1.

introduction to Somaclonal Variation

2. Schemes for obtaining somaclonal variation
3. Detection and Isolation of Somaclonal Variants
4. Factor affecting somaclonal variation
5. Mechanism of Somaclonal Variations
6. Advantages of Somaclonal Variations
7. Disadvantages:
8. Application of somaclonal variation in crop improvement
9. Control of Somaclonal Variation
10. To Increase Somaclonal Variation
11. Targets for Somaclonal Variation
12. References
• It is the term used to describe the
variation seen in plants that have been
produced by plant tissue culture.
• Chromosomal rearrangements are an
important source of this variation
 Two schemes

1.Without in vitro selection

2.With in vitro selection

A flow diagram for generation of somaclonal variation
without in vitro selection
Continue………….
1. Analysis of morphological characters
 Qualitative characters: Plant height, maturity date, flowering
date and leaf size
 Quantitative characters: yield of flower, seeds and wax con-
tents in different plant parts

2. Variant detection by cytological Studies

 Staining of meristematic tissues like root tip, leaf tip with
feulgen and acetocarmine provide the number and morphol-
ogy of chromosomes.

3. Variant detection by DNA contents

 Cytophotometer detection of feulgen stained nuclei can be
used to measure the DNA contents
4. Variant detection by gel electrophoresis
 Change in concentration of enzymes, proteins and hemical products
like pigments, alkaloids and amino acids can be detected by their
electrophoretic pattern
5. Detection of disease resistance variant
 Pathogen or toxin responsible for disease resistance can be used as
selection agent during culture.
6. Detection of herbicide resistance variant
 Plantlets generated by the addition of herbicide to the cell culture
system can be used as herbicide resistance plant.
7. Detection of environmental stress tolerant variant
 Selection of high salt 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
aluminium toxicity)
1.Genotype
2.Explant source
3.Duration of cell culture
4.Culture condition
1. Genetic (Heritable Variations)
• Pre-existing variations in the somatic cells of
explant
• Caused by mutations and other DNA changes
• Occur at high frequency

2. Epigenetic (Non-heritable Variations)

• Variations generated during tissue culture
• Caused by temporary phenotypic changes
• Occur at low frequency
A] Genetic variations are:

1.changes in chromosome

2.chromosome structure

3.DNA sequence
1.changes in chromosome numbers;
 Euploidy- changes in whole chromosome sets
 Aneuploidy- changes in parts of chromosome sets

 Ploidy: number of basic chromosome sets (a diploid has 2 sets;

a hexaploid has 6 sets)
 Euploid: organism containing multiples of the basic chromo-
some set
 Monoploid: organisms with one chromosome set (in essentially
diploid taxa)
 Polyploid: organism containing more than two chromosome
sets
3.DNA sequence (base mutations).
 Change at DNA level
• Detected using restriction enzyme analysis
(altered fragment size)
 Changes at protein levels
• Loss or gain of protein band
• Altered levels of specific proteins
 DNA methylation
• Methylation of a gene inactivates its tran-
scription
B] Epigenetic change
 Due to gene expression not gene alteration
 Often temporary
 Some examples
• Morphological change - leaf shape
• Earlier flowering
• Improved adventitious rooting
• Increased vigor
• Plageotropic growth
 Benefit of somaclonal variation is in plant improvement
 Creation of additional genetic varitions
 Increased and improved production of secondary metabolites
 Selection of plants resistant to various toxins, herbicides, high
salt concentration and mineral toxicity
 Suitable for breeding of tree species
 Characteristics for which somaclonal mutants can be enriched
during in vitro culture include resistance to disease pathotox-
ins, herbicides and tolerance to environmental or chemical
stress.
Disadvantages:
 No control - may change in undesirable direction

 Variations are random in nature

 May not be genetically stable

 Require extensive field testing

 May not occur for complex agronomic traits

 Improve by
1.selecting novel varients
2.Disease resistant
3.Abiotic stress resistance
4.Salt tolerance
5.Aluminium tolerance
6.Herbicide resistance
7.Insect resistance
8.Seed quality
 Crop Pathogen Toxin
Alfalfa Colletotrichum sp. Culture filtrate

Banana Fusarium sp. Fusaric acid

Coffee Colletotrichum sp Partially purified culture
filtrate
Maize Helminthosporium may- T-toxin
dis

Oat* Helminthosporium victo- Victorin

riae

Sugarcane** Helminthosporium sp. Culture filtrate

Potato** Phytophthora infestans Culture filtrate

Rice Xanthomonas oryzae Culture filtrate

Tobacco* Alternaria alternata Partially purified toxin

 *Shown to be heritable through sexual propagation
 **Shown to be stable through vegetative propagation
Somaclonal variation in trees:
 Somatic variation may be a better mans for tree
improvement
 Direct gene insertion or agrobacteruim –mediated
transformation have many technical difficulties that
must be overcome with this methods.
 It have the potential to significantly change tree
breeding programs by reducing the time required for
selecting and improving desired traits .
 Applying soma clonal technology to forest
trees offers an even greater advantage
 because of the long generation times of trees
and the opportunity to introduce desired traits
not possible through traditional breeding.
 Somaclonal technology is simpler to exploit
than other types of genetic engineering.
Control of Somaclonal Variation:
 Regular reinitiation of clones from new ex-
plants might reduce variability over time.
 Avoid 2,4-D in the culture medium, as this
hormone is known to introduce variation.
 The duration of the culture cycle may influ-
ence the variability.
To Increase Somaclonal Variation:
 Callus and suspension cultures for several cy-
cles
 Regeneration of large number of plants from
long-term cultures
 Testing of selected somaclones for genetic sta-
bility
 Multiplication of genetically stable somaclones
for developing new cultivars
 • Specific amino acid accumulators
– Screen for specific amino acid production
– e.g. Lysine in cereals
 • Abiotic stress tolerance
– Add or subject cultures to selection agent
– e.g.: salt tolerance, temperature stresses, etc…
 • Disease resistance
– Add toxin or culture filtrate to growth media
– Examples shown on next slide →