somoclonal variation in plants,introduction, basic features, nomenclature, mechanism, factors influencing, applications, causes, detection and isolation of somoclonal variants, advantages & disadvantages, Gametoclonal Variations-features, production of Gametoclones, Source etc

1. SOMOCLONAL VARIATION AND ITS
APPLICATIONS
Dr. SHILPY SINGH
ASSISTANT PROFESSOR
(MIMT, GR. NOIDA)

2.  Genetic variations in plants that have been produced by plant tissue
culture and can be detected as genetic or phenotypic traits
 Somoclonal variation- The variability generated by the use of a tissue
culture cycle
“Soma”-somatic cells and “clones”-generations
 The term Somoclonal variation was coined by Larkin and Scowcroft
(1981)

3. Variations for Karyotype, isozyme characteristics and morphology
in somaclones may also observed.
• Calliclone-clones of callus
• Mericlone-clones of meristem
• Protoclone-clones of Protoplast
Variation occurs in both qualitative and quantitative traits.
Generally heritable mutation and persist in plant population even
after plantation into the field.
Basic Features of Somaclonal Variations

4. Nomenclature
Chaleff (1981) has labelled the plants regenerated from tissue culture
as R or R0 plants and the self fertilized progeny of R0 plants as R1.
Subsequent generations produced by self-fertilization are termed as
R2, R3, R4 etc.
Larkin and Scowcroft (1981) have referred regenerated plants as
SC1(=R0) and subsequent self fertilized generations as SC2, SC3, SC4
etc.

5. Mechanism of Somaclonal Variations
• Pre-existing variations in the somatic cells of explant
• Caused by mutations and other DNA changes
• Occur at high frequency
• Variations generated during tissue culture
• Caused by temporary phenotypic changes
• Occur at low frequency
 Genetic (Heritable Variations)
 Epigenetic (Non-heritable Variations)

6. SCHEMES FOR OBTAINING SOMOCLONAL VARIATION
 Two schemes, with and without in vitro selection have been generally followed for
getting somoclonal variation in crop plants.
Without in vitro selection
 An explant is cultured on a suitable medium e.g. small shoot segments (1-2 cm) of
sugarcane, cotyledons, hypocotyls, protoplasts, leaves, embryos etc.
 The basal medium is supplemented with growth regulators which support
dedifferentiation stage, i.e. callus. Normally these culture are subcultured and
then transferred to shoot induction medium for plant regeneration.
 The regenerated plants are transferred to pots, grown to maturity and analysed
for variants.
Disadvantage
 This approach is time consuming due to the fertilization step and requires
screening of many plants.

7. Without in vitro Selection
Explant
Shoot regeneration
Plant
Explant derived callus
Transfer to the field
Screening for desirable traits
Agronomic traits

8. With in vitro selection
 The dedifferentiated culture i.e. callus is subjected to selection against inhibitors like
antibiotics, amino acid analogs, pathotoxins etc.
 Different selection cycles are performed to get tolerant cells/callus cultures that are
subsequently regenerated into plants. These plants are then in vivo screened against
the inhibitor.
 If the plants are resistant to the inhibitor, then stable transmission of that character is
analyzed in subsequent generations.
 In this approach, variants for a particular character are selected rather than the general
variation obtained in first case where selection is done at the plant level.

9. Explant
Explant derived callus
Multiplication of callus
Purified culture filtrate
Pathogen
Toxin isolation
Determination of lethal concentration of toxin for
tissue
Tolerant calli
Regeneration
In vivo testing against toxin/pathogen (Putative resistant plants R0
or SC1
Progeny rows from each plant
Test for disease resistance
Small pieces of calli on toxic medium
Selection cycles
Multiplication of callus
Disease resistant plants (R1 or SC2)
Agronomic trials
Self pollination
Vegetative
propagation
With in vitro Selection

10. Factors influencing Somoclonal Variation
 Genotype
 Explant source
 Duration of cell culture
 Culture conditions
 Selection propagule (cells, protoplasts or calli)
 Selective agent (Inhibitors used can be an amino acid analog, herbicide, a
synthetic toxin isolated from fungal liquid culture) Selection technique
 Regeneration of plants
 In vivo testing
 Agronomic analysis
 Resistance stability

11. Application of Somoclonal Variation
As a result of somaclonal variations, several novel variants of existing crops have been
developed e.g. An improved scented Geranium variety named ‘Velvet Rose’, pure thorn-less
blackberries etc.
A list of somoclonal variants obtained from different
crop species with their morphological characters
 Production of agronomically useful plants (Novel Variants)

12.  Abiotic/Biotic stress resistance
 Lazar et al., 1988 developed somoclonal variants for freezing tolerance in Norstar winter wheat. A
significant positive correlation between proline level and frost tolerance has been found in a broad
spectrum of genotypes.
 In vitro selection and regeneration of hydroxyproline resistant lines of winter wheat with increased
frost tolerance and increased proline content has been reported (Dorffling et al., 1997). The results
showed strong correlation of increased frost tolerance with increased proline content.
 Plant tissue culture techniques have been successfully used to obtain salt tolerant cell lines or variants
in several plant species, viz. tobacco, alfalfa , rice, maize, Brassica juncea, Solanum nigrum, sorghum
etc.
 Mandal et al., 1999 developed a salt tolerant somaclone BTS24 from indigenous rice cultivar Pokkali.
In vitro selection and regeneration of salt tolerant plants
 Cold tolerance
 Salt tolerance

13.  In recent years, considerable research has been focused on the understanding of physiological, genetic
and molecular processes that lead to aluminium tolerance.
 Despite the problems encountered in adapting culture media for in vitro selection for aluminium
resistance, cell lines have been isolated in several species eg. Alfalfa, carrot, sorghum, tomato, tobacco.
 Jan et al., 1997 elicited aluminium toxicity during in vitro selection in rice by making several modifications
in the media viz. low pH, low phosphate and calcium concentrations, and unchelated iron and aluminium
along with aluminium sulphate.
 Wang et al., 1997 reported a sorghum somoclonal variant line (R111) resistant to drought stress.
 Drought tolerant rice lines were obtained by in vitro selection of seed induced callus on a media
containing polyethylene glycol as a selective agent which simulated the effect of drought in tissue culture
conditions (Adkins et al., 1995).
 Zemetra et al., 1993 used in vitro selection technique for generation of somoclonal variants for Russian
wheat aphid (Diuraphis noxia) in wheat.
 Croughan et al., 1994 reported variant of Bermuda grass (Cynodon dactylon) called Brazos R-3 with
increased resistance to fall armyworm.
 Aluminium tolerance
 Drought tolerance
 Insect Resistance

14. Disease resistant crop plants obtained without in
vitro selection
 Disease Resistance
The greatest contribution of somoclonal variation towards plant improvement is in the
development of disease resistant genotype in various crop species. Carlson (1973) first tested
the possibility of in vitro selection for disease resistance for wild fire disease of tobacco
caused by Pseudomonas syringae pv tabaci.
Disease resistant crop plants obtained by in vitro
selection

15.  Herbicide resistance
 Through in vitro selection several cell lines resistant to herbicides have been isolated and few have
been regenerated into complete plants.
 Important achievements are tobacco, soyabean, wheat, maize etc resistant to various herbicide
such as glyphosate, sulfonylurea, imidazolinones etc.
 Seed Quality
 Recently, a variety Bio L 212 of Lathyrus sativa has been identified for cultivation in central India
which has been developed through somoclonal variation and has low ODAP (β-N-oxalyl-2-α, β
diamino propionic acid), a neurotoxin (Mehta and Santha, 1996), indication the potential of
somoclonal variations for the development of varieties with improved seed quality.
 Enhancing alien gene introgression into cultivated species
 Important in enhancing inter-specific crosses where the genomes show no homology So the
chromosomal exchange results In new combinations and transfer of alien chromosome segment.
Some examples of somoclonal variants for herbicide resistance

16. Causes of Somoclonal
Variation
Genetic Cause
Physiological
Cause
Biochemical
Cause

17.  Exposure of culture to plant growth regulators
 Culture conditions
a) Change in chromosome number
 Euploidy: Changes chromosome Sets
 Aneuploidy: Changes in parts of chromosome Sets
 Polyploidy: Organisms with more than two chromosome sets
 Monoploidy: Organism with one chromosomes set
Causes of Somaclonal Variations
Physiological Cause
Genetic Cause

18. b) Change in chromosome structure
 Deletion
 Inversion
 Duplication
 Translocation
c) Gene Mutation
 Tansition
 Transversion
 Insertion
 Deletion
d) Plasmagene Mutation
e) Transposable element activation

19. f) DNA sequence
 Change in DNA
• Detection of altered fragment size by using Restriction enzyme
 Change in Protein
• Loss or gain in protein band
• Alteration in level of specific protein
 Methylation of DNA
• Methylation inactivates transcription process
 Lack of photosynthetic ability due to alteration in carbon metabolism
 Biosynthesis of starch via Carotenoid pathway
 Nitrogen metabolism
 Antibiotic resistance
Biochemical Cause

20. Detection and Isolation of Somaclonal Variants
1. 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
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 morphology of chromosomes
3. Variant detection by DNA contents
 Cytophotometer detection of feulgen stained nuclei can be used to measure the
DNA contents

21. 4. Variant 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.
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)

22. Advantages
 Help in crop improvement
 Creation of additional genetic variations
 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
Disadvantages
 A serious disadvantage occurs in operations which require clonal uniformity, as in the
horticulture and forestry industries where tissue culture is employed for rapid
propagation of elite genotypes
 Sometime leads to undesirable results
 Selected variants are random and genetically unstable
 Require extensive and extended field trials
 Not suitable for complex agronomic traits like yield, quality etc.
 May develop variants with pleiotropic effects which are not true.
Advantages & Disadvantages of Somaclonal Variations

23. Gametoclonal Variations
 The variation observed among plants regenerated from cultured gametic cells is
termed gametoclonal variation as compared to somoclonal variation, derived
from somatic cells.
 The term gametoclones (in place of somaclones) is used for the products of
gametoclonal variations.
 As the somatic cells divide by mitosis, the genetic material is equally distributed
to the daughter cells. In contrast, the gametes, being the products of meiosis,
possess only half of the parent cell genetic material.

24. The gametoclonal variations differ from somaclonal variations by three distinct
features:
1. Mutants obtained from gametoclonal variations give rise to haploid plants since a single
set of chromosomes are present.
2. Meiotic crossing over is the recombination process observed in gametoclonal variations.
3. The gametoclones can be stabilized by doubling the chromosome number.

25.  Gametoclones can be developed by culturing male or female gametic cells. The
cultures of anthers or isolated microspores are widely used. Improvements have
been made in several plant species through development of gametoclones e.g.,
rice, wheat, and tobacco.
Production of Gametoclones
Variation among plants regenerated from gametophytic tissue culture

26. Cell culture technique may induce genetic variations.
Variations may be induced while doubling the haploid
chromosomes.
Genetic variations may occur due to heterozygosity of
the diploids.
Source of Gametoclonal Variations

27. Reference
 Introduction to Plant Biotechnology book by H. S. Chawla