Dr. J. Souframanien Nuclear Agriculture & Biotechnology Division Bhabha Atomic Research Centre, Mumbai National seminar

1. Role of induced mutations in legume
improvement

2. Mutation
Radiation induced mutation
Induced variability
New variety development
Achievements of gamma rays
New mutagens for crop improvement
Outlay of the presentation

3. Sources of natural variations
Spontaneous mutation
Occurs in the absence of a
mutation causing agent
Induced mutation
Originates from an
exposure to a mutagenic
agent.
Sudden heritable change in characters of an organism

4. Induced Mutations
Physical mutagens
(Radiations)
X-Rays
Gamma Rays
Neutrons
Beta particles
Alpha particles
Chemical mutagens
EMS
Sodium azide
Acridine orange
etc.

5. Radiation: Energy in the form of particles or
electromagnetic waves`
Ionizing Radiation: Radiation with sufficient energy to
remove an electron from an atom or molecule.
Radiation

6. The spontaneous emission of radiation by unstable atoms
Radioactivity
Paper
Alpha Particles
Beta Particles
Gamma rays
Concrete

7. APPLICATIONS OF RADIATION IN AGRICULTURE AND FOOD
Genetic
improvement
of crop plants
Control of
Insect pest
(SIT)
Fertilizer
use
efficiency
study
Preservation
of
Agricultural
produce

8. Genetic improvement of crops using radiation in Agriculture
Radiation
Gammacell Irradiator
Genetic variation
Parent Mutant
New variety
Induction of genetic variability

9. Effect of Radiation

10. CHROMOSOMAL MUTATIONS

11. Radiation Induced Variability
Narrow leaf mutant

12. Early flowering mutant

13. Terminal pod bearing habit
Mungbean Black gram mutant

14. Blackgram seed colour mutants
Black gram seed size mutants

15. Large seed mutants of mungbean induced by gamma rays
Comparison of Samrat with four LSM’s for pod size
100 seed weight 3.9 6.4 6.2 5.8 5.20

16. Basal branching pigeonpea Determinate cowpea
Mutations for variability… Plant type

17. Yellow mosaic disease resistant mutant
in black gram
Resistant Susceptible

18. TGM 80TGM 78
Mutations for variability… in groundnut

19. Parent TGM 94
Parent TGM 59 TGM 117
Parallel reticulation
Dumb-bell pod
Mutations for variability… Pod type in groundnut

20. Seed mutants…
Parent Mutant
Parent Mutant

21. Mutations for variability… Seeds, Pods in Soybean

22. Simulated water stress
method in the field
Susceptible Tolerant
Long tap root mutant
Long root mutant - mungbean
Long root mutant (71 cm)
Samrat (control) (19.5 cm)

23. Identification of root mutants in in PEG induced water
stress(M2 seedling)

24. Identifying low oligosaccharide mutants in
black gram

25. The raffinose family
oligosaccharides (RFOs), such
as raffinose, stachyose and
verbascose, are synthesized
from sucrose by the
subsequent addition of
galactose moieties donated by
galactinol.
Indigestible in human and monogastric
animals as they lack α-galactosidase
Flatulence causing sugars

26. 0
10
20
30
40
50
60
70
Sucrose Raffinose Stachyose Verbascose Ajugose Total RFOs
Oligosaccharide(mg/gseedmeal)
TU94-2
TU43-1
TU55-1
TU 51
TU 1-820-1-5
P
P
P
(Parent)
Genotype /
Sugar Total RFO (mg/g)
TU 94-2 58.3
TU 1-820-1-5 43.2
TU 43-1 26.6
TU 51 41.8
TU 55-1 36.5
P

27. Development of new crop varieties
Mutant x Mutant
Mutant x cultivar

28. Pigeonpea (Cajanus cajan)
Pigeonpea TT-401
Yield: 1750 kg/ha

29. Trombay Mung varieties
TMB 37
Popular in Madhya Pradesh and
Uttar Pradesh, Early maturing
for summer seasons,
TJM-3
High Yielding & resistant to
PM, YMV & Rhizoctonia root rot
diseases
TM96-2
High yielding, PM resistant
& suitable for rice fallows.

30. Black gram varieties
TAU 1: TAU1 is very popular in Maharashtra covering
maximum black gram area
TU 94-2: Resistant to Yellow Mosaic Virus. Andhra
Pradesh, Karnataka, Kerala, Tamil Nadu

31. Released for Maharashtra, WB, Rajasthan and Karnataka
Trombay groundnut variety, TAG 24 for commercial cultivation
0
1
2
3
4
5
6
7
8
Record yield Most of the
farmers
National
average
Podyieldintonnes/hectare
TAG 24 yield realization on farmers' fields

32. Large seed size in groundnut
Mutant varieties with large seed size which can mature in
shorter time 110-120 days.
TKG-19A, TPG-41, TLG-45, TDG-39
TPG-41
Matures in 115-120 days
High oleic acid (62%)
Seed dormancy of 25 days

33. Defined objective
Use of efficient mutagens.
Seed material
Pure seed, (12-14% moisture content)
LD50 and appropriate dose: one appropriate, second a
little less and third a little more dose
M1 generation
M2 Large population (plant to row)
Rapid screening procedure
The key to the success of a mutation breeding

34. Successes in plant mutation breeding
3218 registered mutant varieties
in 214 plant species
Majority (> 80%) have been developed by nuclear techniques
Most are food security crops
Legumes 492
Others 378
Cereals 1589
Flowers 642
Oil crops 110
Successes in plant mutation breeding

35. Total Number : 3218 Asia: 1937 (60%)
China (810)
India (329)
Russian Federation (216)
Netherland (176)
Mutant Variety Database

36. Mutant cultivars released in India
Total 329 ( 48 crop species)
Group Species No. of mutants % of
total
Cereals 6 71 23.6
Legumes 10 49 16.3
Oil crops 5 33 11.0
Industrial 4 24 8.0
Vegetables 8 11 3.6
Ornamentals 9 102 33.9
Other 6 11 3.6

37. Type of radiation No. of mutants % of
total
Gamma rays 910 64.5
x-rays 311 22.0
gamma chronic 61 4.3
fast neutrons 48 3.4
thermal neutrons 22 1.6
other 59 4.2
Total by radiations 1411 100.0
Mutant varieties developed with
different types of radiation (World)
Source: IAEA

38. New mutagens for crop improvement
Why do we need new mutagens?
What are the new mutagens?
Space radiation
Electron beam radiation
Ion beam radiation

39. Climate Change Impacts

40. New mutagens: Electron and Ion beam radiation
Electrons /Positively charged ions are accelerated at a
high speed (around 20–80%of the speed of light) and
used to irradiate target cells.
As a physical mutagen, ion beams are similar to other
forms of radiation such as X-rays, γ-rays, and
electrons.
It is different from X-rays, γ-rays in that ion beams
have much higher linear energy transfer (LET)
Source: Accelerators

41. Particle accelerators are machines, which are capable of
producing beams of electrically charged particles of kinetic
energy ranging from several hundreds of keV to thousands
of MeV
ION BEAM: cyclotron particle accelerator

42. Centre for Advanced Technology (CAT), Indore, BARC and VECC (Kolkata)

43. Accelerator at Home

44. Ion Beam X-Rays, γ rays
etc.
Mutagen type Physical Physical
Linear energy
transfer (LET)
High Low
LET 10–1000
keV/µm or higher
0.2 keV/µm
Relative biological
effectiveness
High Low
Large DNA alteration
(DSB & deletion)
High Low
Dose rate High (2x105
Gy/sec.)
Low
(10Gy/sec.)
Mutation frequency & Higher Lower
spectrum
Ion Beam Vs Gamma rays

45. LET is the energy deposited to target
material when an ionizing particle passes
through it.
dE is the average energy locally imparted
to the medium.
dx : distance traversed in the medium
Linear Energy Transfer (LET)

46. Different kinds of radiation have
different energy transfer pattern.
Equal doses of different types of
radiation do not produce equal
biological effect.
1 Gy of neutron produces a greater biological effect than 1 Gy
of gamma rays.
The difference lie in the pattern of energy transfer
Relative Biological Effect (RBE)
For low LET » RBE proportional to LET
For high LET » RBE maximum
Over kill effect

47. The optimal LET
The average separation in ionizing events produced by
sparsely ionizing radiation e.g. X-ray is ̴50 A.
LET of 100 keV/µm is optimal in terms of producing
biological effect. Ionizing event=diameter of DNA
double helix leading to double strand breaks (DSBs).
More free radicals are created in short span of time,
which are distributes closely in space.

48. Mutation induced by Ion Beams
48
Energy deposition Irradiation to cell
・ ・・ ・ ・ ・ ・ ・ ・ ・ ・・
g-rays: low LET radiation
spur (~3nm)
300nm
g-rays: 2,000 spurs/Gy
Nucleus
Ion beams: high LET radiation Carbon ions: 4 tracks/Gy
Core
(~3nm) Nucleus
Can ion beams cause different mutation?
Ion-beam induced mutation for plant breeding
1. High mutation rate ：Small samples & spaces for screening
2. Broad mutation spectrum ：Producing new varieties and mutants
3. Minimum No. of DNA damage ：Pinpoint-breeding without bad
characters

49. Carnation varieties developed using ion
beams (Japan).
Parent

50. X-rays
×
Original Var.
Jimba
Ion
beams
Aladdin
Reduced Axillary Buds
Early Flowering
Aladdin 2
Ion
beams
＋
Reduced Axillary Buds
Success of reducing axillary buds:
the greatest goal in chrysanthemum
with Kagoshima Biotechnology Institute
New Varieties

51. Rice breeding suitable for low nitrogen input
- for a solution to eutrophication -
New Varieties
Low-nitrogen-fertilizer
grown rice mutant var.
Original var.
“Akino-uta”

52. Food safely & security:
development of low-cadmium Koshihikari rice
コシヒカリ lcd-kmt1
WT:
Koshihikari
Low-cadmium
mutant variety
Proc. Natl. Acad. Sci. USA (2012)
コシヒカリ lcd-kmt1WT: Koshihikari
Low-cadmium
mutant variety
WT: Koshihikari
Low-cadmium
mutant variety
Field A
(High Cd)
Field B
(High Cd)
Field C
(High Cd)
GrainCd
Maximum
allowed Cd
concent.
Conclusive evidences
1. The same yield and good taste as
original Koshihikari
2. Three independent mutants on the
same gene
(1) One base deletion
(2) Large deletion (277 kbp)
(3) Insertion of transposon (433 bp)
Ion beam breeding is definitely
suitable for staple grain !

53. OrnamentalsModel plants
Low-nitrogen-
fertilizer grown rice
Crops
Novel mutants and varieties induced by ion beams
Trees & fruit
Melon with good
growth in low
temperature
Oyster mushroom
with new character
UV-B resistant
Frilled flower
Flavonoid accumulation
Bacterial-Leaf-Blight
resistant rice
Blast resistant rice
Potato Virus Yellow
resistant tobacco
Yellow Mosaic Virus
resistant barley
Carnation with new
flower color/shape
New flower-color
chrysanthemum
Osteospermum with
new pastel coloration
Ficus with high NO2
assimilation
Chrysanthemum with
reducing axillary buds
53
Hyper-
Yeast producing higher
amount of ethyl caproate
Microorganism
Wax –rich conifer
Thornless mutant of
Yuzu (Citrus junos)
Protease high-producing
Aspergillus
High temperature
resistant rizobium

54. Spectrum and frequency of mutants induced
by electron and ion bean irradiation (India)
Rice
Black gram
1 ?
2?
3?
4?
1 ?
2?
3?
4?

55. Induced mutation stay as an efficient plant breeding
method towards improvement of legumes .
Electron / Ion beams can be utilized as a novel
mutagen to generate new mutants for basic research
and to create new varieties as novel genetic resources.
Mutation induction is producing mutation grids for
gene discovery and gene function analyses an
invaluable resource for genomics, reverse and forward
genetics.
Conclusion

56. Thank you
souf@barc.gov.in