SOMACLONAL VARIATION
In isolation of useful mutants
& commercial production of
secondary metabolites
TOPIC : 6
ANIKA TAHSIN
Roll-01
AFSANA AKTER
Roll-07
SAIMA MUBASHSHIRA
Roll-13
NASRIN AKTER
Roll-42
SHAMSUTTIYEBA SHIFA Roll-38
INTRODUCTION
ANIKA TAHSIN
Roll-01
EXPLOITING THE MODEST
 Mankind had been modifying microbes, plants, even other animals to
obtain desired traits for about 10,000 years. With the evolutionary
changes constantly changing the designs of the living, we have obtained
the now common food species, characteristically different from their
ancestors.
 Modifications of such have led to advantageous outcomes e.g.
increased food production, reliability, and yields; enhanced taste and
nutritional value; and decreased losses due to various biotic and abiotic
stresses, such as fungal and bacterial pathogens
GENETIC MODIFICATION
Genetic Engineering Independent
Genetic Engineering Dependent
• Simple Selection
• Microbial Vectors
• Interspecies Crossing
• Microprojectile Bombardment
• Somatic Hybridization
• Electroporation
• Somaclonal Variation
• Microinjection
• Mutation Breeding
• Transposons/Transposable Elements
PRODUCING SUPERIOR TRAITS WITHOUT ENGINEERING
 Somaclonal variation, a natural phenomenon that can RANDOMLY generate
beneficial traits (from human perspective, of course) in plants; a term coined by
two Australian scientists Larkin and Scowcroft in 1981
 Plants regenerated from tissue culture sometimes had novel features, which they
thought, might provide a new source of genetic variability, new variants carrying
attributes of value to plant breeders
 As independent of genetic engineering, it is especially suitable for application
in developing countries with limited resources to identify gene sequences
HETEROGENEITY ARISING FROM THE HOMOGENEOUS
Somaclonal variation is defined as the variability generated through a
tissue culture cycle - a process that involves the establishment of a
dedifferentiated cell or tissue culture under defined conditions, proliferation
for a number of generations
In general, plants produced in tissue culture are true-to-type, meaning genetically homogeneous, and have a uniform phenotypic appearance.
However, in most cases, a large number of cell cycle results in a genetically
heterogeneous population of cells with variable phenotypes.
SOMACLONES ARISE THROUGH MOLECULAR ALTERATIONS DURING CELL DIVISION CYCLES IN TISSUE CULTURE
DISTINCT
DISTINCT
DISTINCT
DISTINCT
DISTINCT
DISTINCT
HETEROGENEITY ARISING FROM THE HOMOGENEOUS
This heterogeneity is majorly due to either,
 Expression of chromosomal mosaicism in cells of explants, or
 Alterations resulting from culture conditions through spontaneous mutations
The resulting variation is attributed to (heritable) changes in
• Chromosome number (e.g. aneuploidy, polyploidy) or structural rearrangements
• Nucleotide base sequence or copy number of a given gene
• Activation and transposition of transposable elements and
• Stable (non-heritable) changes in gene expression
MOSAICISM REFERS TO THE PRESENCE OF TWO OR MORE POPULATIONS OF CELLS WITH DIFFERENT GENOTYPES IN ONE INDIVIDUAL WHO HAS DEVELOPED FROM A SINGLE FERTILIZED EGG.
GENERATING NOVEL FEATURES
 Despite
being
considered
an
undesirable
consequence in plant tissue culture; as we continue to
limit
chance
of
variability
in
the
available
germplasm and gene pool by exhausting intense
breeding and selection; somaclonal variation has
proven to be effective regardless, in generating
novel features.
 This
process
also
provides
higher
mutation
frequencies over spontaneous variation for genetic
improvement.
HANDLING PLANTS LIKE MICROBES!
 Somaclonal variation has a remarkable
potential to screen mutants in vitro, and
identification of mutants with novel
characteristics
 Protoplast, cell suspension and callus
cultures are handled like microorganisms to
search for biochemical mutants
 ‘Selection agents’ (e.g. herbicide) are
used for successful isolation and detection
of desired trait (e.g. herbicide resistant)
Figure: Generation of herbicide-resistance in
plants using somaclonal variation
THE RED FLASH
 Caladium (Caladium x hortulanum Birdsey), an ornamental plant with dramatic leaves, which come in many
different shapes, colors, and variegation patterns. Reports indicated that somatic variation was common in caladium
and variations include changes in leaf shape, apex, base and margin, color of lamina, spotting, veins, and petiole
attachment and color.
Figure: Leaf morphology of the wildtype (WT) and
ten somaclonal variant types (SVT) of ‘Red Flash’
caladium. These somaclonal variants exhibited
considerable changes in leaf shape, coloring of the
main veins, spots, margins, and leaf size, and could
be separated into 10 somaclonal variant groups.
For the wildtype and each SVT, the photo of the
leaf was taken from: SVT1 from Plant M2, SVT2
from Plant M1, SVT3 from Plant M5, SVT4 from
Plant M36, SVT5 from Plant M187, SVT6 from
Plant M45, SVT7 from Plant M75, SVT8 from Plant
M7, SVT9 from Plant M4, and SVT10 from Plant
M203. Scale bar 3 cm.
Reference: Somaclonal variation in ‘Red Flash’ caladium:
morphological, cytological and molecular characterization, Cao et.
al, Plant Cell Tiss Organ Cult (2016) 126:269–279
VARIEGATION IS THE APPEARANCE OF DIFFERENTLY COLOURED ZONES IN THE LEAVES AND SOMETIMES THE STEMS, OF PLANTS.
THE RED FLASH
Results from this particular study (Cao & Sui, 2016) showed that the type of leaf explants and auxin affected the
occurrence of somaclonal variation in ‘Red Flash’. The highest percentage of variants (25%) was observed among
plants regenerated from mature leaf explants cultured on the media containing 2,4-dichlorophenoxyacetic acid.
Twelve variants contained 1.1–5.4 % less nuclear DNA and appeared to have lost one chromosome. Two variants
contained 5.4–9.2 % less nuclear DNA and appeared to have lost two chromosomes. One variant contained 95.0 %
more nuclear DNA and 2n = 58 chromosomes (aneuploid)
Figure: Micrographs of somatic chromosomes in the
root tips of the wildtype and five somaclonal variants
(M25, M187, M3, M7, and M4) of ‘Red Flash’
caladium obtained from in vitro cultured young or
mature leaf segments. Photographs were taken under
a bright field
microscope at 1000 magnifications. a M45 (2n = 2x 2 = 28), b M187 (2n = 2x - 1 = 29), c wildtype (2n =
2x = 30), d M3 (2n = 2x = 30), e M7 (2n = 2x = 30),
and f M4 (2n = 4x - 2 = 58). Scale bars 10 μm.
Reference: Somaclonal variation in ‘Red Flash’ caladium:
morphological, cytological and molecular characterization, Cao
et. al, Plant Cell Tiss Organ Cult (2016) 126:269–279
ROSE-SCENTED GERANIUM
Pelargonium graveolens produced rose
scents are most commercially important
in the perfume industry and are
cultivated and distilled for their scents.
Pelargonium distillates and absolutes,
commonly known as "scented geranium
oil" are sometimes used to supplement or
adulterate expensive rose oils.
‘Velvet Rose’ is an improved scented
Geranium variety which has been
developed through somaclonal variation
(2002).
THORNLESS BLACKBERRY
An example of heritable somaclonal
variation is the development of pure
thornless blackberries Lincoln Logan (Rubus).
A tissue culture-derived bramble cultivar,
‘Lincoln Logan’ were developed by
separating the histogenic layers of chimeral
‘Thornless Logan’ to form a pure thornless
type (Hall et al., 1986)
This ‘Everthornless’ is a genetically pure form
of ‘Thornless Evergreen’ that was obtained
via somaclonal variation (McPheeters and
Skirvin, 1995; Plant Patent No. 9407)
ISOLATION AND DETECTION
AFSANA AKTER
Roll-07
ISOLATION OF SOMACLONAL VARIANTS
In general, two schemes are followed in isolation of somaclonal
variants:
1. Without in vitro Selection (plant level)
2. With in vitro Selection (cellular level)
WITHOUT IN VITRO SELECTION
1
Unorganized callus and cells, grown in non-selective culture for various
periods on a medium, are induced to differentiate
2
Explant (leaf, stem, root etc) is cultured on a suitable medium,
supplemented with growth regulators
3
These cultures are normally sub-cultured, and transferred to shoot
induction medium for regeneration of plants
4
5
So produced plants are in pots, transferred to field
• Newly generated plants are analyzed for subclonal variation
WITHOUT IN VITRO SELECTION
Limitations:
 Lack of specificity for isolation of somaclones
 The appearance of a desired trait is purely by chance
 Time consuming and require screening of many plants
WITH IN VITRO SELECTION
In this approach, variant of a particular character are selected at
cellular level, rather than the general variation obtained in the first case.
Protoplast, cell suspension and callus cultures are handled like
microorganisms to search for biochemical mutants
WITH IN VITRO SELECTION
1
Dediffentiated culture is subjected to selection against inhibitors like
antibiotics, amino acid analogs, pathotoxins etc
2
The compounds are in optimal concentrations in the medium, such
that some cell population survives and can be further grown on a
selective medium.
3
Selection cycles are carried out to isolate the tolerant callus cultures
and these cali are regenerated into plants.
4
The plants so obtained are in vivo screened against the toxin (or
pathogen or any other inhibitor)
5
The plants resistant to the toxin are selected and grown further by
vegetative propagation or self-pollination
6
The subsequent generations are analyzed for resistant plants
against the specific inhibitory substance
WITH IN VITRO SELECTION
WITH IN VITRO SELECTION
Advantages:
 Specific selection for isolating a desired trait
 Procedure is less time consuming as compared to without in vitro approach
DETECTION OF SOMACLONAL VARIANTS
Detection is carried out by the following schemes:
1. Analysis of morphological characters:
Qualitative character: plant height, maturity date, flowering date and leaf size.
Quantitative character: yield of flower, seed 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 gel electrophoresis:
Change in concentration of enzymes, proteins and chemical products like pigments,
alkaloids.
FACTORS INFLUENCING VARIATION
When attempting to produce somaclones for a new crop plant, some
factors that are valid for both the schemes must be considered.
 Selection propagule (cells, protoplasts, calli)
 Genotype and explant source
There is a greater potential for mutation in shoots regenerated adventitiously
compared to axillary shoot regeneration
FACTORS INFLUENCING VARIATION
Selection agent (toxin, herbicide, amino acid analogue)
 Technique used for selection.
 Duration of cell culture
In general, the frequency of mutation increases with the age of the
tissue cultures
 Growth hormone effects
 Stability of resistant substance
SECONDARY METABOLITE PRODUCTION
SAIMA MUBASHSHIRA
Roll-13
SECONDARY METABOLITES
 Secondary metabolites are chemicals produced by plants for which no role
has yet been found in growth, photosynthesis, reproduction, or other "primary"
functions.
 Unlike primary metabolites, absence of secondary metabolites does not result
in immediate death, but rather in long-term impairment of the organism's
survivability, fecundity or aesthetics, or perhaps in no significant change at all.
 Often play an important role in plant defense against herbivory and other
interspecies defenses.
COMMERCIAL APPLICATION OF SECONDARY METABOLITES
Analgesic
medicine
(codeine,
morphine)
Muscle
relaxant
(atropine)
Pharmaceutical
Industry
Antifertility
drug
(diosgenine)
Anticancer
drug (taxol,
vincristine)
Antimalarial
drug
(quinine)
CRITERIA OF A SOMACLONAL VARIANT
To be of commercial use, a somaclonal variant must fulfill certain
basic requirements:
1. It must involve useful characters.
2. It should be superior to the parents in the character(s) in which improvement
is sought.
3. The improved character(s) must be combined with all other desirable
characters of the parent.
4. The variations must be inherited stably through successive generations by
chosen means of propagation.
TURMERIC : ONE OF THE MAJOR APPLICATION
Turmeric (Curcuma longa L.), known as the ‘Golden Spice’, is one of the most
important ancient spices and a customary item for export. It has been used as a
medicinal plant, is reported to be a therapeutic agent for several major human
diseases.
The primary biological active constituent of turmeric is the curcumin, a secondary
metabolite polyphenol that has potent anti-inflammatory and anti-oxidant
properties.
In turmeric, natural genetic variation is less due to vegetative propagation and lack
of sexual cycle. Hence, the present investigation aims at the isolation of high
yielding somaclonal variants through callus phase in turmeric variety Suguna.
EVALUATION OF SOMACLONES BASED ON
MORPHOLOGICAL TRAITS
In a notable study conducted by Roopadarshini and Gayatri (2012) the somaclones
isolated as V1 generation (first generation following the in vitro phase) were
hardened and transferred to the field to study their morphological traits
1. Plant height,
2. Number of tillers per clump,
3. Number of leaves per clump,
4. Leaf size,
5. Yield of rhizomes per clump and dry recovery
This traits compared with the normal regenerants and control (variety Suguna)
EVALUATION OF SOMACLONES BASED ON
MORPHOLOGICAL TRAITS
v
The somaclone “Narrow elongated
leaf with thick short pseudostem”
(SC1) was found to be superior
with regard to plant height
(110.42 cm) and rhizome yield
(538.87 g)
when compared to the other
somaclones, normal regenerants
and the control plant.
Reference:
Isolation of Somaclonal Variants
for Morphological and Biochemical Traits in
Curcuma longa (Turmeric); Roopadarshini, V*
and Gayatri, MC; Research in Plant Biology
(2012), 2(3): 31-37
EVALUATION OF SOMACLONES BASED ON
BIOCHEMICAL TRAITS
The somaclone “Narrow elongated leaf
with thick short pseudostem” (SC1) was
found to be superior with regard to
biochemical traits, with high curcumin
(5.48%), oleoresin (15.23%) and volatile
oil (7.16%) contents
when compared to other somaclones,
normal regenerants and the control plant.
It was observed that there exist highly
significant differences with regard to
morphological and biochemical traits
among the somaclones.
Reference:
Isolation of Somaclonal Variants for
Morphological and Biochemical Traits in Curcuma longa
(Turmeric); Roopadarshini, V* and Gayatri, MC; Research
in Plant Biology (2012), 2(3): 31-37
SECONDARY METABOLITE PRODUCTION
NASRIN AKTER
Roll-42
PATCHOULI : A SOURCE OF ESSENTIAL OIL
Patchouli, a medium-sized aromatic shrub, is grown mainly in Indonesia, Malaysia,
China, and Brazil for its essential oil.
Pogostemon patchouli whose essential oil is highly valued in perfumery, is exclusively
vegetatively propagated for the absence of flowering and seed set.
Absence of generation of genetic variability through sexual reproduction in patchouli
has resulted in its restricted adaptability. Since patchouli has been propagated only
vegetatively for more than 50 year, it is expected that it may have accumulated a
large number of mutations either in homozygous or heterozygous conditions which
may have remained phenotypically unexpressed in single or a few cells. Somaclonal
variation could thus be expected to release this hidden and unexpressed variability.
EVALUATION OF SOMACLONAL VARIATION
In an eminent study conducted by Seshagirirao et. al, 2012; 40 randomly selected
somaclones were employed for evaluating somaclonal variation. These plants were
multiplied vegetatively through their stem cuttings along with their parental variety
Johore and evaluated in SC2 and SC3 generations (second and third vegetative
generation, respectively, after the in vitro phase).
Plant height and herb yield were recorded at harvest after 4 momths of planting. The
harvested herb was shade-dried for 3 days and the essential oil content in the shadedried herb was estimated.
The essential oil composition with respect to the seven constituent, these are βpatchoulene, β-caryophyllene, α-guaiene, seychellene, α,δ-patchoulene, α-bulnesene, and
patchouli alcohol, was determined by gas chromatography (GC).
COMPARISON AMONG DIFFERENT TRAITS
Significant variation was observed among somaclones for plant height, herb yield, essential oil yield, and
contents of patchouli alcohol, α-guaiene, α ,δ-patchoulene, and α-bulnesene in the essential oil.
Reference: Evaluation of somaclonal variation for genetic improvement of Patchouli (Pogostemon patchouli), an exclusively
vegetatively propagated aromatic plant; Seshagirirao et. al, J. Crop Sci. Biotech. (2012); 15 (1) : 33-39
INTER-TRAIT CORRELATIONS
Essential oil yield was positively
correlated with plant height, herb
yield, and essential oil content, with a
higher correlation between herb yield
and essential oil yield than others.
Correlations between essential oil content
and plant height or herb yield were
positive but relatively low.
The correlations between patchouli
alcohol content in the essential oil and
plant height, herb yield, and essential oil
content were intermediate and negative.
Reference: Evaluation of somaclonal variation for
genetic improvement of Patchouli (Pogostemon
patchouli), an exclusively vegetatively propagated
aromatic plant; Seshagirirao et. al, J. Crop Sci.
Biotech. (2012); 15 (1) : 33-39
PROSPECTS AND LIMITATIONS
SHAMSUTTIYEBA SHIFA
Roll-38
LIMITATIONS OF SOMACLONAL VARIATION
Despite several applications of somaclonal variations, there are certain
limitations/ disadvantages:
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.
LIMITATIONS OF SOMACLONAL VARIATION
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.
CONFUSED?