Somaclonal Variation in Plant tissue culture - Variation in somaclones (somatic cells of plants) Somaclonal variation # Basis of somaclonal variation # General feature of Somaclonal variations # Types and causes of somaclonal variation # Isolation procedure of somaclones via without in-vitro method and with in-vitro method with their limitations and advantages # Detection of isolated somaclonal variation # Application (with examples respectively related to crop improvement) # Advantages and disadvantages of somaclonal variations. Also watch, Gametoclonal variation slides to understand, how to changes occur in gametoclones of plants. https://www.slideshare.net/SharmasClasses/gametoclonal-variation

1. Dr. Divya Sharma
Assistant Professor
Somaclonal Variation and Its
Crop Improvement

2. SOMACLONAL VARIATION
Soma ----- Somatic cells
Clonal ----- Clones ----- Generations
Variation ----- Changes
SOMACLOANL VARIATION ------ SOMACLONES
(variation in somaclones)

3. SOMACLONAL VARIATION
 “Somaclonal variation” term was first coined by Larkin
and Scowcroft (1981).
According to Larkin and Scowcroft (1981), “Somaclonal variation is the
genetic variability which is regenerated during tissue culture” or plant
variants derived from any form of cell or tissue cultures.
Larkin and Scowcroft. 1981

4.  The phenomenon of high variability in individuals from plant cell
cultures or adventitious shoots is called Somaclonal
variation.
 Somaclonal variation is the variation seen in plants that have been
produced by plant tissue culture.
 Genetic variation (genetic mutation) in plants that have been
produced by plant tissue culture and can be used detected as
genetic or phenotypic traits (caused by in-vitro conditions or by
chimeral separation).
 Genetic variations are inherited by the clones of the treated plant.
 Somaclonal variations may lead to desirable characteristics like
increased pest resistance etc. This work performed to achieve some
of beneficial properties of plants like disease resistance, fruit
quality, stress resistance, nutritional quality
improvement, yield improvement, etc.
Larkin, 1981

5.  The recovery of genetic changes in plants generated from somatic
cells thus offers an opportunity to unravel natural variability and to
use this variation for development of new varieties for plenty of
good purpose (Larkin, 1981).
 Plant tissue culture cycle is a process that involves establishment of
a differentiated cells/tissue culture under defined conditions of
proliferation for a number of generation and subsequent
regeneration of plant.
Larkin, 1981
Somaclonal variation in Sugarcane [Saccharum officinarum]

6. Gametoclonal Variation
 If the somatic-issue derived variants have a gametophytic
origin such as pollen or egg cell, then its known as
“Gametophytic variation”.
 It introduced for variation observed among plants
regenerated from cultured gametic cells.
Larkin, 1981

7. Basis of Somaclonal Variation
Somaclonal variation --- occur as a result of genetic heterogeneity in
plant tissue culture (inside the plant or explant) that changes can passes
from one generation to another.
This maybe due to:
 Expression of chromosomal mosaicism or Genetic disorders
 Spontaneous mutations due to culture conditions or Physiological
cause

8. General Features of Somaclonal Variation
 Variation in karyotype, isozyme characteristics and
morphology (number and structure of chromosomes) in
somaclones may be observed.
 Regenerated plants with altered chromosomal changes often
show changes in leaf shapes and color, growth rate and habit,
and sexual fertility.
 Generally heritable mutations and persist in plant population
even after plantation into the field.
 Variations occur in both Qualitative and Quantitative traits
(Larkin, 1981).
Larkin, 1981

9. Types of Somaclonal Variation
•Preexisting variations in the somatic cells
of explant
•Caused by mutations and other DNA
changes
•Occur at high frequency
Genetic
(heritable)
variations
•Variations generated during tissue culture
•Caused by temporary phenotypic changes
•Due to culture conditions, varied
nutrients, etc
•Occur at low frequency
Epigenetic
(non-heritable)
variations
Satyanarayana, 2015

10. Types of Somaclonal Variation
Gametoclonal
variation
[variation observed
the plants
regenerated from
gametic cultures]
(clones of gametes)
Protoclonal
variation
[variation observed
among the plants
protoplast cultures]
(clones of
protoplast)
Calliclonal
variation
[variation
observed among
the plants callus]
(clones of callus)

11. Calliclone
Mericlone
Protoclone
• Clones of callus
• Clones of meristem
• Clones of protoplast

12. Causes of Somaclonal Variation
Physiological cause
Genetic cause
Biochemical cause

13. Physiological Cause
• Exposure of culture to plant
growth regulators
• Culture media conditions
Physiological
cause
Evan et al., 1984

14. Genetic cause
Change in
chromosome
number
• Aneuploidy (gain
or loss set of
chromosomes)
• Polyploidy
(organism with more
than 2 chromosomes
set)
• Monoploidy
(organism with one
chromosomes set)
Change in
chromosome
structure
• Deletion (a segment
of base is deleted)
• Inversion (a segment
of chromosomes is
reversed)
• Duplication
(addition of
chromosomes)
• Translocation (arms
of chromosomes
switched)
Gene mutation
• Transition
• Transversion
• Insertion
• Deletion
Lee and Ronald, 1988

15. Plasma gene
mutation
• Changes in genetic
material which
present inside
mitochondria and
chloroplast
• Mutant character
shows cytoplasmic
inheritance
Transposable
element
activation
• Transposable
element / jumping
genes cause
mutation via
replication,
recombination and
repair
DNA sequence
• Changes in DNA –
Detection of altered
fragment size by
using Restriction
enzyme
• Change in protein
– Loss or gain in
protein band; and
Alteration in level of
specific protein
• Methylation of
DNA – Methylation
inactivates
transcription process
[Base mutation]
Lee and Ronald, 1988

16. Biochemical Cause
• Lack of photosynthetic ability due
to alteration in carbon metabolism
• Biosynthesis of starch via
carotenoid pathway
• Nitrogen metabolism
• Antibiotic resistance
Biochemical
cause

17. Callus tissue
Organogenesis
Regenerated
plants
Hardening
and selfing
Somaclonal
variants

18. Factors Affecting Variation
 Selection agents (toxin, herbicide, amino acid analogue)
 Selection propagule (cells, protoplasts, calli)
 Duration of cell culture
 Growth hormone effects
 Technique used for selection
 Stability of resistant substance
 In-vivo testing procedure
 Genotype and explants source
 Ability for regeneration of plants

19. Isolation of Somaclonal Variation
(via two schemes)
Without in-vitro
selection
With in-vitro
selection

20. Generation of
somaclonal
Without in-
vitro selection
Satyanarayana, 2015

21.  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
grown 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
Without in-vitro technique

22. 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

23. Generation of
somaclonal
With in-vitro
selection
Satyanarayana, 2015
Chaleff, 1981 has
labeled:
Plant regenerated from
tissue culture (R or R0)
Self fertilized progeny of
R0 plant as R1
Larkin and Scowcroft,
1981 have referred:
Regenerated plants as
SC1 (=R0)
Self fertilized
generations as SC2,
SC3, SC4 etc.

24.  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
 The differentiated callus obtained from an explant (any cells,
protoplast or calli) is exposed in the medium to inhibitors like toxins,
antibiotics, amino acid analogs.
 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 (or
pathogen or any other inhibitor)
 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.
With in-vitro technique

25. Advantages of With in-vitro Selection
 Specific approach for isolation of desired trait
 Less time consuming procedure as compare without in-
vitro approach

26. Detection and Isolation of
Somaclonal Variants
Analysis of morphological characters
• Qualitative characters: Plant height, maturity date, flowering
date and leaf size
• Quantitative characters: Yield of flower, seeds and wax contents
in different plants parts
Variants detection by cytological studies
• Staining of meristematic tissues like root tip, leaf tip with
feulgen and acetocarmine provide the number and morphology of
chromosomes
Skirvin et al., 1994

27. Variants detection by DNA contents
• Cytophotometer detection of feulgen stained nuclei can be used
to measure the DNA contents
Variants detection by gel electrophoresis
• Change in concentration of enzymes, proteins and
chemical products like pigments, alkaloids and amino acids can
be detected by their electrophoretic pattern
Detection of disease resistance variant
• Pathogen or toxin responsible for disease resistance can be used
as selection agent during culture
Skirvin et al., 1994

28. Detection of herbicide resistance variant
• Plantlets generated by the addition of herbicide to the cell culture
system can be used as herbicide resistance plant
Detection of environmental stress tolerant
variant
• Selection of high tolerant cell lines in tobacco
• Selection of water-logging and drought resistance cell lines in
tomato
• Selection of temperature stress tolerant in cell lines in pear
• Selection of mineral toxicities tolerant in sorghum plant (mainly for
aluminum toxicity)
Skirvin et al., 1994

29. Occurrence of Somaclonal Variation
 Avoiding long term cultures
 Using axillary shoot induction systems where possible
 Propagating chimeras by other clonal systems
 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).

30. Applications of
Somaclonal Variations
 Production of agronomically useful plants
 Resistance to disease
 Resistance to abiotic stresses
 Resistance to herbicides
 Improved seed quality and geraniums (esp. scented
varieties)
 Woody ornamentals
E x a m p l e s : S u g a r c a n e – s e l e c t i o n s f o r h i g h e r y i e l d & d i s e a s e
r e s i s t a n c e ; P o t a t o e s – y i e l d & d i s e a s e r e s i s t a n c e ; P a u l o w n i a –
s e l e c t i o n f o r l e a f v a r i e g a t i o n .

31. Production of agronomically useful plants
[different crop species with their morphological characters]
Crop Character (s)
Rice Flowering period, panicle size; like number plant height; leaf length, shape and color;
frequency of fertile seed; sterility mutants
Wheat Plant height; tiller number; grain color; seed storage protein
Maize Reduced pollen fertility and male sterility; twin stalks from a single node
Sugarcane High sugar yield; increasing stalk length, diameter, weight and density
Barley Increased grain yield; leaf shape; heading date; ash content
Oats Plant height; heading date; morphology and fertility
Soybean Variable height; maturity; seed protein and oil content
Potato Higher yield; growth habit, maturity and morphology
Tomato Dwarf habit; early flowering; orange fruit color
Carrot Higher carotene content
Pineapple Foliage density; leaf color, width and spine formation
Brassica Multiple branching stem; altered leaf; slow growth; failure to flower or delay in flowering;
large pollen grains
Tobacco Increased yield; plant height; leaf number, shape, width and yield; type of inflorescence
and yield

32. Resistance to disease
 Development of disease resistance in many crops : Rice, Wheat,
Maize, Sugarcane, Tobacco, Apple, Tomato, etc
 Selected crops somaclonal variants, with increasing disease resistance
developed, without in-vitro selection are respectively .
Resistance first reported in Sugarcane for eye spot disease
(Heminthosporium sacchari), downy mildew (Sclerospora sacchari)
and Fiji virus disease by regenerating plants from the callus of
susceptible clones and screening the somaclones.

33. Crop Pathogenic
organism(s)
Rice Helminthosporium oryzae
Maize Helminthosporium maydis
Barley Rhynchosporium secalis
Sugarcane Puccinia melanocephala,
Sclerospora saccharii,
Helminthosporium sacchari
Potato Streptomyces scabie, Alternaria
solani, Phytophthora infestans,
Potato virus X and Y
Tomato Pseudomonas solanacearum,
Fusarium oxysporum
Tobacco Phytophthora parasitica
Apple Phytophthora cactorum
Banana Fusarium oxysporum
Lettuce Lettuce mosaic virus
Alfalfa Fusarium solanii, Verticillium
albo-atrum
Crop Pathogenic
organism(s)
Selection
agent
Rice Helminthosporium oryzae,
Xanthomonas oryzae
Crude toxin
Bacterial cells
Maize Helminthosporium maydis Hm T toxin
Wheat Helminthosporium sativum,
Pseudomonas syringae
Crude toxin
Syringomycin
Barley Helminthosporium sativum,
Fusarium spp.
Crude toxin
Fusaric acid
Sugar-
cane
Helminthosporium sacchari Toxin
Potato Phytophthora infestans,
Fusarium oxysporum,
Erwinia carotovora
Crude filtrate
Crude filtrate
Pathogen
Tomato Pseudomonas
solanacearum, tobacco
mosaic virus X
Crude filtrate
Virus
Tobacco Phytophthora syringae,
Pseudomonas syrinae
Toxin
Methionine
sulfoximine
Alfalfa Fusarium oxysporum Crude filtrate
A list of disease resistant crop plants
obtained by somaclones at the plant level
without in-vitro selection
A list of disease resistant crop plants obtained
by with in-vitro selection

34. Resistance to abiotic stresses
 Freezing tolerance [Eg. Wheat]
 Salt tolerance [Eg. Rice, Maize, Tobacco]
 Aluminium tolerance [Eg. Carrot, Sorghum, Tomato]
 Drought tolerance

35. Herbicide resistance
 Tobacco resistant to Glyphosate, Sulfonyl urea and Picloram.
 Carrot resistant to Glyphosate
 Lotus resistant to 2,4-dichlorophenoxy acetic acid (2,4-D)
Crop Common name Herbicide
Glycine max Soybean Imazethapyr
Nicotina tabacum Tobacco Glyphosate
Gossypium hirsutum Cotton Sulfonylurea, Imidazolinone
Zea mays Maize Sethoxydim, Cycloxydim
Zea mays Maize Glyphosate
Triticum aestivum Wheat Difenzoquat
Beta vulgaris Beet Imidazolinone
Datura innoxia Sacred datura Chlorosulfuron

36. Improved seed quality
New variety of Lathyrus sativa seeds [Lathyrus Bio L212]
with low content of neurotoxin has been developed through
somaclonal variations.

37. Advantages and Disadvantages of
Somaclonal Variations
Advantages Disadvantages
Help in crop improvement Require extensive and extended field trails
Provides additional genetic variability (under in-
vitro conditions)
Sometime lead to undesirable results such as
reduced fertility, growth rate and overall
performance of plant
Increased and improved production of secondary
metabolites
Selected variants are random and genetically
unstable and non-heritable
Selection of plants resistant to various toxins,
herbicides, abiotic stress, high salt concentration,
mineral toxicity; and plant improved quality of
seeds (a new variety Lathyrus sativus)
A series advantage occur in operations which
require clonal uniformity, as in the horticulture and
forestry industries where tissue culture is employed
for rapid propagation of elite genotypes
Suitable for breeding of higher plants Not suitable for complex agronomic traits like yield,
quality etc
Simpler and easier as compared to recombinant
DNA technology
May develop variants with pleiotropic effects which
are not true
Stable cell line can be preserved by cryopreservation Required extensive and extended field trails
Examples: Bio-13, a somaclonal variant of
Citronella java (with 37% more oil an 39% more
citronellon) a medicinal plant
Plants often lost the power of regeneration

38.  Evans D.A., Sharp W .R ., Medina H. “Somaclonal and
Gametoclonal Variation” Am J Botany (1984 ) 759-774.
 Satanarayana U. “Somaclonal Variation, Biotechnology, 9th Ed,
(2015 ) 855 : 546-549.
 Skirvin R .M ., Margaret N. “Sources and Frequency of
Somaclonal Variation” Hor tSci 29.11 (1994 ) 1232-1237 .
 Larkin P .J., Scowcroft R . “Somaclonal Variation -A Novel
Source o f Variability from Cell Cultures for Plant
Improvement” Theor Appl Genet 60.4 (1981 ) 197-214 .
References

39. Sharma’s Classes - Dr. Divya Sharma