Somaclonal Variation in Perennial Horticulture Crops: Importance and Implication

1. Doctoral Seminar-I
Presented to:
Dr. Kiran Kour
Seminar Incharge
Associate Professor
(Division of Fruit Science)
Presented by:
Pradeepti Sharma
Reg. No.: J-22-D-05-HF
Phd. Fruit Science
Topic:
Somaclonal Variation in Perennial Horticulture
Crops: Importance and Implication

2. Somaclonal Variation in Perennial
Horticulture Crops: Importance and
Implication

3. Index ☟
• Introduction
• History
• Mechanism
• Causes
• Analyses
• Somaclonal Variation in Perennial Horticulture Crop
• Advantage
• Disadvantage
• Future Perspective
• Conclusion

4. Soma" mean the somatic cells and
"clones" means the generations.
The term somaclonal variation was
first coined by Larkin and
Scrowcroft in 1981.
According to Larkin and
Scowcroft, "Somaclonal variation
is the genetic variability which is
regenerated during tissue culture"
Genetic variations in plants that
have been produced by plant tissue
culture and can be detected as
phenotypic traits.
SOMACLONAL

5. Cont.
• .
Variations in number and structure
of chromosomes are commonly
observed.
Regenerated plants with altered
chromosomal changes often show
changes in leaf shape, colour,
growth rate, etc.
It is generally heritable mutations
and persist in plant population
even after plantation into the field.

6. History
• The first well-documented incidence of variation among tissue culture regenerants
was reported by Heinz and Mee (1971) who found individual sugarcane
regenerants with heavier tillering and increased erectness.
• Skirvin and Janick (1976) were among the first to emphasize the potential of
clonal variation for improvement of horticultural cultivars.
• Larkin and Scrowcroft (1981) gave the field a previously unknown legitimacy by
coining the term "somaclonal variation" to describe this variation.

7. Cont.
• The notable example could be banana in which occurrence of off-types from tissue
cultured plantlets ranged from 6 to 38 % in cavendish cultivars (Sahijram et al.,
2003).
• Somaclones may itself have numerous applications in plant breeding and genetic
improvements (Jain 2001).

8. Why somaclonal variation is important?
• Somaclonal variations have provided a new and alternative tool to the
breeders for obtaining genetic variability relatively rapidly in annual as well
as perennial fruit crops, which are either difficult to breed or have narrow
genetic base.

9. Mechanism of somaclonal variations

10. • Pre-existing variations in the somatic
cells of ex plant
• Caused by mutations and other DNA
changes
• Occur at high frequency
1. Genetic (Heritable
variations)
• Variations generated during tissue culture
• Due to cultural conditions
• Occur at low frequency
2. Epigenetic (Non-heritable
variations)

11. Causes of somaclonal variation:
Genetic Cause
Physiological
Cause
Biochemical
Cause

12. Genetic Cause
Change in
chromosome
number
• Aneuploidy
• Polyploidy
• Monoploidy
Change in
chromosome
structure
• Deletion
• Inversion
• Duplication
• Translocation
Gene mutation
• Transition
• Transversion

13. Genetic Cause
Plasma gene
mutation
• Changes in
genetic material
which present
inside
mitochondria
and chloroplast
Transposable
element activation
• Transposable
element /
jumping genes
cause mutation
via replication,
recombination
and repair
DNA sequence
• Methylation of
DNA -
Methylation
inactivates
transcription
process

14. Physiological Cause
• Exposure of culture to plant growth regulators
• Culture conditions

15. Biochemical Cause
• Lack of photosynthetic ability due to alteration in carbon metabolism
• Antibiotic resistance

16. Variation occurs in types of cells
Gameto-clonal
variation
(variation observed
the plants regenerated
from gametic
cultures)
(clones of gametes)
Proto-clonal
variation
(variation observed
among the plants
protoplast cultures)
(clones of protoplast)
Calli-clonal
variation
(variation observed
among the plants
callus)
(clones of callus)
Meri-clonal
variation
(variation observed
among the plants
meristem)
(clones of mericlones)

17. Mechanism of somaclonal variation in
micro propagated plants as a result of
oxidative burst upon in vitro culture

18. Oxidative stress
Hyper/hypo-
methylation of
DNA
Chromosomal
rearrangements
Changes in
chromosome number
Free radicals (Superoxide, hydrogen peroxide)
DNA based deletion/
substitution
Explant
preparation (e.g.
wounding and
sterilization)
Media components
(e.g. plant growth
regulators and salts
in vitro culture
environment (physical
state temperature, light
etc)
Mutation under in
vitro environment
Somaclonal
variation

19. Steps involved in induction and selection
of somaclonal variation

22. Isolation of somaclonal variation (via
two schemes)
Without in-
vitro selection
With in-vitro
selection

23. Generation of somaclonal without in-vitro
selection

24. Explant (leaf etc)
Explant derived callus
Shoot regeneration
Plant
Transfer to the field
Screening for desirable traits
Horticultural trails

25. Without in-vitro technique
• Unorganized callus and cells, grown in cultures for various periods on a medium
that contain no selective agents are induced to differentiate whole plants.
• An explant is cultivated on a suitable medium, supplemented with growth
regulators.
• The unorganized callus and cells do not contain any selective agent (toxic or
inhibitory substance).
• These cultures are normally sub-cultured and transferred to shoot induction
medium for regeneration of plants.
• The so produced plants are grown in pots, transferred to field, and analyzed for
somaclonal variants.

26. Limitation of without in-vitro selection
No specific approach for isolation of somaclones
Appearance of desired traits are purely by chance
Time consuming procedure
Require screening in many plants

27. Generation of somaclonal with in-vitro
selection

28. Explant (Leaf, etc]
Explant derived callus
Multiplication of callus
Proliferation and
maintenance of callus
Small pieces of calli in toxin
medium
Isolation of
tolerant calli
Regeneration
In-vivo testing against
toxin/pathogen
Progeny clones from each plant
Test for disease resistance
Generation of disease resistant
plants
Pathogenic organism
Purified culture filtrate
Toxin isolation
Toxicity determination
for lethal concentration
Horticultural trails

29. With in-vitro technique
• Cell lines are analyzed from plant cultures for their capability to survive in
the presence of a toxic substance in medium or under environmental stress
conditions.
• Selection cycles are carried out to isolate the tolerant callus cultures and
these calli are regenerated into plants.
• The plants so obtained are in-vitro screened against the toxin (pathogen or
any other inhibitor).

30. • The plants resistant to the toxin are selected and grown further by vegetative
propagation or self-pollination.
• The subsequent generations are analyzed for disease resistant plants against
the specific pathogenic organism.
Cont.

31. Advantages of with in-vitro selection
Specific approach for isolation of desired trait.
Less time consuming procedure as compare
without in-vitro approach.

32. Detection and isolation of somaclonal
variants
1. Analysis of morphological characters
2. Variant detection by cytological studies

33. 3. Variant detection by DNA contents
4. Variant detection by gel electrophoresis

34. 5. Detection of disease resistance variant
6. Detection of herbicide resistance variant

35. 7. Detection of environmental stress tolerant variant
8. Molecular markers
• 9. Cytological markers

36. Occurrence of somaclonal variation can
be reduced by
• Avoiding long term cultures
• Using axillary shoot induction systems where possible
• Well known that, increasing numbers of subcultures increase the likelihood of
somaclonal variation, so the number of subcultures in micropropagation protocols
should be kept to a minimum
• Regularly reinitiating clones from new explants, which might reduce variability
over time
• Avoiding 2,4-D in the culture medium, as this hormone (Introduce variation).

37. • Plant growth substances: The minimum use of plant growth regulators is
always recommended for conservation through tissue culture.
• The concentration of plant growth regulators has been found to affect the
frequency of somaclonal variation in banana.
Cont.

38. Applications of somaclonal variations
• Production of agronomically useful plants
• Resistance to disease
• Resistance to abiotic stresses
• Resistance to herbicides
• Improved seed quality

39. .
Somaclonal
Variation in
Perennial
Horticulture
Crop
.

41. Martin et al., 2006

42. Distinctive phenotypic features of the Musa sp. cv.
Grande Naine and variant CUDBT-B1
Phenotypic features Grande Naine (normal) CUDBT-B1
Pseudostem height 1.7m 1.5 m
Pseudostem diameter 15 cm 10 cm
Pseudostem color Green--yellow Variegated
Color of lamina, petiole,
peduncle, ovary base of male
flower, and fruits
Green Variegated (cream or pale
yellow)
Midrib dorsal surface color Light green Pale green with variegation or
cream color
Peduncle length 50-60 cm 40-50 cm
Bract color Pink purple Pink purple with variegation
Martin et al., 2006

43. a) Shoots from corm developed on MS medium
b) 5-monh old field-grown CUDBT-B1 with variegated leaves
c) RAPD analysis of CUDBT-B1 and normal (parent) banana. M, DNA marker;
d) initiation of shoots from shoot-tip culture of CUDBT-B1
e) in vitro propagated acclimatized progeny of CUDBT-B1.
Martin et al., 2006

44. Callus was induced from leaf pieces of in vitro grown plants of Actinidia deliciosa (cv Tomuri) on basal
medium supplemented NAA, kinetin and 85.5 mM NaCl for 6 subcultures.
The NaCl concentration was chosen following preliminary results.
Twenty Petri dishes with 10 leaf pieces were prepared cutting off the leaf margins and dividing the leaf into
3-4 segments.
Survived calli were transferred on a regeneration medium consisting in the same basal medium in which only
zeatin was present to induce shoot formation.
Caboni et al., 2003

45. Cont.
• Lines obtained from tolerant calli were subcultured 3 times and evaluated after 4
subcultures for tolerance to NaCl at the same concentration used for callus
selection.
• Rooting was performed in vitro on a MS medium supplemented with IBA
Caboni et al., 2003

46. Cont.
• In conclusion, shoot lines of Actinidia deliciosa regenerated from calli under selective pressure,
showed NaCl tolerance response in vitro. RAPD markers revealed genetic changes in 2 of the
selected tolerant clones. it could be proposed that the tolerance response to NaCl is maintained in
acclimatised plants and preliminary results in open field seem to confirm this behaviour. Thus,
somaclonal variation induced by in vitro organogenesis seems to be utilisable in Actinidia to select
lines tolerant to NaCl.
Caboni et al., 2003

47. Apple rootstock Malling 7 resistant
Modgil et al., 2012

48. Effect of different concentrations of fungal culture filtrate (FF) of Dematophora necatrix on
calli and regenerants of M7.
Serial no. Regeneration
medium
supplemented with
FCF (%)
Frequency of
surviving callus
(After 6 weeks)
Frequency of
surviving of
regenerants (after 2
weeks)
Percent survival of
regenerants (after 4
weeks)
1 Control 100.00 100.00 100.00
2 10 100.00 100.00 100.00
3 20 93.40 96.00 90.67
4 30 81.306 93.67 85.67
5 40 73.30 78.66 73.33
6 50 39.30 75.00 68.00
7 60 12.371 63.33 58.67
8 70 5.188 61.67 40.00
9 72.5 0.00 19.67 0.00
10 75 0.00 0.00 0.00
11 77.5 0.00 0.00 0.00
12 80 0.00 0.00 0.00
13 90 0.00 0.00 0.00

49. (a and b) Surviving and dead shoots on selection medium containing 60 and 70% FCF (c) selected shoots
(regenerants) growing on selection medium with 70% FCF, after 3rd selection cycle (d and e) multiplication and
rooting of selected regenerants (f and g) resistant lines (after pathogenecity test) growing in the pot and the field.
Modgil et al., 2012

50. Perez et al., 2009

51. Perez et al., 2009

52. Kaushal et al., 2005

53. Performance of the variants and parents of Fragaria x ananassa
Duch. of cv. Chandler with respect to the vegetative characters.
Cultivar Type of
variant
Number
of leaves
Leaf area
(cm2)
Petiole
length
(cm)
Plant
height
(cm)
Plant
spread
(cm)
Number
of
runners/
plant
Chandler Parent 3-5 12.53-
17.29
2.1-5.5 4.0-8.1 8.0-23.0 0-6
CP-SCIII 8-9 20.17-
30.5
6.2-7.6 4.0-10.5 14.25-
22.25
3-15
CL-SCIII 7-9 11.42-
15.25
5-5.7 3.5-11 9-17 0-4
Kaushal et al., 2005

54. Advantages
Help in crop improvement
Provides additional genetic variability (under in-vitro conditions)
Selection of plants resistant to various toxins and herbicides
Environmental adaptation: abiotic stress, high salt concentration, mineral toxicity
Disease resistance

55. Disadvantages
Plants often lost the power of regeneration
Unpredictable changes such as reduced fertility, growth rate and overall performance of plant
Regulatory concerns
Epigenetic changes
May develop variants with pleiotropic effects which are not true

56. Future Perspective:
• Development of new crop varieties with desired traits.
• Precision breeding
• Molecular understanding
• Legal and regulatory considerations

57. Conclusion:
Somaclonal variation offers a
rapid and focused approach to
introducing novel traits in crops.
Somaclonal variation holds
promise in shaping a more
productive horticultural future.

58. QUESTIONS:
• Somaclonal variation can be obtained by?
• Difference between genetic and epigenetic variation?
• One drawback of without in vitro selection?
• Name one hormone which induces variation?
• One advantage of somaclonal variation in crops?

60. Reference:
• Brar, D.S., Jain, S.M. (1998). Somaclonal Variation: Mechanism and Applications in
Crop Improvement. In: Jain, S.M., Brar, D.S., Ahloowalia, B.S. (eds) Somaclonal
Variation and Induced Mutations in Crop Improvement. Current Plant Science and
Biotechnology in Agriculture, vol 32. Springer, Dordrecht.
https://doi.org/10.1007/978-94-015-9125-6_2
• https://agrihunt.com/articles/pak-agri-outlook/somaclonal-variation/