Presentation prepared as a part of my Ph.D seminar topic

1. COLCHIPLOIDY AND MUTATIONAL
BREEDING IN TREES
(FOR-602)
Presented by
Mr. Uday kumar
Ph.D. Agroforestry (SAF)

2. colchicine
• Colchicine is a poisonous chemical extracted
from seeds (0.2-0.8%) and bulbs (0.1-0.5%) of
autumn crocus (Colchicum autumnale).
• It is readily soluble in alcohol, chloroform or
cold water, but is relatively less soluble in hot
water.
• Pure colchicine is C22H25O6N.
(Kishor, 2013)

4. Colchiploidy
• Colchiploidy is defined as the polyploidy induced by
application of colchicine.
• Polyploidy is one of the crop improvement tools using
in fruit crops.
• Colchicine treatment is the most effective and the
most widely used treatment for chromosome doubling.
• It has been used with great success in a large number
of crop species belonging to both dicot and monocot
groups.
• This method was already exploited in many fruit crops
for improvement of different traits.
(Kishor, 2013)

5. colchicine
• Colchicine is for inducing polyploidy in plant
cells by inhibiting chromosome segregation
during meiosis.
• Half the resulting gametes contain no
chromosome
• Other half contains double the usual number
of chromosomes.
(Kishor, 2013)

7. Megaspore chromosome doubling in Eucalyptus
urophylla induced by colchicine treatment to
produce triploids
• This study aims to explore a technique to
produce triploid through female gamete
chromosome doubling in Eucalyptus urophylla
• Cytological observation on microsporogenesis
and megasporogenesis were carried out to
guide megaspore chromosome doubling to
investigate the possibility of triploid induction
by colchicine treatment using two different
methods of application
Yang et.al., 2018

8. Materials and methods
• Floral branches used were
selected from Eucalyptus
urophylla clone.
• Flower bud samples were
fixed in FAA (formalin-acetic
acid-alcohol) fixative at 4o c
for 24 hrs.
• The anther containing cells
undergoing meiosis from
each flower bud were
extracted and stained with
2% acetocarmine for
microsporogenesis
observation
Yang et.al., 2018

9. • The ovaries from the same flower bud were
embedded with paraffin then sectioned and
stained with iron hematoxylin for
megasporogenesis.
• After cytological observations the
determination of the developmental process
of the megasporogenesis was identified and
further colchicine treatment was carried on
Materials and methods
Yang et.al., 2018

10. Colchicine treatment
• Floral branches were treated with 0.25% / 0.50%
colchicine solution.
• Colchicine treatment is performed in two
different ways .
1. Colchicine solution was slowly aspirated from
centrifuge tube (ACT) into a cut near a floral
branch.
2. colchicine solution was slowly injected using an
infusion apparatus (II A) into the cut at the base
of floral branch.
Materials and methods
Yang et.al., 2018

11. (a) Colchicine solution was slowly aspirated from a centrifuge tube into a cut near the
floral shoot by capillary action (abbreviated as ACT).
(b) Colchicine solution was injected using an infusion apparatus into a cut at the base
of the floral branch (abbreviated as IIA).
Yang et.al., 2018

12. • Matured seeds both from the treated group and
control group were collected and germinated .
• When the seedlings grew to about 20 cm ht both
flow cytometry and somatic chromosome
counting were used to detect the ploidy level of
offspring.
• A total of 10 phenotypic traits of data were
recorded when all the plants had grown up to 6
months old in the green house
Materials and methods
Yang et.al., 2018

13. Results
Yang et.al., 2018

14. Megasporogenesis stages on different days for colchicine treatments in Eucalyptus urophylla.
Yang et.al., 2018

15. Stomatal characteristics of diploid and triploid plants in Eucalyptus urophylla.
Stomata size and density in Eucalyptus urophylla (scale bar = 20 micro meter).
(a) Triploid plant (b)Diploid plant.
Yang et.al., 2018

16. Descriptive statistics (mean values + SD) of ten quantitative traits measured from the top
mature leaf of Eucalyptus urophylla at 6 months old.
Yang et.al., 2018

17. Ploidy level detection of offspring derived from megaspore chromosome doubling by
colchicine treatment in Eucalyptus urophylla.
(a) Somatic chromosome counting of triploid plant (2n =3x = 33, scale bar = 10 m).
(b) Histogram presenting the flow cytometric analysis results
Yang et.al., 2018

18. Eucalypts grown in greenhouse at 6 months old. (a) Triploid plant. (b) Diploid plant
Yang et.al., 2018

19. Mutational breeding in trees

20. Mutation : Any sudden change occurring in hereditary material of an
organism.
Mutation Breeding : The genetic improvement of plants for various
characters through the use of induced mutation.
Some other terms:
Mutation
Changes in genes and chromosomes
Mutated
Altered genes
Mutant
New organism with a mutated gene or rearranged
chromosomes
Introduction

21. Historical event

23. Mutation
Recessive
Harmful
Random
Recurrent
Pleiotropic
Low
frequency
Imanish et al.,2007,Japan
General Properties of Mutation

24. Physical
mutagens
Chemical
mutagens
Type of Mutation
(Sharma, 2009)

25. mutagens
physical
Ionizing
radiation
Particulate
radiation
Alpha rays
Beta rays
Fast neutrons
Non
particulate
radiation
X-rays
Gamma rays
Non ionizing
radiations
UV rays
Chemical
Alkylating agents
Base analogues
Acridine dyes
Deamination agents

26. IONIZING RADIATION
Particulate radiation : high energy atomic particles generated
due to radioactive decay.
Beta rays: high energy electrons produced by radioactive
decay of H3
, P32
, S35
. And they are negatively charged.
Alpha rays: composed of alpha particles 2 protons +2
neutrons
Fast neutrons : produced in cyclotrons or atomic reactors
due to radioactive decay of heavier elements
Non particulate radiation: high energy radiations composed of
photons
x rays: produced from x ray tubes
Gamma rays: produced by the decay of some radioactive
isotopes C14 , Co60 etc..

27. NON IONIZING RADIATION
• UV rays are the only non ionizing radiation
with mutagenic properties.
Wavelength range: 100-3900 Angstroms
(10-390nm).
Low energy radiation and has a very poor
penetrating power.

28. Induced Mutation
 Mutation which are produced by the use of mutagenic agents(mutagens)
 Mutagens:-Agent which are used for induction of mutation are known as
mutagens
S.N. Type of
radiation
Main properties Mode of action or changes caused
1. X-rays penetrating and non-
particulate.
Induce mutations by forming free radicals
and ions. Cause addition, deletion, transitions
and transversions.
2. Gamma- rays very penetrating and non
particulate.
Induce mutations by ejecting atoms from the
tissues. Cause addition, deletion, transitions and
transversions.
3. Alpha-particles particulate, less penetrating
and positively charged.
Act by ionization and excitation. Cause
chromosomal and gene mutations.
5. Fast & thermal
neutrons
particulate, neutral
particles, highly
penetrating.
Cause chromosomal breakage and gene
mutations.
6. Ultra violet
rays
Non- ionizing, low
penetrating.
Cause chromosomal breakage and gene
mutations.

29. Chemical agents Examples
Mode of action or
changes caused
1) Alkylating agents
2) Base analogues
3) Acridin dyes
4) Others
EMS,EI,
MMS, etc.
5Bromo uracil, 2 Amino
purine.
Acriflavin, proflavin
Nitrous acid,
Hydroxylamine
Sodium azide
Base substitution
AT↔GC Transition
Base substitution
AT↔GC Transition
Frame shift mutation
(addition, deletion of
nucleotides)
GC↔AT Transitions
Cont.…….

32. Effects of mutagens
(Sharma, 2009)

33. Chromosome Mutations
• May Involve:
–Changing the
structure of a
chromosome
–The loss or gain
of part of a
chromosome

34. Chromosome Mutations
• Five types exist:
–Deletion
–Inversion
–Translocation
–Nondisjunction
–Duplication

35. Deletion
• Due to breakage
• A piece of a
chromosome is lost

36. Inversion
• Chromosome segment
breaks off
• Segment flips around
backwards
• Segment reattaches

37. Duplication
• Occurs when a gene
sequence is
repeated

38. Translocation
• Involves two
chromosomes that
aren’t homologous
• Part of one chromosome
is transferred to another
chromosomes

39. Translocation

40. Nondisjunction
• Failure of chromosomes to separate
during meiosis
• Causes gamete to have too many or too
few chromosomes

41. Chromosome Mutation Animation

43. Gene Mutations
• Change in the nucleotide sequence of a
gene
• It includes base substitution and base
addition and deletion

44. Base substitution
• Include:
–Transition
A G
T C
–Transversion
A T/C
G T/C

45. Base Addition and Deletion
• Change of nucleotide sequence
• Includes the deletion and insertion of
nucleotide in a gene
• In addition one or more bases are
added
• In deletion one or more bases are
deleted

46. Frameshift Mutation
• Inserting or deleting one
or more nucleotides
• Changes the “reading
frame” like changing a
sentence
• Proteins built incorrectly

47. Frameshift Mutation
• Original:
–The fat cat ate the wee rat.
• Frame Shift (“a” added):
– The fat caa tet hew eer at.

48. Amino Acid Sequence Changed

49. Gene Mutation Animation

50. Breeding methods

51. Steps of Mutation Breeding
Formulation of objectives of the programme
Selection of the plant part to be used for mutation
Selection of mutagen (physical or Chemical mutagen)
The dosage of mutagen
Giving the mutagen treatment
Handling of mutagen treated population
(Sharma, 2006)

52. Selection of the plant part to be treated

53. Not
(Sharma, 2006)
Pollen grains are not frequently used because:-

54. Procedure

55. Dose and Duration of Mutagen

56. Giving Mutagen Treatment

57. Handling of Mutagenic Population

58. Chimera ?

59. Chimera ?

60. Breeding for oligogenic traits

61. Combining zygotic embryo culture and mutation induction
to improve salinity tolerance in avocado
• In the present work radiation-induced mutation followed by
in vitro culture of zygotic embryos and high osmotic
pressure selection methods to improve salt tolerance in
avocado are investigated
• The in vitro germination, rooting, bud multiplication and
plantlet acclimatization of Cuban avocado varieties were
recorded.
CASE STUDY -2
Fuentus et al., 2009

62. COMBINING MUTAGENIC AND SELECTIVE SALINE
DOSES IN THE SAME APPROACH
The treatments were as follow:
A) Culture of zygotic embryos in MS salt medium for four weeks for
plantlet induction,
B) Culture of zygotic embryos irradiated at mutagenic dose in MS
salt medium for four weeks for plantlet induction,
C) Culture of zygotic embryos irradiated at mutagenic dose in MS
salt medium supplemented with 150 mM of NaCl for four
weeks for plantlet induction,
D) Culture of zygotic embryos in MS salt medium supplemented with
150 mM of NaCl for four weeks for plantlet induction.
Embryos were sub-cultured in fresh medium and grown during eight
weeks and later acclimatization and growing under greenhouse
conditions was done.
Fuentus et al., 2009

68. Conclusion
• The use of in vitro techniques such as anther/microspore
culture, shoot organogenesis and somatic embryogenesis
can overcome some of the limitations in the application of
mutation techniques, such as the lack of effective mutant
screening techniques and the unrealistically large, but
necessary, size of the mutated population.
• Here mutant lines were planted under field conditions for later
segregation analysis for salinity tolerance.

69. Thank you