Plant Gene Transfer

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Plant Gene Transfer
John J. Finer
http//www.oardc.ohio-state.edu/plantranslab/
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• For successful production of transgenic plants,
• DNA delivery
• Marker genes (GFP) are useful
• Regeneration from single cells

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DNADelivery
Agrobacterium
Particle gun
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Agrobacterium Agrobacterium is a soil borne
gram-negative bacterium, that has a unique
ability to introduce part of its DNA into plant
cells.
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Agrobacterium Most of the native transferred
bacterial DNA is replaced with genes of interest.
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Agrobacterium In the laboratory, bacteria are
co-cultured or inoculated with plant tissue and
the bacteria transfer part of their DNA into
plant cells.
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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.
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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.
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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.
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GFP - Jellyfish Green Fluorescent Protein
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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.
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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.
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GFP - Jellyfish Green Fluorescent Protein
GFP is only useful as a marker or indicator of
successful gene transfer. It has little
application beyond this.
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Introduction of the gfp gene into different
target tissues
Petunia petal - cells
Soybean seed whole seedling (on right)
Wheat callus - cells
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GFP expression in wheat seeds (left seed, on
left) and roots (below)
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GFP expression in soybean tissues
No gfp red chlorophyll fluorescence
GFP green fluorescence
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gif animation of GFP expression in soybean
tissue Shows variability in expression
patternstandard illumination on left gfp
illumination on right
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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.
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Plant Regeneration Under the right conditions,
plant tissues regenerate into whole plants via
two distinct processes somatic embryogenesis and
shoot morphogenesis.
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Plant Regeneration For somatic embryogenesis,
embryos or artificial seeds form, which can
germinate into a whole plant.
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Plant Recovery
Starting MaterialImmature seeds
Soybean Embryogenesis
Germination
Induction
Development
Proliferation
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Plant Regeneration For shoot morphogenesis,
shoots form, which must generate roots before
they can be transferred to the soil.
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Production of Transgenic PlantsWhen 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.
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For further information or suggestions,
contact John J. FinerDept Horticulture and Crop
ScienceOARDC/The Ohio State University1680
Madison Ave.Wooster, OH 44691USA Finer.1_at_osu.ed
uhttp//www.oardc.ohio-state.edu/plantranslab/