Introduction to DNA
The underlying reason that transgenic plants can be constructed is the
universal presence of DNA (deoxyribonucleic acid) in the cells of
living organisms. This molecule stores the organism's genetic information
and orchestrates the metabolic processes of life. Genetic information is
specified by the sequence of four chemical bases (adenine, cytosine,
guanine, and thymine) along the length of the DNA molecule. Genes
discrete segments of DNA that encode the information necessary for
assembly of a specific protein. The proteins then function as enzymes to
catalyze biochemical reactions, or as structural or storage units of a
cell, to contribute to expression of a plant trait. The general sequence
of events by which the information encoded in DNA is expressed in the form
of proteins via an mRNA intermediary is shown in the diagram below.
The transcription and translation
processes are controlled by a complex set of regulatory mechanisms,
so that a particular protein is produced only when and where
it is needed. For more information on molecular genetics,
consult any recent genetics text or the web site Access Excellence,
Graphics Gallery http://www.accessexcellence.org/ . Even species that
are very different have similar mechanisms for converting
the information in DNA into proteins; thus, a DNA segment
from bacteria can be interpreted and translated into a functional
protein when inserted into a plant.
Among the most important tools in the
genetic engineer's tool kit are enzymes that perform specific functions on
DNA. The image at left (Voet,
Donald 1995 Biochemistry) shows
the structure of DNA as a double helix with the phosphate backbone in
yellow-green and the bases in white or teal green. The blue and red
figures represent the 3-D structure of a restriction enzyme (EcoR1)
recognizes and cuts the DNA at a specific region of the DNA. Other enzymes
known as ligases join the ends of two DNA fragments. These and
enzymes enable the manipulation and amplification of DNA, essential
components in joining the DNA of two unrelated organisms.
Locating Genes for Plant Traits
Identifying and locating genes for agriculturally important traits is
currently the most limiting step in the transgenic process. We still know
relatively little about the specific genes required to enhance yield
potential, improve stress tolerance, modify chemical properties of the
harvested product, or otherwise affect plant characters. Usually,
identifying a single gene involved with a trait is not sufficient;
scientists must understand how the gene is regulated, what other effects
it might have on the plant, and how it interacts with other genes active
in the same biochemical pathway. Public and private research programs are
investing heavily into new technologies to rapidly sequence and determine
functions of genes of the most important crop species. These efforts
should result in identification of a large number of genes potentially
useful for producing transgenic varieties.
The techniques for locating and sequencing stretches of DNA that control
specific traits are beyond the scope of this web site. The interested
reader is referred to Klug and
Cummings, 1998, Lewin,
Wong, 1997, or
other recent genetics texts.
Designing Genes for Insertion
Once a gene has been isolated and cloned (amplified in a bacterial
vector), it must undergo several modifications before it can be
effectively inserted into a plant.
Simplified representation of a constructed
transgene, containing necessary components for successful
integration and expression.
- A promoter sequence must be added for the gene to be correctly
expressed (i.e., translated into a protein product). The promoter is the
on/off switch that controls when and where in the plant the gene will be
expressed. To date, most promoters in transgenic crop varieties have been
"constitutive", i.e., causing gene expression throughout the life cycle of
the plant in most tissues. The most commonly used constitutive promoter is
CaMV35S, from the cauliflower mosaic virus, which generally results in a
high degree of expression in plants. Other promoters are more specific and
respond to cues in the plant's internal or external environment. An
example of a light-inducible promoter is the promoter from the cab gene,
encoding the major chlorophyll a/b binding protein.
- Sometimes, the cloned gene is modified to achieve greater
expression in a plant. For example, the Bt gene for insect resistance is
of bacterial origin and has a higher percentage of A-T nucleotide pairs
compared to plants, which prefer G-C nucleotide pairs. In a clever
modification, researchers substituted A-T nucleotides with G-C nucleotides
in the Bt gene without significantly changing the amino acid sequence. The
result was enhanced production of the gene product in plant cells.
- The termination sequence signals to the cellular machinery that
the end of the gene sequence has been reached.
- A selectable marker gene is added to the gene "construct" in
to identify plant cells or tissues that have successfully integrated the
transgene. This is necessary because achieving incorporation and
expression of transgenes in plant cells is a rare event, occurring in just
a few percent of the targeted tissues or cells. Selectable marker genes
encode proteins that provide resistance to agents that are normally toxic
to plants, such as antibiotics or herbicides. As explained below, only
plant cells that have integrated the selectable marker gene will survive
when grown on a medium containing the appropriate antibiotic or herbicide.
As for other inserted genes, marker genes also require promoter and
termination sequences for proper function.
Transformation is the heritable change in a cell or organism brought about
by the uptake and establishment of introduced DNA. There are two main
methods of transforming plant cells and tissues:
- The "Gene Gun" method (also known as microprojectile
or biolistics). This technique, which is shown and explained in the
animated demo section of this web site, has been especially useful in
transforming monocot species like corn and rice.
- The Agrobacterium method, which is described below.
via Agrobacterium has been successfully practiced in dicots (broadleaf
plants like soybeans and tomatoes) for many years, but only recently has
it been effective in monocots (grasses and their relatives). In general,
the Agrobacterium method is considered preferable to the gene gun, because
of the greater frequency of single-site insertions of the foreign DNA,
making it easier to monitor.
Agrobacterium Method of Plant Transformation
Agrobacterium tumefaciens is a remarkable species of soil-dwelling
bacteria that has the ability to infect plant cells with a piece of its
DNA. When the bacterial DNA is integrated into a plant chromosome, it
effectively hijacks the plant's cellular machinery and uses it to ensure
the proliferation of the bacterial population. Many gardeners and orchard
owners are unfortunately familiar with A. tumefaciens, because it
crown gall diseases in many ornamental and fruit plants.
Crown gall of raspberry caused by
Source: Ohio State University
Diagram of Agrobacterium
The DNA in an A. tumefaciens cell is contained in the bacterial
as well as in another structure known as a Ti (tumor-inducing) plasmid.
The Ti plasmid contains
- a stretch of DNA termed T-DNA (~20 kb long) that is transferred to
the plant cell in the infection process.
- a series of vir (virulence) genes that direct the infection
A tumefaciens can only infect a plant through wounds. When a plant
or stem is wounded it gives off certain chemical signals. In response to
those signals, the vir genes of A. tumefaciens become
a series of events necessary for the transfer of the T-DNA from the Ti
plasmid to the plant's chromosome. Different vir genes
- Copy the T-DNA.
- Attach a product to the copied T-DNA strand to act as a leader.
- Add proteins along the length of the T-DNA, possibly as a
- Open a channel in the bacterial cell membrane, through which the
The T-DNA then enters the plant cell through the wound. It is not clear
how the bacterial DNA moves from the cytoplasm to the nucleus of the plant
cell, nor how the T-DNA becomes integrated into the plant chromosome.
Remember that most of the time plant DNA does not exist as an exposed
strand, but is wrapped with histone proteins and is in a supercoiled
state. One speculation is that the T-DNA waits until the plant DNA is
being replicated or transcribed, then inserts itself into the exposed
plant DNA (Galun and Breiman,
To harness A. tumefaciens as a transgene vector, scientists have
the tumor-inducing section of T-DNA, while retaining the T-DNA border
regions and the vir genes. The transgene is inserted between the T-DNA
border regions, where it is transferred to the plant cell and becomes
integrated into the plant's chromosomes (Wong,
Selection and Regeneration
Selection of successfully transformed tissues. Following the gene
insertion process, plant tissues are transferred to a selective medium
containing an antibiotic or herbicide, depending on which selectable
marker was used. Only plants expressing the selectable marker gene will
survive, as shown in the figure, and it is assumed that these plants will
also possess the transgene of interest. Thus, subsequent steps in the
process will only use these surviving plants.
When grown on
selective media, only plant tissues that have successfully
integrated the transgene construct will survive.
Regeneration of whole plants. To obtain whole plants from
tissues such as immature embryos, they are grown under controlled
environmental conditions in a series of media containing nutrients and
hormones, a process known as tissue culture. Once whole plants are
generated and produce seed, evaluation of the progeny begins. This
regeneration step has been a stumbling block in producing transgenic
plants in many species, but specific varieties of most crops can now be
transformed and regenerated.
Tissue culture of
transgenic plants in a controlled environmental chamber.
Future Developments in Transgenic Technology
New techniques for producing transgenic plants will improve the efficiency
of the process and will help resolve some of the environmental and health
concerns. Among the expected changes are the following:
- More efficient transformation, that is, a higher percentage of
plant cells will successfully incorporate the transgene.
- Better marker genes to replace the use of antibiotic resistance
- Better control of gene expression through more specific promoters,
so that the inserted gene will be active only when and where needed.
- Transfer of multi-gene DNA fragments to modify more complex
Plant Breeding and Testing
Intrinsic to the production of transgenic plants is an extensive
evaluation process to verify whether the inserted gene has been stably
incorporated without detrimental effects to other plant functions, product
quality, or the intended agroecosystem. Initial evaluation includes
- Activity of the introduced gene
- Stable inheritance of the gene
- Unintended effects on plant growth, yield, and quality
If a plant passes these tests, most likely it will not be used directly
for crop production, but will be crossed with improved varieties of the
crop. This is because only a few varieties of a given crop can be
efficiently transformed, and these generally do not possess all the
producer and consumer qualities required of modern cultivars. The initial
cross to the improved variety must be followed by several cycles of
repeated crosses to the improved parent, a process known as backcrossing.
The goal is to recover as much of the improved parent's genome as
possible, with the addition of the transgene from the transformed parent.
The next step in the process is multi-location and multi-year evaluation
trials in greenhouse and field environments to test the effects of the
transgene and overall performance. This phase also includes evaluation of
environmental effects and food safety. For more information on these
aspects, please proceed to the Evaluation & Regulation portion
of this web site.
Ann Fenwick, formerly a research associate in the
Department of Soil and Crops Sciences at Colorado State
University, contributed to the content on this page.
Page last updated : March 11, 2004
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