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Newsletter
- July 1998
Inside This Issue
Version française
John Doornbos
This newsletter is designed to support the theme for our Annual
Meeting this fall, Getting Ready for the 3rd Millenium, where
we will look at genetics and biotechnology. Biotechnology has
become a very important and high profile issue, particularly in
agriculture. It has the potential to become very important in
forestry as well. With a shrinking productive land base and demands
to practice more extensive forestry, trees that are genetically
engineered to grow faster, resist disease and insects or withstand
herbicides could become useful in meeting the demands for wood
supply and less intrusive forestry. However, some risks and significant
ethical issues must be resolved before we will be allowed to take
advantage of these technological opportunities. As we all know,
high profile is not always good profile. But good science can
ensure that high profile is good profile. The articles in this
newsletter are designed to prepare or introduce us to some of
the topics and science that will be discussed at the Annual Meeting.
I look forward to seeing you there.
Top
Jim Richardson
Technical Director
The forest industry in several regions of Canada has an increasing
interest in intensively managed plantations to provide their timber
and fibre supply, as natural forests available for harvest are
increasingly distant from conversion plants and are becoming increasingly
reserved for conservation and environmental reasons. Many companies
which currently utilize natural stands of aspen and poplar are
looking to develop superior genotypes of hybrid poplar and aspen
as an alternative source of supply. Individual companies and other
organizations wishing to pursue genetic improvement of poplar
have become involved with cooperatives, primarily U.S.-based,
designed to maximise cooperation in the required research.
The Canadian Forest Service has articulated the need for a specifically
Canadian cooperative or consortium which would address particular
Canadian research priorities such as the introduction of genes
for fungal resistance and which could deal with the Canadian regulatory
process. As one of the first steps towards establishing a Canadian
poplar biotechnology cooperative, the Canadian Forest Service
asked the Poplar Council of Canada to undertake a survey of Canadian
poplar-using industries to determine more precisely the level
of interest there might be in such a cooperative. The survey was
carried out through personal visits by the Council’s technical
director to corporate industrial members of the organization,
as well as other Canadian poplar-using companies (a total of 22
visits), to explore the idea of a cooperative and to determine
the level of interest and the research priorities perceived by
industry.
The most significant outcome of the survey is the finding that
most companies visited expressed positive interest in the possibility
of a Canadian poplar biotechnology cooperative. No company said
they would not be interested in such a cooperative under any circumstance.
Almost all said they would seriously consider participating in
a cooperative at one level or another, although for some it was
more a question of wanting to monitor developments in this field
rather than wanting access to improved material at the earliest
opportunity.
However, the expression of interest was, in most cases, hedged
with one or more reservations. The cooperative must be very clearly
focussed on issues of relevance to industry and should not be
driven by "science for science’ sake", but rather
have clear, achievable goals. The cooperative must address issues
of regional importance. The size and geographic diversity of growing
conditions and concerns in Canada is an important factor. The
cooperative must offer more than existing, primarily U.S.-based,
cooperative arrangements can provide or are providing. The traits
of interest for improvement in poplar vary considerably, but improved
growth is almost universally considered important.
Cost of participation in a Canadian poplar biotechnology cooperative
is not generally a major factor, and all companies would be glad
to provide in-kind contributions to the operation of a cooperative.
The question of whether a cooperative should have a single level
of membership or allow for both full membership and an information-only
level is a divisive one. Some interviewees believe there must
be a single level of membership only. Others made it clear they
would only consider participating, at least initially, at an information
or ‘observation’ level.
In terms of the objective of a cooperative, greatest industry
interest was expressed in the possibility of obtaining concrete
products - specifically, improved planting material. There was
a general recognition of the need for help that a cooperative
might provide with the intricacies of the Canadian regulatory
processes. Raising forest sector and general public awareness
of the benefits of poplar biotechnology was similarly recognized
as a need that a Canadian cooperative could help fill, but some
of the larger companies felt that their own corporate public relations
groups could handle this.
For many companies, poplar biotechnology is clearly not a high
priority in relation to their immediate operational concerns.
There was in fact some confusion as to the kind of help that a
cooperative might provide. It is evident in this regard that an
organization which could, in the short term, provide practical
assistance with current tree improvement programs, including clonal
selection and establishment of more conventional test plantations,
would win many friends in industry.
The primary conclusion to be drawn from the survey is that there
is clearly sufficient potential industrial support to justify
further pursuit of the establishment of a Canadian poplar biotechnology
cooperative. However, more in-depth discussion and debate on the
objectives, structure and operation of the organization will be
required with all parties (industry, science organizations, and
biotechnology companies) before a cooperative can be successfully
launched.
The Canadian Forest Service continues to lead the effort towards
the establishment of a cooperative. The Poplar Council is ready
to continue working with the Canadian Forest Service towards this
end. We believe that the Council could have a role in the coordination
of such a cooperative, with benefits to all parties.
Further information on the results of the industry survey can
be obtained from the Technical Director.
Top
Pierre J. Charest
A/Director, Planning and Evaluation, Science Branch, Canadian
Forest Service
What is Biotechnology?
Biotechnology in its largest meaning is the use of living organisms
or their processes for the production of goods and services. The
word itself is not new and was first used in 1919 by Karl Ereky,
a Hungarian engineer to indicate the production of goods from
raw materials using living organisms. From that point of view,
there are many foods that are the product of biotechnology such
as beer, wine, cheese and bread (a perfect meal!!) that are all
produced using living organisms such as yeast and bacteria. Furthermore,
genetic improvement of plants (crops and trees) which is essentially
the action of combining sets of genes to obtain desired characteristics
could be considered biotechnology.
However, these later examples are ones that would fit better under
what is often referred to as conventional biotechnology. The era
of new biotechnology started in the 1970s with the manipulation
of DNA, the essence of the genetic code of all living organisms.
DNA is the acronym for Deoxy riboNucleic Acid which is a sugar
molecule derivative. DNA encodes for RNA (RiboNucleic Acid) which
is then translated into proteins by the cellular machinery. The
proteins serve as enzymes or as structural molecules. The DNA
molecule both ensures the expression of individual organism characteristics
and the transmission of its hereditary material.
This new biotechnology was first applied to bacteria like Escherichia
coli (an organism that is ubiquitous) to decipher metabolic pathways
and to use DNA manipulation for the production of biochemicals.
The manipulation of DNA was rendered possible by the discovery
of cellular enzymes that cut, modify and ligate pieces of DNA.
Essentially, scientists copied the cellular machinery to enable
them to manipulate DNA. In a way, it is biotechnology applied
to biotechnology. Scientists are working with nature to produce
desired characteristics.
What is Genetic engineering?
With the increased knowledge of genes, of the cellular processes
and of the proteins that they encode, came the possibility to
design organisms for specific purposes. Genetic engineering or
recombinant DNA techniques allowed the transfer of specific genetic
information or hereditary information (or traits) from one organism
to another. Once the DNA is transferred, the modified organisms
express the genes that give it a new characteristic.
Genetic engineering is a refined way to perform traditional breeding;
although the difference is with the possibility to transfer genes
between species and even between kingdoms. It permits us to bypass
the life cycle of living organisms and the potential for genetic
manipulation is limitless. The term engineering is used to refer
to the act of planning and designing the genes for specific purposes.
Examples of genetic engineering in plants include the development
of crop varieties that are herbicide or insect resistant.
Is genetic engineering really limitless?
The only limit to genetic engineering is human imagination and
creativity. In addition to being able to transfer genes from one
living organism to another, it is possible to synthesize chemically
genes (they are chemical molecules) that will encode characteristics
that are not found in nature. This work is often done using computer
simulation to look at the kinds of modifications that a given
synthetic DNA sequence will bring about in the protein translated
from the RNA. This type of technology is called protein engineering.
A well-known example of protein engineering is the Bacillus thuringiensis
d-endotoxin gene that has been chemically synthesized and optimized
for increased specificity and efficacy.
Why is there so much hype about genetic engineering (or the new
biotechnology)?
Genetic engineering (or the new biotechnology) is new and brings
tremendous promises that have started to be realized. Furthermore,
because it can be used on humans, it raises serious ethical issues.
Like any new technology, the general population does not know
much about it and the information that is out in the media tends
to present the sensational aspects of its application such as
the cloning of Dolly the sheep. Particularly, some people are
concerned about potential adverse effects on human and ecosystem
health. Government and industry are providing more and more information
on the subject to ensure a fruitful debate around the issues surrounding
the use of biotechnology.
Two public opinion polls (1998 and 1994) sponsored by the federal
government have shown that the public is supportive of the use
of forest biotechnology, in particular, for the protection of
our forests against pests.
Are the safety, ethical and, social issues associated with biotechnology
being addressed?
The issues associated with biotechnology are addressed at different
levels. For the safety issues, the federal government has a comprehensive
regulatory framework that is science based and that takes into
account the environmental and industrial aspects of the application
of biotechnology. Also, the federal government conducts research
that attempts to determine the potential effects of the new biotechnology
products on human and ecosystem health.
The ethical and social issues are addressed at the political level
in Canada by the ministers responsible for files associated with
biotechnology such as health and agriculture. A National Advisory
Board on Biotechnology will be created shortly in Canada to provide
advice to the federal government on all issues related to biotechnology.
Why use biotechnology?
There are several reasons why countries like Canada and commercial
enterprises are investing in biotechnology. It provides powerful
tools and new products to improve the profitability of commercial
ventures, and contribute to the development of a new economy based
on knowledge. Furthermore, with the looming problems associated
with overpopulation, biotechnology could contribute to increased
productivity in agriculture and forestry.
What about biotechnology in forestry?
Biotechnology in forestry has already made a significant impact
with the use of Bacillus thuringiensis as a biological insecticide
and the use of tissue culture (the use of pieces of plants to
regenerate new ones identical to the parent plant) to propagate
elite tree genotypes. It is also being used to help in the treatment
of pulp mill effluents and in biopulping. Potentially, it can
be used in:
- forest regeneration e.g. tree improvement, tree propagation
and weed control;
- forest protection e.g. trees resistant to pests and biopesticides;
- wood product processing e.g. biopulping and wood preservation;
- mill effluent treatments e.g. enzymes and microorganisms to
degrade pollutants;
- bioremediation e.g. use of trees for removing or containing
contaminants (heavy metals, organic chemicals) in soils;
- site reclamation or restoration e.g. use of trees to colonize
devastated sites.
What is the state of research in forest biotechnology in Canada?
Canada as a whole has been a leader in scientific research associated
with biotechnology. In particular, Canadian research in tree tissue
culture, biopesticides, and genetic engineering of both trees
and biopesticides has led the way around the world. However, the
forest sector has been relatively slow to take up these new technologies.
Top
Cees van Oosten
Pacifica Poplars
Pacifica Papers Inc.
Parksville BC
Since 1987, Pacifica Poplars (formerly MB Poplar) has been managing
hybrid poplars, first on an experimental basis (till 1993) and
since then on an operational basis. Approximately six years ago,
I was approached by Dr. Steve Strauss of Oregon State University
to see if there was interest in genetic engineering of poplar.
When a critical mass of interested companies was willing to support
the concept, TGERC was formed and we became a member. Companies
and organizations supporting TGERC consist of active poplar managing
companies (Pacifica Poplars, Boise Cascade, Potlatch, Fort James)
and companies with scientific interest that consider cooperative
research as very cost effective (the cooperative approach leverages
company’s contributions many times through outside competitive
grants). Poplars are the best tree species to work with, as they
are relatively easy to transform, have a small genome, grow fast
and can be propagated asexually. In other words, an ideal species
to learn the science of transforming trees.
From my viewpoint there are five dimensions to genetic engineering:
1. the biological/technical
2. the environmental
3. the political/social
4. the regulatory
5. the morass of intellectual property rights (IP’s)
1. The biological and technical dimension is the domain of highly
specialized scientists who speak a language of their own. For
the uninitiated customer (the one who is paying the bill) this
subject can be highly confusing and can lead to poor control of
the entire process. This is where enthusiasm and drive of the
scientists could spiral out of control and where good communication
and project control are absolutely critical to the long term viability
of this work. Since TGERC is a scientific cooperative and the
aims are to do research, Dr. Steve Strauss spends considerable
time communicating with the membership to ensure the cooperative
stays on the rails. There is at least one scientific session per
year where member companies can be brought up to speed on the
progress and where priorities can be set. These plans and priorities
are then discussed at business meetings (sometimes twice a year)
where decisions are made regarding the direction and budgets for
the coming year(s).
From a biological and technical viewpoint, TGERC has exceeded
all expectations! For instance, there already are Roundup® resistant
poplars, using the Agrobacterium method to insert the responsible
gene. This gene is owned by Monsanto and was made available for
this research. TGERC has identified floral homeotic genes in poplar
that could play a role in sterility or could force earlier flowering,
useful in breeding work.
Progress is astounding; the technology moves forward at breakneck
speed and TGERC scientists are able to capitalize on this in productive
ways.
2. The environmental dimension concerns itself with the relationship
of the transgenic organism and the environment in which it lives
and is released (planned or unplanned). One of the objectives
of TGERC has always been to determine what happens to the genes
once released into the environment. A large project is currently
underway to do exactly that. This project was initially funded
through TGERC and thus through membership fees. The project is
so successful that funding now comes from outside TGERC membership
through competitive grants. This is an example of how our contributions
to TGERC are leveraged many times, which is extremely efficient.
To meet the challenges of potential environmental concerns TGERC
decided early on to concentrate on sterility issues and a large
part of its efforts are directed to this.
3. The political/social dimension is where the rubber hits the
road. As most already know, political and social concerns are
raised regarding transgenic organisms. We already have the FlavourSaver
tomato, the Roundup Ready® soybeans and canola, BT cotton etc.
There is a lot of misinformation and a lot of concern through
ignorance. Will a transgenic crop like poplar raise similar concerns,
or is it only related to edible crops? Is paper made from glyphosate
resistant poplars acceptable?
4. The regulatory dimension deals with federal regulators. Since
we do not deal with a food crop, I expect the regulatory process
to be simpler. TGERC realized early on that biosafety could be
enhanced by making the transgenic crop sterile. This may not be
a requirement, but it would be unacceptable to me as a customer
to step to the regulators with a crop that does all we want it
to do, except being sterile, when sterility turns out to be an
overriding concern. We cannot afford to go back to square one.
5. "The morass of intellectual property rights" dimension
is real and of great concern. Biotechnology companies are fighting
each other in the courts over whose patents are being used, infringed
upon, etc. Litigation in the courts could take years and could
slow progress. From my simplistic point of view, getting a glyphosate
tolerant poplar crop would be great, but can we afford it? Since
the trees have been transformed using the Agrobacterium method
(for research purposes), will there be an issue between the company
that owns the patents to this technology and the company that
owns the glyphosate gene? What if we want to insert that sterility
gene and also that BT gene or more than one BT gene. Do we still
have a ballgame and can we afford the entrance fees?
Looking into the future, there is another development where we
can equip elite varieties of hybrid poplar with stable disease
resistance. The Poplar Molecular Genetics Cooperative (PMGC) run
by Dr. Toby Bradshaw of the University of Washington, of which
we are also a member, will shortly be able to isolate and clone
major poplar rust resistance genes that can then be transferred
to elite clones, using TGERC technology. Here we can use genes
from the Populus genus itself and will be able to achieve the
desired results without the time consuming work of traditional
breeding methods.
Top
Anne-Christine Bonfils
Science Advisor
Canadian Forest Service
One of the most recent advances of biotechnology is the ability
to transfer genes between unrelated organisms through genetic
engineering. Techniques for genetically engineering trees are
being optimized and allow the transfer of single gene traits into
superior genotypes, leading to the integration of desired traits
such as shortened growth cycles, pest and disease tolerance, herbicide
resistance, and high quality fibre. While genetically modified
organisms may offer considerable benefits, they also raise a number
of important questions regarding safety and genetic diversity
within species, whether they are derived from genetic engineering
or not. Strong science-based regulations are thus required to
ensure that the products of biotechnology will meet high standards
for human health and environmental safety. At the same time, these
regulations must be efficient and responsive so that they do not
become an unnecessary burden to a developing industry.
In January 1993, Federal Regulatory Departments have agreed to
put in place a Regulatory Framework for biotechnology-derived
products. It is based on basic principles aimed at building up
an effective regulatory system for the protection of the environment
and human health and safety. The framework addresses Canada's
international commitments under the United Nations Commission
on Sustainable Development and the United Nations Convention on
Biological Diversity. Harmonization with member countries of the
Organization of Economic Cooperation and Development (OECD) is
also a major consideration. The principles of the regulatory framework
include:
- maintaining Canada’s high standards for the protection
of the health of workers, the general public and the environment;
- using existing legislation and regulatory institutions to
clarify responsibilities and avoid duplication;
- continuing to develop clear guidelines for evaluating products
of biotechnology which are in harmony with national priorities
and international standards;
- providing for a sound scientific database on which to assess
risk and evaluate products;
- ensuring both the development and enforcement of Canadian
biotechnology regulations are open and include consultations
with stakeholders and the public; and
- contributing to the prosperity and well-being of Canadians
by fostering a favorable climate for investment, development,
innovation and adoption of sustainable Canadian biotechnology
products and processes.
Biotechnology-derived products and processes are thus regulated
by the departments and agencies that are responsible for regulating
equivalent products developed using conventional techniques and
processes. The federal legislative authority for health and environmental
assessment of forest biotechnology lies under several acts. The
Seeds Act for trees, the Plant Protection Act for imports, the
Fertilizers Act for biofertilizers and mycorrhizae, the Pest Control
Products Act for microbial pest control agents and the Canadian
Environmental Protection Act for microorganisms are used in the
pulp and paper industry. The Seeds Act, the Plant Protection Act,
and the Fertilizers Act are administered by the Canadian Food
Inspection Agency (CFIA), while the Pest Control Products Act
is administered by the Pest Management Regulatory Agency, and
the Canadian Environmental Protection Act by Environment Canada.
An interdepartmental committee meets regularly to ensure a coordinated
approach in the development, ongoing improvements, and implementation
of these regulations. The Canadian Forest Service contributes
to this process by providing scientific and technical expertise
to the above departments and agencies for the products of biotechnology
in the forest sector.
The Seeds Act for example, regulates the inspection, testing,
quality and sale of seeds in Canada. Regulations under the Seeds
Act were amended in early 1997 to clarify the information requirements
for environmental safety of plants with novel traits, including
trees, prior to release. Confined field trials are authorized
subject to conditions that minimize environmental interactions
and require close monitoring of the materials. Unconfined release
is authorized, with or without conditions, following thorough
case-by-case environmental safety assessments. These include precise
characterizations of the novel proteins and the modified plant,
considerations of weediness and invasiveness, ability to transfer
genetic information to related species, potential to become a
pest, potential to cause unwanted interactions with other organisms
in the environment and potential to cause negative impact on biodiversity.
The Canadian Forest Service, in collaboration with provincial
regulatory agencies, are currently assessing regulatory guidelines
under the Seeds Act to ensure that they can adequately be applied
to trees.
The first confined field trial of genetically modified trees
was authorized under the Seeds Act by the CFIA in 1997, in Valcartier
near Quebec City. It consists of a 900m2 plot of poplar trees
(Populus alba x grandidentata) genetically modified to contain
two marker genes, and is carried out by the Laurentian Forestry
Centre of the Canadian Forest Service.
The Canadian Environmental Protection Act plays a "safety
net" role in the Canadian regulatory framework for biotechnology-derived
products. It covers any new biotechnology products that are not
assessed for health and environmental impacts under other federal
legislation, thus ensuring that there are no gaps in the framework.
Additional information on Canada’s Regulatory Framework
for biotechnology-derived products, and in particular on the regulation
of genetically modified trees, is available by contacting the
Office of Biotechnology, Canadian Food Inspection Agency, 59 Camelot
Dr., Nepean, Ontario K1A 0Y9 (tel: 613
225-2342, fax: 613 228-6604).
Top
Armand Sequin
Research Scientist, Laurentian Forestry Centre, Canadian Forest
Service
As previously mentioned, forest biotechnology comprises several
technologies, including in vitro culture (somatic embryogenesis),
microbiology and the use of recombinant DNA, or genetic engineering.
In this section, I will describe the leading-edge techniques employed
to introduce an economically important gene into poplars, along
with the advances achieved so far in this regard.
Historical overview
Poplar breeding programs have been under way for a number of years,
with the primary goal of identifying genotypes that have superior
physiological characteristics, such as growth and other silvicultural
traits, or superior resistance to various biotic or abiotic stresses.
Although tremendous progress has been made through traditional
breeding, this type of approach can be fairly time-consuming.
Traditional genetic crosses permit the exchange of chromosome
segments carrying one or more desired genes, and this genetic
mixing produces individuals that have superior characteristics
for a given phenotype. However, exchanged chromosomal regions
may at times have negative nontarget effects. Until recently,
it was difficult to determine what part of the genome had been
modified. With the advent of molecular markers, however, researchers
can now gain insight into recombination events, track genotype
pedigrees and greatly expand possibilities for accessing genes
[3].
On another front, genetic engineering can now be used to introduce
a gene conferring some beneficial trait, such as insect resistance,
into a particular genotype. A desired trait can be obtained in
a specific genotype within a relatively short time period. Furthermore,
multiplication of the transgenic (genetically transformed) material
does not require sexual reproduction, since trees can be propagated
by in vitro culture. This also holds true for material that has
not been genetically transformed. Finally, a gene conferring a
given trait can be transferred even if the organisms involved
are not genetically compatible (for example, from a bacterium
to a tree).
Production of transgenic plants began in the late 1970s, following
discoveries made with the soil bacterium Agrobacterium tumefaciens
[7]. This organism infects a number of plant species, inducing
tumour formation. It has the natural ability to transfer a tiny
bit of its DNA, containing all the genetic information required
to initiate the development of tumours. European and U.S. laboratories
discovered that the region responsible for tumour formation can
be replaced with selected genes, thereby providing a natural means
of introducing the genes into plants [12, 15]. Development of
this technology has permitted the production of several transgenic
plant species, almost all of them agricultural crops [8, 29].
In the case of trees, the first positive results were obtained
in 1987 in poplars [11, 24]. Before describing the different stages
in the genetic transformation of poplar, I would like to mention
a recent work, published by the USDA Forest Service and edited
by N.B. Klopfenstein et al. The book covers several aspects of
genetic engineering of poplar, including in vitro culture and
biotechnological applications [18].
Genetic transformation
There are two basic steps in genetic transformation. First, the
DNA being introduced has to reach the nucleus of the cell that
is to be transformed. This foreign DNA will not replace part of
an existing chromosome, but will instead be randomly inserted
into a chromosomal region. In the vast majority of cases, the
introduction of a new gene will not affect the plant’s normal
genetic expression. Second, the introduced DNA may either be broken
down by the cell or be integrated into the plant’s chromosomes.
In the latter case, a stable genetic transformation occurs, and
the introduced DNA is transmitted to the daughter cells through
mitosis. This same DNA can be transmitted to subsequent generations
during gamete formation [22].
Selecting transformed cells is a key part of genetic transformation.
To permit this selection following the incorporation of foreign
DNA, a gene conferring resistance (usually to the antibiotic kanamycin)
is inserted with the DNA material to provide a selectable marker.
Only the cells containing this resistance gene will be able to
survive when the transformed in vitro material is cultured with
the marker. This screening method makes it possible to recognize
genetically transformed cells. Other molecular tools can be used
subsequently to detect the presence of the introduced DNA and
confirm the genetic transformation.
Agrobacterium-based method of introducing DNA
For poplar trees and most plants, genetic transformation using
the Agrobacterium tumefaciens is a simple and effective approach
[16, 29]. As mentioned previously, this soil bacterium has the
ability to transfer part of its DNA to the infected plant. The
plasmid of Agrobacterium DNA that is thus incorporated into the
plant causes tumours. More specifically,the genes that are incorporated
into the plant cells induce the production of enzymes that play
a role in synthesizing or modifying plant hormones. When these
bacterial genes are replaced with the genes of choice, infection
of the plant with Agrobacterium does not result in tumour formation.
The genetic engineering method employing A. tumefaciens [14, 17,
26] works with several poplar genotypes, but there is still room
for improvement, since some genotypes resist genetic transformation.
Plant material used and selection of transformants
Genetic transformation is not limited to introducing a particular
plasmid into the cell nucleus. The cell has to be able to survive
the shock of transformation and selection for the antibiotic marker.
Furthermore, the entire plant has to be regenerated. This is where
in vitro culture comes into the picture. This technique involves
propagating plant tissues (or single cells) in a controlled environment
free of micro-organisms. The resultant in vitro culture lines
can be used to obtain uniform (clonal) material of the desired
quantity. In fact, a whole tree can even be regenerated from a
single cell. Organogenesis, the preferred method in the case of
poplar trees, consists in regenerating the entire plant using
organs derived from tissue or from isolated cells. These in vitro
techniques are also employed in the vegetative propagation of
elite trees.
The main stages in genetic transformation and in vitro culture
of poplar are summarized in the figure below. This process generally
takes 8 to 12 months and involves 6 steps. Two initial components
are required: a specific poplar clone, cultivated under sterile
in vitro conditions, and the compatible Agrobacterium strain whose
plasmid carries the genes to be introduced. The inoculation with
the Agrobacterium is the first step; it allows the bacterium to
enter and infect the plant cells. After that, the DNA from the
Agrobacterium plasmid is either broken down or integrated into
the tree genome (integration). To promote the growth of genetically
transformed cells, an antibiotic is added to the plant cell culture
as a selectable marker. This is the selection step. Once the transformed
cells have been identified, they are multiplied in order to regenerate
plantlets. Then, the in vitro stock is transferred to a culture
medium for rooting. After the plantlets are well-rooted, hardening
is induced so they can be transferred to the greenhouse.
Concrete examples
Resistance to insects
Use of the bacterium Bacillus thuringiensis (B.t.) for controlling
insect pests has been the subject of exhaustive descriptions by
several authors [6, 13, 19]. Genetic transformation allows us
to use this biological control tool for other applications. In
fact, the gene that codes for the B.t toxin was modified so the
genetic code would be readily recognized by target plants, and
a specific promoter was added to the plasmid to ensure effective
control over toxin production in the plants. Genetic constructs
of this type, employing specific B.t. genes for different insect
pests (lepidopterans, coleopterans, etc.), have been introduced
into a number of plant species. Several laboratories have conducted
research on poplar [10, 20, 21], permitting the production of
transgenic trees with greater resistance to insect pests.
Resistance to forest pathogens
Fungal and bacterial infestations cause substantial losses in
poplar stands. Without giving an exhaustive list of these disease
problems, I should mention that poplar trees are susceptible to
cankers caused by fungi of the genera Septoria and Hypoxylon,
to leaf-spot disease caused by Marssonina sp. and Septoria sp.,
and to rusts caused by Melampsora sp. Recent advances in plant
biotechnology have permitted the creation of new plant varieties
that are more resistant to various pathogens. The genes introduced
to obtain this resistance may actually produce antifungal or antibacterial
proteins [9]. Various approaches, which are currently at the experimental
stage, will be used to assess the effectiveness of these strategies
for forest trees.
Modification of lignin
Extracting lignin from wood fibre is a costly and polluting stage
in pulp and paper production. Development of transgenic trees
that have a lower lignin content, but do not have unfavourable
physiological characteristics, is now within the reach of present
technology. Lignin is the most abundant organic compound in the
biosphere, after cellulose, and makes up 15 to 35% of the dry
weight of trees. Biochemical pathways in lignin synthesis have
been the subject of numerous investigations, and several genes
responsible for the enzymes involved have been characterized [2,
32]. Based on the findings from this work, genetic engineering
technology has been harnessed to modify the metabolic routes concerned,
with highly promising results [5]. Some of the transgenic trees
contain a modified type of lignin which may be easier to extract
[1].
Tolerance to herbicides
In poplar trees, herbicide resistance would make it much easier
to control competing vegetation in new plantations. There are
two main strategies for conferring tolerance to a particular herbicide:
introducing a mutant gene whose corresponding protein is no longer
a target for the herbicide, or inserting a gene to allow the production
of an enzyme that detoxifies the herbicide. The possibility of
conferring herbicide tolerance through genetic engineering has
been widely exploited in agriculture. So far, transgenic poplars
have been created with resistance to several types of herbicides,
including glyphosate and phosphinotricine. Field tests are currently
being conducted with herbicide-tolerant transgenic poplars to
determine their actual levels of tolerance.
Other applications
Remediating contaminated soil through the use of living organisms
is a new environmental technology that has progressed considerably.
Most bioremediation methods are based on the use of micro-organisms
(bacteria) that are capable of breaking down toxic substances.
Another strategy involves using plants as a filtering and decontamination
system. Genetic engineering can be used to increase the resistance
of certain plants to toxic metals such as cadmium [4, 23]. Similarly,
using genetically modified poplars to eliminate chemical contaminants
in the soil is now feasible. In one application, a gene from a
bacterium that has the ability to degrade toxic substances (chlorophenols)
has been transferred to poplars. The transgenic trees have the
ability to achieve sustained growth in chlorophenol-contaminated
soil [27]. Furthermore, over time the presence of the poplars
helps to reduce concentrations of the chemical contaminants in
the soil. Finally, the new vegetation cover prevents soil erosion
and dispersion of the contaminants and creates a favourable micro-environment
for soil micro-organisms.
Deployment of transgenic poplars
After the first transgenic poplars were developed, field tests
were conducted by various research groups in the United States
and Europe [28]. Overall, the results have shown that transgenic
trees do not have any abnormal characteristics. These tests, along
with studies on the potential environmental effects of transgenic
trees, are of paramount importance in ensuring full public acceptance
of the new genetic material [25].
In related avenues of research, major efforts have been devoted
to enhancing understanding of what occurs at the molecular level
during flower development in forest trees. The goal of these studies
is to identify some of the genes involved in cone or flower formation.
Once this information is obtained, genetic engineering tools can
be used to inhibit flower development. This will make the use
of transgenic trees more acceptable, because the modified DNA
will not be able to spread in the natural environment.
A similar approach, using various genes that regulate flower development,
can speed the development of floral organs. These so-called homeotic
genes were isolated from the plant Arabidopsis thaliana, which
is related to mustard (family Brassicaceae). One of the genes,
LEAFY, plays a role in triggering flower development in Arabidopsis
[30]. A genetic construct permitting strong expression of the
LEAFY gene was introduced into poplar trees; it substantially
reduces the time frame for flower maturation [31]. Developing
trees that are capable of reproducing faster is obviously advantageous
for accelerating breeding programs.
Outlook for the future
Genetic engineering of forest trees entails a major investment
that can generate considerable benefits over the long term. This
includes the increased protection that tree biotechnology offers
against forest insect pests. In the present article, I have given
a brief description of the advances achieved with poplar trees.
Clearly, the gains made so far have come about through riding
on the coattails of technologies originally developed for agronomically
important crops. The research undertaken in plant molecular biology
in the field of agriculture is not at all like that being done
in forestry. Nevertheless, many genes involved in various physiological
processes specific to trees are being studied at present. Knowledge
of these processes is essential for effectively integrating biotechnologies
into tree breeding work.
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