be backcrossed to the parent variety to remove these defects. This extends the programme and adds time, labour, and cost.
• Pleiotropy: Mutations often produce pleiotropic effects.
• Quantitative mutations go against selection history (Brock's principle): This means if a variety has been selected for high yield over many generations, most induced mutations of yield genes will DECREASE yield (reverting toward the original unselected state), not increase it further. This limits the improvement achievable in highly selected polygenic traits.
• Problems in variety registration: It may be difficult to convincingly demonstrate that a mutant variety is distinct from the parent variety — PBR (Plant Breeders' Rights) laws require the DUS standard (Distinctness, Uniformity, Stability) and a mutant variety that differs in only one character may not pass the Distinctness test under some DUS systems. It may also attract royalty liability if the parent variety was protected.
• Recessive mutations in polyploids and clones are difficult to detect: In polyploid species (tetraploid, hexaploid), a recessive mutation at one of the multiple copies of a gene may be completely masked by the other normal copies at homoeologous loci. Larger doses and larger populations are required. Mutagenesis is most suitable for diploid, self-pollinated species.

Indian Mutant Varieties

Crop	Variety	Mutagen / Method	Parent / Institute	Improved Trait
Wheat	Sharbati Sonora	Gamma rays	Sonora 64; M.S. Swaminathan (IARI)	Amber grain colour; higher protein content; first premium-quality Indian wheat mutation variety
Wheat	NP 836	Irradiation (type uncertain — verify)	NP 709; IARI
Wheat	Pusa Lerma	Gamma rays	Lerma Rojo; IARI	Higher yield
Rice	Jagannath	Gamma rays or X-rays (sources differ)	T141; CRRI Cuttack	Semi-dwarf, early maturity, lodging resistance
Rice	GFB 24	Spontaneous mutant	Konamani variety	Natural sport
Rice	MTU 20	Spontaneous mutant	MTU-3	Natural sport
Rice	IIT-48, IIT-60	Mutation (specific mutagen unclear)	Demo Notes; early maturity	Early maturing
Mungbean	Pant Mung-2	Gamma rays	Demo Notes cite GBPUAT Pantnagar	High yield
Blackgram	TAU-1	EMS treatment	TNAU	High yield; disease resistance
Groundnut	TAG-24, TG-37A	X-rays / Gamma rays	Demo Notes cite BARC Trombay	Bold pods; high oil content
Castor	Aruna (NPH1)	Fast neutrons	HC 6; TNAU	Dwarf, early-maturing castor
Cotton	Indore 2	Irradiation (X-rays per Demo Notes)	Malwa Upland 4	Higher yield
Cotton	MLU 7	Gamma rays	Culture 1143 EE
Cotton	MLU 10	Gamma rays	MLU 4
Cotton	DB 3-12	Spontaneous mutant	G. herbaceum variety Western 1
Sorghum	CO 18	Spontaneous mutant	CO 2
Sugarcane	Co 8152	Gamma rays	Co 527	Higher cane yield
Mustard	Primax (white)	Induced mutation	1950
Banana	Klue Hom Thong KU1 (Thailand)	Gamma rays	Thailand	Panama disease resistance
Barley (global)	Golden Promise	Gamma rays	UK

IFoS 2018 (Q2b, 10M) — Distinguish between Heterosis breeding and Mutation breeding. IFoS 2020 (Q3c, 10M) — Describe the role of mutation in development of disease resistant varieties.