Documente Academic
Documente Profesional
Documente Cultură
Technology
(BSB 3163)
Part 2: Plant Cell Technology
Topic 3: Applications to Plant Breeding ~
Somaclonal Variation
3.0 Somaclonal
Variation
3.0 Somaclonal
Variation
3.2 Type of somaclonal variation
(a) Genetic changes:
3.0 Somaclonal
Variation
3.0 Somaclonal
Variation
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
Variation
3.0 Somaclonal
Variation
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
Variation
3.0 Somaclonal
Variation
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
Variation
3.0 Somaclonal
Variation
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
Variation
3.0 Somaclonal
Variation
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
Variation
3.0 Somaclonal
Variation
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
Variation
3.0 Somaclonal
Variation
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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3.5 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
Variation
3.0 Somaclonal
Variation
3.6 Detection and isolation of somaclonal variants
Two basis:
3.0 Somaclonal
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3.0 Somaclonal
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3.6 Detection and isolation of somaclonal variants
Cell selection
Selection pressure is applied which permits the preferential
survival/growth of variant cells
Selection methods:
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3.0 Somaclonal
Variation
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
Variation
3.0Somaclonal
Somaclonal
Variation
3.0
Variation
Selection methods:
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
Variation
3.0 Somaclonal
Variation
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
3.0 Somaclonal
Variation
3.0 Somaclonal
Variation
3.7 Applications
3.0 Somaclonal
Variation
3.0 Somaclonal
Variation
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
Variation
3.0 Somaclonal
Variation
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
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.
Questions
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?
100 paddy
Seeds
(F0)
Gamma irradiation
(200Gy)
Some die