A genetic revolution in the Canadian Forest Service

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.

BIOTECHNOLOGY, A YOUNG SCIENCE ?

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.

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.

HOW LONG UNTIL WE HAVE GMTs ?

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.

	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.

STERILE GMTs

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.

BETTER KNOWLEDGE OF ENVIRONMENTAL EFFECTS

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.

	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.

SOMATIC EMBRYOGENESIS: A BASE FOR GENETIC ENGINEERING

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.

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.

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.

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.

SPRUCE ABLE TO PRODUCE ITS OWN INSECTICIDE

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.

GENETIC ENGINEERING, MORE THAN GMOs

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.

ECONOMIC ADVANTAGES OF TRANSGENIC TREES

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.

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.