APPLICATION OF MUTATION BREEDING IN FIELD CROPS

2. Master’s Seminar Presentation
on
“APPLICATION OF MUTATION BREEDING IN
FIELD CROPS ”
Presented by
V. S. PAWAR
Reg. No: 2015A32M.
Department of Agricultural Botany ,
Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani

3. INTRODUCTION
OBJECTIVES OF MUTATION BREEDING
TYPES OF MUTATION
MECHANISM.
APPLICATION.
PROCEDURE FOR MICRO MUTATIONS BREEDING / SELECTION.
HANDLING OF SEGREGATING POPULATION
SCREENING / SELECTION
BREEDING FOR A BIOTIC AND BIOTIC STRESS.
BREEDING FOR QUALITY.
ACHIEVEMENTS AND LIMITATIONS OF BREEDING .
CONCLUSION.

4. Mutation : Changes in the nucleotide sequence of DNA
Mutation Breeding Term coined by Freisleben and Lein (1944)
MILESTONES IN MUTATION BREEDING
1901-04 de vries : to induced mutation in plants and animals.
1907 Cramer : publishes extensive of spontaneous mutants in crop plants
1922 Alberto : X-rays & ultra-violet rays used for inducing mutation.
1927 Muller : Induced mutation in Drosophila fly used X-rays.
1928 Stadler : Induced mutation in Barley and Maize.
Plant breeders are handicapped due to lack of availability or non-existence
of desired genotypes.
The purpose of induced mutations is to enhance the mutation frequency
rate in order to select appropriate variants for plant breeding.
Mutations are induced by physical and chemical mutagen treatment of
both seed and vegetatively propagated crops.
Induced mutations has over the past 50 years played a major role in the
development of superior crop varieties translating into a tremendous
economic impact on agriculture and food production.

5. To study the relative effects of physical and chemical mutagens on some
biological parameters in various generation of field crops.
To estimate the frequency and spectrum of chlorophyll & viable mutations
in field crops.
To study the extent of induced genetic variability for quantitative characters
in various generations.
To isolate the desirable mutants for both qualitative and quantitative traits
in various generation.
To study the breeding behaviour of selected mutant lines and their analysis
for protein and oil content in various generation
To induced mutations for enhancing variability in field crops .
To study the qualitative and quantitative traits of various field crops .
To isolate and identify desirable mutant in field crops .
To improve the genotype .
To improving both oligogenic as well as polygenic characters.

6. A. Spontaneous mutation : Mutation occurs in natural populations.
B. Induced mutation : Mutation may be artificially induced by
various mutagenic agents.
Induced mutations are of two types :
Mutations called micro and macro mutations depending on the magnitude
of phenotypic effect produced by them.
I . Macro-mutation : Mutation with distinct morphological changes in the
phenotype produces a large phenotypic effect
Easily recognizable on individual plant basis.
Oligogenic in nature.
It can be easily selected in M2 generation.

7. II. MICROMUTATION : Mutation with invisible phenotypic changes
Produces small phenotypic effect.
It cannot be recognizable.
Detected only in group of plants and need treatment of statistical data.
Polygenic nature and selection delays till M3.

8. MUTATION ARE INDUCED BY USING VARIOUS MUTAGENE
Mutagens Physical or chemical agents which greatly enhance the frequency
of mutation
TYPES OF MUTAGENENS
I. Ionizing radiation
a. Particulate radiation : alpha rays, beta-rays, Fast neutrons and
thermal neutrons
II. Non –ionizing radiations : X-rays and gamma rays
I. Alkaylating agents : EMS , MMS etc
II. Acridine dyes : Proflavin, Acridine orange , Acridine yellow and
Ethidium bromide
III. Base analogues : 5 bromo Uracil, % Chlorouracil
IV. Other Mutagens : Nitrous acid , Sodium Azide

9. A. Physical mutagene
Animation : Mechanism action of radiation.

10. Transition :-
Transition is the transfer of one purine (adenine, guanine) by
another Purine or one Pyramidine (thymine, cytosine) by another in a DNA
molecule.
 Out of four bases of DNA, adenine pairs with thymine and guanine with
cytosine but in a mutated state a rare adenine can pair with cytosine creating
an abnormal DNA molecule, which will mutate.
Transversion :-
Transversion is substitution of purine bases by pyramidine and vice-
versa and leads to mutation.
Deamination :-
Replacement of amino group by hydroxil group is called
Deamination . Deamination of cytosine, for example, produces uracil, which
pairs as thyarnine . This type of defective pairing leads to mutation. Many of
the mutagenic damages in cell can be repaired back but most mutagens
inactivate the repair enzymes.

11. Development of improved varieties : All over the world,3222 improved
varieties have been developed through induced mutations in different
crops (IAEA, 2015).
Induction of male sterility : CMS mutants have been induced in barley,
sugar beets, pearl millet and cotton.
Production of haploids : Use of X-rays irradiated pollens has helped in
production of haploids in many crops.
Creation of genetic variability: for increasing the range of genetic
variability in barley, oats, wheat and many other crops
Asexually propagated crops like sugarcane and potato, somatic mutations
may be useful
Overcoming self incompatibility : Mutation of S gene by irradiation
leads to restoration of self fertility in self incompatible species.
Improvement in adaptation: of Hibiscus subdarifa. Induced mutants
of this species could be successfully grown 1600 kilometer north of its
normal habitat (Gregory).

13. It should be the best
variety available in
the crop
Seed should be
pure
Source : Oladosu et. Al., 2016

14. Seeds
Pollen grain
Vegetative Propogation
Corns
Bulbs
Complete plants

15. Mutagen treatments Effects :-
I. Reduce germination
II. Growth rate
III. Vigour &
IV. Fertility
Mutagens generally induce a high frequency of chromosomal changes
and meiotic and mitotic irregularities .
Optimum mutagen dose is one , which produces maximum frequency
of mutations and causes the minimum killing .
Close to mLD50 dose is optimum.
Varies with crops Eg. 46 krad for Vicia faba,
120-140 krad for Brassica napus
Varies with mutagens
Eg. EMS 0.3 -1.5%, FOR 2-6 HOURS

16. PHYSICAL MUTAGENS
FAST
NEUTRONS
3-8Gy 2-6Gy 2-5 GY
X-RAYS 95-250Gy 150-250Gy 150-400Gy
GAMMA RAYS 100-350Gy 50-350Gy 150-400Gy
CHEMICAL MUTAGENS
EMS 0.2-0.5% X 8-20 h 0.01-0.04% x 10-30 h 0.02-2.5 % x 8-20 h
DMS 0.01-0.05 % x 4-6 h 0.005-0.04% x 5 h 1.0-1.5 % x 8-20 h
NaN3 0.5-2 mM x 4-6 h 0.5-2 mM x 5h 0.5- 1.5 mM x 5 h
EI 0.01-0.03 x 3-6 h 0.04-0.09 % x 3-5 h 0.03-0.06% x 8-12 h

17. M1 GENERATION
Seed treated with chemical mutagens should be washed thoroughly and be
planted as soon as possible
Large M1 generation is raised from treated seeds (Wider spacing)
Eg. 25,000 plants are to be grown to obtain a useful mutation in M1
generation
Mutagens with high mutation frequency M1 generation size can be reduced.
The M1 plants should not be allowed to cross pollination
M1 population should be planted 75-100 m apart from parental or other
genotypes of the same crop species.
Mechanical isolation
M1 generation Dominant mutations are selected each plant selfed and
harvested separately for M2
M2 GENERATION
Two methods of sowing M2 generation can be followed
M1 plant to row where all seeds produced from a single plant are grown in row
M1 spike or branch to row.
Oligogenic mutants with distinct features are identified and selected.

18. Mainly three types screening / selection techniques in M2 and subsequent
generation
i. Visual
ii. Mechanical/ physical
iii. Other methods
I. VISUAL SCREENING :
Most effective & efficient methods for identifying mutant phenotypes .
Visual selection often is the prime basis for selection for disease
resistance, earliness, plant height, colour changes , ion –shattering,
adaptation to soil, climate growing period etc.

19. II. MECHANICAL/PHYSICAL
Very efficient for seed size , shape , weight, density, etc. using appropria
te sieving machinery.
III. OTHER METHODS
Chemical, Biochemical, Physiological
Eg. low alkaloid content mutants can be selected using colorimetric tests.
Chromatographic or electrophoresis techniques may be used to selected
isolate protein variants.

20. METHODS FOR GENERATING MUTANT VARIETIES
MUTAGENESIS
FORWARD GENETICS
CHEMICALS
RADIATION
REVERSE GENETICS
INSERTIONAL MUTAGENESIS
Agro bacterium mediated transformation.

21. Potential line 1000 Grains Weight (g) Panicle length (cm)
MK-Control 18.3 18.87
MK- D 300Gy 19.87 19.51
MK-E 300Gy 21.6 20.99
MK-F 300Gy 21.72 20.52
Soe hay Marn et al , November 2015

22. Numerous mutants have been developed through mutation in
duced , showing enhanced resistance to various diseases
(virus , bacterial , and some extent fungi)
Eg.
Induced mutations at the locus confers resistance to powdery
mildew and barley yellow mosaic virus

23. Dose (kGy)
Radial colony growth ( μ m) at diff erent incubation times (h)
Alternaria alternata Aspergillus flavus
24h 48h 72h 24h 48h 72h
Control 250.0 980.0 1430.0 280.0 1120.0 1510.0
0.5 149.1 738.0 1211.2 156.5 910.0 1258.3
1 20.6 169.3 328.6 98.8 746.6 1152.3
1.5 12.0 137.2 214.5 66.9 569.7 1054.4
2 0 67.1 132.3 32.9 373.6 927.1
2.5 0 0 0 30.9 356.6 821.3
3 0 0 0 0 0 0
Jyoti Prakash Maity et al , November 2011

24. Treatment
100 Grains Weight (g) LLS Rust (1-9 scale)
M3 M4 M3
TFGRG 5 PARENT
Control
52 45 6
200Gy + 3mM NaN3 64.1 57.6 1
300Gy + 3mM NaN3 63.2 52.3 7
Survendu Mondal et al , october , 2007

25. Starch, protein, fatty acid , vitamins ,etc elimination of undesired
substances such as anti-nutritional factors
Raising or lowering the concentration of specific substances such as fatty
acid .
Through mutation is by inducing knock-outs in genes involving in the
metabolic pathways.
Eg. High quality edible oil quality of soybean gamma ray irradiation.

26. M. N. Hajos et al , November 2011
Treatment Dose (kR Gy) Oil content (%)
KA Control 0 21.9
T1 100 23.9
T2 150 22.9
T3 200 23.6
T4 300 21.1

27. JL 12
Control 2097 ± 26.1 44 ± 2.8
100 kR 2174 ± 21.2 52.3 ± 2.05
200 kR 1834 ± 121.62 48.02 ± 5.1
300 kR 1346 ± 16.9 43.8 ± 1.03
JL 24
Control 2073 ± 38.8 47 ± 1.8
100 kR 2358 ± 289.9 55.5 ± 1.2
200 kR 2190 ± 62.2 48.05 ± 1.9
300 kR 1650 ± 76.3
48.5 ± 5.5
L. Tshilenge-Lukanda et al. American Journal of Plant Sciences, 2013

28. Mutagenic Treatment With Gamma Rays.
Uniform 200 pure dry seeds with about 10 ± 1 per cent moisture each
of two varieties viz., JS-97-52 and MAUS-47, along with their F1 (JS -97-52
x MAUS-47) were exposed to 10, 20 and 30 kR dose of gamma rays (CO60)
with a dose rate of 2.39 kR per minute at Nuclear and Agriculture Division,
B. A. R. C. Trombay, Mumbai-400 085 and the same number of untreated
seeds of each varieties served as control.

29. JS 97-52
Control 94.16 14.03 ± 0.33 116.42 ± 2.79 10.19 ± 0.15 11.85 ± 0.33
10 kR 90.37 17.38 ± 0.81 117.52 ± 5.49 10.39 ± 0.26 12.24 ± 0.56
20 kR 85.92 12.46 ± 0.91 105.03 ± 7.19 10.14 ± 0.24 10.84 ± 0.84
30 kR 81.03 17.84 ± 0.68 120.62 ± 5.54 10.53 ± 0.29 12.66 ± 0.67
MAUS 47
Control 93.86 19.41 ± 0.47 103.33 ± 2.07 10.56 ± 0.17 10.92 ± 0.29
10 kR 89.28 24.46 ± 0.92 116.20 ± 4.61 10.84 ± 0.20 12.51 ± 0.53
20 kR 86.64 27.18 ± 1.36 147.22 ± 8.97 10.89 ± 0.31 15.58 ± 0.94
30 kR 82.03 29.66 ± 1.17 157.7 ± 7.96 10.90 ± 0.29 16.83 ± 0.75

30. GROWTH HABIT MUTANTS

31. IAEA mutant database, http://mvgs.iaea.org (2015)

32. IAEA mutant database, http://mvgs.iaea.org (2015)

33. Country Registration date No. of Released Varieties
CHINA 1957-2011 810
INDIA 1950-2010 330
JAPAN 1961-2008 481
VIET NAM 1975-2011 55
PERU 1995-2006 3

34. IAEA mutant database, http://mvgs.iaea.org (2015)

35. VARIETY RELEASE
YEAR
PARENTAGE
JAGANNATH / BSS-873 1975-76 MUTANT OF T-141
HUR-105 2008 MPR 7-2
PTB-58 2009 PTB-20
Gamma radiation induced rice mutants were released in India as high-
yielding varieties under the series ‘PNR’
The two ripening and aromatic mutation – derived rice varieties
‘PNR381’ and PNR 102 currently popular in Haryana and UP.

36. VARIETY MAIN IMPROVED ATTRIBUTES
PUSA-408
(AJAY)
Resistant to Ascochyta blight , high yield, profuse
branching, semi erect,
maturity 140-155 DAS.
PUSA-413
(ATUL)
Resistant to fusarium wilt , Stunt virus and foot rot,
high yield, profuse branching,
semi erect, maturity 130-140 DAS.
PUSA-417
(GIRNAR)
High resistance to fusarium wilt and moderate
resistance to Ascochyta blight , Stunt virus , high yield,
profuse branching, maturity 110-130 das

37. IARI , New Delhi.
BARC, Mumbai
Tamil Nadu Agricultural University, TN
National Botanical Research Institute, Lucknow ,UP

38. Possible to achieve instsnt progress in elite material.
Single trait improvement can be made to an established variety
preferred by producers, processors and / or consumers.
Limited breeding effort required.
Novel variation can be produced.
Single gene mutants with no negative pleiotropic effects are
possible.
For some mutagenic treatment such as gamma and X- ray ,
there is neither residual radiation nor chemical contamination
of the treated material.
The treated material is safe to handle.
Specific genes / traits can be targeted.
Possible to calculate chances of success (Mutation frequency)

39. The process is generally random and unpredictable.
Useful mutant are rare and predominantly rare.
Mutant have strong negative pleiotropic effects on
their traits.
Required lot of space for trail.
Most mutants are of no useful to breeding.
Large population sizes and effective mass screening
methods are required to select rare mutants .

40. CONCLUSION
In the present investigation, it was observed that irradiated and chemically
treated hybrid generated more variability than that of hybridization and
cultivar population.
Single gene mutants with no negative Pleiotropic effects are possible.
Produced dwarf and semi dwarf varieties.
Development of hybrid and synthetic varieties in field crops.
Mutation Breeding helpful in development of disease , pest , resistance
variety.
Induced mutagenesis and its breeding strategies are potential tools for
improving both quantitative and qualitative traits in crops.