Jan 26 Kim
HO: Somaclonal Variation- a Novel Source of Variability from Cell Cultures
for Plant Improvement
Genetic variability is a vital component of plant breeding. Plant cell culture is a significant
addition to plan improvement. It is a new technique for increasing genetic variability quickly and
without sophisticated technology. A tissue culture cycle is a dedifferentiated tissue culture under
defines culture conditions, proliferation for several generations, and regeneration of plants.
Starting explants can come from almost any plant organ or cell type.
Somaclones: plants derived from any cell culture
2 Variation in Cultures
Variation in subclones is frequently observed. Such variation encompasses auxin and cytokinin
habituation, varying alkaloid and secondary metabolite production, and resistance to anti-
metabolites. Interestingly, there has not yet been a good explanation for this variation. The
authors posit that the culture heterogeneity comes from drastic genetic rearrangements.
3 Substantive Examples of Somaclonal Variation
3.1 Sugar Cane
Sugar cane was the first plant for which the usefulness of somaclonal variation was seen. After
results of variability, it was used to find a resistance to Fiji disease. In 1970, somaclones were
screened for their reaction to Fiji disease and Downey Mildew. There was a shift in increased
resistance. Other diseases studied included mosaic virus disease and eyespot disease. A wealth of
research support the idea that somaclonal variation is arises easily in sugar cane.
Although there have been lots of breeding programs in North America and Europe, these areas
have not used new releases for complex reasons. Simmonds argued that this is due to the “old”
varieties, while not remarkable in one area are also not defective in any area. About 22% of the
world’s potato crop is lost due to disease. Shepard et al. argues that it might be easier to improve
a popular variety than create a new one. About 60 somaclones have been found that have
significant advantages to the parent cultivar.
Cultivar: cultivated variety of a plant that has been deliberately selected for specific desirable
There have been reports showing genetic variation in regenerated plants. Variation was seen in
CO2 absorption, chlorophyll content, yield, stem diameter, height, leaf number, and total
alkaloids. Somaclones showed higher resistance to tobacco mosaic virus and root knot resistance.
Barbier and Dulieu showed that frequency of mutation was at a maximum in plants from early
callus cultures and protoplasts. The frequency of mutation stayed steady in plants from older
In an extensive study by Oono, 800 somaclones were studied and only 28.1% were considered
parental. There was a high variety in seed fertility, plant height and heading date.
Jan 26 Kim
In addition to somaclonal variation, there is also a high rate of cytogenetic abnormality. The
most frequent changes showed deletions and non-homologous exchanges.
Abphyl syndrome: twice the number of leaves on a normal number of nodes
Somaclonal variation has also been seen in the mitochondrial genome.
Barley from microspore culture of homozygous material demonstrate phenotypic variation that is
unexpected. They are tetraploid, which makes the sterility of their progency unexpected.
3.8 Brassica sp.
Cauliflower propagation by adventitious meristems show somaclonal variation. There is also
unexpected variation in doubled haploids of Brassica napus.
There is a remarkable amount of variability in Pelargonium cultivars. When the somaclones
were compared to plants from stem cuttings, there were almost indistinguishable from parental
plants. There was a small amount of ploidy changes in the varients.
3.10 Other species
The literature shows that other species also show abnormalities. Some of them are: carrot, lily,
clover, sorghum, and pineapple.
4 Enhances Variation in Interspecific Hybrids
Chromosome breakage and reunion are thought to be responsible for some of the somaclone
variability. Interestingly, somaclonal plants from a sterile hybrid showed enhanced multivalent
formation. Exchange between the genomes is possible if breakage and reunion occur.
5 The Origin of Somaclonal Variation
There are a number of proposed mechanisms for somaclonal variation, all speculative. The
desirable goal is to understand the mechanism and control them.
5.1 Karyotype Changes
In the past, varients were seen as caused by gross karyotype changes. This theory was weakened
when 5 protoclones of potato showed normal karyotype. The same is true in sugar cane,
sorghum, and Pelargonium. This leads to the conclusion that gross karyotype changes are
possible from cultures, they do not explain the occuance of somaclonal variation.
5.2 Cryptic Changes Associated with Chromosome Rearrangement
Cryptic chromosome rearrangements may explain genetic variation found in cultured cells.
These cryptic rearrangements include receiprocal translocations, deletions, insertions, and ring
chromosomes. Such changes can cause the loss of genetic material.
Jan 26 Kim
5.3 Transposable Elements
Transposable elements are pieces of DNA that can move independently from one locus of a gene
to another. There is evidence to suggest that certain mutants can be explained by transposable
elements. It is possible that the tissue culture environment may be favorable for transposition.
5.4 Somatic Gene Rearrangements
Mouse Ig genes are a good example of somatic gene rearrangements. During differentiation, the
embryonic cells undergo rearrangement. It is likely that this type of rearrangement is in higher
plants. If this is true, plants from somatic cell lines through tissue culture would allow
rearrangements to continue in the new germ line.
5.5 Gene Amplification and Depletion
Some genes in higher organisms are able to amplify themselves. This could mean increased
mRNA and protein production. Research on soybean cultures imply that DNA sequence
depletion an occur in plant tissue cultures.
5.5 Somatic Crossing Over and Sister Chromatid Exchange
Twin-spotting occurs when somatic crossing over occurs in heterozygotes. Estimated frequency
of sister chromatid exchange is high. Symmetric sister chromatid exchange can lead to
duplication and deletion of genetic material.
5.6 Cryptic Virus Elimination
Examples abound in which prior virus infection makes a plant more susceptible to a fungal
disease. Viruses can be in plants but cause no symptoms. Tissue culture is a technique to free
plants from viruses. Somaclones that show disease resistance may have been freed from the
This article showed the widespread nature of somaclonal variation, and examined possible
mechanisms. The authors view somaclonal variation as having a potential effect in plant
improvement. Genetic variability should be used with the utmost care.
F3: Biology of Cultured Cells
3.1 The Culture Environment
The cell often doesn’t have the correct in vivo phenotype because of changes in the
microenvironment. These are the ways in which the environment influences the culture (1)
nature of the substrate, (2) degree of contact with other cells, (3) constitution of the medium, (4)
constitution of the gas phase, and (5) incubation temperature
3.2 Cell Adhesion
Most cells grow as adherent monolayers. Cell surface receptors in the extracellular matrix
mediate cell adhesion. Spreading may be preceded by extracellular matrix protein secretion,
which the cells bind to through receptors. Given this information, vessels that have been
conditioned with previous cell growth provides a better surface.
Jan 26 Kim
Earlier, it said that it was found that cells would attach to a slight net negative charge. Does the
conditioned glass/plastic have a slight negative charge as well? Or was it just coincidence?
Cell Adhesion Molecules
There are 3 classes of transmembrane proteins responsible for cell-cell and cell-substrate
interaction. Self-interactive cells include cell-cell adhesion molecules, CAMs, and cadherins.
Cell-substrate interaction comes mostly from integrins. Cells must resynthesize matrix proteins
before attaching, or have a matrix-coated substance provided.
Intercellular junctions have a variety of functions: mechanical, sealing space between cells (tight
junctions), and allowing molecule flow (gap junctions). Proliferating epithelial cells will form an
increasing number of desmosomes and can even form complete junctional complexes. This is
one reason they are difficult to disaggregate after being confluent for too long.
The extracellular matrix fills the intracellular spaces in tissue. The make-up of the matrix
depends on the cell type. If adjacent cell types are different, both will contribute to the ECM
composition, producing a basal lamina. Overall, cultured cell lines are left to create their own
ECM, but some specialized cells require ECM to be provided.
There are 2 components of substrate interaction: adhesion (allows for attachment and spreading
needed for cell proliferation) and specific interactions. Using ECM elements can increase cell
survival, proliferation, or differentiation. However, there is a risk of adventitious infection unless
recombinant molecules are used.
The three components of the cytoskeleton are microfilaments (associated with signaling between
the nucleus and cell surface), intermediate filaments (structural and possible signaling function),
and microtubules (function in cell motility and intracellular movement of micro-organelles).
I remember learning this in FB. It’s cool to see how all this builds on the foundation we learned
Fibroblasts at low cell density are the most motile and epithelial monolayers at high cell density
are the least mobile. Movement occurs erratically until cell density is confluent.
(confluent: blended into one)
3.3 Cell Proliferation
The M (mitosis), G1 (Gap 1), S (synthesis), and G2 (Gap 2) phases are the four steps that make
up the cell cycle. In the M phase, chromatin condenses and the chromatids segregate to each
daughter cell. In G1, the cell continues to DNA synthesis or exits the cell cycle (G0). In S, DNA
replication occurs. In G2 the cell prepares to reenter into mitosis. There are checkpoints at the
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beginning of S and G2 that verify DNA integrity and will halt the cycle if DNA repair is needed.
The cell will then be repaired, or undergo apoptosis if repair is not possible.
Control of Cell Proliferation
Signals from the environment determine if the cell will enter the cell cycle. A low cell density
means cells have free edges and are able to spread. They will enter the cycle if mitogenic growth
factors are present. A high cell density will prevent normal cell growth (but not the proliferation
of transformed cells). This inhibition is started by cell contact, and continues due to crowding,
change in cell shape and decreased spreading.
The conditions to induce differentiation may often oppose the conditions needed for
proliferation. Therefore we need to outline two sets of conditions: one to maximize cell
proliferation and the other to maximize cell differentiation.
Maintenance of Differentiation
Specific function is maintained longer when the 3D structure of the tissue is kept the same, like
organ culture. However, there are disadvantages of organ culture: cannot be propagated, needs to
be prepared de novo each time, and is more difficult to quantify than cell cultures. Instead of
organ culture re-creating a 3D structure may be better.
Dedifferentiation is the idea that differentiated cells lose their specialized properties in vivo,
explaining why cell lines were unable to express the in vivo phenotype. It is speculated that these
properties are lost because (1) the wrong cell lineage was selected, (2) undifferentiated cells
overgrow terminally differentiated cells, or (3) the absence of inducers.
Dedifferentiation and deadaptation are different terms. Dedifferentiation means that cell
properties are lost by changing to a more phenotype. Deadaptation means that synthesis of
specific products is under regulatory control and can be synthesized if the conditions are correct.
Animal tissue culture seems to be a lot more complicated than plant tissue culture.
3.5 Cell Signaling
A variety of cellular function is controlled by cell-cell and cell-matrix interaction and nutritional
and hormonal signals. Endocrine signals are signals that reach the cell through the bloodstream.
Paracrine signals reach the cell from adjacent cells without going through the bloodstream.
Homotypic paracrine or homocrine signals are those that come from and target the same type
of cell. If a cell generates its own signal and is the target of those signals, it is autocrine. Only
autocrine and homocrine signaling will occur in vitro. Because many cultures fail to plate with
high efficiency at low density, conditioned media or feeder layers may be used.
3.6 Energy Metabolism
Most media contains added glucose to serve as a carbon source for glycolysis, resulting in lactic
acid. Under normal conditions, adding O2 will create free radicals (toxic to the cell), so
anaerobic conditions are used. The citric acid cycle continues to function, and amino acids can
Jan 26 Kim
be used as a carbon source. This has the disadvantage of producing ammonia as a byproduct,
which is also toxic to the cell. However, using dipeptides seems to decrease ammonia
3.7 Initiation of the Culture
A culture is derived from the outgrowth of migrating cells from a tissue fragment or the dispersal
of tissue. Primary culture is the first step in a series of selective processes. Selection occurs when
cells migrate from the explants (in primary explantation) or cells that survive disaggregation and
adhere to the substrate (dispersed cells). When primary culture is continued for several hours,
another step of selection occurs. There are 3 possible outcomes. (1) Cells that are able to
proliferate will increase, (2) some cells will survive but not proliferate, and (3) some will be
unable to survive.
When cells are confluent (cells are in close contact with one another and all the growth area is
used, cells sensitive to density limitation and contact inhabitation will stop growing.
Transformed cells will overgrow because they are insensitive to density limitations.
3.8 Evolution of Cell Lines
The primary cell becomes the cell line after the first subculture. The cell line can be subcultured
a couple times. After every subsequent subculture, the part of the population with the ability to
rapidly divide will eventually predominate. By the third passage the culture becomes a resilient
and quickly proliferating cell. One of the big problems in tissue culture is how to prevent
overgrowth of slower growing cells. However, there has been a lot of progress made in using
selective media and substrates.
Normal cells can only divide a set number of times, and will die out eventually. This is called
senescence, and is believed to be decided by the inability of telomeres to replicate.
3.9 The Development of Continuous Cell Lines
Some cell lines will give continuous lines. This ability most likely shows its ability for genetic
variation. A common trait of human continuous cell lines is a subtetraploid chromosome number.
Transformation: alteration in growth characteristics that usually correlates with tumorigenicity;
this term is also applied to forming a continuous cell line
Continuous cell lines are usually aneuploid. There is a variety in chromosome number between
the diploid and tetraploid balues.
Most cell will not give continuous cell lines.
3.10 Origin of Cultured Cells
The cellular components of the culture are important to consider because most people work with
finite or continuous proliferating cell lines. One can imagine that a cell culture being a balance
between multipotent stem cells, undifferentiated precursor cells, and mature differentiated cells.
The balance shifts depending on the environment. The source of the culture will affect which
cellular components are present.
Jan 26 Kim
1. What are some variation in subclones?
2. Explain cryptic virus elimination.
3. What is the difference between dedifferentiation and deadaptation?
4. What are the advantages and disadvantages of using ECM constituents?
5. Define transformation.