The Public Controversy Should we develop transgenics? Should we
release transgenics? Are transgenics safe? Are transgenics a threat
to non-transgenic production systems? Are transgenics a threat to
natural eco-systems?
Title Page: Biotechnology: Principles, Applications, and Social
ImplicationsPart II: From Protein to Product
NOTE: In Slide Show format, click on the slide. You will notice
that on many pages a series of text boxes or images will appear
sequentially on the same slide. This is intended to provide you
with a progression of concepts. Remember to study the slide notes
to get a better understanding of what is being presented in the
slides.
The general definition is very broad. Many individuals prefer
this definition because they can claim process such as plant
breeding or mutagenesis are actually biotechnology. The detailed
definition points to the fact that a foreign gene needs to be
inserted for a product to be considered a biotech product.
Biotechnology involves the modification of a whole range of
organisms. This foreign gene can be inserted into plants, animals,
fungi, bacteria or viruses. The key for me is that a foreign gene,
or an engineered gene from the same species is added back into the
organisms. The modified organisms would not have these traits
without the intervention of man.
As we are all aware, most species have an abundance of
variation. The photograph of bean seeds is a great illustration of
the variation in nature. You can notice not only many different
colors, but also many different patterns. A large array of
interacting genes are responsible for this variation.
But man can always dream of a new use for an organism. These
dreams often involve asking the species to do something it does not
now normally do. Biotechnology involves added new traits to a
species. Two of the most dramatic examples of mans dreams of
improving the utility of plants is shown here. At the top is the
picture of bananas. The goal is to express vaccines in these fruits
so that individuals who eat them will receive the vaccine and
become immune to the disease.
The rice photograph illustrates a recent invention called Golden
Rice. This strain of rice has been engineered to express enzymes
required for the vitamin A pathway that dont normal exist in this
species. By the goal is to provide this as crop as a dietary
product that will improve the nutrition and health of those who eat
it.Before we can understand who man goes about using biotechnology
approaches to modify a species, we must understand basic genetic
principles and terminology. All traits are controlled by genes. A
gene can have different forms. These forms are called alleles.
It is important that you become fluent with these terms and the
differences they imply. It is simple as remember that genes have
alleles. Or, alleles are alternate forms of a gene.This slide is
intended to help you understand the difference between genes and
alleles. Genes control specific traits. Here are two traits of pea
that Gregor Mendel, the father of genetics, studied.
Plant height is a trait. That trait is controlled by the plant
height gene. The plant can be either tall or short. Different
alleles of the plant height gene determine if the plant will be
tall or short. If you remember from your genetics class, the allele
for tall plant height is dominant to the short allele. This means
that heterozygous individuals carrying both the tall and short
allele will appear to be tall. Using the genetic terminology, this
also means that the short allele is recessive to the tall allele.
Similarly, seed shape is controlled by a specific gene. The
alternate shapes, smooth or wrinkled, are controlled by different
allelic forms of the seed shape gene. Similarly, smooth is dominant
to the recessive wrinkled phenotype.As the previous slide
demonstrated, each gene has a specific function. The fact that two
alleles control alternate forms of a trait means they must also be
very similar to each other, but different enough to produce
different phenotypes. In modern terminology, each allele will have
very similar DNA sequences and will encode the same protein
product. But the sequence of the alleles will differ at some point,
and that difference is directly responsible for the various
phenotypes generated by the different alleles. So what about the
genes encoding the two traits that Mendel studied. This slide
provides the essential information regarding the plant height gene.
This gene encodes and important enzyme in the gibberllic acid
biosynthetic pathway. If you remember, gibberellic acid is
responsible for node elongation in plants. If a plant has a longer
nodes, it will be taller than a plant with the same number of
nodes, but whose nodes are shorter. The specific gene product is
gibberellin 3--hydroxylase. This protein adds a hydroxyl (-OH)
group to the gibberellin called GA20 and this process makes the
biologically active GA1 gibberellin.
The dominant allele contains the normal form of this gene, and
the result of its expression will be tall plants. The recessive
allele is very similar to the dominant allele. In fact, the only
difference is a change in a single nucleotide that results in the
change of an alanine to a threonine in the final protein. This may
seem like a trivial change, but the result of the change is a
20-fold reduction in the active of the protein encoded by this
mutant allele. This comparison highlights the type of information
that is gathered from these sequence analysis of different
alleles.So what about the smooth vs. wrinkled phenotypes expressed
by the seed shape gene? This gene encodes the strach branching
enzyme (SBE) isoform 1. The recessive allele has no activity, and
the result in the seed is that it contains much more sucrose than
starch. The result of higher sucrose levels is a higher water
content. As these seeds dry they lose more water and the seed takes
on a wrinkled appearance. Unlike the recessive plant height allele
that is the result of a single nucleotide change, the recessive
seed shape allele is the result of the insertion of a large piece
of DNA into the coding region. This large piece of DNA is a
transposable element. This element is similar to the elements that
Barbara McClintock discovered in corn.
Now that we understand the relationships between alleles at a
gene, it is time to place this understanding in a large context.
You should become very fluent with this concept: the Central Dogma
of Molecular Genetics. This concept is a unifying principle that
describes the manner in which the sequence information in the gene
is eventually expressed as a trait (or phenotype). DNA is the
biochemical molecule of all genes.
DNA contains the genetic code that will eventually be converted
into the protein that will control the phenotype expression. But
DNA is not directly used for phenotypic expression. Instead, the
information in the DNA molecule is used to create an intermediary
molecule (RNA). The process to produce RNA is called transcription.
RNA is an active molecule in another process called translation
that is used to create the protein. The protein itself can have
many different functions. It could be an enzyme in a metabolic
pathway. Alternatively, it could act as a regulator transcription.
Finally, it could serve as a structural component of the cell.
Whatever it role is, it will control the final phenotype or the
outward appearance of plant.Remember our definition of
biotechnology??? Here is the detailed one again: The application of
the technology to modify the biological function of an organism by
adding genes from another organism. As the definition implies, we
first need to isolate a gene that will be added to our organism of
interest. For example, we may wish to add a gene from a bacteria
into a plant species. Another approach would be to isolate a gene
from our species of interest, modify that gene to change its
function, and then reinsert the modified form back into the
species. This is the first major biotechnology step, and it is
called gene manipulation.
Once we have isolated or modified the gene of interest, the next
step is gene introduction. This step involves the addition of the
gene to our species of interest. In all cases, this introduction
must be accompanied by stable integration of that gene into the
genome of the target species. Once the gene is stably integrated,
it is passed along to all subsequent generation in the same manner
as all other genes in the genome. The technique used to integrate
the gene into the species is called transformation, and the
modified organism is called a transgenic organism.We will discuss
gene manipulation and gene introduction separately. The Central
Dogma of Molecular Genetics was introduced several slides back. As
a remember, it states that the information stored in DNA is
transcribed into RNA, the RNA molecule is used in a process called
translation to produce translation to produce a protein, and the
protein is involved in some process that actually produces the
final phenotype of the organism. The dogma implies we need to
manipulate the DNA of gene that encodes for the protein if we are
going to develop a transgenic organism. The DNA itself is a
double-helix molecule that is stored inside the nucleus of the
cell. Every cell inside the organism has exactly the same DNA
molecule.We normally think of the DNA in the form of a chromosome.
Chromosomes are the condensed form of DNA. The simplest form of DNA
is the double-stranded molecule. These two strands are
complementary to each other. This complementarity is based on the
fact that if one strand has an adenine at one nucleotide residue,
the complementary strand has a thymine residue at the same
location. And if one strand has a guanine at a specific residue,
the complemenntary strand has a cytosine residue. This is an
important concept because it is the basis of an important screening
process called hybridization.
The double-strand molecule then undergoes a series of
condensation steps to produce the chromosome. Each of this
different steps are illustrated here in this slide. It is important
to remember that throughout the life-cycle of the cell, DNA is in
an uncondensed form. The chromosome only appears during the process
of cell division.All of genes reside on a specific location on the
chromosome. Any chromosome will contain thousands of genes. To
illustrate this point, the human genome consists of about 35,000
genes which are spread over 22 chromosomes. In contrast, the model
plant species Arabidopsis thaliana contains about 30,000 genes on
just five chromosomes. This illustrates the point that although
plants and animals contain about the same number of genes, the have
a different number of genes on their chromosomes. The goal of gene
manipulation is to isolate that one gene of interest from among the
many genes in the genome.To understand biotechnology, it is
important to have a general appreciation of how genes are cloned.
These fall into three gene approaches. The first approach is based
on the fact that genes between related species have similar DNA
sequences. This procedure is called homology cloning. The second
procedure is based on the direct relation that exists between the
DNA sequence and the final protein sequences. If you have an idea
of the protein sequence you can develop a probe to get at the gene.
This approach is called complementary genetics. The third procedure
relies on experiments that place the gene at a specific genetic
location. This procedure is called map-based cloning.We will first
discuss homology cloning. As mentioned earlier, this procedure is
based on the fact that the genes between two closely related
species have very similar DNA sequences. To take advantage of this
procedure, you need a probe from a species similar to the one you
are working on. Let's say you know that someone has cloned a gene
from mouse that you are interested in as a human geneticist. You
would then contact the person and request a copy of that gene. That
will be used as a probe in your experiments.
The first step is for you to grow out a clone library. This
library contains all of the genes from your species. Therefore a
human clone library contains all of the genes in the human genome.
The clones are then transferred to a special filter where the DNA
for each clone binds. They bind at a spot that is directly
analogous to their position on the plate in which they are grown.
This relationship is illustrated on this slide.
The next step is to add the probe to the library. The probe is
normally radioactively labeled. The importance of this will be seen
shortly. The probe then will bind by base-pair complementarity to a
clone to which it is very similar. This is the hybridization step
that was mentioned on an earlier slide. Excess probe is washed off,
and the washed filter is then exposed to an x-ray film. The film is
then read, and any hot-spot is the location of a clone that is
similar to the probe you used. It is hot because the probe was
radioactievely-labelled. You then go back to the original plate
containing your genes and select the clone containing your gene.The
second cloning procedure is called complementary genetics. And as
with that procedure, homology cloning requires a probe from some
source. The difference with homology cloning though is that we
develop our own probe based on the protein sequence.
Probe development is based on two simple facts. The first is
that the sequence of the gene in its DNA form will predict the
protein sequence. This also implies that if you know the protein
sequence you can derive the DNA nucleotide sequence. The second
fact is that the placement of an amino acid into a growing protein
chain is directed by a sequence of three nucleotides. This fact is
illustrated in the slide. You should notice that some amino acids
are encoded by one triplet sequence, some by two sequences, and
others by three, four or six. As you will see this is a minor issue
for complementary genetics approaches.
The probe is actually synthesized using a procedure called PCR,
or polymerase chain reaction. The DNA synthesis procedure requires
DNA primers. One primer is designed to a region in the amino (NH3+)
part of the gene. As you can see, the sequence of the forward
primer is based on the nucleotide sequence that corresponds to the
amino acid sequence. When you look at this sequence you will notice
the sequence denoted as A/T. This means the primer will actually
consist of a mixture primers some with an A at that position and
some with a T. The symbol N means the primer pool will contain a
collection of primers each with one of the four nucleotides at that
position. Therefore, this primer pool will consist of 8 (2x4)
different primers.
The reverse primer is synthesized using the same principles, but
it is complementary to the genetic code sequence. (It is
complementary because of the fact that DNA consists of two
complementary strands. Therefore, the PCR synthesis process is
designed to produce both strands.) So, as you can see the first
nucleotide is complementary to the A/G bases that encode the lysine
(Lys) amino acid, and the primer instead consists of T (thymine) or
C (cytosine). As with the forward primer, the reverse primer is
actually a pool that consists of 32 [2 (T or C) x 4 (A, T, C or G)
x 2 (C or G) x 2 (T or A)] primers.PCR is a DNA synthesis process.
The previous slide described the first step in PCR, the development
of the primers. The primers satisfy one of the two requirements of
DNA synthesis, a primer from which the new chain grows. The second
requirement is a DNA template. To generate a DNA template, the
double-stranded DNA molecule must be converted into a
single-stranded state. This is done by heating the DNA to a high
temperature. Step 3a in the slide shows this process. Once the DNA
is single stranded, the temperature is lowered so that the primer
can anneal. Once annealed, the temperature is raised to the optimum
temperature required by a special DNA polymerase enzyme called Tag
polymerase. Once the temperature is reached, the DNA is replicated.
As you can see in step 3c, now have two strands of DNA.This is an
animation of one step in the PCR process. Take a few minutes and
let the animation run through a number of times. It will recycle on
its own. This step will show the denaturation (converting the DNA
from single- to double-stranded state). The second step is
annealing (the binding of the primer to the single-stranded DNA).
The final step is extension (the duplication of a strand from the
end of the primer).As the previous two slides illustrated, the many
feature of the PCR process is the replication of one double-strand
DNA molecule into two. But the PCR process does not involve just a
single replication cycle. Rather, the step is repeated many times
(35-50 times). This repetition leads to an exponential increase in
the amount of DNA. At the end, a large amount of DNA is produced
that can be used for a number of purposes.
One of those purposes, and the one we are interested with here,
is the development of a DNA probe. Remember that we started with a
protein sequence of interest, and we used that information to
design primers to amplify a DNA fragment that would encode that
fragment. Therefore at the end of the PCR replication, we have
sufficient DNA to use as a probe for library screening.The final
step in using complementary genetics for cloning involves screening
a library. The steps are exactly the same as we described for
homology cloning. The only difference is that we now use the PCR
synthesized DNA as our probe. The final result will be the
isolation of a DNA clone from the library. That clone will contain
DNA sequences that will encode the gene for the protein in which we
are interested. The final technique of method of obtaining genes is
called map-based cloning. This procedure combines genetic
information that locates a gene to a small region of a chromosome.
The position of the gene is located to that region by the use of a
DNA marker that resides very close to the gene. The first two steps
illustrate this point. Typically a marker is discovered at a short
distance away from the gene of interest. That marker is then used
to discover another marker that is very close that cosegregates
with the gene of interest. That cosegregating marker is very close
to the gene (closer than the first marker) and is used to isolate a
series of overlapping clones or contig.
A series of steps is then used to identify ORFs or open reading
frames. An ORF is a DNA sequence that has all the characteristics
of a gene. Since the DNA marker (and by association the gene of
interest) resides on the contig, one of the ORFs will be the gene.
We wont go into the details, but the ORF that is a gene is
eventually identified by one of two procedures. Transformation is
one approach. As defined earlier, transformation involves the
addition of DNA to an organism and changing that organisms
phenotype. For map-based cloning, the ORF is added to a mutant
organism, and if the resulting transgenic plant expresses the wild
type phenotype then the ORF is the gene you are trying to clone.
For example, if you had and ORF that encoded the gene responsible
for plant height in pea, that gene could be added to a short pea
plant. If the addition of the ORF is the gene for plant height,
that transgenic plant would be tall.
An alternative approach is to sequence many mutant phenotypes of
a specific ORF. That analysis may provide useful information that
would allow you to determine a particular ORF is the gene of
interest. This is the approach used in human genetics.
The development of the transgenic organism uses some gene
isolated by the procedures that were just outlined. But it is
important that the appropriate gene is used to obtain the specific
phenotype you wish to develop. We are going to spend a bit of time
concentrating on two important phenotypes: glyphosate (RoundUp)
resistance and increased vitamin A content. Each of the phenotypes
can be achieved by adding one or several genes to a plant.The
RoundUp Ready technology is the most visible plant biotechnology
product on the market. To better understand plant biotechnology in
general, it is important to understand the development of these
transgenic organisms. RoundUp is a brand name herbicide
manufactured by Monsanto Corp. The active ingredient in this
herbicide is glyphosate. The chemical binds to the active site of
the EPSP synthase enzyme. This enzyme is a key to the development
of a group of amino acids called the aromatic amino acids. When
this enzyme is bound by glyphosate, it can not synthesize those
amino acids, and the plants die because protein synthesis is
severely disrupted. Glyphosate will not bind the to a particular
genetically-engineered version of EPSP synthase. Therefore RoundUp
Ready crops with this altered enzyme will survive when sprayed with
the herbicide.This slide shows the actual biochemical pathway that
we discussed in the previous slide. EPSP synthase synthesizes
3-enolpyruvly shikimic acid-5-phosphate. This is the essential
precursor to aromatic amino acids. When plants are sprayed with a
glyphosate-containing herbicide, such as RoundUp, this important
precursor is not synthesized, and consequently the plant is starved
of aromatic amino acids. The result is plant death.RoundUp
Resistant plants have a very simple solution. An engineered version
of EPSP synthase, one that was discovered in a bacteria, is
introduced into the plant. This enzyme can not be bound by
glphosate. Therefore, if a field is sprayed with the herbicide, the
introduced version of the gene produces a functional enzyme. The
3-enolpyruvl shikimic acid-5-phosphate precursor is synthesized
normally, and the plant produces enough aromatic amino acids to
survive. The second major plant biotechnology product is more
recent and was developed to address the vitamin A deficiency
problems prevalent throughout the world. This vitamin deficiency is
very critical because it can cause blindness and affects the
severity of many diseases including diarrhea and measles. This is a
severe problem that affects more than 100 million children
worldwide. A simple solution would be to distribute vitamins to the
affected children. Unfortunately, many countries where the
deficiency is chronic do not have the necessary infrastructure to
deliver the vitamin tablets to the most needed.
The solution that is currently being promoted is to improve the
vitamin content in widely-consumed, and readily available to the
consumer. Transgenic rice plants were developed that contain
elevated levels of the precursor to vitamin A. This GMO is called
Golden Rice because of its color: it is yellow rather than white.
It is yellow because -carotene, a yellow precursor to vitamin A is
abundant in the seed.Unlike the single-step RoundUp Ready pathway,
the carotene synthesis pathway involves multiple enzymes. This
important vitamin A precursor cannot be synthsized in rice because
it lacks four of the key enzymes. Therefore, the precursor is not
made, and the plant contains white kernels.In a major feat of
genetic engineering, scientists inserted a complete functioning
-carotene biosynthetic pathway into the rice plant. They did this
by inserting genes from daffodil the produce functioniong versions
of the first and last enzymes of the pathway. In addition, a single
bacterial gene that provides the same function as the second and
third enzymes of the pathway, was also introduced. With a
functioning pathway, the transgenic rice is able to produce the
vitamin A precursor -carotene. It is this product that gives
"Golden Rice" its characteristic yellow color.The Golden Rice story
illustrates a key point: it is very important to industry metabolic
pathways. These pathways are very important for our understanding
of specific products are produced in the organism. Only by
understanding this pathways will we be able to create novel new
products.This diagram shows in general the interrelationship
between the many different pathways. A key point to understand is
that the different sub-pathways interconnect. Therefore modify one
component of the pathway may affect the production of a product in
a separate sub-pathway.
Keeping this in mind, we can now envision how to engineer plants
so they produce novel products. We have already seen how modifying
a vitamin biosynthetic pathway can positively affect vitamin
production. We could also improve nutrition in other ways. For
example, if we were to focus our attention on the amino acid
pathways we could, for example, increase the lysine content in
typically lysine-poor grains. Conversely, someday we might be able
to improve legumes by introducing the correct genes necessary to
enrich the metionine content.
We could also envision new products if we modify other pathways.
Oils are a key component to both the food and manufacturing
industries. A better understanding of the genes in the complex
lipid pathway may allow us to produce better industrial oils.This
slide illustrates the variety of different traits that have been
modified in plants. It also shows the particular gene that was
introduced into the plant to obtain the specific trait. As you can
see, scientists have successfully introduced many different genes
and produced many different results. For example, it was discovered
that expressing the in the plant a particular protein of a virus,
the coat protein, the plant would then become resistant to that
virus. This technique has been widely credited with saving the
papaya industry in Hawaii, where the papaya ringspot virus nearly
eliminated the papaya growing industry. This is a success story
that is often overlooked, probably because the problem was to a
crop of limited production value.It is now time to cover the
development of transgenic crops in greater depth. The two major
steps are creating a transformation cassette that contains the gene
of interest, and then successfully introducing the cassette into
the plant.All transformation cassettes contain three regions. The
gene of interest region contains the actual gene that is being
introduced into the plant. This is the gene that provides the new
function to the plant. In this diagram, the region is shown in
red.
Many plant tissues are treated with the transformation cassette
during the transformation step. Not all of these tissues actually
receive the cassette. To distinguish those that contain the gene
from those that dont, it is necessary to use a selection process.
The selectable marker is a gene that provides the ability to
distinguish transformed from non-transformed plants. This is shown
by green.
The most common method to introduce the transformation cassette
is by using the plant pathogen Agrobacterium. For this system to
work it is necessary that the cassette contain insertion sequences
that are used by the bacteria. These are shown by the gray.All of
these components of the transformation cassette contain multiple
components. In addition to the coding region that encodes the
protein product, the gene of interest region also contains two
important controlling regions. The promoter region resides just
before the coding region and determines when, where, and to what
degree the gene of interest will be expressed.
In general, two types of promoter regions are used. A
constitutive promoter turns the gene on in all tissues at all
times. In general, this leads to a relatively high level of gene
expression. The most often used constitutive promoter controls the
expression of the 35S RNA of the cauliflower mosaic virus. It is
abbreviated as CaMV35S promoter. Other promoters direct a very
specific expression pattern. For example, the glutelin 1 promoter
directs that the expression of the glutelin storage protein at a
specific time of seed development. It also ensures the protein is
only expressed in the rice endosperm. If the gene of interest is
preceded by the CaMV35S promoter, it will be expressed in all
tissues at all times. Conversely, the expression of the target gene
could be limited to the endosperm if it is controlled by the
glutelin 1 promoter.
Some, but not all genes, encode protein that function in the
plant organelles. These organelles are the chloroplast and the
mitochondria. For example, photosynthesis, and part of the carbon
and lipid metabolism pathways are carried out in the organelles. To
ensure these protein are delivered to the appropriate organelle, a
transit peptide is required. This is a short amino acid sequence
that is found directly before the coding region. This sequence is
recognized by proteins in the outer membranes of the appropriate
organelle. This recognition process leads to the import of the
protein into the organelle. Therefore, if you are gene of interest
functions in the organelle, an appropriate transit peptide must be
included in the transformation cassette
As stated above, the selectable marker is a gene that encodes a
protein product. For it to be expressed, it also needs a promoter
region. It is typical to use the constitutive CaMV35S RNA promoter.
The gene it controls encodes a protein that enables a transformed
plant to survive in the presence of normally toxic compound. The
most often used selective agents are kanamycin and hygromyin, two
bacterial antibiotics, and the herbicide glufosinate. The protein
encoded by the selectable marker genes generally renders these
selective agents harmless to the transgenic plant.This slide shows
the effect of the selectable marker.The insertion sequences
straddle the gene-of-interest coding region and the selectable
marker. These are use by Agrobacteria to create a DNA molecule that
is sent out of the bacteria into the plant where it is eventually
inserted into the nucleus of a cell in the recipient plant tissue.
If the cell follows the proper developmental pathway that leads to
a new plant, every cell in that plant will contain the sequences in
between the insertion sequences.This slide demonstrates one of the
transformation cassettes used to develop Golden Rice was developed.
Slowly click through this slide, and you will see each of the
components of the cassette.Two techniques are used to deliver DNA
found in the transformation cassette into plant tissues during the
plant transformation process. One is a biological system based on
the plant pathogen Agrobacterium tumefaciens. The second is a
mechanical method where the DNA is shot into plant cells using a
gene gun.
Regardless of the delivery method, the delivery system must use
a plant tissue source that can be manipulated to produce new
plants.This slide shows the basic steps of plant tissue culture.
Some plant part is placed is on a defined culture media. That media
induces the the tissue to develop callus. Callus is an
undifferentiated mass of cells. These cells then grow into plant
shoots, which are later rooted. The small seedling will then grown
into a mature, seed-producing plant. When developing transgenic
plants, the transformation cassette is introduced into that plant
part that can be induced to grow new plants. In many ways,
Agrobacterium, has been the most successful method of delivering
DNA into plants. It is a naturally occurring plant pathogen. It
inserts DNA into the nucleus of a plant cell. That DNA contain
genes that encode hormones and food products the bacteria uses to
support its own growth. Here you can see the gall growth on the
plant tissue. This is the natural result of the infection.This
photograph shows exactly how large the gall can grow.The
interaction between the bacteria, Agrobacterium, and the host plant
is very complex. It has been studied in great depth and many of the
major details of the interaction have been described. It is this
understanding that allowed other scientists to convert
Agrobacterium into a plant transformation vector that delivers the
cassette.
It works this way. An Agrobacterium strain in which the
phytohormones have been removed is used. The transformation
cassette is then introduced into that Agrobacterium strain. The
source tissue for plant transformation is infected with the strain.
Then the steps described previously are applied. The tissue is
grown in the presence of the selective agent. That tissue culture
media also contains the hormones necessary to allow the plant
tissue develop new plants. Because of the selection pressure placed
on that tissue, those new shoots will contain the important genes
of interest found in the transformation cassette. For simplicity
sake, many details are being left out. But these are many
steps.Early on it was known that tissues infected with
Agrobacterium could not be coaxed to regenerate new plants. Soon it
was realized that the plant hormone balance was not correct. To
over come this effect, the genes encoding the phytohormones were
removed. Once removed, plant tissues infected with the modified
Agrobacterium could produce the regenerated plant. With this
realization, it was a simple step to envision how to deliver genes
of interest into a plant: include these genes in the cassette.The
second method currently in use is the gene gun. The principle is
very simple. The transformation cassette DNA is coated onto a
particle. That particle is then accelerated (using ballistics or an
air stream). The particle then enters the plant cell. At that
point, the transformation cassette DNA is eluted off the particle,
and by a process that is not known, the DNA becomes integrated into
the nucleus of the cell.
The same basic principles guiding plant transformation with
Agrobacterium is used with the gene gun. A tissue source that is
capable of being manipulated to produce new plants is treated; in
this case it is shot with particles containing the transformation
cassette. The tissue is then placed under selection, and those
shoots that develop contain the gene of interest.This slide
summarizes the steps necessary for plant transformation.And this
slide illustrates those steps.This and the next slide illustrate
the types of traits that can be obtain using genetic engineering of
plants. Notice the particular types of genes that were used to
obtain these traits. Some genes encode a protein that directly
provides the trait. This is illustrated by the gene that encodes
the Bt-toxin protein that is harmful to plant insect pests. Other
genes encode protein that regulate the expression of a trait. Cold
tolerance can be added to a crop by introducing gene that encode a
transcription factor. These factors interact with other genes to
turn on their expression.More recently, such varied traits as salt
tolerance and mercury resistance have been introduced into plants
transferring genes for specific proteins. The last step in plant
genetic engineering is field testing. This slide shows a field that
contains herbicide resistant and tolerant plants.What is needed is
for the public to accept these crops. Examples such as these, were
a corn crop is freed of weed pressure make a compelling case for
acceptance of these new agricultural products. But, it should be
noted that these traits are all producer orientated. The public, in
general, is interested in the consumer perspective. Here are the
general questions that drive the controversy.
