|Overview: How to make a Transgenic Plant
|Click on Image to view large size
Step 1: Extracting
the desired D.N.A
First before we are able to extract the desired D.N.A gene we must be able to identify the particular gene. Unfortunately in this day and age we do not know very much about which genes are responsible for what traits
and therefore the hardest part of extracting the desired D.N.A gene is the identifying part.
Most of the time, identifying one gene involved with the trait is insufficient because scientists must understand how
the gene is regulated, what other effects it might have on the plant, and how it interacts with other genes. Scientists today still know very little on which genes are responsible for enhancing yield potential, improving
stress tolerance, modifying chemical processes of the harvested crop or any other plant characteristics. Most of the research in transgenic is focused on identifying and sequencing these certain genes. Isolating a specific gene is not that hard to understand. The
two main tools involved with isolating a gene are the restriction enzymes and the D.N.A ligase. It will be easier to think of the restriction enzymes as “scissors” and the D.N.A ligase as
“glue scissors”. The restriction enzymes recognize and cut the D.N.A
at a specific region of the D.N.A, much like scissors. Note that there are different
restriction enzymes for different regions of D.N.A that is required to be cut. The
ligase then attaches the two ends of D.N.A fragments together, much like glue. These
two enzymes along with many more allow for manipulation and amplification of DN.A which are essential components in joining
the D.N.A of two unrelated organisms. Before the specific D.N.A region can be
inserted into another organism, we must obtain the D.N.A in a significant amount. This
brings us to the next step, cloning the gene.
Step 2: Cloning the gene of interest
The first step in cloning is to extract the D.N.A gene that is required using
the restriction enzymes and the D.N.A ligase. After the D.N.A is extracted from
the cells it is placed in a bacterial plasmid. A plasmid is molecular biological
tool that allows any segment of D.N.A to be put into a carrier cell (usually a bacterial cell such as E. coli) and replicated
to produce more of the D.N.A. Along with the desired D.N.A, an antibiotic- resistance
gene is also inserted into a bacterial plasmid, which in turn is inserted into a carrier cell.
This allows for the carrier cell to be successfully amplified through a process called transformation. Transformation will amplify the carrier cells but at the same time only amplifying the carrier cells with
the desired D.N.A. Transformation involves the carrier cell being placed
into two mediums; one medium would have a specific antibiotic while the other medium would not have the antibiotic. The carrier cells are then placed onto both mediums. The medium
that did not have the antibiotic would grow substantially. While the medium with
the antibiotic would only grow slightly. This is because the carrier cells that
did not have the desired D.N.A and antibiotic- resistance gene will not grow on the medium with the antibiotic on it. This ensures that the carrier cells are all going to have the desired D.N.A in it. So as the carrier cells grow the D.N.A inside the cell will grow with it, and therefore
be amplified or cloned to a considerable amount. Once the D.N.A has been amplified
it is now almost ready to be inserted into the desired crop.
Step 3: Designing a gene so it can be easily inserted into a crop.
Before a gene can be successfully inserted into a crop, it must be slightly
modified. First a promoter sequence must be added to the gene so that it can
be correctly expressed (ex. So that it can be successfully translated into a protein product).
This is considered an on/off switch which controls when and where the specific gene will be expressed. A common promoter is CaMV35S, which is from the cauliflower mosaic virus.
This promoter generally results in a high degree of expression in plants. Sometimes
a gene must also be modified so that it can achieve a greater expression in plants.
For example, the BT gene was modified by replacing A-T nucleotides with G-C nucleotides (which are preferred in plants)
without significantly changing the sequence of the gene. This resulted in more
production of the gene product in plant cells. Another thing that must be added
to a gene is a terminator sequence, which sends a signal to the cellular machinery that the end of a gene has been reached. The last thing that must be added to the gene is a selectable marker gene. This marker gene is added in order to identify plant cells or tissues that have successfully been inserted
with the desired D.N.A gene. The marker gene can also encode proteins that provide resistance to toxins, such as herbicides
and antibiotics. After the gene has been successfully modified it is now ready
to be inserted into the plant.
Step 4: Transformation
Transformation is a change in a cell or organism brought on
by the introduction of new D.N.A. There are two main methods of accomplishing
this 1: The gene gun method, and 2: The Agrobacterium method.
1: The Gene Gun method. This is also known as the micro-projectile bombardment
method. This method is mainly used in corn and rice. This involves high velocity micro-projectiles that deliver the desired D.N.A into living cells using a “gun”.
The desired D.N.A is attached to the micro-projectiles and fired into the cell.
This method is much like a universal delivery system and it can eliminate problems
such as the gene being rearranged when it enters the cell.
2: The Agrobacterium method. This method involves the use of soil-dwelling
bacteria known as Agrobacterium tumefaciens. This bacterium has the ability to infect plant cells with a piece of its D.N.A. The piece of D.N.A that is integrated into the plants chromosomes is a tumor inducing plasmid. This plasmid will take control of the plants cellular machinery and uses it to make copies of its own bacterial
D.N.A. On this plasmid there is also a region where the scientist can insert
the desired D.N.A, which will be transferred to the plant cell. This plasmid
is also activated when the plant has been wounded because when the plant is wounded it sends off chemical signals, and these
signals activate the plasmid. When the plasmid is activated it enters the plant
cell through the wound. It is still unknown how the D.N.A moves from the cytoplasm
to the nucleus of the plant cell or how it is integrated into the plant chromosome.
To be able to use Agrobacterium tumefaciens successfully as a vector, the tumor inducing part of the plasmid
has been removed so that it will not harm the plant as it is inserted. This method
is useful because it can allow for large fragments of D.N.A to be transferred very effectively but the limitations are that
not all crops can be infected by this bacterium.
Step 5: Plant Breeding
After the D.N.A has been successfully inserted the plant tissues are then transferred to a selective medium which
contains an antibiotic or herbicide that matches the marker gene. As in the cloning
process only plants expressing the selective trait will survive and it is assumed that these posses the desired gene. To obtain whole plants from these tissues, a process known as tissue culture is used. This process is when the plant tissues are grown under controlled environments in
a series of mediums that contain nutrients and hormones. To be sure that these
plants have the desired gene in them, they undergo a series of test. These tests
pay specific attention to the activity of the gene, inheritance of the gene, and unintended effects on plant growth, yield,