Plant transformation based on direct DNA delivery

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• Plant transformation based on direct DNA delivery
• Polyethylene glycol (PEG)-mediated protoplast
  transformation.
• Particle bombardment
• Electroporation
• Microinjection
• Ultrasound mediated
• Pollen-mediated

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Electroporation
Use of high voltage electric current (1-1.5 kV)
to permeabilize cell membranes to facilitate DNA
uptake.
DHalluin et al. (1992) Plant Cell 4 1495-1505
Immature Embryos (maize)
Transgenic plant
electroporation
Embryogenic callus
Advantage there is no need to establish callus
or suspension culture first. Explants (embryos)
were directly electroporated.
Transgenic plant
electroporation
Pollinate flowers
Pollen
Electroporated pollens can be germinate at 30
efficiency. However, no transgenic plant has so
far been reported using this concept, even though
it has been shown that pollen grains can be
permeated with macromolecules such as DNA.
Electroporation method is very efficient in
permeating DNA into cells and protoplasts,
therefore this method is very effective for
transient expression studies
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Pollen mediated transformation
Nicotiana langsdorfii X Nicotiana glauca Hybrid
(develops genetic tumors)
In 1976, attempts were made to use pollen to take
up foreign DNA and then cross fertilize a related
species. glauca pollen were incubated in DNA
isolated from langsdorfii. The DNA treated pollen
were used to pollinate emasculated glauca plants.
One group claimed that the sexual progeny thus
obtained formed tumors on the stem. However,
these experiments were not reproduced in other
labs.
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Microinjection
Injection (sometimes it is simply pipetting) of
DNA into cavities containing meristems or sexual
organs. Some studies claimed success while
others reported negative results. The
verification was difficult due to the potential
contamination. The transformed tissue was not
regenerated into plant. In 1987 a report was
published in Nature that described the generation
of transgenic rye plants by injecting DNA into
floral tillers. Authors reported kanamycin
resistant plants and Southern analysis to prove
the integration of npt gene. But they didnt
carry out progeny analysis. A large number of
labs tried to repeat this method but to no
avail. Two more similar reports (irreproducible)
were made in barley in 1990s. Current status
unreliable, tricky, low (or no) success rate.
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Ultrasound-induced (sonication) DNA uptake
Ultrasound treatment causes the formation of
bubbles with generation of high pressure and
temperature and violent-flow or streaming of
fluids. This method has been used to introduce
DNA into plant protoplasts. First report
described the introduction of DNA into tobacco
protoplasts but no transgenic plant was obtained.
Subsequent reports described generation of
transgenic tobacco and wheat but insufficient
molecular evidence was provided. It appears
that this method may be effective in introducing
DNA into cell but not to the nucleus. Further,
it does not present much advantage over other
methods and therefore it hasnt been explored
much.
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PEG-mediated protoplast transformation
It is the oldest (direct DNA) reliable method for
plant transformation. In the first report (Krens
et al. 1982 Nature 29672), Agrobacterium Ti
plasmid was introduced into petunia protoplasts.
Formation of tumors, opine synthesis and Southern
blot provided the verification, which is an
extensive and complete analysis to show success
of transformation. The first report of
generating transgenic plants using this method
was provided by Paszkowski et al. (1984). They
regenerated transformed protoplasts into plants
that were kanamycin (drug) resistant. This
method has been very useful and applied to
several plant species. But it is a tedious
procedure!
Protoplasts are treated with DNA in the presence
of PEG and Ca
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Particle bombardment
PDS-1000 gene gun Particle inflow gun ACCELLTM
technology
PDS-1000 First particle gun was developed by
Sanford and colleagues in 1987. They introduced
CAT (chloremphenicol acetyl transferase) gene
into onion epidermal cells and detected transient
gene activity. First transgenic plants were
produced in 1988 using soybean and tobacco tissue
culture. Christou (1988) and Klein (1988)
bombarded soybean shoot meristem and tobacco
leaf, respectively. Christou recovered chimeric
transgenic soybean plants that transmitted the
gene into next generation. Particle bombardment
is the method of choice for chloroplast
transformation.
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Helium particle gun
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ACCELL technology
It uses high voltage electric discharge to
vaporize a water droplet which produces a
controlled shock wave. Initial shock wave is
reflected to produce secondary shock wave, which
in turn accelerate a mylar sheet coated with
particles. A screen stops the mylar sheet and
allows the particles to hit the target.
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Particle Inflow gun
Particles are supported by a screen in a syringe
filter unit and accelerated in a stream of helium
without the need of macrocarrier. The target
tissue is held in a chamber under vacuum. A
solenoid controlled by a timer relay is used to
generate a burst of low pressure helium.
Advantages Not patented, easy to design, gives
good results. Disadvantages not commercially
available, some safety concerns
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Design of Particle Inflow gun
DNA coated particles are suspended in ethanol and
deposited on a metal sieve plate. Since the
particles are not dried up and stuck to plastic
like in PDS-1000 system, low pressure He is able
to accelerate them.
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Gold or tungsten particle are used
gold
tungsten
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Factors affecting particle bombardment-mediated
transformation
Selectable marker genes choosing a proper
selection marker is critical.
Markers selection agent npt II (Tn5) G418
(geneticin), kanamycin, neomycin,
paromycin hpt hygromycin B Gentamycin acetyl
transferase gentamycin SPT (Streptomycin
phosphotransferase) streptomycin,
spectinomycin dhfr mutant form methotraxate bar
(PAT) PPT, bialaphos aroA (EPSP
synthase) glyphosate (roundup) als (Acetolactate
synthase mutant form) sulphonyl urea Bromoxynil
nitrilase (bxn) bromoxynil
A mutant form of dhfr isolated from mouse is
resistant to methotrexate drug, which eventually
interferes with DNA synthesis. Thus this gene
serves as a selectable marker. Glyphosate
inhibits photosynthesis by competitive inhibition
of EPSP synthase, which is involved in shikimate
pathway for amino acid synthesis. EPSP synthase
is encoded by nucleus and localized in
chloroplast. aroA gene was isolated from
Salmonella typhimurium strain resistant to
glyphosate.
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Reporter genes
First reporter gene CAT chloremphenicol acetyl
transferase (chromatography and
autoradiography) nos or ocs nopaline or
octopine synthase (chromatography) uidA or gusA
(GUS) MUG or X-gluc (color or fluorescence)
Jefferson (1987) luc (firefly luciferase)
luciferin (bioluminiscence) Ow et al. (1986) gfp
(green fluorescent protein) no substrate, UV
irradiation.
positive
positive
negative
positive
positive
positive
GFP expression in rice callus
LUC tobacco
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Gene copy number
Number of copies of the introduced DNA, the
pattern of integration, and the chromosomal
location of integration have profound effect on
the expression of the introduced genes. Direct
DNA transfer methods generally produce more
complex integration patterns than those produced
by Agrobacterium-mediated transformation.
Several people are studying the parameters that
can simplify integration patterns such as amount
of DNA to be introduced. It has been reported
that by reducing the amount of introduced DNA,
one can produce higher number of transgenic lines
containing simple integration pattern. Since
Agrobacterium generates more single copy
integrations than say biolistics, a novel
approach called Agrolistics was developed by
Novartis Co. to facilitate production of single
copy transgenic plants of maize, wheat and other
such crops that are not amenable to Agrobacterium
transformation.
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Agrolistic
All major monocots were transformed by biolistic
method by 1992. Agrobacterium produces simple
integration patterns Biolistic produces complex
patterns
T-DNA
LB
RB
Binary vector
Co-combardment of binary vector with VirD1 and
VirD2 expression cassettes produced integration
patterns just like the ones generated by
Agrobacterium.