Genetically modified plants
are created by adding genes
to specifically change
a particular trait of the plant.
Plants that have been
genetically modified
are sometimes referred to
as transgenic plants.
To make a genetically modified plant,
you need the following components:
- the DNA for the gene
you want to express in the plant,
including a promoter so the DNA
is turned into the protein product;
- a way to tell which plants
contain the DNA modification;
- a way to get the DNA into the plant;
- and of course, a plant.
The first item needed to make
a genetically modified plant
is a piece of DNA
that encodes the desired trait.
These genes can come from
the same species or other species,
or can be modifications
of existing genes.
Genetic modifications can improve
a wide variety of plant features.
Papaya Ringspot Virus almost
wiped out papayas in Hawaii,
but the expression of a ringspot protein
gives Sunspot papayas
resistance to the virus,
much like an immunization.
Bt products give corn and cotton
resistance to borers and boll worms,
insects that drill holes in the plant
and kill it.
Golden Rice was engineered
to prevent blindness
caused by vitamin A deficiency.
Other modifications
make the plan herbicide resistant,
so a farmer can tend to a much larger acreage
by spraying herbicides
instead of plowing or manual weeding.
There are also genetic modifications
to facilitate farming
under harsh conditions,
like drought or salinity.
Finally, some genetic modifications
modify the properties of the plant itself,
such as increasing the size or decreasing
the growing time of the plant,
or changing qualities like browning
when an apple is cut.
In addition to the DNA that codes
for the desired trait,
a plant specific promoter sequence
is required.
The promoter acts
as the start point for transcription.
The promotor that is most commonly used
in genetically modified plants
is derived from Cauliflower Mosaic Virus,
and is abbreviated CaMV35S.
We are going to need a lot of
DNA to make a transgenic plant.
The easiest way to make a large number of
copies of your target DNA
is to use bacteria as a copy machine.
To do this,
the DNA must be in a circular form,
and contain an origin of replication.
This circular DNA that can be replicated
is called a plasmid,
and is often represented as a circle,
with key features indicated
with color blocks and arrows.
So now we have the DNA
for the desired trait,
the promoter so the trait
will be transcribed into RNA,
all in a plasmid that can be
replicated in a bacterial cell.
But how are we going to tell
if the DNA is in the cell?
The most common strategy
is to add an antibiotic resistance gene
to the plasmid.
If the bacteria is grown on plates
containing the antibiotic,
only the cells that contain
the plasmid will grow.
Let's review the components we need
to make a transgenic plant.
We need the DNA that codes
for the desired trait.
Check.
With a promotor so the sequence
will be transcribed and translated.
Check.
We need a way to select for cells
that contain our DNA;
a plasmid with an antibiotic resistance
gene meets this requirement.
Check.
Now all we need is a way
to get the DNA into the plant.
There are two different ways
that are commonly used
to get DNA into a plant.
The first technique is a brute force method:
a gene gun.
Gene guns fire DNA coated gold particles
at the plant cells,
and hope that some of them
end up in the right place.
Let's look at how a gene gun works up close.
The gold particle is coated with DNA
that includes the promoter,
the sequence for the desired trait,
and a selection marker.
The gold particles are fired at the plant
cells
with enough force to go through the cell wall
and plasma membrane,
and hopefully land in the nucleus.
This strategy has the advantage
of not needing extra DNA from the plasmid,
but it is damaging to cells
and hard to control.
The DNA is randomly incorporated
into the chromosomes
through DNA repair mechanisms,
but could alter the expression
of necessary plant genes,
or end up in the mitochondrial
or chloroplast genome
instead of the nuclear genome.
Obviously,
another approach would be beneficial.
Like many sophisticated techniques
in biology,
nature provides the model system.
Do you see the tumor growing
on this tree?
It is caused by a bacterial infection
with Agrobacterium tumefaciens,
often referred to as Agrobacterium
or just Agro.
The Agrobacterium transfers a segment of DNA
from a plasmid into a plant cell,
causing the plant cells to grow
(thus causing the tumor)
and produce a food product
for Agrobacterium caused opines.
Let's look more closely at the
tumor inducing (or Ti) plasmid.
This is a very large plasmid,
containing almost 200 genes
and over 200,000 base pairs.
Like all plasmids,
it contains an origin of replication,
so that the plasmid can be copied.
Like most bacteria,
many of the genes are organized into operons,
so that activation of one promoter
triggers the transcription and translation
of several gene products
involved in the same process.
There are a set of virulence genes required
for infecting and transferring DNA
to the plant.
There is a separate operon containing genes
required to metabolize the opines
the plant will make for food for the bacteria.
A section of the plasmid
contains the sequence of DNA
that will be transferred to the plant.
This segment is called the Transfer or T DNA,
and is defined by a left border
and a right border sequence.
The Transfer DNA contains two main sections,
one that contains the genes
that trigger tumor formation
and a section that contains the genes
to make the opines to feed the Agrobacterium.
The ends of the sequences
to be transcribed in the plant
are marked with nopthaline synthesis
or NOS termination sequences.
To use this plasmid to make transgenic plants,
the sections that are harmful to the plant
are removed
and the genes to be transferred
into the plants are added.
The opine synthesis and
breakdown genes are removed,
as well as the tumor inducing genes.
The gene for the trait as well as a
plant promoter is added in its place.
Most of the time a very strong promoter,
such as the
35S Cauliflower Mosaic Virus Promoter,
is used to ensure that the production
of the desired gene is high.
A second selectable marker
is introduced with a bacterial promoter,
to allow for selection of Agrobacterium
that contain the Ti plasmid.
The virulence genes remain
as they are necessary to
transfer the T DNA
from the Agrobacterium
into the plant.
The transfer of the Ti plasmid
from the Agrobacterium involves
the expression of a number of genes
in the Agrobacterium cell.
Plant cells release a variety of small molecules.
When one of these small molecules
is detected by the Agrobacterium,
it triggers the expression
of a transcription factor
that initiates the transcription of the
virulence genes on the Ti plasmids.
One of these gene products
leads to the production of the T DNA sequence,
which then moves through another
virulence protein into the plant cell.
Like the gene gun method,
the T DNA is randomly incorporated
into the chromosomal DNA,
but there is much less damage to the cell,
and no chance of incorporation into
the chloroplast or mitochondrial DNA.
So now we have the DNA for our desired trait,
a selection method,
and the choice of two different ways
to get the DNA into the plant.
Once the DNA is successfully integrated
into the plant genome,
the plant is described as transformed.
But how do you grow a transgenic plant
from the few cells
that incorporated the new DNA?
Under sterile conditions,
the plant cells are grown on media that
contains both the antibiotic for selection
and plant hormones.
The antibiotic ensures that only cells
that were transformed will grow,
and the plant hormones
allow for the generation
of an entire plant
from just a few plant cells.
After the plant has generated both the
root and shoot portions of the plant,
it can be transferred to regular soil,
and the new properties of the plant
can be tested.
So today we have seen
that to make a transgenic plant,
first the DNA sequence for
a particular trait must be identified,
and a promoter sequence added
to ensure the transcription of the
DNA into RNA once it is in the plant.
This sequence is integrated
into a plasmid
containing a selectable marker
such as an antibiotic resistance gene.
The gene for the trait
and the selectable marker
are transferred into a plant cell
with either a gene gun or
Agrobacterium mediated transformation,
and then an entire plant is regenerated
using sterile culture conditions
with antibiotic selection and plant hormones.
