Transgenic Plants

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Transgenic Plants
a.k.a. Genetically Modified Organisms
(GMO) Genetically Engineered Plants Genetically
Transformed Plants Transgenic Plants Recombinant
DNA Plants Gene-Spliced Plants Bioengineered
or Biotech Plants
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What are Transgenic Plants?
Plants that contain a source gene inserted from a
species outside of the target plant species. The
gene may have a similar function to a gene that
is already present but with a different DNA
sequence. Alternatively, and more often, it is a
gene not already present in that specie.
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What are Transgenic Plants?
Formerly, sexual cross barriers were a problem
for moving genes between species. Now these
barriers are not a problem for mixing gene pools.
Animal, fungus, and bacteria genes can now
become plant genes.
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Other Important Cell Characteristics
1. To generate transgenic plants, we need a
population of embryonic cells, anyone of which
can reorganized to a somatic embryo and
ultimately a plant. When transformation is
attempted on a group of plant cells, only a small
percentage are successfully transformed.
Therefore, a large population of cells is needed.
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Other Important Cell Characteristics
2. Individual cells in culture, protoplasts, can
divide and produce somatic embryos. Eventually,
somatic embryos can produce callus and plantlets.
(Tissue and Cell Culture) 3. Two protoplasts
from different species may fuse, exchange DNA,
and produce what is called a somatic hybrid.
This is not too different from GMO, but
is. Somatic means body cell and not germline.
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Three Steps of Genetic Engineering
1. Isolating a genetic segment (DNA), and
constructing a DNA recombinant DNA molecule
(includes promoter). 2. Finding a vector that
moves the DNA into the cell. 3. Placing the
vector in the plant cell.
Hopkins, 1999
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Three Steps of Genetic Engineering
3. Placing the vector in the plant cell. The
plant cell must have the potential to regenerate
into a whole plant. To be useful, it needs to be
sexually fertile in order to pass the gene on to
subsequent generations.
Hopkins, 1999
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Isolating DNA
1. One can digest DNA from the source species
(using restriction endonucleases) and clone genes
from those fragments. 2. One can isolate mRNA
from the source species and carry out
mRNA-directed DNA synthesis via reverse
transcriptase (cDNA).
Hopkins, 1999
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Inserting the DNA into the Vector
1. Open up the plasmid, which is a small circular
piece of DNA, to allow the foreign DNA to
splice into the plasmid via complementary sticky
ends. 2. At this point, the plasmid is ready
for introduction into the host or target species
cell.
Hopkins, 1999
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How Are Transgenic Plants Generated?
First Approach
Use of Agrobacterium a. A gene is
selected (usually with a promoter, marker). The
promoter is often a constitutive 35S promoter
from cauliflower mosaic virus. The marker can
be visual plus selection antibiotic or
herbicide resistance. b. Once the
gene is selected, it is inserted into the
Ti-plasmid of the bacterium. It is usually
Agrobacterium tumefaciens).
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How Are Transgenic Plants Generated?
First Approach
Use of a Agrobacterium c. Once
the gene is in the bacterium, it is cocultivated
with plant tissue, the DNA is transferred,
Agrobacterium is subject to disinfestation, the
transgenic cells are selected, and cultured to
regenerated plants.
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Hopkins, 1999
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Hopkins, 1999
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How Are Transgenic Plants Generated
Second Approach
Use of a Gene Gun a. A gene is selected (usually
with a promoter, marker). b. The gene is
inserted into plasmids. Gold particles are
coated with the plasmids. The gold particles
are loaded into a gene gun and shot into
tissue culture. c. Biolistic gun developed by
Cornell U. (Sanford), can be powered by gun
powder or helium.
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An Example From Agronomy
Roundup Ready Soybean
The herbicide glyphosate (active ingredient in
Roundup) is usually toxic to plants but the
addition of one gene not present in the plant
kingdom allow the plant to fight off the
glyphosate effects. Consequently, the plant can
survive higher glyphosate concentrations.
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EPSPSa.k.a. enolether-5-enolpyruvylshikimate
3-phosphate synthase
This enzyme is part of the shikimate/arogenate
pathway. EPSPS is critical in the synthesis of
tyrosine, phenylalanine, and tryptophan. There
are actually eleven enzymes that are required to
complete the shikimate/arogenate pathway.
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Biochemical/Physiological Strategy
It happens that glyphosate inhibits EPSPS because
it is a PEP analog. How do we produce a
glyphosate-resistant plant? One approach is to
overproduce EPSPS so that many copies of the
enzyme EPSPS are present. Another approach is to
insert a different version of the protein that is
insensitive to glyphosate.
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How Does RR Technology Work?
Roundup Ready soybean has a single added
protein a Roundup-tolerant enzyme (CP4-EPSPS).
Roundup herbicides kill plants and bacteria by
inhibiting EPSPS (enolpyruvylshikimate-phosphate
synthase). A soybean plant with the added
CP4-EPSPS gene thus makes two different EPSPS's
one plant EPSPS (inhibited by Roundup) and one
bacterial CP4-EPSPS (not inhibited by Roundup).
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How Does RR Technology Work?
The presence of the Roundup-tolerant CP4-EPSPS
allows the plant to continue making the amino
acids derived from the EPSPS pathway even in the
presence of Roundup. As a result, the plant isn't
affected by Roundup herbicide. The added
CP4-EPSPS is everywhere in the plant, so Roundup
has no opportunity to cause harm to the plant.
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Roundup Ready Technology
Erythrose-4-P PEP
From Monsanto web site
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Roundup Ready Soybean Chronology
From Monsanto web site
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Other Approaches to GM Plants

• Antisense Gene - Insert a copy of a gene that has
  an exact reverse DNA sequence to the gene of
  interest. Once mRNA is made from the antisense
  gene, it will hybridize with the normal mRNA
  which disables the normal mRNA. This was done
  with polygalacturonase in tomato to produce the
  Flavr-Savr and extend shelf life.