Transcript bYTEBoss bly-217-transgenic-crops

APPLICATION OF MOLECULAR
BIOLOGY TO AGRICULTURE
• WHAT ARE TRANSGENIC CROPS?
• STEPS IN THE DEVELOPMENT OF TRANSGENIC
CROPS
• EXAMPLES OF TRANSGENIC CROPS
• MERITS AND DEMERITS OF TRANSGENIC
CROPS
INTRODUCTION TO BIOTECHNOLOGY
LECTURE NOTES BY:
PROF. E.H. KWON-NDUNG
DEPARTMENT OF BOTANY
FEDERAL UNIVERSITY LAFIA
WHAT ARE TRANSGENIC CROPS?
Transgenic crops are simply crops that contain
a gene that has been artifically inserted into
that crop, instead of being inserted through
pollenation. The gene that is inserted into the
crop is called a transgene. For example the
transgene inserted into corn is called Bacillus
Thuringiensis. This transgene is a soil
bacterium that produces a protein that is
poisonous to insects.
STEPS IN THE DEVELOPMENT OF TRANSGENIC
CROPS
Step 1: Extracting the desired D.N.A.
Step 2: Cloning the gene of interest.
Step 3: Designing the gene so it can
easily be inserted into the plant.
Step 4: Transformation.
Step 5: Plant breeding
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.
Steps in creating transgenic crops:
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.
Steps in creating transgenic crops:
• 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.
Steps in creating transgenic crops:
•
•
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.
Steps in creating transgenic crops:
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, and quality.
Steps in creating transgenic crops:
EXAMPLES OF TRANSGENIC CROPS
Genetically Engineered Corn - BT Corn
What is Bt?
Bt stands for Bacillus thuringiensis, which is a spore forming soil bacterium
that produces protein crystals that are toxic to many types of insects. Bt can
be found about almost anywhere. It is distributed in the soil sparsely but
frequently, and that is why it can be found almost anywhere. Bt has been
found in all type of environments from beaches to the desert to tundra type
habitats.
• There are also over a thousand types of Bt that produce over 200 types of
protein crystals which are toxic against a wide variety of insects and some
other invertebrates. Bt belongs to the bacteria family, Bacillus cerus, which
cause food-poisoning in humans. Bt does not cause food poising to humans
because it contains a plasmid that produces the certain protein crystals that
are toxic to insects.
• Proteins crystals bind specifically to certain receptors in the insect’s intestine.
Not all insects have these certain receptors, which allow for high species
specificity. Humans and other vertebrates also do not have these receptors
and therefore the toxin does not affect us.
History of Bt
Bt was first discovered in 1901 by a Japanese biologist, Shigetane Ishiwatari. He
was investigating the cause of the sotto disease (sudden collapse disease) that
was killing large populations of silkworms. Bt was then rediscovered in 1911 by
Ernst Berliner when he had isolated a bacteria that had killed a Mediterranean
flour moth. Berliner had mentioned the existence of protein crystals but the
activity of the crystals was not discovered until much later.
By 1920 farmers were using Bt as a pesticide to kill moth larvae, since that was
the only strain of Bt that was known at the time. But in 1956 Fitz-James Hannay
and Angus Hannay had discovered that the reason Bt killed the moths was due
to the protein crystals. Research had begun on Bt and the Bt crystals. So by
1977 there were 13 different strains of Bt, all still only effective against moths.
But also in 1977 the first strain was found that was toxic to flies. The next strain
was found in 1983 that was toxic to beetles. Today there are thousands of
strains and many encode for crystals that are toxic to a wide variety of insects.
Also because of Bt’s ability to be effective and not harm the environment the
government and private industries have funded research on Bt.
How does Bt work?
• The only way that Bt can be poisonous is that if it is eaten.
The toxin becomes active when it is dissolved in the high
pH insect gut. These toxins then attack the gut cells of the
insect by creating holes in the lining. Bt spores and
bacteria then spill out into the gut which cause the insect
to stop eating and die in a couple of days. Even though the
toxin does not kill the insect immediately, parts of the
plants that have been treated with Bt will not be affected
because the insect stops eating within hours. Note that Bt
does not spread to other insects and it does not cause
disease outbreaks on its own. The Bt toxin is very specific
though because of the many different strains of Bt. Each
Bt strain is specific to different receptors inside the insect
gut. So the toxicity of the Bt depends on the receptors
involved, and damage to the gut upon binding of the
toxins to the receptors. Each species of insects has certain
receptors inside their gut that will match only certain
toxins, much like a lock and a key.
How Bt toxins work :
1. Insect eats the Bt toxin (crystals and spores)
2. The toxin binds to certain receptors in the
gut and the insect stops eating
3. The crystals cause the gut wall to break
down which allow the spores and normal
gut bacteria to enter body
4. The insect dies as spores and gut bacteria
proliferate into body.
Merits of transgenic crops
•
As of 2003 U.S grew 63% of the world’s transgenic crops, while Argentina grew 21%,
and Canada grew 6%. Other countries that grew transgenic crops include China and
Brazil that both grew 4% and South Africa that grew 1%. The future seems that we will
see an exponential growth in transgenic crops with researches gaining more
information about the process and the traits involved. As the new technologies are
being produced there are many merits and demerits surrounding the issue of
transgenic crops.
1.
2.
Transgenic cropscan protect itself against predators that feed off of the crops
Transgenic crops allows for less tillage, especially if the herbicide resistance gets
added into crop. The herbicide resistance enables the plants to be sprayed with
herbicides and they will not be killed. This will enable the farmers to spray
herbicides and remove only the unwanted plants such as weeds. This will also
conserve fertility through minimizing soil damage through compressions
3.
No insecticidal sprays will be needed on Transgenic crops, because the insecticide
is already engineered into it. Transgenic crops are also likely to give rise to lower
levels of mycotoxins in the final food product. Also less damage from organisms
mean less opportunity to develop fungi to infect the plant and bring toxic
substances.
Transgenic crops only targets the pests that attack the crop, because the pest is
only effective if it is eaten. So it will only affect the pests that that eat the
transgenic crops. For example, it has been proven that the Bt gene in corn is
expressed in more leaves and stems than in Bt corn pollen, and therefore the risk
to butterflies through pollen drift onto their plants is diminished.
4.
4.
Transgenic crops can also be produced more often and
more quickly. More genes can be engineered into the crop.
5.
Transgenic crops has also been tested for genetic stability,
substantial equivalence, nutritive properties, toxicity and
allergenicity. It is also known that conventional breeding can
introduce increased levels of plant toxins into a new variety
or can modify its digestibility or nutritiousness. It has also
been proven that some organic crops have higher levels of
toxic substances.
6.
Transgenic crops can be engineered with the ability to resist
pests and can also be engineered to have more vitamins,
minerals and anti- cancer substances.
7.
Transgenic crops can help solve hunger in the world
because transgenes can have a higher growing yield and
effective utilization of scarce land because of better pest
resistance and nutrient utilization
Demerits of Bt Corn
1.
The introduction of transgenic crops has raised a number of
possible negative consequences.
2.
The low tillage is also a prime spot for a monoculture in the fields.
A monoculture is where all of the crops are the same, so with this
it is a perfect situation for weeds.
3.
Genetic pollution from transgenic crops is another concern. It is
thought that the genes can spread into other organisms through
pollen, seeds and microbial process. This is different from other
types of pollution because once the genes are out they cannot be
re-called. An example is canola seeds in Canada, when some
canola seeds were contaminated with unapproved genetically
modified rapeseed and accidentally shipped to the UK. The UK had
already planted these seeds and the crops had to be destroyed.
4.
The total herbicides used with the herbicide resistant crops will kill
all weeds and reduce the biodiversity. This in turn will effect the
crops because if one species is taken out completely, you cannot
successfully predict what will happen to the other species
3.
Transgenic crops can also kill other beneficial
organisms and thus affect other forms of life in the
ecosystem.
4.
Inserting organisms into others will subject them to
natural genetic resistance to the toxins.
5.
Transgenic crops can produce unpredictable toxins
and allergens into food plants and therefore into the
final product. Thus, transgenic crops are considered
unstable as the number of copies of an inserted
gene changes through later generations.