Plant and Mammalian Cell

Technology
(BSB 3163)
Part 2: Plant Cell Technology
Topic 3: Applications to Plant Breeding ~
Somaclonal Variation

Sept - Dec 2011

3.0 Somaclonal Variation

3.1 Introduction

Somaclonal variation is a general phenomenon of all plant regeneration

systems that involve a callus phase
Somaclonal variation : soma refers to somatic tissue and clonal as
difference observed within the clones.
Variations ~ phenotypic differences; choromosomal rearrangements,
biochemical and molecular changes.
Definition: Genetic or epigenetic changes induced during the callus
phase of in vitro cultured plant cells -- sometimes visible as a changed
phenotype in regenerated plants.
In tissue cultures, such changes can be a problem as the main objective of
tissue culture is to raise genetically stable cultures.
However, investigations have shown that plant tissue culture undergo
frequent genetic changes and they are expressed in the form of variant traits
in regenerated plants

3.0 Somaclonal Variation

3.1 Introduction
There are two general types of somaclonal variation
(a) Genetic changes, heritable (alter the DNA)
Transmitted to the next generation
Important for crop improvement
Analysis of R0, R1, R2 progenies leading to true breeding
variants

(b) Epigenetic ~ stable, but non-heritable changes (alter gene

expression)
Temporary changes and ultimately reversible, e.g. changes in
gene expression such as hormone habituation of cell cultures
e.g. cold resistance in Nicotiana sylvestris.
Might persist through the life of the regenerated plant

3.0 Somaclonal
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3.0 Somaclonal
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3.2 Type of somaclonal variation
(a) Genetic changes:

Point mutations (e.g. Adh mutants in wheat)

Cytoplasmic (maternal inheritance)
Gene amplification (e.g. gene copy number)
Activation of transposable element
Cytogenetics (changes to genome structure)
Aneuploidy gain or loss of 1 or more chromosomes
Polyploidy gain or loss of an entire genome
Translocation arms of chromosomes switched
Inversion piece of chromosome inverted

3.0 Somaclonal
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3.0 Somaclonal
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3.2 Type of somaclonal variation
(b) Epigenetic:
Change in phenotype that isnt stable during sexual
propagation
May or may not be stable during asexual propagation
Usually undesirable in a breeding program, not always
undesirable in propagation
Habituation (most studied epigenetic change)
Define as loss of exogenous requirement for a growth
factor (usually a PGR); e.g., auxin or cytokinin habituation
Detection: callus may lose requirement for a PGR in the
process of several transfers to fresh medium

3.0 Somaclonal
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3.0 Somaclonal
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3.2 Type of somaclonal variation
(b) Epigenetic:
Habituation (most studied epigenetic change)
Characteristics
Often occur gradually
Are regularly reversible (especially in regenerated
plants)
Are not seed-transmitted

3.0 Somaclonal
Variation
3.0 Somaclonal
Variation
3.3 Possible causes
Pre-existing cellular differences
If mother plant is originally a chimeras, i.e. composed of cells of
different genetic origin, different cell layers if the meristematic
tissue might have different genetic composition
Common from callus initiated from explants containing
differentiated and matured tissues with specialized functions
Polyploid cells give more variability than diploids

3.0 Somaclonal
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3.0 Somaclonal
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3.3 Possible causes
Tissue culture induced variation
The dedifferentiation and redifferentiation process:
Axillary shoot proliferation vs. organogenesis and
embryogenesis
Hypothesis of DAmato
Somaclonal variants are rare in micropropagated plants
(when multiplication is by axillary branching of shoot tips /
buds)
More common during shoot organogenesis and somatic
embryogenesis (especially with callus phase)

3.0 Somaclonal
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3.0 Somaclonal
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3.3 Possible causes
Tissue culture induced variation
The culture environment
Tissue culture is inherently stressful to cultured plant cells
Temperature
Length of culture: ploidy changes increase with increase
lengths of culture
Nutrient depletion favor the development of abnormal
cells (shortage of precursor necessary for rapid nucleic
acid biosynthesis)
Composition of culture medium
Some growth regulators trigger polyploidy in vitro

3.0 Somaclonal
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3.0 Somaclonal
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3.3 Possible causes
Tissue culture induced variation
The culture environment
Environment stress is known to cause:
DNA methylation: methylation of cytosine is known to
cause gene inactivation; this may occur during the
redifferentiation process
Most mutational events directly or indirectly related to
alternations in the state of DNA methylation
A decrease in methylation correlates with increased gene
activity

3.0 Somaclonal
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3.0 Somaclonal
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3.3 Possible causes
Tissue culture induced variation
The culture environment
Environment stress is known to cause:
Gene amplification: can result in increase gene
expression
Transpositional changes
Inadequate control of the cell cycle (errors in microtubule
synthesis, spindle formation)
Importance of PGRs
Scant evidence of direct mutagenic action
More evidence for transient modifications of
phenotype (e.g. dwarfing)

3.0 Somaclonal
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3.0 Somaclonal
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3.4 Mutagens
Physical Mutagens (irradiation)
Neutrons, Alpha rays
Densely ionizing (Cannon balls), mostly chromosome
aberrations
Gamma, Beta, X-rays
Sparsely ionizing (Bullets), chromosome aberrations &
point mutations
UV radiation
Non-ionizing, cause point mutations (if any), low
penetrating
Chemical Mutagens (carcinogens)
Many different chemicals
Most are highly toxic, usually result in point mutations

3.0 Somaclonal
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3.0 Somaclonal
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3.5 Traditional mutation breeding procedures
Treat seed with mutagen (irradiation or chemical)
Target: 50% kill
Grow-out 1st generation (R1) plants
Evaluation for dominant mutations possible, but most are
recessive
Grow-out R2 plants
Evaluate for recessive mutations
Expect segregation
Progeny test to select true mutants
Prove mutation is stable and heritable

3.0 Somaclonal
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3.0 Somaclonal
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3.6 Detection and isolation of somaclonal variants
Two basis:

Visual analysis of cultures: morphologically distinct cells such

as non-green cells or cells that accumulate anthocyanins and
other plant pigments are detected visually.
Experimental analysis of cultures: isolate herbicide and
antibiotic resistance variants, plant cells grown on media
containing wild type cells of culture. Surviving cells are
subcultured and retested for growth on a herbicide or
antibiotic supplemented medium. This will eliminate remaining
wild type cells that may have inadvertently survived on the
first round of selection.

3.0 Somaclonal
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3.0 Somaclonal
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3.6 Detection and isolation of somaclonal variants

Examples of experimental analysis of cultures:

Screening

Observation of large number of cells or plants for the detection of variants

Normally for mutants for yield or yield traits (production for biochemicals)
1st generation plant (R1) are scored for the identification of variant plants
2nd generation plant (R2) are evaluated for confirmation

Cell selection
Selection pressure is applied which permits the preferential
survival/growth of variant cells
Selection methods:

Direct selection, e.g. resistant to herbicides

Rescue method, e.g. low temperature resistant cells
Stepwise selection, e.g. salt resistant cells
Double selection, e.g. antibiotic resistance with chlorophyll developed

3.0 Somaclonal
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3.0 Somaclonal
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Selection methods:
In direct selection, the cells resistant to the selection pressure
survive and divide to form colonies; the wild type cells are killed by
the selection agent. This is the most common selection method. It is
used for the isolation of cells resistant to toxins (produced by
pathogens), herbicides, elevated salt concentration, antibiotics,
amino acid analogues etc.
In the rescue method, the wild type cells are killed by the selection
agent, while the variant cells remain alive but, usually, do not divide
due to the unfavourable environment. The selection agent is then
removed to recover the variant cells. This approach has been used
to recover low temperature and aluminium resistant variant cells.

3.0 Somaclonal
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3.0Somaclonal
Somaclonal
Variation
3.0
Variation
Selection methods:

The selection pressure, e.g., salt concentration, may be gradually increased

from a relatively low level to the cytotoxic level. The resistant clones isolated at
each stage are subjected to the higher selection pressure. Such a selection
approach is called stepwise selection. It may often favour gene amplification
(which is unstable) or mutations in the organelle DNA.

In some cases, it may be feasible to select for survival and/or growth on one
hand and some other feature reflecting resistance to the selection pressure on
the other; this is called double selection. An example of double selection is
provided by the selection for resistance to the antibiotic streptomycin, which
inhibits chlorophyll development in cultured cells. The selection was based on
cell survival and colony formation in the presence of streptomycin (one feature)
as well as for the development of green colour in these colonies (second
feature; only green colonies were selected).

3.0 Somaclonal
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3.0 Somaclonal
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3.7 Applications

Herbicide Tolerance
Tolerance: able to grow in the presence of the herbicide
either the target enzyme or altered form of enzyme
Most successful application of somaclonal variation have
been herbicide tolerance
Glyphosate tolerant petunia, carrot, tobacco and tomato
[elevated EPSP (enolpyruvyl shikimate phosphate
synthase)]
Imazaquin (Sceptor) tolerant maize

Disease Resistant variants

Plant cell cultures are exposed to lethal concentrations of
toxins involved in disease development (Add toxin or
culture filtrate to growth media)
Fiji disease resistant sugarcane, exposed plant to culture
filtrate of Helminthosporium sp.
Late blight resistant potato, exposed plant to culture
filtrate of Phytophthora infestans

3.0 Somaclonal
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3.0 Somaclonal
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3.7 Applications

Stress resistant variants

Add or subject cultures to selection agent
Salt tolerant alfalfa and citrus fruits to be cultivated at saline soil
Aluminium resistant, e.g. N. plimbaginifolia
Low temperature resistant e.g. chilies
High yield variants
Alfalfa variety Sigma, tobacco, corn, tomato etc
Variants for efficient nutrient utilization
Tomatoes with increase rate of phosphate uptake so can grow in phosphate
deficient condition
Specific product accumulators
Screen for specific product produced
Lysine in cereals
Variants for morphology
Potato of different tuber shape
Rice for plant height
Geranium for better flower

3.0 Somaclonal
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3.0 Somaclonal
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3.8 Advantages & Disadvantages
Advantages:
Frequency of useful somaclonal variations is high
New mutations may be isolated which were not available in the
germplasm or through mutagenesis
Time frame is shorter as compared to conventional mutation breeding
Free from undesirable features e.g. sterility
Effective selection at cell level
Relatively small effort, time, cost and space requirements
Only approach for the isolation of biochemical mutants, especially
auxotrophic (plant that is unable to synthesize a particular organic
compound required for its growth) mutants in plants
Not subject to regulatory requirements (or consumer attitudes) of
genetically engineered plants

3.0 Somaclonal
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3.0 Somaclonal
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3.8 Advantages & Disadvantages
Disadvantages:
Many mutations are non-heritable
Requires dominant mutation (or double recessive mutation); most
mutations are recessive
Can avoid this constraint by not applying selection pressure in culture,
but you loose the advantage of high through-put screening have to
grow out all regenerated plants, produce seed, and evaluate the R2
Alternative: perform on haploid cell lines

Many selected plants show undesirable features e.g. reduced

fertility, growth and overall performance

Students
Activities
3
Students
Activities
3
(Directed-self Learning)
Case Study: Application of somaclonal variation and in vitro
selection to rice improvement (By: J. Bouharmont, A.
Dekeyser, V. Van Sint Jan and Y. S. Dogbe In RICE
GENETICS II Proceedings of the Second International Rice
Genetics Symposium).

Task:
Read the abstract provided and based on the knowledge or
applications that you have learnt on somaclonal variation
topic, answer the questions given.

Case Study: Application of somaclonal variation and in vitro

selection to rice improvement
Abstract: Somaclonal variation was observed in the greenhouse and in the field
on plants regenerated from calli and on their progeny. Many plantlets were
recovered from cells cultivated on medium with 1.5% NaCl. Several selected
plants showed even higher salt tolerance. A few cell lines survived at a
sublethal NaCl concentration (1.75%). However, the progeny of one plant
from such selection did not express improved salt tolerance. Adding Al2(SO4)3
to the culture medium reduced cell proliferation. Calli were also affected by
other medium modifications required for Al solubilization. Some plants
regenerated from calli selected on a modified medium, with or without Al,
expressed a degree of Al tolerance. Selection for cold tolerance was
attempted by long-term culturing of calli at sublethal temperatures (11-13 C).
Some plants regenerated from cell selection and tested under hydroponlc
conditions showed improved cold tolerance. Progeny of plants regenerated
for the different stresses are being field-tested in Africa. Although incomplete,
these experiments confirm the potential of somaclonal variation in rice
improvement and the applicability of in vitro selection for stress tolerance.

Questions

How many type of selection pressures were implemented to the rice

cultivars?

Based on the abstract, if you are given rice cultivars A and B; design
an experiment on how are you going to implement the selection
pressures mention above in the format of a flow chart.

Tips: In the lecture note, the sources of genetic variability can be due
to pre-existing cellular differences and / or tissue culture induced
variation. Which of the above was used in the experiment?

Which method of cell selection did the researchers use?

Traditional Mutation Breeding

100 paddy
Seeds
(F0)

Gamma irradiation
(200Gy)

Grow under normal water condition

Grow under water stress
No seeds survived
(Drought resistant gene
is influenced by
dominant allele)

50 seeds survived / growth

Adult plants (F1)
Obtained seeds

Grow under water stress

Some can grow
Adult plants (F2)

Some die

Test for stability

Continue growing the plants (water stress) until F3 and F4 generations