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Plant Gene
Transfer
John J. Finer
http://www.oardc.ohio-state.
edu/plantranslab/
For successful production of
transgenic plants,
1) DNA delivery
2) Marker genes (GFP) are useful
3) Regeneration from single cells
DNA
Delivery
Agrobacterium
Particle gun
Agrobacterium
Agrobacterium is a soil borne gram-
negative bacterium, that has a unique
ability to introduce part of its DNA into
plant cells.
Agrobacterium
Most of the native transferred
bacterial DNA is replaced with
genes of interest.
Agrobacterium
In the laboratory, bacteria are cocultured or inoculated with plant
tissue and the bacteria transfer part
of their DNA into plant cells.
Particle gun
For particle bombardment, tungsten or
gold particles are coated with DNA
and accelerated towards target plant
tissues. In the early days, the force
used to accelerate the particles was
a .22 caliber blank. Today, most
devices use compressed helium.
Particle gun
The particles punch holes in the plant
cell wall and usually penetrate only
1-2 cell layers. Particle
bombardment is a physical method
for DNA introduction and the
biological incompatibilities
associated with Agrobacterium are
avoided.
Particle gun
The DNA-coated particles can end up
either near or in the nucleus, where
the DNA comes off the particles and
integrates into plant chromosomal
DNA.
GFP - Jellyfish Green
Fluorescent Protein
GFP - Jellyfish Green
Fluorescent Protein
GFP is a marker gene used in DNA
transfer studies. The jellyfish green
fluorescent protein gene has been
modified for optimum expression in
plants. The protein from the gene will
fluoresce green when illuminated
with high intensity blue light.
GFP - Jellyfish Green
Fluorescent Protein
Chlorophyll can fluoresce red under the
same conditions which cause GFP
to appear green. GFP fluorescence
occurs as spots if individual cells are
targeted or the whole tissue can be
green if all of the cells within the
tissue contain the gfp gene.
GFP - Jellyfish Green
Fluorescent Protein
GFP is only useful as a marker or
indicator of successful gene
transfer. It has little application
beyond this.
Introduction of
the gfp gene into
different target
tissues
Petunia petal - cells
Soybean seed – whole seedling
(on right)
Wheat callus - cells
GFP expression in
wheat seeds (left
seed, on left) and
roots (below)
GFP expression in soybean tissues
No gfp – red
chlorophyll
fluorescence
GFP – green
fluorescence
gif animation of GFP expression in
soybean tissue
Shows variability in expression pattern
standard illumination on left – gfp illumination on right
Plant Regeneration
Plants can be generated from single
cells using “tissue culture”, where
parts of the plants are placed in Petri
dishes. The specific response of the
plant tissue depends on the starting
plant materials and the medium in
the Petri dish.
Plant Regeneration
Under the right conditions, plant
tissues regenerate into whole
plants via two distinct processes;
somatic embryogenesis and
shoot morphogenesis.
Plant Regeneration
For somatic embryogenesis,
embryos or artificial seeds form,
which can germinate into a
whole plant.
Plant Recovery
Starting Material
Immature seeds
Soybean
Embryogenesis
Germination
Development
Induction
Proliferation
Plant Regeneration
For shoot morphogenesis, shoots
form, which must generate roots
before they can be transferred to
the soil.
Production of Transgenic Plants
When DNA delivery, plant regeneration
and selection for transgenic cells are
merged, transgenic plants can be
produced. The idea is to introduce DNA
into cells, which can be selected and
generated into whole transgenic
organisms.
For further information or
suggestions, contact:
John J. Finer
Dept Horticulture and Crop Science
OARDC/The Ohio State University
1680 Madison Ave.
Wooster, OH 44691
USA
[email protected]
http://www.oardc.ohio-state.
edu/plantranslab/