Full information about mutation for Agriculture students

1. Submitted by:-
Name:- Bhumi A. Gameti
An assignment on
MUTATION AND IT’S DETECTION

2. MUTATION
 The term 'mutation' refers to sudden heritable changes in
the genome, excluding those resulting from incorporation
of genetic material from other organisms.
 In the molecular term mutation is defined as the
permanent and relatively rare change in the number or
a sequence of nucleotides.
 Mutation was first discovered by Wright in 1791 in male
lamb which had short legs. Later on mutation was
reported by Hugo de Vries in 1900 in Oenothera,
Morgan in Drosophila (white eye mutant). The term
mutation was coined by Hugo de Vries.

3. Terminology
 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.
 Antimutator genes :- Decrease the frequency of spontaneous mutation of
other genes of same genome.
 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 within a gene.
 Gene mutations or point mutations: The changes which alter the
chemical structure of a gene at molecular level.

4. Characteristics of Mutations
1. Mutations are mostly recessive and very rarely dominant.
2. Most mutations have harmful effects and very few (less
than 0.1 %) are beneficial.
3. Mutations may be due to a change in a gene, a group of
genes or in entire chromosome.
4. If gene mutations are not lethal, the mutant individuals
may survive.
5. If mutation occur at both loci simultaneously, the mutants
can be identified in M1 generation. However, if it is
restricted to one locus only, (dominant to recessive) the
effect can be seen only in M2 generation.

5. Characteristics of Mutations
7. Many of the mutants show sterility.
8. Most mutants are of negative selection value.
9. Mutations are random i.e. they can occur in any tissue or
cell of an organism.
10. Mutations are recurrent i.e. the same mutation may occur
again and again.
11. Induced mutations commonly show pleiotropy often
due mutation in closely linked genes.

6. TYPES OF MUTATION

7. Based on source
Spontaneous Mutations
 Occurs spontaneously.
1. Due to error during DNA
replication.
2. Due to mutagenic effect
of natural environment
E.g. UV rays from sunlight

8. Based on source
 Induced Mutations
 Mutation that originates in response to mutagenic
treatment

9. Based on direction
Forward mutation Reverse mutation
 Any change from wild
type into mutant allele
 A change from mutant
allele to wild type.
Based on site
Nuclear mutation Cytoplasmic mutation
 Mutation in nuclear gene.  Mutation in cytoplasmic
gene.

10. Based on tissue
Germinal Mutations
 A gene is altered in a germ cell.
 Because germ cells give rise to gametes, some gametes
will carry the mutation and it will be passed on to the next
generation.
 Typically germinal mutations are not expressed in the
individual containing the mutation.

11. Based on tissue
Somatic Mutations
 Mutations in somatic cells.
 To maintain this mutation,
the individual containing
the mutation must be
cloned.
 Two example of somatic
clones are….
navel oranges are seedless
oranges cloned by cutting and
grafting
Red delicious apples. high in dietary fiber
and contain vitamin C

12. Based on character
Morphological
Biochemical
 Alters Morphological character.
 Also known as visible
mutation.
 i.e. Dwarf peas, short legged
sheeps, curly wings in
Drosophila
 Alters production of
biochemicals by the
organism.

13. Based on visibility
Macro-mutations Micro-mutations
 Mutations with large no.
of changes in phenotypes.
 Easily detected
 Found in qualitative
characters.
 Mutations with invisible
phenotypic changes.
 Not easily detected.
(special techniques
requires)
 Observed in quantitative
characters.

14. Based on survival
 Lethal mutation:- Kill all the individuals
 Sub lethal mutation:- Kill most of the individual
 Sub vital mutation:- Kill some of the individual
 Vital mutation:- Do not affect the survival
 Super vital mutation:- Increase the survival

15. Based on cytology
 Chromosomal mutation:-
Changes in either chromosome number or structure.
 Gene mutation:-
Alterations in base sequences of concerned genes.
 Cytoplasmic mutation:-
Changes in chloroplast DNA (cpDNA) and mitochondrial
DNA (mtDNA).

16. Based on the effect on the functions
 Loss of function mutation
Reduce function
 Gain of function mutation
Change in function

17. Mutants
The product of mutation.
 Morphological mutants detected by outward
appearance of an individual. i.e. shape, size and
colour.
 Lethal mutants cause mortal or lethal effect on the
organism.
 Conditional lethal mutants that expresses
characteristics of the wild type when grown under
certain conditions
 Biochemical mutants For bacteria, biochemical
mutants need to be grown on a media supplemented
with a specific nutrient. Such mutants are called
auxotrophs.

18. Mutagens
“Agents which greatly enhance the frequency of mutation.”
Physical mutagens Chemical mutagens
“Include various types of
radiations”
 X-rays
 Gamma rays
 Alpha particles
 Beta particles
 Fast and thermal neutrons
 UV rays
 Alklyting agents
 Base analogues
 Acridine Dyes
 Other Mutagens
 Nitrous acid
 Hydroxylamine

19. Materials used for treating with mutagens
 Seeds
 Pollen
 Vegetative Buds
 Whole Plants
 Bulbils
 Tubers
 Suckers

20. Significance of mutations in Plant Breeding:
1. When a desirable character is linked with an undesirable
character.
2. To know source of resistance gene in the available
germplasm
3. To create variability
4. To develop male sterile lines
5. To create variations in vegetatively propagated plants

21. DETECTION OF MUTATIONS:-
 Scoring of some types of mutations in certain organisms is
relatively easy. For example, mutations for antibiotic
resistance in bacteria are simply detected by plating the
bacterial cells on a medium containing a lethal
concentration of the concerned antibiotic (selective
medium). The colonies that develop on such a medium
will be produced by cells resistant to the antibiotic.
 But detection of morphological mutations in eukaryotes
requires examination of each individual of the population
for the mutant phenotype. This is not only tedious
requiring much time, but is also a source of errors.

22. Detection of Mutations in Prokaryotes

23. 1. Detection of Auxotrophic Mutations
 Auxotrophic mutants are unable to synthesize some
biochemical essential for their growth and development
that wild type cells are capable of producing. Therefore,
such mutants cannot be detected using the simple
approach described before; these mutants are detected by
replica-plating.
 The replica-plating technique was developed by Joshua
and Esther Lederberg in 1952 for direct selection of
bacterial mutants.

24.  In this technique, the cells are
first plated onto the complete
medium (inoculation) to obtain
distinct colonies; this is called
the master plate.
A block of wood or cork of a
size suitable for the master plate
is covered with velvet cloth.
This block is sterilized, and
then lowered into the master plate
till the velvet touches all the
colonies.
Generate the mutants by treating a culture with a mutagen.

25. Now the block is withdrawn
and gently lowered onto a plate
containing the selection medium
so that the bacterial cells
sticking onto the velvet are
transferred onto the medium;
such a plate is known or replica
plate.
For detection of auxotrophic
mutants, the selection medium is
the minimal medium in which
only wild type cells can grow.

26. A reference point is marked both on the master plate and on the
replica plate. This makes it possible to locate in the master plate any
colony of the replica plate.
A single master plate can be used to produce several replica plates.
The colonies that develop on the selection medium plate are due
to wild type cells.
In contrast, those colonies of master plate that fail to grow on the
minimal medium are auxotrophic mutants.

27.  The mutant colonies can be isolated from the master plate
and used for further investigations, e.g., confirmation of
their auxotrophic nature, identification of the deficient
biochemical, etc.
 This approach can be used for detection of other types
of mutants, e.g., antibiotic resistance, etc.

28. 2. The Ames Test
 This test was developed by Bruce Ames (1974) and
coworkers and is based on histidine-requiring (his-)
auxotrophic mutants of Salmonella typhimurium.
 The routine Ames test addresses to both these needs as
follows.
 The his- cells are plated onto a medium that contains
traces of histidine, which is enough to allow a few cell
divisions, but inadequate for visible colony formation.

29. Salmonella typhimurium
Bacteria which is his-ve
Bacteria + Minimal medium (contains traces of histidine)
Chemical Agent
Completely Non-Mutagenic Non Mutagenic Mutagenic
Experiment should be repeat

30.  Ames test is routinely used to investigate the
mutagenicity of various chemicals. But, some of the
chemicals may become mutagenic only when they are
acted upon by liver enzymes.
 For example, nitrates themselves are neither mutagenic
nor carcinogenic. But in eukaryotic cells, nitrates are
converted to introsamines, which are highly mutagenic
and carcinogenic.
 The test chemical is incubated with rat liver extract
containing the liver enzymes, i.e., the microsomal
fraction. This allows modification of the chemical in the
same way as it would be in the liver of animals.

31. In order to increase the efficiency of the test, the his-ve strains used in the test are defective
in DNA repair, and have increased permeability to chemicals. It has been observed that
more than 90% of the chemicals that are mutagenic are also carcinogenic.

32. Mutation Detection in Drosophila
 In Drosophila, several genetic stocks have been
constructed for the detection of lethal and visible
mutations in the X-chromosome and in autosomes.
 The two genetic stocks most commonly used for mutation
detection in X-chromosome are,
 CIB and
 Attached-X stocks.

33. 1. CIB Stock of Drosophila
 This method involves use of a ClB
stock which carries…
(i) an inversion in heterozygous state
to work as crossover suppressor (C),
(ii) a recessive lethal (l) on X-
chromosome in heterozygous state,
and
(iii) a dominant marker, Barred (B)for
the barred eye (narrow eye).
 One of the two X-chromosomes in a
female fly carried all these three
features and the other X-chromosome
was normal. Male flies irradiated for
induction of mutations were crossed to
ClB females. Male progeny receiving
ClB X-chromosome will die.

34.  The ClB female flies obtained in
progeny can be detected by barred
phenotype. These are crossed to
normal males.
 In the next generation 50% of males
receiving ClB X-chromosome will
die.
 The other 50% males will receive X-
chromosome, which may or may not
carry the induced mutation.
 In case lethal mutation was induced
no males will be observed.
 On the other hand, if no lethal
mutation was induced, 50% males
will survive.
 Thus, the ClB method was the most
efficient method for detecting sex
linked lethal mutations.

36. 2. Attached-X Chromosome Technique
 This technique is based on
attached-X (X-XY) females.
 It is designed to study visible sex-
linked mutations in Drosophila.
Mutagen-treated males are mated
with attached-X females.
 The X-XX (super female) and YY
progeny produced from such
crosses do not survive. Only the
X-XY (female) and XY (male)
progeny are recovered.
 All the male (XY) progeny
receive their Y chromosome from
the attached-X female parent,
while their X chromosome is
contributed by the mutagen-
treated male parent.

37. The frequency (%) of a visible mutation
in the X chromosome of the
mutagen-treated males
If a visible mutation was induced in the X chromosome of the sperm (produced by
the mutagen-treated male), it will be expressed in the male progeny.
Therefore, all the male progeny obtained from the cross are scored for visible
mutations. The frequency of a visible mutation is expressed as the ratio between
the number of males showing a mutation and the total number of males in the
progeny.

38. Russell's Test for Dominant Lethals
 The dominant lethal assay in mice was initially used (in
1954) by Russell and coworkers and by others to detect
radiation-induced mutations. Subsequently, it was
extended to study the mutagenicity of chemicals.
Therefore this assay system is often called Russell's test
or dominant lethal test system (DLTS).

39. Detection of Mutations in Plants
 Techniques for detection of mutations are relatively poorly developed for
plant species. The following three approaches are generally adopted for this
purpose.
1. In some species, e.g., maize, strains homozygous for several recessive
genes as well as those for dominant alleles of these genes are available.
 In such a case, seeds or plants of a strain homozygous for several
dominant genes are treated with a mutagen.
 These plants (M1, the generation treated with mutagen) are crossed with
the strain having the recessive alleles of the same genes in homozygous
state (tester strain).
 The treated plants are generally used as female parent due to partial male
sterility in mutagen-treated plants. In the progeny of this cross, plants
exhibiting recessive forms of the concerned characters are counted.

40.  The frequency of mutation for a gene is estimated as follows.
 The plants showing the recessive form of the trait will receive one recessive
allele from the tester parent having the recessive traits. But the other
recessive allele would have been produced due to mutation in the mutagen-
treated parent as this parent had the dominant allele of the gene.

41. 2. In most plant species, however, such strains are not readily available. In
such species, seeds of a variety or strain are treated with a mutagen
and grown to obtain the M1 generation.
 The M1 plants are selfed to avoid out crossing due to partial male sterility
in M1, plants. The seeds thus obtained represent the M2 generation. M2
plants are grown and plants having mutant features are scored.
 The frequency of a given mutation is estimated as per cent ratio between
the number of plants exhibiting a mutation in M2 and the total number of
plants in M2.

42. 3. The above procedures are applicable when mutations are to be
detected in qualitative traits (macro mutations).
In crop improvement, mutations in quantitative traits (micro
mutations) are equally, often more, important.
 For detection of micro mutations, M2 generation is grown as
described above. All M2 plants showing a visible macro
mutation and/or partial pollen sterility are rejected. Seeds from
all normal-looking fully fertile M2 plants are harvested
separately.
 They are planted in individual plant progeny rows in M3
(preferably in a replicated trial).
 Data are recorded on quantitative traits. Means and variances
of M3 progenies are compared with those of the parental
variety to ascertain if an M3 progeny significantly from the
parental variety for a quantitative trait. A significant deviated
deviation from the parent variety will indicate the occurrence
of micro mutations for the concerned trait.

43. REFERENCES
 B. D. Singh, 1990. Fundamentals of Genetics, Kalyani
Publishers, Page no.: 290-308
 Phundan Singh, 2004. Genetics, Kalyani Publishers, Page no.:
208-216
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