Mutation BreedingMutation Breeding

TaryonoTaryono

Faculty of AgricultureFaculty of Agriculture

Gadjah Mada Gadjah Mada UniversityUniversity

Mutation - MutantMutation - Mutant

MutationMutation

Changes in genes and chromosomesChanges in genes and chromosomes

MutatedMutated

Altered genesAltered genes

MutantMutant

New organism with a mutated gene or New organism with a mutated gene or rearranged chromosomesrearranged chromosomes

Mutation BreedingMutation Breeding

AdvantagesAdvantages– Screen very high populations (cell based)Screen very high populations (cell based)– Can apply selection to single cellsCan apply selection to single cells

DisadvantagesDisadvantages– Many mutations are non-heritableMany mutations are non-heritable– Requires dominant mutation (or double recessive Requires dominant mutation (or double recessive

mutation); most mutations are recessivemutation); most mutations are recessive Can avoid this constraint by not applying selection pressure Can avoid this constraint by not applying selection pressure

in culture, but you loose the advantage of high through-put in culture, but you loose the advantage of high through-put screening – have to grow out all regenerated plants, produce screening – have to grow out all regenerated plants, produce seed, and evaluate the Mseed, and evaluate the M22

Alternative: perform on haploid cell linesAlternative: perform on haploid cell lines

MutationMutation

May involve any traitMay involve any trait All kind of transition are encountered, All kind of transition are encountered,

from drastic morphological changes from drastic morphological changes deviations in physiology so minute as deviations in physiology so minute as to be almost indiscernibleto be almost indiscernible

Harmful or even lethalHarmful or even lethal

Type of mutationType of mutation

Spontaneous (natural) mutationSpontaneous (natural) mutation1.1. Some have played an outstanding role in Some have played an outstanding role in

development of valuable crop cultivars and development of valuable crop cultivars and hybridshybrids

2.2. Unfortunately, it can not form the basis of Unfortunately, it can not form the basis of modern plant breeding due to its low modern plant breeding due to its low frequency and difficulties in detectionfrequency and difficulties in detection

Induced mutationInduced mutation

Genetic structure changesGenetic structure changes Gene (point mutation)Gene (point mutation)

ChromosomeChromosome GenomeGenome

Gene (point) mutationGene (point) mutation→→ a change in specific sequence of nucleotides in DNA a change in specific sequence of nucleotides in DNA

molecules leading to the formation of a new type of molecules leading to the formation of a new type of protein or preventing that of the normally proteinprotein or preventing that of the normally protein

→→ take place at the molecular or sub-microscopic leveltake place at the molecular or sub-microscopic level

→→ Such change may be accompanied by the emergence Such change may be accompanied by the emergence of a new trait inherited in accordance with Mendel’s of a new trait inherited in accordance with Mendel’s LawsLaws

Chromosomal mutationChromosomal mutation Mutation associated with splitting and subsequent Mutation associated with splitting and subsequent

changes in the structure of the chromosomeschanges in the structure of the chromosomes The end of the split chromosomes may fuse to The end of the split chromosomes may fuse to

form structure again, but the new chromosomes are form structure again, but the new chromosomes are not always exactly what the used to benot always exactly what the used to be

The microscopic structures of chromosomes may The microscopic structures of chromosomes may be characterized by deletion or deficiency (loss of a be characterized by deletion or deficiency (loss of a chromosomal segment), duplication (doubling of a chromosomal segment), duplication (doubling of a chromosomal segment), inversion (rearrangement chromosomal segment), inversion (rearrangement of a group of genes in a chromosomal segment in a of a group of genes in a chromosomal segment in a such a way that their order is reversed; such a way that their order is reversed; rearrangement of genetic material in a rearrangement of genetic material in a chromosome results from loss of segment, its chromosome results from loss of segment, its rotation by 180°, and fusion of the separated ends) rotation by 180°, and fusion of the separated ends) and translocation (change in a position of a and translocation (change in a position of a chromosome or more often exchange of segments chromosome or more often exchange of segments between different chromosomes) between different chromosomes)

Genome mutationGenome mutationChanges in sets of chromosomesChanges in sets of chromosomes

Remarks:

1. Breeders are more interested in gene mutation, because chromosomal rearrangement usually produce negative results, such as lower fertility of the offspring

2. Mutant are aften of great value for breeding as sources of new, previously unknown useful characters

3. Mutagenesis may be instrumental in obviating the technical difficulties arising in the crossing of such a small flowered crops such as milled

Story of induced mutationStory of induced mutation

1.1. X rays can significantly promote mutation in fungi (1925)X rays can significantly promote mutation in fungi (1925)2.2. X rays produced pronounced mutagenic effect on the X rays produced pronounced mutagenic effect on the

fruit fly Drosophila (1927)fruit fly Drosophila (1927)3.3. Artificial mutants can serve as good source material in Artificial mutants can serve as good source material in

plant breeding; X rays induce mutations in Maize and plant breeding; X rays induce mutations in Maize and barley (1928)barley (1928)

Today there are three groups of breeders:Today there are three groups of breeders:1)1) Mutation breeding is useless, we can accomplish the Mutation breeding is useless, we can accomplish the

same thing with conventional methodssame thing with conventional methods2)2) Mutation breeding will produce a breakthrough given Mutation breeding will produce a breakthrough given

enough effortenough effort3)3) Mutation breeding is a tool, useful to meet specific Mutation breeding is a tool, useful to meet specific

objectivesobjectives

Technique for inducing mutationTechnique for inducing mutation

Physical mutagensPhysical mutagens Chemical mutagensChemical mutagens

Physical mutagens

1.1. Various sources of ionizing radiations are explored, most Various sources of ionizing radiations are explored, most often X and gamma rays, UV radiation, fast and slow often X and gamma rays, UV radiation, fast and slow neutron, alpha ray, beta rayneutron, alpha ray, beta ray

2.2. Radioactive isotopes P-32 and S-35 are not convenient for Radioactive isotopes P-32 and S-35 are not convenient for use due to the storage and application difficultiesuse due to the storage and application difficulties

3.3. The usual sources of gamma rays in laboratories are The usual sources of gamma rays in laboratories are radioactive cobalt (Co-60) and Cesium (Cs-137) placed in radioactive cobalt (Co-60) and Cesium (Cs-137) placed in cobalt bombcobalt bomb

Physical mutagensPhysical mutagens4. 4. The object can be irradiated in two ways:The object can be irradiated in two ways: With an aid of a powerful source of a short-duration gamma With an aid of a powerful source of a short-duration gamma

rays for short duration radiation. Need special units for rays for short duration radiation. Need special units for irradiating living objectirradiating living object

A much weaker radiation but operating continuously (gamma A much weaker radiation but operating continuously (gamma field). field).

5.5. the dosage must be varied depending not only on the plant the dosage must be varied depending not only on the plant species whose seeds/organs are irradiated, but also on many species whose seeds/organs are irradiated, but also on many other factorsother factors

6.6. plant must be irradiated heavily enough to ensure as many plant must be irradiated heavily enough to ensure as many inherited changes as possible but without seriously affecting inherited changes as possible but without seriously affecting the germination, growth and fertility of plant directly emerging the germination, growth and fertility of plant directly emerging from the irradiated seeds or vegetative organs (from the irradiated seeds or vegetative organs (critical critical radiation doseradiation dose:: dosage which strong enough to assure many dosage which strong enough to assure many mutation not yet so strong as to kill plantsmutation not yet so strong as to kill plants))

Chemical mutagensChemical mutagens

Mutagenic substances belonging to different classes of Mutagenic substances belonging to different classes of chemical compounds, such as ethylene imine, diethyl chemical compounds, such as ethylene imine, diethyl sulfate, dimethyl sulfate, N-nitrosoethyl urea, N-sulfate, dimethyl sulfate, N-nitrosoethyl urea, N-nitrosomethyl urea, methal sulfonate, diepoxy butane, nitrosomethyl urea, methal sulfonate, diepoxy butane, ethyleneoxideethyleneoxide

Most are highly toxic, usually result in point mutationsMost are highly toxic, usually result in point mutations Use in solution in the concentration ranging from tenth – Use in solution in the concentration ranging from tenth –

hundredths even thousandths of percenthundredths even thousandths of percent Many chemical mutagens are much more effective than Many chemical mutagens are much more effective than

physical one. If irradiation of crops produces 10 – 15% of physical one. If irradiation of crops produces 10 – 15% of viable inherited changes, chemical mutants do the same viable inherited changes, chemical mutants do the same at a rate of 30 to 60%at a rate of 30 to 60%

They often exert more specific and finely tuned action on They often exert more specific and finely tuned action on the cellthe cell

Chemical mutagensChemical mutagens Some substances (supermutagen) are capable Some substances (supermutagen) are capable

of causing inherited changes in plants at a rate of causing inherited changes in plants at a rate up 100%up 100%

Chemical mutagens aim at the most vulnerable Chemical mutagens aim at the most vulnerable spot of a living organism (DNA) to induce spot of a living organism (DNA) to induce changes in nucleotides and alter the genetic changes in nucleotides and alter the genetic information (Sometimes causes specific information (Sometimes causes specific mutation)mutation)

It provides a powerful tool to induce desire It provides a powerful tool to induce desire changes in a traitchanges in a trait

Use of mutations in sexually Use of mutations in sexually reproduced cropsreproduced crops

More valuable in self than cross pollinated. More valuable in self than cross pollinated. The probability of producing desirable The probability of producing desirable mutations and genetic variability is mutations and genetic variability is theoretically highertheoretically higher

SeedsSeeds Very young seedlingVery young seedling

Use of mutations in asexually Use of mutations in asexually produced cropsproduced crops

It has been much easier and quicker to obtain variant It has been much easier and quicker to obtain variant plant typesplant types

Specific location of the mutation event (segmental Specific location of the mutation event (segmental chimera) becomes important. chimera) becomes important.

The mutant must be in meristematic tissue that will The mutant must be in meristematic tissue that will produce faithfully through cutting or other vegetative produce faithfully through cutting or other vegetative meansmeans

BudBud ScionScion CuttingCutting TuberTuber bulbsbulbs

Traditional Mutation Breeding ProceduresTraditional Mutation Breeding Procedures

Treat seed with mutagen (irradiation or Treat seed with mutagen (irradiation or chemical)chemical)

Target: 50% killTarget: 50% kill Grow-out MGrow-out M11 plants (some call this M plants (some call this M00))

– Evaluation for dominant mutations possible, Evaluation for dominant mutations possible, but most are recessivebut most are recessive

Grow-out MGrow-out M22 plants plants– Evaluate for recessive mutationsEvaluate for recessive mutations– Expect segregationExpect segregation

Progeny test selected, putative mutantsProgeny test selected, putative mutants– Prove mutation is stable, heritableProve mutation is stable, heritable

Mutation breeding scheme for seed Mutation breeding scheme for seed propagated croppropagated crop

Mutagenic applicationMutagenic application Growing the plants (M1 generation)Growing the plants (M1 generation) Identification of induced mutation, seed harvest Identification of induced mutation, seed harvest

from mutated plants (M2)from mutated plants (M2) Continue the identification and selection of Continue the identification and selection of

induced mutation (M3)induced mutation (M3) First agronomic evaluation. Propagation of First agronomic evaluation. Propagation of

promising lines (M4)promising lines (M4) Multilocation trials of stable mutant and Multilocation trials of stable mutant and

recombinant lines (M5 – M8)recombinant lines (M5 – M8) Official testing and releasing of mutant (M9)Official testing and releasing of mutant (M9)

Mutation breeding scheme for Mutation breeding scheme for vegetative propagated cropvegetative propagated crop

Mutagenic applicationMutagenic application Cutting back the M1V1 shoot, bud grafting, or in vitro Cutting back the M1V1 shoot, bud grafting, or in vitro

propagation via axillary budspropagation via axillary buds Isolation of induced somatic mutation, establishment of Isolation of induced somatic mutation, establishment of

clones, cutting back of non-mutant shoots from chimeric clones, cutting back of non-mutant shoots from chimeric plants (M1V2)plants (M1V2)

Further isolation of somatic mutations, vegetative Further isolation of somatic mutations, vegetative propagation of mutant plant (in vivo or in vitro), preliminary propagation of mutant plant (in vivo or in vitro), preliminary evaluation of mutants (M1V3)evaluation of mutants (M1V3)

Evaluation of mutant clone performance, assesing Evaluation of mutant clone performance, assesing segregation from mutant crosses and reselection of desired segregation from mutant crosses and reselection of desired recombinants. Released of improved mutant (M2V4)recombinants. Released of improved mutant (M2V4)

Requirements for Mutation BreedingRequirements for Mutation Breeding Effective screening procedureEffective screening procedure

– Most mutations are deleteriousMost mutations are deleterious With fruit fly, the ratio is ~800:1 deleterious to beneficialWith fruit fly, the ratio is ~800:1 deleterious to beneficial

– Most mutations are recessiveMost mutations are recessive Must screen MMust screen M22 or later generations or later generations Consider using heterozygous plants?Consider using heterozygous plants?

– But some say you should use homozygous plants to be sure But some say you should use homozygous plants to be sure effect is mutation and not natural variationeffect is mutation and not natural variation

Haploid plants seem a reasonable alternative if possibleHaploid plants seem a reasonable alternative if possible– Very large populations are required to identify Very large populations are required to identify

desired mutation: desired mutation: Can you afford to identify marginal traits with replicates & Can you afford to identify marginal traits with replicates &

statistics? Estimate: ~10,000 plants for single gene statistics? Estimate: ~10,000 plants for single gene mutantmutant

Clear ObjectiveClear Objective– Can’t expect to just plant things out and see what Can’t expect to just plant things out and see what

happens; relates to having an effective screenhappens; relates to having an effective screen– This may be why so many early experiments failedThis may be why so many early experiments failed

Mutation detectionMutation detection

Detection, isolation and testing mutants are Detection, isolation and testing mutants are extremely difficult extremely difficult

Due to the sporadic nature of viable useful Due to the sporadic nature of viable useful mutations, it is advisable to have larger plant mutations, it is advisable to have larger plant populationpopulation

When mutagens are used in breeding, the When mutagens are used in breeding, the biological nature of the trait (dominance or biological nature of the trait (dominance or recession of the mutation) and crops must recession of the mutation) and crops must be taken into account be taken into account

Trend in plant breeding based Trend in plant breeding based on mutation on mutation

Mutagens are used to induce mutations within a broad Mutagens are used to induce mutations within a broad range and at a high frequency to obtain ample source of range and at a high frequency to obtain ample source of material for selectionmaterial for selection

Mutant with a specific changes in certain characters are Mutant with a specific changes in certain characters are created in order to correct some defects in crop varieties. It created in order to correct some defects in crop varieties. It is important that the other economic characters remain is important that the other economic characters remain unalteredunaltered

Mutagens can be used to solve special problems in plant Mutagens can be used to solve special problems in plant breeding for instance by increasing the number of genetic breeding for instance by increasing the number of genetic recombinations and breaks of undesirable linkages, recombinations and breaks of undesirable linkages, transferring chromosomal fragment from one plant species transferring chromosomal fragment from one plant species into chromosomes of another during hybridization, into chromosomes of another during hybridization, obtaining homozygous mutant through irradiation of obtaining homozygous mutant through irradiation of haploids with subsequent doubling of chromosome number haploids with subsequent doubling of chromosome number

Mutation useful for crop Mutation useful for crop improvement improvement

Useful mutationUseful mutation

Any mutational change in a character which can be put to Any mutational change in a character which can be put to practical usepractical use

1.1. Improve nutrition value of crop productImprove nutrition value of crop product

2.2. Short stemShort stem

3.3. High lodging resistanceHigh lodging resistance

4.4. Disease resistanceDisease resistance

Successes of Mutation BreedingSuccesses of Mutation Breeding

Herbicide Resistance and ToleranceHerbicide Resistance and Tolerance Resistance:Resistance: able to break-down or metabolize the able to break-down or metabolize the

herbicide – introduce a new enzyme to metabolize the herbicide – introduce a new enzyme to metabolize the herbicideherbicide

Tolerance:Tolerance: able to grow in the presence of the able to grow in the presence of the herbicide – either herbicide – either ↑ the target enzyme or↑ the target enzyme or altered form altered form of enzymeof enzyme– Most successful application of somaclonal breeding have been Most successful application of somaclonal breeding have been

herbicide toleranceherbicide tolerance– Glyphosate Glyphosate resistantresistant tomato, tobacco, soybean (GOX enzyme) tomato, tobacco, soybean (GOX enzyme)– Glyphosate Glyphosate toleranttolerant petunia, carrot, tobacco and tomato petunia, carrot, tobacco and tomato

(elevated EPSP (elevated EPSP (enolpyruvyl shikimate phosphate synthase)(enolpyruvyl shikimate phosphate synthase))) But not as effective as altered EPSP enzyme (bacterial sources)But not as effective as altered EPSP enzyme (bacterial sources)

– Imazaquin (Sceptor) Imazaquin (Sceptor) toleranttolerant maize maize Theoretically possible for any enzyme-targeted Theoretically possible for any enzyme-targeted

herbicide – it’s relatively easy to change a single herbicide – it’s relatively easy to change a single enzyme by changing a single geneenzyme by changing a single gene

Wild type

Mutagen

(chemical, radiation, T-DNA,…)A Mutagenesis

M1 plants

Pools of M2 seeds

Pool 2 Pool 1

Harvested In pools

“Recessive phenotypes appear here”

Strain A/A

Strain A/B

Strain B

Pool 3, etc.

B Screening

Screen M2 pools (1, 2, etc.) for mutant phenotypes

Mutant mut-1

Propagate mutant from mut1/mut-1 or from its mut-1/+ heterozygous siblings

Re-screening

Establish segregation ratio

- Recessive or dominant?- Monogenic or polygenic?- Penetrance?

Initiate mutant characterization

M3 seedlings

M2 seedlings

Mutant phenotype

Wild-type phenotype

Legend

M3 plantmut-1/mut-1

xDBackcrossing

Wild type+/+

Multiple backcrosses to remove background mutations

BC1 plantmut-1/+

x

Wild type+/+

ECareful phenotypic study

BC2 plantmut-1/+ “What exactly is wrong?”

M3 plantmut-1/mut-1

x

M3 plantmut-2/mut-2

CAllelism Tests

mut-1/mut-2

AllelismSingle gene

mut-1/+mut-2/+No allelismTwo genes

Case 1: Case 2:

“How many genes are involved?”

G Gene Cloning

xWild type +/+Strain B

mut-1/mut-1Strain A

OutcrossF Mapping

mut-1/+Strain A/Strain B

Examine co-segregation of mutant phenotype versus strain-specific (visible or molecular) traits

+/+ mut-1/mut-1

x Option 2: Selfing

mut-1Strain A

mut-1/mut-1

x

Option 1:Backcross

Mapping population Mapping population

“How does the gene function?”

“Where is the gene located?”

Mutagenesis - Overview

Wild type

Mutagen

(chemical, radiation, T-DNA,…)

A Mutagenesis

M1 plants

Pools of M2 seeds

Pool 2 Pool 1

Harvested In pools Mutant phenotype

Wild-type phenotype

Legend

Pool 3, etc.

B Screening

Screen M2 pools (1, 2, etc.) for mutant phenotypes

Mutant mut-1

Propagate mutant from mut1/mut-1 or from its mut-1/+ heterozygous siblings

Re-screening

Establish segregation ratio

- Recessive or dominant?- Monogenic or polygenic?- Penetrance?

Initiate mutant characterization

M3 seedlings

M2 seedlings

Mutant phenotype

Wild-type phenotype

Legend

M3 plantmut-1/mut-1

x

M3 plantmut-2/mut-2

C Allelism Tests

mut-1/mut-2

AllelismSingle gene

mut-1/+mut-2/+

No allelismTwo genes

Case 1: Case 2:

“How many genes are involved?”

Mutant phenotype

Wild-type phenotype

Legend

M3 plantmut-1/mut-1x/x

x

D Backcrossing

Wild type+/+X/X

Multiple backcrosses to remove background mutations

BC1 plantmut-1/+x/X

x Wild type+/+X/X

E Careful phenotypic study

BC2 plantmut-1/+X/X

BC2 plantmut-1/mut-1

G Gene Cloning

x

Wild type +/+Strain B

mut-1/mut-1Strain A

Outcross

F Mapping

mut-1/+Strain A/Strain B

Examine co-segregation of mutant phenotype versus strain-specific (visible or molecular) traits

+/+ mut-1/mut-1

x Option 2: Selfingmut-1Strain A

mut-1/mut-1

x

Option 1:Backcross

Mapping population Mapping population

“How does the gene function?”

“Where is the gene located?”

Strain A/A

Strain A/B

Strain B

Mutant

Wild type

Heterozygote