Plant transformation based on
direct DNA delivery
1.Polyethylene glycol (PEG)-mediated
protoplast transformation.
2.Particle bombardment
3.Electroporation
4.Microinjection
5.Ultrasound mediated
6.Pollen-mediated
7.Cell penetrating peptides
PEG-mediated protoplast transformation
It is the oldest (direct DNA) reliable method for plant transformation. In the first
report (Krens et al. 1982 Nature 296:72), 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 resistant.
Protoplasts are treated with DNA in the presence of PEG and Ca++
This method has been very useful and applied to several plant species.
But it is a tedious procedure!
And it is necessary to be able to produce protoplasts and to to regenerate from
them.
Electroporation
• Use on cells without walls
Use on cells without walls
(plant protoplasts or animal
cells).
• High-voltage pulses cause
pores to form transiently in
cell membrane; DNA pulled
in by electrophoresis and
diffusion.
• Drawback - its more
cumbersome to regenerate
plants from single
protoplasts than from the
tissue transformations with
Agrobacterium
animal cells).
• High-voltage pulses
Electroporation
Use of high voltage electric current (1-1.5 kV) to permeabilize cell
membranes to facilitate DNA uptake.
D’Halluin et al. (1992) Plant Cell 4: 1495-1505
Immature
Embryos
(maize)
electroporation
Embryogenic callus
Transgenic
plant
Advantage: there is no need to establish callus or suspension culture first.
Explants (embryos) were directly electroporated.
electroporation
Pollen
Pollinate flowers
Transgenic
plant
Electroporated pollen can supposedly 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.
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.
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 contamination. The transformed tissue was not regenerated into a
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 the npt gene. But there was no 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.
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 plants were 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 a cell
but not into the nucleus. Further, it does not present much advantage over
other methods and therefore has not been much explored.
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.
Method:
1.Precipitate DNA onto small tungsten or gold particles.
2. Accelerate particles to high speeds and aim them at
cells or tissues.
3. Selective growth and regeneration of transgenic plants
as described for Agro-mediated transformation
Gold or tungsten particle are used
gold
tungsten
Original 22-caliber biolistic gun
DNA is bound to the microprojectiles, which impact
the tissue or immobilized cells at high speeds.
J. Sanford & T. Klein, 1988
The Helium Gas Gun – Circa 2000
Helium particle gun
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.
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
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.
Factors affecting particle bombardment-mediated transformation
Selectable marker genes: choosing a proper selection marker is critical.
Markers
npt II (Tn5)
hpt
Gentamycin acetyl transferase
SPT (Streptomycin phosphotransferase)
dhfr mutant form
bar (PAT)
aroA (EPSP synthase)
als (Acetolactate synthase mutant form)
Bromoxynil nitrilase (bxn)
selection agent
G418 (geneticin), kanamycin, neomycin, paromycin
hygromycin B
gentamycin
streptomycin, spectinomycin
methotraxate
PPT, bialaphos
glyphosate (roundup)
sulphonyl urea
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.
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.
negative
positive
positive
positive
positive
positive
GFP expression in rice callus
LUC+ tobacco
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.
“Agrolistic”
All major monocots were transformed by biolistic method by 1992.
Agrobacterium produces simple integration patterns
Biolistic produces complex patterns
LB
T-DNA
Binary vector
RB
Co-combardment of binary vector with
VirD1 and VirD2 expression cassettes:
produced integration patterns just like the ones
generated by Agrobacterium.