An overview of mutations its importance and drawbacks with its application in the field of Plant Breeding and crop Improvement.

1. Mutation And Its Role In
Crop Improvement
By:-
Ankur Kumar
MSc.
Genetics & Plant Breeding
Birsa Agricultural University, Ranchi.

2. INTRODUCTION:-
 The current day world scenario is really an alarming one. The whole of
natural cycles are disturbed.
 The earth is facing a drastic change in its climatic attributes and yet
another problem is the ever increasing human population.
 With the change in climatic cycles ,there are a lot of problems like global
warming, recurrent floods and droughts, disturbed cycles of rainfall,
increase in ambient temperature of earth etc.
 These climatic imbalance cause various biotic stress and are also one of
the cause of various pests attack in form of both insects and pathogens
constituting to abiotic stress to the plants.
 It’s the task of we agricos to get to the solution and despite these harsh
challenges of the day keep the production and productivity of our crops
High. This can only be achieved through better stable varieties of the
crops who not only give better yields but is also resistant and tolerant to
various biotic and abiotic stresses.

3.  The improvement of the crop varieties which can be better yielders along
with a capability of sustaining both biotic and abiotic stress is really
important.
 Crop Improvement includes development of new varieties over the pre
existing varieties in terms of absolute yield, stability of yield, improved
quality and market.
 In traditional plant breeding technique we have a number of crop
improvement techniques, but they work on selection and segregation
techniques.
 While Mutation becomes an important aspect of crop improvement as it
focuses on creation of variations.
 Many studies have supported , mutation as one of an important tool to
develop varieties resistant in terms of stress conditions both biotic and
abiotic in nature.

4. Mutation and its Historical background:-
 Mutation:- Mutation can be defined as a sudden heritable change in the
character of an organism which is not due to either segregation or
recombination.
 This change in the character of the individual is due to change in number
or sequence of nucleotides.
 The term mutation was first used by Hugo de Vries to describe the sudden
phenotypic changes which were heritable, while working with Oenothera
lamarckiana. (1900)
 But the earliest record of mutations dates back to 1791 when Seth Wright
noticed a male lamb with unusually short legs in his flock of sheep. This
lamb served as a source of short leg trait for the development of Ancon
breed of sheep.
 The systematic studies on mutations were started in 1910 by T.H. Morgan
who used Drosophila melanogaster (white eye mutant) for his studies.

5.  In 1927, H.J. Muller demonstrated for the first time the
artificial induction of mutations by using x-rays in Drosophila.
 Similarly in 1928, L.J. Stadler demonstrated an increase in the
rate of mutations due to x-rays in barley and maize.
 Induction of mutations by chemicals in fungus Aspergillus was
demonstrated by R.A. Steinberg in 1939.
 C. Auerbach and J.N. Robson in 1946 used chemicals to
induce mutations in Drosophila.
 The first plant breeding programme using mutations
(mutation breeding) was initiated in 1929 in Sweden, Germany
and Russia.
 In India it was initiated in early 1930s.

6. Important terms related to Mutation:-
 Muton: The smallest unit of gene capable of undergoing
mutation and it is represented by a nucleotide.
 Mutator Gene: A gene which causes another gene or genes to
undergo spontaneous mutation.
 Mutable Genes: Genes which show very high rates of mutation
as compared to other genes.
 Mutant: An organism or cell showing a mutant phenotype due
to mutant allele of a gene.
 Mutagen: A physical or chemical agent which induces mutation.
 Hot Spots: Highly mutable sites with in a gene.
 Gene mutations or Point mutations: The changes which alter the
chemical structure of a gene at molecular level.

7. Classification Of Mutation:-
Based on direction of mutations :-
a) Forward mutation : Any change from wild type
allele to mutant allele.
b) Backward mutation or Reverse mutation: A change
from mutant allele to wild type.
Based on source / cause of mutations :-
I. Spontaneous mutation: Mutation that occur
naturally.
II. Induced mutation: Mutation that originates in
response to mutagenic treatment.

8. Based on tissue of origin :-
a) Somatic mutation: A mutation occurs in the somatic tissue of an
organism constitute somatic mutation.
b) Germinal mutation: A mutation in germline cells or in reproductive
tissues of an organism constitute germinal mutation.
Based on trait or character effected :-
a) Morphological mutation: A mutation that alters the morphological
features of an individual are called morphological mutation.
b) Biochemical mutation: A mutation that alters the biochemical function
of an individual is called morphological mutation.

9. Based on effect on survival :-
a) Lethal mutation: Mutation which kills the individual that carries it.
(survival is 0%)
b) Sub-lethal mutation: When mortality is more than 50% of individuals
that carry mutation
c) Sub-vital mutation: When mortality is less than 50% of individual that
carry mutation.
d) Vital mutation: When all the mutant individuals survive (survival-100%)

10. Based on visibility or quantum of morphological effect
produced :-
a) Macro-mutations: The mutations which produce a distinct
morphological change in phenotype (which can be detected easily
with out any confusion due to environmental effects) Generally
found in qualitative characters. Eg : colour of flowers, height of plant
etc. are counted as macro mutations.
b) Micro-mutations: Mutations with invisible phenotypic changes,
(which can be easily confused with effects produced due to
environment). Generally observed in quantitative characters. These
mutations can be distinguished and identified under micro
mutations.

11. Based on the site of mutation or on cytological
basis :-
a) Chromosomal mutations: Mutations associated with detectable
changes in either chromosome number or structure.
b) Gene or point mutations: Mutations produced by alterations in
base sequences of concerned genes.
c) Cytoplasmic mutations: Mutations associated with the changes in
chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA).

12. Mechanism Of Mutation

13. Chromosal Mutation:-
 An abnormal change in the
structure of all or part of a
chromosome, OR in the number of
chromosomes an organism has
 Ex: normal humans have 46
chromosomes
 Humans with Down Syndrome
have 47

14. Trisomy 21

15. Down syndrome (Trisomy 21)
 Extra 21 chromosome
 Effects 1/700
 Alters child’s phenotype–
characteristic facial features, short
stature
 Usually some degree of mental
retardation

16. An Abnormality in Tiger

17. Molecular basis of mutations:-
 The term mutation is presently used to cover only those changes
which alter the chemical structure of the gene at molecular level.
Such changes are commonly referred to as “point mutations”. Point
mutations involve a change in the base sequence of a gene which
results in the production of a mutant phenotype.
 Point mutations can be subdivided into the following three classes
on the basis of molecular change associated with them.
1. Base substitution
2. Base deletion
3. Base addition

18. Base substitution:-
When a single base in a DNA molecule is replaced by another base it
is known as base substitution. This can be of two types.
I. Transition: Replacement of a purine by another purine or a pyrimidine by
another pyrimidine. (or) The substitution of a purine by another purine or
of a pyrimidine by another pyrimidine base in DNA or RNA is known as
transition.
(A G or C T)
I. Transversion: Replacement of a purine by a purimidine and vice versa. (or)
The substitution of a purine by a pyrimidine or of a pyrimidine by a purine
in DNA or RNA is known as transversion.
(A or G C or T or U)

19. Base deletion : In base deletion, one or more bases
are altogether deleted
Base addition: There is insertion of one or more
bases.
If the number of bases added or deleted is not a multiple of
three, a frameshift mutation is obtained, as the reading frame
in such case is shifted from the point of addition or deletion
onwards. Hence, in a frameshift mutation, all the amino acids of
a polypeptide chain located beyond the site of mutation are
substituted / altered.

20. Frameshift mutations: The mutations which arise
due to addition or deletion of nucleotides in mRNA
are known as frameshift mutations, because the
reading frame of base triplets (codons) beyond the
point of addition or deletion is altered as a
consequence of such mutations.

21. Normal Transcription:-
DNA (antisense strand)
mRNA
Polypeptide
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
The antisense strand is the DNA strand which acts as the
template for mRNA transcription.

22. Mutations: Substitutions
Substitution mutation
GGTCACCTCACGCCA
↓
CCAGUGGAGUGCGGU
↓
Pro-Val-Glu-Cys-Gly
Substitutions will only affect a single codon
Their effects may not be serious unless they affect an amino acid that
is essential for the structure and function of the finished protein
molecule (e.g. sickle cell anaemia)
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids

23. The genetic code is degenerate
A mutation may have no effect on the phenotype
Changes in the third base of a codon often have no
effect.

24. No change
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Substitution mutation
GGTCTTCTCACGCCA
↓
CCAGAAGAGUGCGGU
↓
Pro-Glu-Glu-Cys-Gly

25. Disaster
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Substitution mutation
GGTCTCCTCACTCCA
↓
CCAGAAGAGUGAGGU
↓
Pro-Glu-Glu-STOP

26. Mutations: Inversion
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Inversion mutation
GGTCCTCTCACGCCA
↓
CCAGGAGAGUGCGGU
↓
Pro-Gly-Glu-Cys-Gly
Inversion mutations, also, only affect a small part of the
gene

27. Mutations: Additions
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Addition mutation
GGTGCTCCTCACGCCA
↓
CCACGAGGAGUGCGGU
↓
Pro-Arg-Gly-Val-Arg
A frame shift mutation

28. Mutations: Deletions
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Deletion mutation
GGTC/CCTCACGCCA
↓
CCAGGGAGUGCGGU
↓
Pro-Gly-Ser-Ala-Val
A frame shift mutation

29. Characteristic features of mutations:-
 Mutations are mostly recessive and very rarely dominant.
 Most mutations have harmful effects and very few (less than 0.1 %) are
beneficial.
 Mutations may be due to a change in a gene, a group of genes or in
entire chromosome.
 If gene mutations are not lethal, the mutant individuals may survive.
However, chromosomal mutations are generally lethal and such mutants
do not survive.
 If mutation occur at both loci simultaneously, the mutants can be
identified in M1 generation. However, if it is restricted to one locus only,
the effect can be seen only in M2 generation.
 Macro-mutations are visible and can be easily identified, while micro-
mutations can not be seen with naked eye and need special statistical
tests (or statistical analysis).

30.  Many of the mutants show sterility.
 Most mutants are of negative selection value.
 Mutation for altogether new character generally does not occur.
 Mutations are random i.e. they can occur in any tissue or cell of an
organism. However some genes show higher mutation rate than others.
 Mutations are recurrent i.e. the same mutation may occur again and again.
 Induced mutations commonly show pleiotropy often due mutation in
closely linked genes.

31. Mutation As A Source
Of Crop Improvement

32. Mutations of useful in case of Crop Improvement:-
 Spontaneous mutations: Spontaneous mutations occur naturally without
any apparent cause. There are two possible sources of origin of these
mutations.
1. Due to error during DNA replication.
2. Due to mutagenic effect of natural environment Eg : UV rays from sunlight
The rate of spontaneous mutations is very low. 1 in 10 lakhs i.e. 10−6 But
different genes may show considerably different mutation rates. In crop plants
some varieties were developed through spontaneous mutations. They are-
Crop Variety
1. Rice GEB-24, Dee-Geo-Woo-Gen
2. Wheat Norin
3. Groundnut TMV-10
4. Sorghum Co-4 (coimbatore 4)

33.  Induced Mutation:-
Mutations can be induced artificially through treatment with either physical
or chemical mutagens. The exploitation of induced mutations for crop
improvement is called mutation breeding. The rate of induced mutations is
very high. The induced mutations did not differ from spontaneous
mutations in expression.

34.  Artificial induction of mutations: Mutations can be induced
artificially using
1. Physical mutagens or radiations
2. Chemical agents

35. 1. Physical mutagens:-
Include various types of radiations, viz., x-rays, g-rays, a-rays, ß-rays, fast
neutrons, thermal or slow neutrons, UV rays etc. The physical mutagens are
classified into
 Ionizing radiations: They work through the release of ions. They have
deep penetrating capacity. Eg : x-rays, g-rays, a -particles etc. For
irradiation special units are used. With an aid of a powerful source of a
short-duration gamma rays for short duration radiation. A much weaker
radiation but operating continuously (gamma field).
 Non-ionizing radiations : They function through excitation and have a
very low penetrating capacity. Eg : UV rays.(U V Rays works on the
principal of formation of Pyrimidine dimers and Pyrimidisation.

36. 2. Chemical mutagens :-
1. Alkylating agents: This is the most powerful group of mutagens. These
are the chemicals which are mainly used to induce mutations in
cultivated plants. They induce mutations especially transitions and
transversions by adding an alkyl group (either ethyl or methyl) at various
positions in DNA. Alkylation produces mutation by changing hydrogen
bonding in various ways. Eg: Dimethyl sulphonate (DMS), Ethyl methane
sulphonate (EMS),Nitrosomethyl Urea (NMU), Nitrosoethyl Urea (NEU),
Methyl methane sulphonate (MMS).

37.  Base analogues: These are
chemicals which are very similar to
DNA bases, such chemicals are
sometimes incorporated in DNA in
place of normal bases during
replication. Thus they can cause
mutation by wrong base pairing. An
incorrect base pairing results in
transitions or transversions after
DNA replication. Eg: 5–
bromouracil, 3-bromodeoxy uridine,
2 -amino purine.

38.  Antibiotics: A number of antibiotics like mitomycin and streptomycin have
been found to possess chromosome breaking properties. Their
usefulness for practical purposes is very limited.
 Acridine dyes: Acridine dyes Eg: proflavin, acriflavin, acridine orange, etc.
are very effective mutagens. These are positively charged and they insert
themselves between two base pairs of DNA. This is known as
intercalation. Replication of intercalated DNA molecules results in
addition or deletion of one or few base pairs which produces frame shift
mutations.
 Intercalating Agents: These are the compounds that can slide between
the nitrogenous bases in a DNA molecule.This tends to cause a greater
likelihood for slippage during replication, resulting in an increase in
frameshift mutations. Example (Sodium Azide)

39. Procedures of Mutation Breeding
 Choice of material
 Choice of mutagen
 Part of the Plant to be Treated
 Dose of mutagen
 Handling of segregating generations

40.  Choice of material : It should be the best variety available in crop and
seed should be pure.
 Choice of mutagen : Generally chemical mutagens are more preferred
for seed treatment and radiations for the treatment of vegetative parts.
 Part of the Plant to be Treated :
Seeds
Pollen grains
Vegetative propagules
Corns
Bulbs
Complete plant

41.  Dose of mutagen
 Mutagens generally induce a high frequency of chromosomal changes
meiotic and mitotic irregularities.
 Optimum mutagen dose is one, which produces maximum frequency of
mutations and causes the minimum killing.
 Close to LD50 dose is optimum. LD50 is the dose of mutagen that kills
50% of the treated individuals.
 Varies with mutagens eg:- EMS – 0.3-1.5 %, for 2-6 hours
 Handling of treated Materials.

42. The handling procedure for selection of a recessive
mutant allele of an oligogene.

43.  M1. A good number of seeds are treated with a mutagen and are spece
planted. In general, the number of treated seeds is so adjusted as to give
to good lot of fertile M1 plants at the harvest. Care should be taken to avoid
outcrossing. M1 plants will be chimeras for the mutations present in
heterozygous state. About 20 to 25 seeds from each M1 spike are harvested
separately to raise the M2 progeny rows.
 M2. Careful and regular observations are made on the M2 rows. But only
distinct mutations are detected in M2 because the observations are based
single plants. All the plants in M2 rows suspected of containing new
mutations are harvested separately to raise individual plant progenies in M3.
if the mutant is distinct, it is selected for multiplication and testing. However,
most of the mutations will be useless for crop improvement. Only 1-3 per
cent of M2 rows may be expected to have beneficial mutations.
 Alternatively, M2 may be grown as a bulk produced by compositing one or
more, but equal number of, seeds from each M1 spike/fruit/branch.
plants are then selected in M2 and individual plant progenies are grown in
M3.

44.  M3. Progeny rows from individual selected plants are grown in M3. Poor
and inferior mutant rows are eliminated. If the mutant progenies are
homogeneous, two or more M3 progenies containing the same mutation
may be bulked. Mutant M3 rows are harvested in bulk for a preliminary
trial in M4.
 M4. A preliminary yield trial is conducted with a suitable check, and
promising mutant lines are selected for replicated multilocation trials.
 M5-M7. Replicated multilocation yield trials are conducted. The out-
standing line may be released as a new variety. The low yielding mutant
lines, however, should be retained for use in hybridization programmes.

45. Mutation breeding for polygenic traits:-
Mutagenesis does produce genetic variation in polygenic traits; this variation is
usually as much as 50% of that generated in F2 generation, but sometimes it
may be as much as or even greater than the latter.
 M1 and M2. M1 and M2 are grown in the same way as in the case of oligogenic traits.
In M2, vigorous, fertile and normal looking plants that do not exhibit a mutant
phenotype are selected and their seeds are harvested separately to raise individual
plant progeny rows in M3.
 M3. Progeny rows from individual selected plants are grown. Careful observations are
made on M3 rows for small deviations in phenotype from the parent variety. Inferior
rows are discarded. Few rows may be homogeneous and would be harvested in bulk.
Selection in done in M3 rows showing segregation; a majority of M3 rows would show
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 in high frequencies that approach
1% or even high, so that their isolation becomes quite cost effective.

46.  M4. Bulked seed from homogeneous M3 rows may be planted in a
preliminary yield trial with a suitable check; superior progenies are selected
for replicated multilocation yield trials. Individual plant progenies from M3
are critically observed. Progenies showing segregation may be subjected to
selection only if they are promising. Superior homogeneous progenies are
harvested in bulk for preliminary yield tests in M5.
 M5-M8. Preliminary yield trials and / or multi-location trials are conducted
depending upon the stage when the progenies become homogeneous.
Outstanding progenies may be released as new varieties.

47. Screening/selection
Mainly two types screening/selection techniques in M2 and subsequent
generation.
 Visual
 most effective and efficient method for identifying mutant phenotypes.
 Visual selection often is the prime basis for selecting for disease
resistance, earliness, plant height, colour changes, adaptation to soil,
climate, growing period etc.
 Mechanical/Physical
Very efficient for seed size, shape, weight, density etc., using appropriate
sieving machinery

48. Achievements Of Mutational
Breeding

51. List of varieties developed in India through
mutation breeding :-

52. 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 increase
the target enzyme or altered form of enzyme.
 This aspect is useful as we can grow weed free plots, as the herbicide
tolerant and resistant plants of ours don’t get affected by the applied
herbicides, while the weeds get killed.
 Glyphosate resistant tomato, tobacco, soybean (GOX enzyme)
 Glyphosate tolerant petunia, carrot, tobacco and tomato (elevated
EPSP (enolpyruvyl shikimate phosphate synthase))
 Imazaquin (Sceptor) tolerant maize

53. Advantages:-
 Mutation breeding is a cheap and rapid method of developing new
varieties.
 Induction of desirable mutant alleles, which is not present in germplasm.
 Induced mutagens is used for the induction of CMS. Ethidium bromide
(EB) has been used for induction of CMS in barley (Minocha et al., 1983)
and pearlmillet (Burton and Hanna, 1976).
 Mutation breeding is more effective for the improvement of oligogenic
characters such as disease resistance.
 Mutation Breeding is very important if we wish to transfer desirable
recessive characters from wild type or for characters which are linked
with undesirable characters.

54. Limitations:-
 The frequency of desirable mutants is very low.
 The process is generally random and unpredictable.
 Identification of micro mutation, which are more useful to a plant breeder
is usually very difficult.
 Mutants have strong negative pleiotropic effects on other traits.
 Health risks: handling, chemical mutagens; radiations, fast neutrons
treatments.
Ways to Mitigate These Limitations:-
 We can mitigate these problems of unpredictable fate of mutations by
increasing the size of population so as to get a good number of mutants.
 Efficient handling of the mutagens should we focused on to eradicate
any health hazards if any.

55. Conclusion:-
 At present genetic variability is narrowed using conventional breeding
approaches for a long period, induced mutagenesis are one of the most
important approaches for broadening the genetic variation and diversity
in crops.
 It has many comparative advantages: it is cost effective, quick, proven
and robust. It is also transferrable and environmentally friendly.
 Crop varieties generated through the exploitations of mutation breeding
are significantly contributing to global food and nutritional security and
improved livelihoods.