1. SOMOCLONAL VARIATIONS

2. • The term somaclonal variation by Larkin and Scowcroft (1981) was
given for the variability generated by the use of a tissue culture cycle.
• Somaclonal variation is defined as genetic variation observed among
progeny plants obtained after somatic tissue culture in vitro.
• Theoretically all progeny plants regenerated from somatic cells should
be identical clones. However, variations might occur in number of
progeny which are known as somaclones and they are genetically
variable from their explant.
• The initiating explant for a tissue culture cycle may come virtually from
any plant organ or cell type including embryos, microspores, roots,
leaves and protoplasts. So, all somatic tissue culture can result in
somaclonal variation.
• Somaclonal variation is a phenotypic changes as a result of
chromosomal rearrangement during tissue culture.

3. Basic Features of Somaclonal Variations
• 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.

5. Nomenclature of somaclonal variation:
• Though different letters and symbols have been used, two symbols
are generally used.
• 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
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

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

7. 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.

9. 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.

11. 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

13. Causes of Somaclonal Variations
• Physiological Cause
• Exposure of culture to plant growth regulators
• Culture conditions
• Genetic Cause
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

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

15. 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
Biochemical Cause
• Lack of photosynthetic ability due to alteration in carbon metabolism
• Biosynthesis of starch via Carotenoid pathway
• Nitrogen metabolism
• Antibiotic resistance

16. Applications of Somaclonal Variation:
i. Production of Novel variants:
• An implication of somaclonal variation in breeding is that novel variants can arise and
these can be agronomically used.
• A number of breeding lines have been developed by somaclonal variation.
• Example: An enhanced scented Geranium variety named ‘Velvet Rose’ has been
generated. An example of heritable somaclonal variation is the development of pure
thornless blackberries Lincoln Logan (Rubus), Hasuyume, a protoplast derived rice
cultivar, and somaclone T-42 has been generated.
• In India two varieties namely Pusa Jai Kisan in mustard Brassica and CIMAP/Bio13 in
Citronella have been released for cultivation.
• An improved variety of rice ‘DAMA’ has been released through pollen haploid
somaclone method which combined microspore culture with somatic tissue culture
(see Heszky and Simon-Kiss, 1992).
• Somaclonally derived mutants in tomato with altered color, taste, texture and shelf life
are being marketed in USA by Fresh World farms

17. Food crops Pathogen
Barley Rhynchosporium secalis
Maize Helminthosporium maydis
Rice Helminthosporium oryzae
Rape Phoma lingam, Alternaria brassicicola
Sugar–cane
Fijivirus, Sclerospora saccharii, Helminthosporium
sacchari
• The greatest contribution of somaclonal variation towards plant improvement is in the
development of disease resistant genotypes in various crop species.
• Resistance was first reported in sugarcane for eye spot disease
(Helminthosporium sacchari), downy mildew (Sclerospora sacchari) and Fiji virus
disease by regenerating plants from the callus of susceptible clones and screening the
somaclones.
• Following table lists few successful examples of somaclonal variants obtained without
in vitro selection at the plant level with an increased disease resistance:

18. iii. Production of abiotic stress resistance variety:
• Somaclonal variation has resulted in several interesting biochemical mutants, which are
being successfully used in plant metabolic pathway studies, i.e. amino acid and
secondary metabolic pathways.
• Investigations have shown that the level of free amino acids, especially proline,
increases during cold hardening.
• In vitro selection has also been used to obtain plants with increased acid soil, salt,
aluminium and herbicide resistance.
iv. Production of Cold tolerance:
• Lazar et al. (1988) developed somaclonal 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.

19. v. Production of Salt tolerance:
• 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.
• In most cases, the development of cellular salt tolerance has been a
barrier for successful plant regeneration, or if plants have been obtained
they did not inherit the salt tolerance.
• Only in a few cases it was possible to regenerate salt tolerant plants.
Mandal et al. (1999) developed a salt tolerant somaclone BTS24 from
indigenous rice cultivar Pokkali.
• This somaclone yielded 36.3 q/ha under salt stress conditions as
compared to 44 q/ha under normal soil.
• Some of the reports for successful production of healthy, fertile and
genetically stable salt tolerant regenerants from various explants.

20. vi. Production of Aluminium tolerance:
• Plant species or cultivars greatly differ in their resistance to aluminium stress.
• 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, e.g.
alfalafa, carrot, sorghum, tomato, tobacco.
• Aluminium tolerant somaclonal variants from cell cultures of carrot were selected
by exposing the cells to excess ionic aluminium in the form of aluminium chloride
(Arihara et al., 1991).
• 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.
• After selection on aluminium toxic media, callus lines were maintained on
aluminium toxic free media and 9 tolerant plants were obtained. Transmission of
aluminium resistance character was identified till the fourth generation.

21. vii. Production of Drought tolerance:
• Wang et al. (1997) reported a sorghum somaclonal variant line (R111) resistant to
drought stress.
• A novel hybrid was developed by crossing R111 with several sterile lines,
suggesting that selection of somaclonal variants is an effective method for
creating new sources of genetic variation.
• 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.
viii. Production of Herbicide resistance:
• Through in vitro selection several cell lines resistant to herbicides have been
isolated and a few have been regenerated into complete plants.
• Among the important achievements are tobacco, soybean, wheat, maize, etc.
resistant to various herbicides such as glyphosate, sulfonylurea, imidazolinones,
etc.

22. • ix. Production of Insect resistance:
• Zemetra et al. (1993) used in vitro selection technique for generation of
somaclonal variants for Russian wheat aphid (Diuraphis noxia) in wheat.
• Calli from susceptible wheat cultivar “Stephens’’ were exposed to an extract from
aphid.
• Resistance to aphid was observed in both R2 and R3 generations.
• x. Seed quality improvement:
• Recently, a variety Bio L 212 of lathyrus (Lathyrus sativa) has been identified for
cultivation in central India which has been developed through somaclonal
variation and has low ODAP (β-N-oxalyl -2-α, β diamino propionic acid), a
neurotoxin (Mehta and Santha, 1996), indicating the potential of somaclonal
variations for the development of varieties with improved seed quality.

23. xi. Introgression of Alien gene:
• The increase in genome rearrangement during tissue culture provides a new
opportunity for alien gene introgression which can help widen the crop
germplasm base, particularly by culturing immature embryos of wide crosses
where crop and alien chromosomes cannot replicate through meiosis.
• Introgression of genes may be better achieved by imposing one or more cell
culture cycles on interspecific hybrid material.
• The enhanced somatic genome exchange is likely to produce regenerants where
part of the alien genome has been somatically recombined into the
chromosomes of crop species.

24. Advantages & Disadvantages of Somaclonal Variations
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.

25. 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.

26. 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
Production of Gametoclones
• 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.

27. Source of Gametoclonal Variations
 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.

28. Reference
• Introduction to Plant Biotechnology book by H. S. Chawla