Transcript Metoda Pemuliaan Tanaman Secara Khusus

Mutation Breeding
Taryono
Faculty of Agriculture
Gadjah Mada University
Mutation - Mutant
 Mutation
Changes in genes and chromosomes
 Mutated
Altered genes
 Mutant
New organism with a mutated gene or
rearranged chromosomes
Mutation Breeding
 Advantages
– Screen very high populations (cell based)
– Can apply selection to single cells
 Disadvantages
– Many mutations are non-heritable
– Requires dominant mutation (or double recessive
mutation); most mutations are recessive
 Can avoid this constraint by not applying selection pressure in
culture, but you loose the advantage of high through-put
screening – have to grow out all regenerated plants, produce
seed, and evaluate the M2
 Alternative: perform on haploid cell lines
Mutation
 May involve any trait
 All kind of transition are encountered,
from drastic morphological changes
deviations in physiology so minute as to be
almost indiscernible
 Harmful or even lethal
Type of mutation
 Spontaneous (natural) mutation
1.
Some have played an outstanding role in
development of valuable crop cultivars and
hybrids
2. Unfortunately, it can not form the basis of
modern plant breeding due to its low
frequency and difficulties in detection
 Induced mutation
Genetic structure changes
 Gene (point mutation)
 Chromosome
 Genome
Gene (point) mutation
→ a change in specific sequence of nucleotides in DNA
molecules leading to the formation of a new type of
protein or preventing that of the normally protein
→ take place at the molecular or sub-microscopic level
→ Such change may be accompanied by the emergence of a
new trait inherited in accordance with Mendel’s Laws
Chromosomal mutation
 Mutation associated with splitting and subsequent changes
in the structure of the chromosomes
 The end of the split chromosomes may fuse to form
structure again, but the new chromosomes are not always
exactly what the used to be
 The microscopic structures of chromosomes may be
characterized by deletion or deficiency (loss of a
chromosomal segment), duplication (doubling of a
chromosomal segment), inversion (rearrangement of a
group of genes in a chromosomal segment in a such a way
that their order is reversed; rearrangement of genetic
material in a chromosome results from loss of segment, its
rotation by 180°, and fusion of the separated ends) and
translocation (change in a position of a chromosome or
more often exchange of segments between different
chromosomes)
Genome mutation
Changes 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 mutation
1.
2.
3.

X rays can significantly promote mutation in fungi (1925)
X rays produced pronounced mutagenic effect on the
fruit fly Drosophila (1927)
Artificial mutants can serve as good source material in
plant breeding; X rays induce mutations in Maize and
barley (1928)
Today there are three groups of breeders:
1)
2)
3)
Mutation breeding is useless, we can accomplish the same thing
with conventional methods
Mutation breeding will produce a breakthrough given enough
effort
Mutation breeding is a tool, useful to meet specific objectives
Technique for inducing mutation
 Physical mutagens
 Chemical mutagens
Physical mutagens
1. Various sources of ionizing radiations are explored, most
often X and gamma rays, UV radiation, fast and slow
neutron, alpha ray, beta ray
2. Radioactive isotopes P-32 and S-35 are not convenient for
use due to the storage and application difficulties
3. The usual sources of gamma rays in laboratories are
radioactive cobalt (Co-60) and Cesium (Cs-137) placed in
cobalt bomb
Physical mutagens
4. The object can be irradiated in two ways:


5.
6.
With an aid of a powerful source of a short-duration gamma
rays for short duration radiation. Need special units for
irradiating living object
A much weaker radiation but operating continuously (gamma
field).
the dosage must be varied depending not only on the plant
species whose seeds/organs are irradiated, but also on many
other factors
plant must be irradiated heavily enough to ensure as many
inherited changes as possible but without seriously affecting
the germination, growth and fertility of plant directly emerging
from the irradiated seeds or vegetative organs (critical
radiation dose: dosage which strong enough to assure many
mutation not yet so strong as to kill plants)
Chemical mutagens
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
Mutagenic substances belonging to different classes of
chemical compounds, such as ethylene imine, diethyl
sulfate, dimethyl sulfate, N-nitrosoethyl urea, Nnitrosomethyl urea, methal sulfonate, diepoxy butane,
ethyleneoxide
Most are highly toxic, usually result in point mutations
Use in solution in the concentration ranging from tenth –
hundredths even thousandths of percent
Many chemical mutagens are much more effective than
physical one. If irradiation of crops produces 10 – 15% of
viable inherited changes, chemical mutants do the same
at a rate of 30 to 60%
They often exert more specific and finely tuned action on
the cell
Chemical mutagens
 Some substances (supermutagen) are capable
of causing inherited changes in plants at a rate
up 100%
 Chemical mutagens aim at the most vulnerable
spot of a living organism (DNA) to induce
changes in nucleotides and alter the genetic
information (Sometimes causes specific
mutation)
 It provides a powerful tool to induce desire
changes in a trait
Use of mutations in sexually
reproduced crops
 More valuable in self than cross pollinated.
The probability of producing desirable
mutations and genetic variability is
theoretically higher
 Seeds
 Very young seedling
Use of mutations in asexually
produced crops








It has been much easier and quicker to obtain variant
plant types
Specific location of the mutation event (segmental
chimera) becomes important.
The mutant must be in meristematic tissue that will
produce faithfully through cutting or other vegetative
means
Bud
Scion
Cutting
Tuber
bulbs
Traditional Mutation Breeding Procedures
 Treat seed with mutagen (irradiation or
chemical)
 Target: 50% kill
 Grow-out M1 plants (some call this M0)
– Evaluation for dominant mutations possible, but most
are recessive
 Grow-out M2 plants
– Evaluate for recessive mutations
– Expect segregation
 Progeny test selected, putative mutants
– Prove mutation is stable, heritable
Mutation breeding scheme for
seed propagated crop
 Mutagenic application
 Growing the plants (M1 generation)
 Identification of induced mutation, seed harvest
from mutated plants (M2)
 Continue the identification and selection of
induced mutation (M3)
 First agronomic evaluation. Propagation of
promising lines (M4)
 Multilocation trials of stable mutant and
recombinant lines (M5 – M8)
 Official testing and releasing of mutant (M9)
Mutation breeding scheme for
vegetative propagated crop
 Mutagenic application
 Cutting back the M1V1 shoot, bud grafting, or in vitro
propagation via axillary buds
 Isolation of induced somatic mutation, establishment of
clones, cutting back of non-mutant shoots from chimeric
plants (M1V2)
 Further isolation of somatic mutations, vegetative
propagation of mutant plant (in vivo or in vitro), preliminary
evaluation of mutants (M1V3)
 Evaluation of mutant clone performance, assesing
segregation from mutant crosses and reselection of
desired recombinants. Released of improved mutant
(M2V4)
Requirements for Mutation Breeding
 Effective screening procedure
– Most mutations are deleterious
 With fruit fly, the ratio is ~800:1 deleterious to beneficial
– Most mutations are recessive
 Must screen M2 or later generations
 Consider using heterozygous plants?
– But some say you should use homozygous plants to be sure effect is
mutation and not natural variation
 Haploid plants seem a reasonable alternative if possible
– Very large populations are required to identify desired
mutation:
 Can you afford to identify marginal traits with replicates &
statistics? Estimate: ~10,000 plants for single gene mutant
 Clear Objective
– Can’t expect to just plant things out and see what
happens; relates to having an effective screen
– This may be why so many early experiments failed
Mutation detection
 Detection, isolation and testing mutants are
extremely difficult
 Due to the sporadic nature of viable useful
mutations, it is advisable to have larger plant
population
 When mutagens are used in breeding, the
biological nature of the trait (dominance or
recession of the mutation) and crops must
be taken into account
Trend in plant breeding based
on mutation
 Mutagens are used to induce mutations within a broad
range and at a high frequency to obtain ample source of
material for selection
 Mutant with a specific changes in certain characters are
created in order to correct some defects in crop varieties. It
is important that the other economic characters remain
unaltered
 Mutagens can be used to solve special problems in plant
breeding for instance by increasing the number of genetic
recombinations and breaks of undesirable linkages,
transferring chromosomal fragment from one plant species
into chromosomes of another during hybridization,
obtaining homozygous mutant through irradiation of
haploids with subsequent doubling of chromosome number
Mutation useful for crop
improvement
 Useful mutation
1.
2.
3.
4.
Any mutational change in a character which can be put to
practical use
Improve nutrition value of crop product
Short stem
High lodging resistance
Disease resistance
Successes of Mutation Breeding
Herbicide Resistance and Tolerance
 Resistance: able to break-down or metabolize the herbicide –
introduce a new enzyme to metabolize the herbicide
 Tolerance: able to grow in the presence of the herbicide –
either ↑ the target enzyme or altered form of enzyme
– Most successful application of somaclonal breeding have been herbicide
tolerance
– Glyphosate resistant tomato, tobacco, soybean (GOX enzyme)
– Glyphosate tolerant petunia, carrot, tobacco and tomato (elevated EPSP
(enolpyruvyl shikimate phosphate synthase)
)
 But not as effective as altered EPSP enzyme (bacterial sources)
– Imazaquin (Sceptor) tolerant maize
 Theoretically possible for any enzyme-targeted herbicide – it’s
relatively easy to change a single enzyme by changing a single
gene
Mutagenesis - Overview
x
Legend
Wild type
M3 plant
mut-1/mut-1
M3 plant
mut-2/mut-2
Case 1:
D
Backcrossing
x
M3 plant
mut-1/mut-1
Wild type
+/+
Case 2:
Mutagen
A Mutagenesis
x
(chemical, radiation, T-DNA,…)
Mutant phenotype
M1 plants
mut-1/mut-2 mut-1/+
mut-2/+
No allelism
Allelism
Single gene Two genes
Harvested
In pools
Pool 1
Pool 2
Pool 3, etc.
Wild-type phenotype
Strain A/A
Pools of
M2 seeds
Strain A/B
C
Allelism Tests
BC1 plant
mut-1/+
Wild type
+/+
E
Careful
phenoty
pic
study
Multiple
backcrosses
to remove
background
mutations
BC2 plant
mut-1/+
“How many genes are involved?”
“What exactly is wrong?”
Strain B
“Recessive phenotypes appear here” F Mapping
Screen M2 pools (1, 2, etc.)
+/+
for mutant phenotypes
“Where is the
gene located?”
M2 seedlings
B Screening
Mutant
mut-1
Re-screening
Establish segregation ratio
mut-1/mut-1
mut-1/mut-1
x
Wild type +/+
Strain B
Propagate mutant from
mut1/mut-1 or from its
mut-1/+ heterozygous siblings
M3 seedlings
Outcross
mut-1/mut-1
Strain A
G Gene
Cloning
Option 1:
Backcross
mut-1/+
Strain A/Strain B
x
mut-1
Strain A
- Recessive or dominant?
- Monogenic or polygenic?
- Penetrance?
x
“How does the
gene function?”
Option 2: Selfing
Examine
co-segregation of
mutant phenotype
versus strain-specific
(visible or molecular)
traits
Initiate mutant characterization
Mapping population
Mapping population
A Mutagenesis
Wild type
Mutagen
(chemical, radiation, T-DNA,…)
Legend
M1 plants
Harvested
In pools
Mutant phenotype
Pool 1
Pools of
M2 seeds
Pool 2
Pool 3, etc.
Wild-type phenotype
B Screening
Screen M2 pools (1, 2, etc.)
for mutant phenotypes
Legend
M2 seedlings
Mutant
mut-1
Propagate mutant from
mut1/mut-1 or from its
mut-1/+ heterozygous siblings
M3 seedlings
Mutant phenotype
Re-screening
Establish segregation ratio
- Recessive or dominant?
- Monogenic or polygenic?
- Penetrance?
Initiate mutant characterization
Wild-type phenotype
C Allelism Tests
“How many genes
are involved?”
x
M3 plant
mut-2/mut-2
Case 1:
M3 plant
mut-1/mut-1
Legend
Case 2:
Mutant phenotype
mut-1/mut-2
mut-1/+
mut-2/+
Allelism
Single gene
No allelism
Two genes
Wild-type phenotype
D Backcrossing
x
M3 plant
mut-1/mut-1
x/x
BC1 plant
mut-1/+
x/X
Multiple
backcrosses
to remove
background
mutations
Wild type
+/+
X/X
x
Wild type
+/+
X/X
BC2 plant
mut-1/+
X/X
BC2 plant
mut-1/mut-1
E Careful phenotypic study
F Mapping
“Where is the
gene located?”
Outcross
+/+
x
Wild type +/+
Strain B
mut-1/mut-1
mut-1/mut-1
Strain A
Option 1:
Backcross
mut-1
Strain A
G Gene
Cloning
mut-1/+
Strain A/Strain B
x
Strain A/A
mut-1/mut-1
x
“How does the
gene function?”
Option 2: Selfing
Strain A/B
Examine
co-segregation of
mutant phenotype
versus strain-specific
(visible or molecular)
traits
Strain B
Mutant
Heterozygote
Wild type
Mapping population
Mapping population