Somaclonal Variation: Discovery and Application

Article shared by Garima G

In this article we will discuss about: 1. Discovery and Definition of Somaclonal Variation, 2.
Techniques to Generate Somaclonal Variations, 3. Selection and Isolation, 4. Screening, 5.
Applications.

Discovery and Definition of Somaclonal Variation:

Improvement of plants through alterations or additions of traits are some of the main
objectives in plant biotechnology. Plant breeders have recognized the potential sources of ge-
netic variability and introduction of new novel genes into the desirable plants.
Plant cell and tissue culture are often referred to as the most useful methods for the
introduction of variation. In vitro cell and tissue culture provides ideal conditions for the
production of genetically variant novel plants.
Regeneration of plants via callus mediated processes often exhibits genetic variation. The
term somaclonal variation was first coined by Larkin and Scowcraft in 1981. According to
Scowcraft genetic variation occurring in tissue culture regenerated plants through somatic
tissues are commonly referred as somaclonal variation. According to Bajaj, any variation
which occurs in tissue culture plants is also referred to as somaclonal variation.
Several terminologies like calliclones and protoclones are referred to the plants, which are
regenerated from stem callus and leaf protoplast, respectively. Similarly, gametoclones are the
plants which are regenerated from haploid cells in culture. Much earlier, it was presumed that
tissue culture plants maintain clonal uniformity after regeneration. But later conclusions
drawn pertaining to clonal uniformity was found to be a myth.
Induction of genetic variation through tissue culture and its applications are the real hot spot
areas in plant modification technology. Totipotency of the plant cells makes much easier for
the plant scientists to recover somaclones through various modes of regeneration.
Plants arising from tissue culture should be exact copies of the parental plants. Phenotypic
variations were frequently observed among regenerated plants. Somaclonal variations may be
neither tissue specific nor organ specific and variation among somaclones has been observed
for a wide range of characters.
Somaclonal variation has potential impact on crop productivity. In vitro developed
somaclonal variant plants often display drought, cold, temperature, salinity and toxic tolerant
characters and sometimes even enhanced tolerance. Therefore, these phenotypic variants
observed from regenerated plants cannot be dismissed as artifacts of tissue culture and instead
can be potentially exploited in the improvement of crops much efficiently.

Techniques to Generate Somaclonal Variations:

i. A Long Term Culture:
The cultivars can be established in vitro and maintained for a considerable period of time by
frequent subcultures on culture media. Generally, subculture is carried out once in four weeks.
The long term maintenance of culture may attract genetic variation. It has been shown that
alteration in karyotypic structure occurs with increasing time in culture.
But, regenerated plants do not display full range of abnormalities, probably due to some kind
of selection pressure prior to regeneration. There have been substantial reports on the
problems concerning genetic stability of plants regenerated from long term cultures. In several
cases abnormal type of callus was shown to maintain inadvertently for a considerable duration
in vitro.
In tobacco, for example, plants regenerated from callus maintained for prolonged period
exhibits floral and leaf abnormalities, but were normal from the plant regenerated from short
term culture. Long term cultures of garlic (Allium sativum) plants show alteration in bulb size
and shape and even plant height.

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Long term maintenance of chrysanthemum leaf callus for nine years was highly variable.
Generally, cultures maintained upto two years and above show more than one chromosomal
alterations. However, exceptions seen in corn plants displayed excellent genetic stability,
maintained upto eight months in culture.

ii. Callus Culture:

Callus contains heterogenous mass of cells and is susceptible to genetic instability. The plant
regenerated from dedifferentiated callus tissue shows genetic variation. When genetic
variation is required in the process of crop improvement programme, the most widely em-
ployed technique for obtaining variations is the tissue culture, particularly, callus culture
cycle.
There are voluminous reports on the production of genetically variant plants obtained from
callus culture cycle. Production of somaclones from rice callus have shown that only a limited
number of plants displayed normal inheritance and majority tend to produce characters altered
genetically. Spontaneous production of polyploids, aneuploids and other chromosomal
rearrangements were observed in callus cultures of Hordeum vulgare.

iii. Plant Genotype:

Nature of plant parts used in tissue culture for regeneration reflects on the direction of genetic
variation. The degree of variation can be seen with specific type of explant. For example,
when pineapple plant regenerates from the callus derived from four types of explants exhibit
wide array of variations depending upon the explant. Only a single character or multicharacter
is altered from plants regenerated from specific tissues.

iv. Hormonal Factors:

Selection of specific hormones at higher concentration can induce genetic variation. Auxin
such as 2,4-D is the most promising candidate for the induction of genetic variation.
Regenerated plants from the callus obtained in presence of 2,4-D show genetic instability.
High concentration of 2,4-D in culture media increases the degree of variation. In several
plants, specific concentration of hormones decides the nature of variation, for example, in
barley plant; variation occurs only at 18 µM of 2,4-D for leaf shape and albino characters.
In the callus cultures of barley, genetic variations are not observed up to 18 µM
concentrations of 2,4-D in the media. The effect of 2, 4-D in inducing chromosomal anomally
was found to be higher than NAA. Addition of coconut milk along with 2,4-D and kinetin
further increases polyploid conditions in the callus cultures of Asparagus racemosus.

Selection and Isolation of Somaclones:

i. Biochemical Selection and Isolation:
Induction and recovery of somaclones are possible based on the display of unique selective
characters of plant cells. Resistance to certain toxic chemicals is the unique characteristic
feature of certain plant cells during regeneration process in vitro, suggesting simple or
multiple alterations within the selected variant cells. For example, certain variant cell lines are
resistant to isonicotinic acid hydrazide (INH) in irradiated cell cultures of haploid tobacco
plant.
Regenerated tobacco shows variations in their leaf shape, root formation including growth
habit. Another somaclone, Alfalfa, was recorded based on the selection for growth on
ethionine containing medium. Careful examination revealed that both resistant and non-
resistant cell lines were developed for ethionine toxicity.
Production of somaclones is also possible by exposing plant cells to toxic chemicals like ethyl
methonate sulphate (EMS). Plants regenerated from the exposed cells exhibit genetic
variation. Addition of chemical mutagens to the callus induction media increased genetic
variability in rice tissue.
Composition of differentiation media also influenced in the induction of genetic variation. For
example, high concentration of benzyladenine (BA, 30 mg/L) increased genetic variation.
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This is 50 times stronger than that of lowest concentration of BA used in the culture medium
of 2 mg/L.

ii. In Vitro Selection of Saline Tolerant Cell Lines:

In vitro selection of salt resistant cell lines are possible by exposing callus or free cells to
different strengths of salt. In the entire process, friable callus system was cut into pieces of
uniform size and transferred to liquid media containing different concentrations of sodium
chloride (NaCl). Callus cells are able to grow and proliferate under low concentration of salt
in the culture media.
However, as concentration increases it may hinder proliferation rate and eventually lead to
death of cells under high salt concentration. Careful observation suggested that few cells are
able to survive high salinity in culture conditions and proliferate sporadically. These saline
resistant cell lines are isolated, cultured and regenerated for salinity tolerant somaclones.

iii. In Vitro Selection of Disease Resistance Cell Lines:

In vitro selection of somaclones resistant to toxins of pathogenic microbes has been evi-
denced. Toxin resistance somaclones cell lines can be induced in vitro by growing cells in
presence of toxin environment. For example, plant pathogenic fungal resistance cell lines can
be established by adding various concentrations of fungal culture filtrate into the media and
followed by culturing callus or free cells. The fungal filtrate presumed to contain toxin range
can influence the growth rate of cells.
Exposure of cells from lowest to highest toxin range decides the fate of cell growth where,
higher concentration of toxin effectively destroys the cells. Resistant cell lines however,
survive and are able to proliferate in culture media. These toxin resistant somaclones cell lines
are selected, maintained, and plants are regenerated in vitro. Field tests are carried out to
establish disease resistant trait in the plants.

Screening of Somaclonal Variants:

Several methods are employed in the screening of genetically variant plants, of which
widely used techniques are as follows:
i. Cytological Screening:
This deals with microscopic assessment of chromosomal variations in cultured cells and
regenerated plants. Some of the preliminary cytological techniques like stained squash prepa-
ration of mitotic cells provides information about chromosomal variations. Certain observa-
tions like chromosomal number, chromosome length and location of nucleolus organizer
regions (NOR) highlighted the nature of genetic variation.
Assessment of meiotic cells can be a vital contribution associated with tissue culture.
Additional advantages of meiotic observations are evaluating pairing relationship among
homologous chromosomes and chromosome behaviour through several stages of meiosis.
Linear differentiation or minor differentiation of chromosome can be detected by employing
more refined technique such as G-banding technique which provides sophisticated evaluation.
For example, Giemsa C-banding has been particularly in formation with Vicia faba and
certain cereals except in the case of Zea mays.

ii. Biochemical Screening:

Many isoenzymes are used as biochemical markers in the screening of somaclonal variant
plants. Isoenzymes such as peroxidases, esterases and dehydrogenases can be employed in the
screening of somaclones. Any minor genetic variation can be visualized by analyzing
isoenzyme protein profiles.

iii. Molecular Screening:

Genetic variation takes place either at single nucleotide level and alteration or mutation of
several base pairs in DNA sequence. Assessment of any genetic variation by regular
cytological methods may not provide substantial evidence, may be due to minor variation at
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few nucleotides sequence in the genome. Certain molecular methods like restriction fragment
length polymorphism (RFLP) and RAPD apparently can elucidate variation at molecular
level.
The genomic DNA obtained from genetically variant plants show polymorphism after
restriction enzyme treatment. The restriction enzyme recognized sites are altered due to
mutation in the nucleotide sequence as a consequence various sizes of the DNA fragment can
be seen in their DNA profiles as polymorphic DNA. Screening of somaclones on several
occasions by RFLP approaches are used for plants obtained by tissue culture.

iv. Gametoclonal Variation:

As the name indicates, gametoclonal variation is derived from gametic cells. The processes of
mitosis are responsible for distributing genetic material in somatic cells and tissues. In meiotic
process however, gametes recover half of the gametic complements with allelels after
following Mendel’s law of segregation and independent assortment.
Gametoclones can be observed in the in vitro grown haploids due to the expression of
recessive and dominant variation in haploids. This is totally different from somaclonal
variation. Another difference is recovery of recombinational events in gametoclones. It is due
to the result of meiotic crossing over rather than non-mitotic crossing over as in the
somaclones.

Applications of Somaclonal Variation:

Several significant applications of somaclonal variations have been envisaged in view of its
role in crop improvement programme.

These are highlighted as follows:

i. Somaclonal variant plants derived from in vitro selection process are well known in
showing stress tolerance character. Somaclones can be grown in wide range of adverse
environmental conditions in the soil as well as in the surrounding environment.
Adverse conditions such as soil pH, temperature and water logging conditions will have
meagre influence on the growth of somaclones. Genetically variable somaclonal plants are
also adapted well to the high salinity soil. Several salt stress tolerable plants have been
produced through cell culture system.
ii. One of the most significant features of the somaclones is the presence of disease resistant
characters against plant pathogenic fungi, bacteria and viruses. In vitro plants have shown to
exhibit wide range of tolerance against microbial toxins.
iii. Somaclones are feasible in the cleaning up of soil contaminated with toxic metals. There-
fore, greater degree of access could be seen in the decontamination of soil by somaclones in a
process popularly known as phytoremediation.
iv. Recovery of somaclones shows resistance to antimetabolites such as amino acids analo-
gous, antibiotic drugs, and pathotoxins.
v. Herbicide tolerant potentials of somaclones have been well documented. These genetically
variant plants can detoxify many of the commonly used herbicides which is of immense value
in cleaning up of soil contaminated by recalcitrant herbicide chemical.
vi. Somaclonal variation offers improvement in the yield of crop plants and significantly
contributing in crop productivity. In addition, somaclones improved productivity in plantation
crops where generation cycle is long. Therefore, somaclonal plants have several defined edge
over conventional plants in acquiring useful traits.

Somaclonal Variation: Isolation and Selection Techniques

Article shared by Srinibas Kumar

In this article, we will discuss about the isolation and selection techniques of somaclonal
variation.

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Isolation and selection of somaclonal variation is an important task. Since several changes are
involved in producing somaclonal variation in different plant species, it is very difficult to sort
out the somaclonal variants using a single selection system. A number of selection systems
are now being used to select the variants.

A brief outline of commonly used techniques for detecting variations is given.

Analysis of Phenotypic Characters:
Phenotypic variations may arise among the regenerates during culture. Such variant characters
Eire observed thoroughly. A detailed report on observed characters is to be prepared for each
variant plants at the active level. After that, variants are transferred from culture flask to the
field.
In the field, such variants plants are observed during their successive growth and
development. Such qualitative and quantitative phenotypic characters viz., plant height, matu-
rity date, leaf size, flowering date, yield, seed fertility, waxiness in different plant parts,
flower morphology etc. are used as a parameter to sort out the variants.
Variants are also compared thoroughly with parental plants in all possible qualitative and
quantitative phenotypic characters. Several consecutive seed generations of the variants are
analysed to peruse whether the variant characters persist or not among the progeny (Fig 14.1).

Cytological Study of the Variants:

The traditional methods of acetocarmine and Feulgen-stained squashes of meristematic tissue
(root tip, leaf tip cells) of the variants permit the study of the number and gross morphology of
chromosomes. So any change in chromosome number or gross structures of chromosomes can
be detected by this method. To have a better assessment of minor structural changes of chro-
mosomes, banding techniques can be used.

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DNA Content of the Variants:
DNA content of the Feulgen stained inter-phase nuclei can be measured by cytophotometer.
An uniformly diploid state of cells always maintains its fixed amount of DNA. Any numerical
changes of chromosome will show either higher or lower values of DNA content. So the
measurement of DNA content can be used as a parameter for rapid screening of variants.

Gel Electrophoresis of Proteins or Enzymes:

A somaclone could be variant for a number of biochemical characters. Among them gel
electrophoresis of the proteins or enzyme extract from the homogenized plant parts is a
reliable parameter for detecting the variants. Any alternation in electrophoretic pattern of
proteins or enzymes indicates that the variants have lost or gained some specific proteins or
enzyme fractions.
The relative concentration of particular proteins or enzymes may alter relative to the parental
proteins or enzymes. Assay of other biochemical products like pigments, alkaloids, amino
acids etc. using the sophisticated instruments have also revealed the extent of variations
among the regenerates.

Selection for Disease Resistance:

Sometimes, disease resistance character may appear among the somaclonal variants where the
parent is highly susceptible to a particular disease. The pathogen or its toxin can be used as a
selection agent during culture.
If the in vitro selection is not feasible on cell, tissue or protoplast culture level, screening at
the seedling level is frequently possible. Behnke (1979) regenerated potato plants from callus
selected for resistance to the toxin culture filtrate of Phytopthora infestans. Field resistance of
some of the sugarcane variants has also been established.

Selection for Herbicide Resistance:

Selection for herbicide resistant phenotypes among the somaclonal variants has also been
made. The herbicide is generally added to the cell culture system and the regenerated plantlets
showing the tolerance to herbicide are selected. Chaleff (1980) has reported the selection of
several Nicotiana tabacum mutants with increased tolerance to picloram.

Selection for Environmental Stress Tolerance:

Salt, water-logging and drought, low and high temperatures and mineral toxicity and defi-
ciency are frequently cited as environmental stresses. Many attempts have been made to iso-
late stress-tolerant phenotypes in tissue culture. The persistence of stress-tolerant trait in
regenerated plants has also been demonstrated.
Selection of high sodium chloride tolerant cell lines in tobacco and the regeneration of plants
have been reported by Nabors et al. (1975). Regenerated plants showed salt tolerance through
two successive sexual generations. But this may be due to physiological adaptation rather than
genetic modification. Few attempts have been made to select for water-logging and drought
resistance in cell culture. Handa et al. (1983) has reported somaclonal variation for resistance
to polyethylene glycol in tomato cells.
Temperature stress is an unattractive in vitro selection agent. Breidenbach and Waring (1977)
studied the chilling response in tomato suspension culture and cell lines with enhanced
chilling tolerance have been demonstrated by Dix and Street (1976) in tobacco. Wu and
Wallner (1983) studied the heat response in suspension culture of pear cells in an effort to
provide a biochemical basis of in vitro selection for tolerance to high temperature.
Mineral toxicities and deficiencies are frequently problems in strongly acidic and strongly
alkaline soils. Aluminium toxicity at low pH is one of the best known examples. Meredith
(1978) reported the stable aluminium resistant variant cell lines in tomato tissue culture.
Sorghum plants have also been regenerated after selection against aluminium (Smith et al.,
1983).

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Somaclonal Variations: Basis, Isolation, Factors and Limitations
Article shared by Nandkishor Jha

Read this article to learn about the basis, isolation and limitations of somaclonal
variations and also factors affecting the production of somaclonal variants.
The genetic variations found in the in vitro cultured cells are collectively referred to
as somaclonal variations.
The plants derived from such cells are referred to somaclones. Some authors use the
terms calliclones and proto-clones to represent cultures obtained from callus and
protoplasts respectively.
The growth of plant cells in vitro is an asexual process involving only mitotic division of
cells. Thus, culturing of cells is the method to clone a particular genotype. It is therefore
expected that plants arising from a given tissue culture should be the exact copies of the
parental plant.
The occurrence of phenotypic variants among the regenerated plants (from tissue cultures)
has been known for several years. These variations were earlier dismissed as tissue culture
artefacts. The term somaclonal variations was first used by Larkin and Scowcraft (1981) for
variations arising due to culture of cells, i.e., variability generated by a tissue culture. This
term is now universally accepted.
As described elsewhere the explant used in tissue culture may come from any part of the
plant organs or cells. These include leaves, roots, protoplasts, microspores and embryos.
Somaclonal variations are reported in all types of plant tissue cultures.
In recent years, the term gametoclonal variations is used for the variations observed in
the regenerated plants from gametic cells (e.g., anther cultures). For the plants obtained
from protoplast cultures, proto-clonal variations is used.

Basis of Somaclonal Variations:

Somaclonal variations occur as a result of genetic heterogeneity (change in chromosome
number and/or structure) in plant tissue cultures.

This may be due to:

i. Expression of chromosomal mosaicism or genetic disorders.
ii. Spontaneous mutations due to culture conditions.
The genetic changes associated with somaclonal variations include polyploidy, aneuploidy,
chromosomal breakage, deletion, translocation, and gene amplifications, besides several
mutations. In fact, the presence of several chromosomal aberrations—reciprocal
translocation, deletions, inversions, chromosomal breakage, multi-centric, acentric fragments
have been found among the somaclones of barley, garlic and oat.
The occurrence of mutations in cultures is relatively low. Mutations may be due varied
nutrients, culture conditions and mutagenic effects of metabolic products that accumulate
in the medium. Somaclonal variations due to transposable elements, mitotic crossing over
and changes in the cytoplasmic genome have also been reported.

Nomenclature of somaclones:
The somaclones that are regenerated from tissue cultures directly are regarded as R0 or R
plants. The self-fertilized progeny of R0 plants represent R1 plants. R2, R3, R4 etc. plants are
the subsequent generations. Some workers use other nomenclature — somaclones (SC1 = R0),
SC2, SC3, SC4 etc. for subsequent generations.

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Isolation of Somaclonal Variants:
There are two procedures commonly used for obtaining the crop plants with
somaclonal variations:
1. Without in vitro selection
2. Within vitro selection.

1. Without in Vitro Selection:

An explant (leaf, stem, root etc.) is cultured 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 (Fig. 46.1).

Somaclonal variants of several crops have been successfully obtained by this approach e.g.,
sugarcane, potato, tomato, cereals etc. A selected list of disease resistant crop plants
obtained from somaclonal variations without in vitro selection along with the pathogenic
organisms is given in Table 46.1.
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Limitations of without in vitro selection approach:
There is no directed and specific approach for the isolation of somaclones without in vitro
selection. Consequently, the appearance of a desired trait is purely by chance. Further,
this procedure is time consuming and requires screening of many plants.

2. With in Vitro Selection:

Isolation of somaclones with in vitro selection method basically involves handling of plant
cells in cultures (protoplast, callus) like microorganisms and selection of biochemical
mutants. The cell lines are screened from plant cultures for their ability to survive in the
presence of a toxic/inhibitory substance in the medium or under conditions of
environmental stress.
A diagrammatic representation of in vitro protocol for the isolation of disease
resistance plants with in vitro selection approach is given in Fig. 46.2.

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The differentiated callus, obtained from an explant 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 analysed for disease resistant plants
against the specific pathogenic organism.
A selected list of disease resistant crop plants obtained from somaclonal variations by in
vitro selection along with pathogenic organisms and selection agents is given in Table 46.2.

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Besides the disease resistant plants, plants with herbicide resistance and antibiotic
resistance have also been developed with in vitro selection approach.

Environmental stress tolerance:

High salt concentration in the soil is the major constraint limiting the crop development
and yield. Many plants with salt tolerance (salinity) have been developed e.g., rice,
tobacco. Several attempts are being made by plant biotechnologists to develop cold tolerant
crops, although the success rate has been very limited.

Advantages of with in vitro selection approach:

The major advantage of with in vitro selection method is the specific selection of the
desired trait rather than a general variation found at the plant level. This procedure is less
time consuming when compared to without in vitro selection approach.

Factors Affecting Production of Somaclonal Variants:

Some of the important factors that influence development of somaclonal variants by
without in vitro selection and within vitro selection are briefly described.

Genotype and explant source:

The nature of genotype of the plants influences the frequency of regeneration and frequency
of production of somaclones. Explants can be taken from any part of plant — leaves, roots,
internodes, ovaries etc. The source of explant is very critical for somaclonal variations. For
instance, potato plants regenerated from callus of rachis and petiole are much higher
(~50%) compared to those regenerated from callus of leaves (~12%).

Duration of cell culture:

In general, for many plant cultures, somaclonal variations are higher with increased
duration of cultures. For example, it was reported that genetic variability increased in
tobacco protoplasts from 1.5% to 6% by doubling the duration of cultures.

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Growth hormone effects:
The plant growth regulators in the medium will influence the karyotypic alterations in
qultured cells, and therefore development of somaclones. Growth hormones such as 2,4-
dichlorophenoxy acetic acid (2,4-D) and naphthalene acetic acid (NAA) are frequently used
to achieve chromosomal variability.

Besides the factors discussed above, selection of somaclones with in vitro selection
are dependent on the following parameters:
i. Selection propagule (cells, protoplasts, calli).
ii. Selection agent (toxin, herbicide, amino acid analogue).
iii. Technique used for selection.
iv. Stability of resistant substance.
v. In vivo testing procedure.
vi. Ability for regeneration of plants.

Limitations of Somaclonal Variations:

Despite several applications of somaclonal variations, there are certain
limitations/ disadvantages also:
i. Most of the somaclonal variations may not be useful.
ii. The variations occur in an unpredictable and uncontrolled manner.
iii. Many a times the genetic traits obtained by somaclonal variations are not stable
and heritable.
iv. Somaclonal variations are cultivar-dependent which is frequently a time
consuming process.
v. Somaclones can be produced in only those species which regenerate to complete plants.
vi. Many cell lines (calli) may not exhibit regeneration potential.

Gametoclonal Variations:
The variations observed while culturing the gametic cells are regarded as gametoclonal
variations. This is in contrast to the somaclonal variations detected in the cultures of
somatic tissues.
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.

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. A selected list of plants regenerated
from gametoclonal variations is given in Table 46.4. Improvements have been made in
several plant species through development of gametoclones e.g., rice, wheat, and tobacco.

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Source of Gametoclonal Variations:
There are three sources of genetic variations in the gametoclones:
1. Cell culture technique may induce genetic variations.
2. Variations may be induced while doubling the haploid chromosomes.
3. Genetic variations may occur due to heterozygosity of the diploids.

Applications of Somaclonal Variations

Article shared by Nandkishor Jha

This article throws light upon the five applications of somaclonal variations.
The five applications are: (1) Production of agronomically useful plants (2) Resistance to
diseases (3) Resistance to abiotic stresses (4) Resistance to herbicides and (5) Improved seed
quality.
Somaclonal variations (and also gametoclonal variations, described later) are highly useful in
plant breeding programmes. The genetic variations with desirable (or improved characters),
besides the existing favourable characters can be introduced into the plants.
In general, the methodology adopted for induction of somaclonal variations are simpler and
easier compared to recombinant DNA technology. Hence, they are preferred by some
workers. The important applications of somaclonal variations are briefly described.

Application # 1. Production of agronomically useful plants:

As a result of somaclonal variations, several novel variants of existing crops have been
developed, e.g., pure thorn-less blackberries. In Table 46.3, somaclonal variations in a
selected list of crops with useful and improved morphological characters are given. The crops
include rice, wheat, maize, sugarcane, potato, carrot etc.

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Application # 2. Resistance to diseases:
Somaclonal variations have largely contributed towards the development of disease resistance
in many crops e.g. rice, wheat, maize, sugarcane, tobacco, apple, tomato. Selected crops
somaclonal variants, with increased disease resistance developed, without in vitro selection
and within vitro selection are respectively given in Tables 46.1 and 46.2.

Application # 3. Resistance to abiotic stresses:

It has been possible to develop biochemical mutants with abiotic stress resistance.
i. Freezing tolerance e.g. wheat.
ii. Salt tolerance e.g., rice, maize, tobacco.
iii. Aluminium tolerance e.g., carrot, sorghum, tomato.

Application # 4. Resistance to herbicides:

Certain somaclonal variants with herbicide resistance have been developed. Selected
examples are given
i. Tobacco resistant to glyphosate, sulfonylurea and picloram.
ii. Carrot resistant to glyphosate.
iii. Lotus resistant to 2,4-dichlorophenoxy acetic acid (2,4-D).

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Application # 5. Improved seed quality:
A new variety of Lathyrus sativa seeds (Lathyrus Bio L 212) with a low content of neurotoxin
has been developed through somaclonal variations.

Examples of Somaclonal Variation

Article shared by Garima G

The following are the some of the examples of Somaclonal Variation: 1. Sugarcane 2.
Potato 3. Tobacco 4. Rice 5. Barley.

1. Sugarcane:
Application of somaclonal variation in crop improvement programme was established in
sugarcane. The Hawaian Sugar Planters Association Experimental Station recorded genetic
variation among sugarcane plants regenerated from tissue culture. Variations were
recorded based on cytogenetic, morphological and enzyme profiles.
After Hawaian success, work was initiated in Fiji in the development of resistant to Fiji
disease virus and several somaclones resistant to Fiji disease and powdery mildew have been
identified and established using tissue culture techniques. As many as 4000 independent
series of somaclones have been produced in Hawai to screen for Fiji disease. Several of these
variants are resistant to the toxin.
In Philippines, several somaclones from sugarcane exhibit significant characters which are
different from parental variety in cane yield, staik length etc. Lieu and Chen (1976) in
Taiwan have found significant genetic variations among sugarcane yield, staik number,
length, percent fibre, etc.
Detailed studies in sugarcane revealed that it is easy to obtain variation in sugarcane
which provides greater promise for the improvement of varieties.

2. Potato:
The commercial potato is autotetraploid. Nearly 20% of the world potato production lost due
to disease. Several popular varieties are subjected for improvement in North America in
order to produce disease resistant somaclones.
Screening over few hundred somaclones exhibit different altered characters and
particularly somaclones were obtained which were resistant to Alternaria solani toxin.
About 2% somaclones screened were resistant to Phytophthora infestans causes late blight,
some of which were resistant to multiple races of the pathogen.

3. Tobacco:
Several reports established the occurrence of genetic variation among regenerated potato
plants. Some of these variant characters are CO2 adsorption and chlorophyll content.
Improvement of 10% of the somaclones exhibit yield variability. The somaclones lines
shows completes difference from itself as well as parental lines in several prominent
characters. Anther derived dihaploids from heterozygous for each of three single genes
controlled character showed more resistant to viral disease.

4. Rice:
Plants regenerated from rice callus shows phenotypic characters. These somaclones exhibit
variations such as number of tiller per plant, panicle size and frequency of fertile plant.
About 800 somaclones were derived from seed callus. Among these variants chlorophyll
content, plant height, fertility and morphology were considered normally in all these
characters. In addition, wide variation in seed fertility and plant height were noticed.

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5. Barley:
The barley plants derived from pollen culture of homozygous sample show phenotypic
variation. The reason for variation was drawn in favour of chromosomal breaks and reciprocal
translocation instead from chromosome number alterations. In the media composition the
con-centration of 2,4-D enhanced genetic variation.

Application of Somaclonal Variation

Article shared by Srinibas Kumar

In this article, we will discuss about the application of somaclonal variation in some
crop plants and horticulturally plants.
In tissue culture, somaclonal variation has been presented a lot of significant contributions to
plant science. Somaclonal variation among regenerated plants from callus and protoplast
cul-ture has been suggested as a useful source of potentially valuable germplasm for plant
breed-ing and plant improvement.
The major benefit of somaclonal variation is to create variation in adapted genotypes.
Recognition of new genotypes at the whole plant level and their efficient exploitation would
however be very useful in breeding programme. Agronomically desirable many traits in
several crop plants have been raised from tissue culture. Many of these traits will find a
place in new improved varieties.

Some examples of somaclonal variation in crop plants as well as in some

horticulturally important plants has been discussed below.

Rice:
The occurrence of somaclonal variation in rice (Oryza sativa) had been independently reported
several times. Numerous variations have been observed in somaclones derived from an assumed
homozygous parent; a dihaploid derivative (a self-doubled haploid or homo-zygous diploid)
from anther culture.
About 1,121 somaclones, selected progeny were examined immediately in three successive
selfed generations and these segregated for characters such as plant height, maturity, heading
date and grain yield. Within two generations of selfing, many of the variants had become
true breeding. Based on progeny analysis of five characters, 72% of the regenerates differed
from the parent in at least one character; 28% differed for two or more traits.
Schaeffer et al. (1984) found significant variation among anther-derived dihaploid of rice.
Significant improvements relative to the parent were observed for seed weight, seed proteins
percentage, tiller number, panicle length and time of flowering. At the International Rice
Research Institute, recent research has involved analysis of somaclonal variation in several
rice cultivars.
Mutants were observed for many characters such as panicle, grain and leaf morphology and
tiller arrangement. Several kinds of chlorophyll-defective mutants were also observed. The
progeny have also been screened for tolerance to salinity and aluminium toxicity.

Wheat:
It has recently become possible to regenerate variant plants from tissue cultures of wheat.
Variations were manifested for both morphological characters and for traits under simple
genetic control such as gliadin proteins in seed, grain colour etc. and polygenic control
such as plant height, heading date and yield. Both heterozygous and homozygous mutants
were screened in the primary regenerates of wheat.

Maize:
The classical genetic, chromosomal and recently molecular research in maize has enabled a
penetrating analysis of somaclonal variation. In one analysis of 77 somaclones regenerated

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from maize tissue cultures, 17 defective endosperm or seedling mutants were identified. In
another analysis of 51 somaclones, eight segregated for recessive kernel mutations and one
segregated for a mutation which caused premature wilting.
Maize studies have also provided conclusive evidence that the mitochondrial genome can
undergo genetic changes during cell culture. Cytoplasmic male sterile line of maize is very
sensitive to T-toxin produced by the causal organism of southern corn leaf blight, Drechslera
maydis. Normal cytoplasm plants are male sterile and resistant to the T-toxin.
The toxin has been used as a selective agent in tissue culture of T-cytoplasm maize lines.
Plants regenerated from the selected cell lines were resistant both to the toxin and to
infection by D. maydis. Toxin resistant, male fertile plants were regenerated from Cms – T
maize culture grown without exposure to toxin. The conversion during tissue culture to toxin
resistance, male fertility was maternally inherited and shown to be associated with the
mitochondria.

Potato:
For the improvement of potato crop Shepard et al (1980) suggested that it will be more
profitable to improve a popular variety selectively rather than to create a new one. The
potato somaclones were also screened for both late and early blight resistance. The parent
‘Russet Burbank’ is highly susceptible to both these diseases. From among more than 800
plants, a range of variation to late blight (Phytophthora infestans) was found.
About 2% of the somaclones were also able to transmit the disease resistant character
through subsequent tuber generations. In addition, several other disease resistant variants
were recovered. Such variants are resistant to early blight (Alternaria solani) and to multiple
races of Phytophthora infestans.

Tomato:
A large number of somaclonal variations have been raised from leaf derived callus tissue of
tomato. Variants were screened for a number of characters such as male sterility, joint-less
pedicel, fruit colour, indeterminate growth etc.

Oat:
Many somaclonal variations have been observed among plants regenerated from cultured
immature embryo, apical meristem of oat. Variants were selected for plant height,
heading date, leaf striping, awns, etc.

Brassica spp.:
The occurrence of somaclonal variation in Brassica spp. has been independently reported
several times. Variants were found which affected flowering time, growth habit, waxiness
glucosinolates, Phoma lingam tolerance.

Nicotiana sp.:
In Nicotiana sp., plant regeneration was possible from anther culture, protoplast culture and
leaf callus culture. From regenerated plant population, somaclonal variants were selected for
a number of characters such as plant height, leaf size, yield grade index, alkaloids, reducing
sugars, specific leaf chlorophyll loci etc.

Lolium:
A triploid hybrid was obtained by crossing a diploid Lolium perenne with a tetraploid L.
multiflorum. Callus tissue derived from this hybrid produced more than 2,000 plants in
five years. These somaclones exhibited a wide variation in leaf shape, size, floral
development, growth vigour and longevity.
Some variants possessed characteristics of both the parents which were agronomically
valuable and their progenies also showed the same variations, whereas these
characteristics were not observed in triploid hybrids reared up in a conventional way.
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Pelargonium:
A high degree of variability in tissue culture regenerated plants from 5 cultivars of
Pelargonium zonale was observed. Changes were found in plant and organ size, leaf
and flower morphology, essential oil constituents, fasciation, pubescence and
anthocyanin pigmentation. One of the variants had been released, as a new cultivar
known as ‘Velvet Rose’.

Geranium:
Tokumasu and Kato (1976) using somatic callus cultures of a dihaploid Geranium plant had
obtained homogenous plants amongst which two were variants for essential oil constituents.
The potentiality of somaclonal variation has increased dramatically with clear evidence of
ex-pression of selected traits in selected plants and seed progeny.
Among the economically important traits which have been selected in vitro and recognized
as having potential impact are now being employed to enhance the efficiency of plant
breeding. Somaclonal variations provide also a powerful option for plant improvement and
this may be the best approach of plant improvement over conventional methods.

Application of Somaclonal Variation

1. Somaclonal variation and gametoclonal variation represent useful source of introducing
genetic variations that could be of value to plant breeders.
2. Single gene mutation in nuclear or organelle genome may give the best available variety in
vitro that has a specific character.
3. Gametoclonal variation, induced mostly by meiotic recombination during the sexual cycle
of F1 hybrid, results in transgressive segregation to uncover unique gene combination.
4. Various cell lines selected in vitro may prove potentially applicable to agriculture and
industry like resistance to herbicide, pathotoxin, salt or aluminium.
5. Variability in cell cultures has played a useful role in synthesis of secondary metabolites
on a commercial scale.
6. Technique employed for Somaclonal and gametoclonal variation are relatively easier than
recombinant DNA technique.

Somaclonal variants for agronomically desirable traits in several crop plants have been raised
from tissue culture. Some examples of Somaclonal variation in crop plants as well as in some
horticulturally important plants are given below:

Rice:
Significant improvements relative to parent were observed for seed weight, seed proteins
percentage, tiller number, panicle length and time of flowering. At IRRI, mutants were
observed for many characters such as panicle, grain, and leaf morphology and tiller
arrangement.

Wheat:
Variations were manifested for gliadin proteins in seed, grain colour, plant height, heading
date and yield.

Maize:
Plants regenerated from selected cell lines were resistant both to T-toxin and to infection to
Drechslera maydis causing southern leaf blight. Cytoplasmic male sterile lines are very
sensitive to the T-toxin produced by Drechslera maydis.

Potato:
Somaclonal variants were selected for resistance to Phytopthora infestans and to its multiple
races and resistance to early blight.
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Tomato:
Somaclones were isolated with variant phenotypes, such as recessive mutation for male
sterility, resistance to Fusarium oxysporum, jointless pedicel, tangerine virescent leaf, flower
and fruit colour.

Sugarcane:
Somaclonal variants have been isolated by different workers for cane yield, sugar yield and
resistance to smut disease caused by Ustilago scitamini, downey mildew caused by
Helminthosporium sacchari.

Geranium:
Skirvin and Janick (1976) developed an improved scented geranium called ‘Velvet rose’
from Pelargonium species by isolating Somaclonal variant. The new cultivar has symmetrical
flowers with large fertile stamens, five paired stigma and sets seed. The parential cultivar, on
the contrary has asymmetrical flowers with reduced-sterile anthers, a two-paired stigma and
never sets seeds.

Somaclonal
Crop Parent variety Remarks
variety

Pusa Jaikisan 17.4% yield advantage (by

Brassica juncea Varuna
(BIO 902) IARI)

More oil & citronellol yields

Citronella java Bio-13 -
(by CIMAP, Lucknow)

Geranium Velvet Rose Robert’s Lemon Rose Polyploid Somaclone

Low neurotoxin (by IARI,

Lathyrus sativus Ratan (Bio L-212) -
New Delhi)

Linseed (Flax) Andro McGregor Salt tolerant

Sugarcane Ono Pinder Resistance to Fiji disease

Darker & more suitable skin

Sweet potato Scarlet -
colour

Altered colour, taste, texture

Tomato - -
& colour

Increased resistance to fall

Cynodon dactylon Brazos R-3 -
armyworm

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