Recent media reports have said
that a genetic revolution is occurring and that the 21st century
will be marked by discoveries in genetics. However, many groups
do not concur with this statement because they view genetic
manipulation as a threat to life on earth as we know it. Who
is right and who is wrong? We will not debate that question
in this article. There is indeed cause for concern considering
that DNA from one species can now be inserted into another
species. An example of this is the use of transgenic mice
to produce human hormones. At the same time, it is incredible
to think that the transgenic animals created through the new
technology can help in the fight against disease. Whether
we like it or not, a genetic revolution is under way. It is
both welcomed and rejected by various groups. Genetic advances
have already transformed the fields of agriculture and medicine,
and they are the basis of an emerging multibillion-dollar
industry, in which Quebec has taken a leading role. This genetic
revolution encompasses all areas of endeavour, including forestry,
and the Laurentian Forestry Centre of the Canadian Forest
Service (CFS- LFC) is playing an active role in this regard.
Drs. Krystyna Klimaszewska, Armand Séguin and Robert
Rutledge are all developing new techniques and products derived
from genetic engineering.
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BIOTECHNOLOGY,
A YOUNG SCIENCE ? |
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Contrary to popular opinion, biotechnologies have been around
for some time. When humans first established settlements around
8000 AD, they began to cultivate the land and select the most
productive and resistant plant species. Some humans even used
fungi to create new types of food, such as bread.
![DNA molecular analysis.](/web/20061103021219im_/http://www.cfl.scf.rncan.gc.ca/CFL-LFC/images/publi-reportages/revolutiongenetique01.jpg)
DNA molecular analysis. |
However, genetic engineering did not appear on the scene
until the 1980s. During this period, scientists discovered
how to transfer a piece of genetic information from one organism
to another. This technique can be used to introduce a particular
character into the receiving organism. For instance, genetic
engineering has resulted in transgenic pigs that can produce
human insulin. This is an example of a genetically modified
organism, or GMO.
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HOW
LONG UNTIL WE HAVE GMTs ? |
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Although going from GMOs to GMTs (genetically modified trees)
is not a big leap, caution is called for. That is why CFS
– LFC researchers are striving to better understand
the mechanisms underlying the genetics of our trees and to
develop indicators of genetic safety. Although genetic improvement
programs for agricultural plants have attained several thousand
generations and breeding cycles, similar programs for forest
trees are only at the second or third generation.
The studies being done by Dr. Séguin's team are aimed
at enhancing basic knowledge of the mechanism whereby genes
initiate resistance behaviours following disturbances or attacks
by forest pests, in the case of white spruce and hybrid poplars.
The knowledge derived from this endeavour will go toward developing
strategies for improving the natural defence systems of trees
by creating individuals that have resistance genes such as
rust and canker resistance genes in poplars, and spruce budworm
and other disease resistance genes in spruce. To this end,
Dr. Séguin's team must identify the mechanisms responsible
for inducing responses to pathogens and isolate the genes
responsible for this type of response so that the genes can
be transferred to other trees.
Similarly, Dr. Rutledge and his team are currently developing
a more effective method for the genetic transformation of
spruce. They are conducting in-depth studies of the mechanisms
associated with genetic transformation through particle bombardment
of somatic embryos.
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DNA, Genes and Chromosomes
Deoxyribonucleic acid, or DNA, is composed of four
bases: adenine, cytosine, guanine and thymine (A,
C, G, T). It is the basic building block of genes.
Genes in turn are grouped to make up chromosomes.
Humans have 23 pairs of chromosomes, or 46 chromosomes
in total. A single human chromosome can contain up
to 175,000 genes. The chromosomes are located in the
cell nucleus.
![DNA: essential information.](/web/20061103021219im_/http://www.cfl.scf.rncan.gc.ca/CFL-LFC/images/publi-reportages/revolutiongenetique02.jpg)
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STERILE
GMTs |
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The transfer of genes from genetically modified trees to
unmodified trees in the natural environment is an abiding
concern of the genetic engineering researchers at the CFS
– LFC. This transfer could be a major limitation on
the commercialization of transgenic trees. We hope to confine
GMTs in order to prevent adverse impacts on the genetic diversity
of natural forests. The ideal way to avert such gene transfers
is to make genetically modified trees sterile. To achieve
this goal, Dr. Rutledge’s group is analyzing the genetic
factors of spruce cone production and the initiation of their
development. In addition to preventing the propagation of
GMTs in nature, inhibiting cone production could be economically
beneficial because it could increase tree growth, since trees
would no longer invest in reproduction. This could represent
added value for transgenic trees.
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BETTER
KNOWLEDGE OF ENVIRONMENTAL EFFECTS |
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Besides this work on conifer sterility, the CFS – LFC
researchers are studying the environmental impacts of GMTs.
Hence, Dr. Séguin’s team has established the
first field test of transgenic poplars in Canada. Genetic
modification of poplars involved adding two marker genes (marker
genes are those that are readily identifiable). These markers
serve, firstly, to check whether the genetic transformation
has been successful, that is whether the genes are actually
present in the poplars. Secondly, the markers are used in
annual monitoring to determine whether the genes end up in
the soil as a result of the decomposition of dead leaves and
branches. The plantation was established using a rigorous
pattern with two rows of nontransgenic poplars among which
some transgenic poplars were interspersed. Once this work
is completed, the CFS – LFC will have accumulated important
data on the persistence of DNA in the soil and on the expression
of the modified genes. The experimental plantation fully complies
with the requirements of the Canadian Food Inspection Agency
(CFIA), which is responsible for overseeing field tests and
the use of plants containing new genetic material in Canada.
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What is a Genetically Modified
Organism?
Modifying an organism entails inserting a new DNA
(deoxyribonucleic acid) sequence into the organism
to cause it to develop a specific character. The usual
method involves cleaving a DNA chain at a specific
location with a restriction enzyme (these enzymes
serve as molecular scissors). Then, the restriction
enzyme is used to cut a plasmid or vector (a molecule
of circular DNA found in certain bacteria). The DNA
fragment is inserted into the plasmid at the site
of the cut. The resulting molecule is called a construct.
Now that we have the construct, it can be introduced
into a cell. There are two ways to do this. The biological
method involves using a bacterium with the ability
to introduce its own DNA into a host (the most commonly
used bacterium, Agrobacterium tumefaciens, infects
numerous plant species), whereas the mechanical method,
particle bombardment, involves bombarding a cell with
microscopic metallic pellets coated with the DNA that
we want to insert. Upon entering the cell, these microbeads
lose their DNA load, and the cell incorporates the
foreign DNA into its own genetic code.
The inserted DNA may subsequently break down in the
cell or become integrated into the plant’s chromosomes.
In the latter case, stable genetic transformation
occurs. As a result, the introduced DNA will be transmitted
the same way as the cell’s own DNA to other
cells during mitosis (cell division) and can be retransmitted
during the formation of reproductive cells and thus
be transferred to subsequent generations.
The introduced DNA does not replace a section of
existing chromosome but instead becomes randomly incorporated
into a chromosomal region. In rare cases, insertion
in a specific gene will inactivate the gene, resulting
in a mutation by insertion. However, in most cases,
the introduction of a new gene will have no effect
on the plant’s normal gene expression.
This manipulation makes it possible to bypass the
DNA barriers of the different species. Indeed, all
living organisms, whether they are mice, humans or
others, are made up of DNA. It is a genetic code that
tells the cells what proteins they need to produce.
Hence, whether the DNA to be introduced comes from
a flower or a bacterium, it has the same basic composition.
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SOMATIC
EMBRYOGENESIS: A BASE FOR GENETIC ENGINEERING |
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Genetic transformation involves more than introducing a specific
plasmid into a cell. The cell also has to be able to survive
the shock of this transformation. Furthermore, it is important
to be able to regenerate the entire plant from transformed
cells. That is why in vitro cell and tissue culture plays
a key role in genetic transformation. The cell and tissue
culture encompasses proliferation of cells in a specific environment
conducive to obtaining a desired quantity of clones. A single
cell or a group of cells can be used to generate a whole tree.
Two methods are available to this end: organogenesis and somatic
embryogenesis.
![Test on transgenic poplar.](/web/20061103021219im_/http://www.cfl.scf.rncan.gc.ca/CFL-LFC/images/publi-reportages/revolutiongenetique05.jpg)
Test on transgenic poplar. |
Organogenesis is a method for regenerating a plant via regeneration
of organs (shoots, roots, etc.) from a tissue or isolated
cells.
Somatic embryogenesis is a leading edge technique and a method
of choice that can be used to propagate plants using a tissue
culture. Starting with an embryo derived from a seed, the
technique makes it possible to produce an unlimited number
of genetically identical embryos that will become trees. The
term "somatic" denotes that the embryos have been
obtained asexually.
Plant propagation through in vitro culture rather than from
cuttings or seeds holds considerable promise for reforestation
and tree genetic improvement programs. The main advantage
of in vitro culture is that large quantities of plants can
be propagated and the production of improved tree stock is
accelerated. In a breeding program, we select the best trees
and then take grafts in order to obtain sufficient quantities
and accelerate production. This approach only becomes truly
productive after 7 to 10 years. With somatic embryogenesis,
however, we can obtain sufficient quantities for field operations
in just 18 months.
Although the long-term objective of in vitro culture and
of molecular genetics is to produce improved stock for reforestation,
the greatest utility of these techniques at present is in
basic research to help us gain insight into the molecular
and cellular mechanisms of our forest species.
![Embryo tissue.](/web/20061103021219im_/http://www.cfl.scf.rncan.gc.ca/CFL-LFC/images/publi-reportages/revolutiongenetique06.jpg)
Embryo tissue. |
At the CFS – LFC, research studies by Dr. Klimaszewska have
led to the development of a somatic embryogenesis protocol (a
recipe) for eastern white pine and western white pine. Her studies
have resulted in a joint patent application for a maturation
method for somatic embryos of conifers. Although pines can now
be reproduced through somatic embryogenesis, a protocol must
be established for inducing the resulting embryo clones to develop
into trees. Such a protocol has already been established for
white spruce, and it permits the rapid development of copies
of trees derived from a single seed. This is how the millennium
trees were produced: more than a million seedlings were produced
from a few seeds.
![Mature somatic embryo.](/web/20061103021219im_/http://www.cfl.scf.rncan.gc.ca/CFL-LFC/images/publi-reportages/revolutiongenetique07.jpg)
Mature somatic embryo. |
Research on pines will enable us to rapidly produce breeding
lines, which will facilitate the development of new varieties
with increased resistance to various pathogens, such as white
pine blister rust.
Dr. Klimaszewska is also developing a method for the long-term
preservation of embryos in liquid nitrogen (cryopreservation).
This research will support efforts to preserve the genetic
resources of forests (organs and tissues). Cryopreservation
is also part of the commercial application of somatic embryogenesis,
since it permits conservation of the best lines and their
propagation whenever desired. We will thus be able to create
a bank of good quality seeds that can be reproduced in large
quantity when necessary.
![Small somatic plants.](/web/20061103021219im_/http://www.cfl.scf.rncan.gc.ca/CFL-LFC/images/publi-reportages/revolutiongenetique08.jpg)
Small somatic plants. |
Tissue culture can play a key role in conventional tree improvement
programs, but it also provides an essential source of plant
tissues for genetic engineering studies on conifers. Through
genetic engineering, we can insert genes into trees to confer
desirable traits such as resistance to disease, insects and
herbicides. Tests aimed at validating the expression of the
new introduced genes and their persistence in transformed
trees are currently under way.
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SPRUCE
ABLE TO PRODUCE ITS OWN INSECTICIDE |
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More than 25 years ago, researchers of the Laurentian Forestry
Centre of the Canadian Forest Service developed formulations
of Bacillus thuringiensis (B.t.), a biological
insecticide used in controlling the spruce budworm. Recently,
Dr. Séguin’s team succeeded in inserting into
spruce the gene that controls the production of B.t.
toxin, thereby enabling the trees to produce the toxin themselves.
The long-term goal is to ensure that the toxin is produced
only when the trees are attacked by the budworm. The gene
will be present, but will not be expressed until necessary.
Tests to validate the integration of the B.t. toxin
gene are being carried out on white spruce to reduce its vulnerability
to the spruce budworm. The testing is being done in the greenhouse
in a controlled environment, and in the field.
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GENETIC
ENGINEERING, MORE THAN GMOs |
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Dr Séguin’s team is also endeavouring to develop
molecular markers for monitoring climate change and its associated
effects on trees. By identifying the genes in trees that respond
to extreme climate events such as drought, we will be able
to develop molecular markers similar to those used to identify
mechanical traits like wood density. These markers of stress
in trees will provide a way to assess the physiological state
of an entire forest.
Another aspect of the research consists in developing molecular
techniques for measuring the genetic variability of tree species
and soil-dwelling micro-organisms, in order to evaluate species
biodiversity.
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ECONOMIC
ADVANTAGES OF TRANSGENIC TREES |
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Over the long term, intensive silviculture of transgenic
trees may benefit the lumber and pulp and paper industries.
Well-designed plantations of these trees could produce a larger
volume of wood per hectare than at present, thus helping to
reduce pressure on natural forests. The GMTs may even contain
less lignin and be more cold resistant.
![DNA extraction process.](/web/20061103021219im_/http://www.cfl.scf.rncan.gc.ca/CFL-LFC/images/publi-reportages/revolutiongenetique10.jpg)
DNA extraction process. |
Biodiversity would be maintained because the genetically
modified trees would be sterile and future plantations of
transgenic trees would only cover a small land area.
Some of the key advantages would be reduced reforestation
costs, decreased pesticide use and more rapid forest renewal
and associated wildlife recovery, as well as alternatives
to using chemical insecticides.
The future is promising for transgenic tree species, and
scientists are already acquiring valuable knowledge about
the genetic components of trees. This knowledge will be put
to use in other biotechnology studies.
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