Alvin D. Yanchuk is Program Manager and Senior
Scientist in the Research Branch of the
British Columbia Forest Service, Victoria,
Canada. He wrote this article while at FAO
as a consultant in the Forest Resources
Division under the Partnership Agreement
with Research and Academic Institutions.
The current direction of research, and issues to be considered in future decision-making regarding the use of biotechnology in forestry.
Biotechnology has witnessed such rapid advancements in the past decade that the forest sector cannot fail to consider its potential impact, and it may be impossible to guess what specific techniques and options will be feasible in the next several decades. Although specific techniques are sure to raise specific critical issues, it is important to evaluate the basic forest genetic management principles that should be followed, irrespective of the technology itself.
This debate in agriculture regarding the possible effects of products from biotechnology in terms of food safety and the environment - which has created confusion in the public and even among professionals - has been and will continue to be very instructive to forestry. Many of the techniques applicable to forestry are similar to the ones used in agriculture, although, as shown in this article, the issues in forestry can be somewhat different and will require special attention.
This article summarizes the biotechnologies currently used and being developed in forestry. It explores the main issues and controversies related to their use, stressing the importance of evaluating these scientifically and within socially accepted decision-making processes. In conclusion, it looks briefly at the direction that research and the application of biotechnology may take in the future, and at the degree to which governments can or should assist the development of biotechnology and regulate its use.
The United Nations Convention on Biological Diversity defines biotechnology as "any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use". Biotechnologies used in forestry fall into three main areas: the use of vegetative reproduction methods, the use of molecular genetic markers, and the production of genetically modified organisms (GMOs)1 or transgenic trees.
Vegetative reproduction comprises a broad range of techniques involving the manipulation of plant tissue samples (e.g. sections of stems, leaves, roots, seeds or even cell cultures) which ultimately allows for complete vegetative "repro-pagation" of the whole plant. The end results are genetically identical individuals - clonal varieties or lines.
The simplest forms of vegetative propagation are the familiar techniques of grafting and the rooting of stem sections or "cuttings". Some species root or graft easily on to rootstock of other genotypes or related species. Others require substantial and elaborate treatment in order to be rooted or propagated from various tissue types, i.e. cloned, successfully. Genetic alterations are not expected to occur with vegetative tissue cloning methods, but somaclonal variations can be induced from time to time with some of the more advanced techniques (outlined below).
In forestry, most cloning methods have been developed to produce trees directly for reforestation purposes, but these techniques are also now a basic prerequisite for the production of genetically modified (GM) trees. The biotechnologies related to vegetative reproduction in forestry fall into the three main categories of micropropagation, cryopreservation and in vitro storage, and in vitro selection.
Micropropagation. Micropropagation is the development of clonal lines from small tissue samples such as buds, roots and embryos extracted from seeds. The last, referred to as somatic embyrogen-esis, will likely be the most promising micropropagation technology for long-term storage and large-scale production of trees from selected clonal lines. Somatic embyrogenesis can be particularly useful once the genetic value of the clones has been determined, which can take several years of evaluation under field conditions. Trees that display normal growth patterns have been developed through somatic embryogenesis for several conifer species, but some of the most successful results to date have been in Picea species (Fan and Grossnickle, 1999). However, the development costs of such advanced tissue culture technologies are high compared with those of simpler techniques (e.g. rooted cuttings).
Trees developed through somatic embryogenesis: a) initiation of somatic embryogenesis from immature megagametophyte in Pinus strobus; b) mature somatic embryos of P. strobus after 12 weeks of culture on abscisic acid (ABA) and gellan gum medium; c) P. strobus somatic embryos after 11 days on germination medium; d) P. strobus somatic seedlings after 12 weeks on germination medium, prior to transfer to a greenhouse; e) P. strobus somatic seedlings in the greenhouse after two months of growth; f) white spruce (Picea glauca) somatic seedlings after four months of growth in the greenhouse
- Canadian Forest Service\K. Klimaszewska
Cryopreservation and in vitro storage. With this technique small tissue samples are maintained at very cold temperatures in order to maintain their current physiological condition. In many conifer species, physiologically juvenile plant material is typically cryopreserved. Trees developed from older tissue can show reduced growth potential later on, but this characteristic varies greatly among species and genotypes. Therefore, the evaluation of genotypes for efficient production of plant material later on is usually one of the main goals of cryopreservation. It is also important that the storage process itself does not induce chromosomal instability which can result in variable and undesirable plant forms (Tremblay, Levasseur and Tremblay, 1999). While the technology is useful and necessary for many applications in clonal forestry, it may have a special role in programmes for species with recalcitrant seed, for which basic seed storage is a major problem (Englemann, 1997).
In vitro selection. This technique involves selecting for a particular trait (e.g. heavy metal or salt tolerance) at an early stage in the micropropagation phase. The trait must also show stable expression for the life of the derived plant. Although in vitro selection has been used to some degree in crop plants, attempts in forest trees have been limited to selection of GM trees. In vitro selection techniques are a basic requirement in the process of screening for successfully GM-transformed clonal lines. Other traits can therefore be combined with basic in vitro selection criteria required for GM screening (e.g. mercury tolerance in yellow poplar [Rugh et al., 1998]).
Genetic markers serve as "random locators" on nuclear or organelle DNA. The challenge to the geneticist is to look for relationships between these markers and characteristics of trees from pedigrees or specific populations. When they are interpreted correctly, genetic markers are invaluable for examining genetic variation among and within populations, assessing levels of outcrossing and inbreeding, and genetic identification or "fingerprinting" of varieties or pedigrees.
Genetic marker data can also be used for assisting with early selection of better genotypes, rather than waiting for the tree to express the trait much later. There have been many developments in both agriculture and forestry in associating marker data with physical traits. However, marker-assisted selection (MAS) is not yet routinely applied in tree breeding programmes, largely because of time and cost constraints. It will also be necessary to show a clear economic advantage over conventional selection approaches. Thus it is likely that MAS will only be applied to a few species and situations, e.g. some of the major commercially used pine (Frewen et al., 2000) and Eucalyptus species.
Most of the markers in use today are based on systems in which DNA is cut by enzymes, after which the DNA sections are multiplied (amplified) in reactors and then separated by electrophoretic techniques - i.e. DNA bands are separated out by their molecular weight and charge, in the presence of an electrical current on a gel medium (Photo 2). Rapid advances in DNA markers, mapping and sequencing technologies are increasing the basic understanding of the role of genes and their products in cellular processes. Investigations in this field, known as "genomics", are well under way in humans as well as in animals, plants and a few tree species (Merkle and Dean, 2000).
Molecular markers are primarily an information tool. They do not contribute directly to plant products, but they provide information that is critical to locating and working with the DNA that can be of interest for genetic transformation, or information on population structure, mating systems and pedigree confirmation.
Chloroplast DNA repeat sequence markers for 16 coastal Douglas fir parents. Lanes marked "hae" are standards, and each numbered lane is a gene marker for each clone. Several clones share common bands, but some (30, 26, 18, 20 and 22) have unique genetic marker bands (alleles); such variable genetic markers are highly useful for genetic fingerprinting
- British Columbia Forest Service\M. Stoehr
The term "biotechnology" is often associated entirely with genetic transformation, the most controversial of all these technologies as it involves the introduction of selected "foreign" genes into the plant genome. Well-characterized genes from other species are used, which typically allows for the expression of a novel trait in a new genetic background. "Biolistics" (i.e. the introduction of DNA into the cell nucleus on small metal particles using high-speed projection techniques) or microorganism vectors that carry the specific gene(s) of interest are typically used to introduce the gene(s). Of course, long-term or adequate expression of the gene in the GM plant is also critical; this is sometimes referred to as "stability".
Genetic transformation techniques are fundamentally different from conventional plant breeding. At least in the initial generations, conventional breeding and selection bring about an increase in the frequency of desirable genes affecting some trait already present in the species. It is presumed that this occurs across many gene loci and that each gene has a small effect, although a few genes of large effect will also be important to the response in early generations. Later, new favourable mutations - most mutations are unfavourable and will probably be selected against - will accumulate as their frequency is increased through breeding (sexual recombination), testing and reselection.
Most forest tree species are still quite close to their wild relatives and have high levels of genetic variation for most traits of interest. Therefore, to be of value, GM trees must offer unique features that cannot be economically delivered through conventional breeding programmes and that are capable of offsetting the costs and time expended in developing the technology.
The traits that have so far been considered for potential genetic modification of forest trees are herbicide resistance, reduced flowering or sterility, insect resistance and wood chemistry. While conventional tree breeding programmes have paid attention to insect resistance and wood quality (but not many of the specifics of wood chemistry), they are likely to have little ability to incorporate traits such as herbicide resistance or reduced flowering.
Two techniques commonly used to create new sources of genetic variation in crops, horticulture and fruit trees, which are not easily classified as either tissue culture or genetic modification, are protoplast fusion (i.e. the hybridization of species by fusion of protoplasts and genomes from related species but with reproductive barriers) and haploi-dization (i.e. the creation of completely homozygous clonal lines made by doubling haploid cells, such as pollen). Neither technique is well developed in forest tree species, but protoplast fusion research is under way in a few woody plants (Guo and Deng, 1999). Another technique routinely used in crops and fruit trees is mutagenesis: a directed or quasi-random mutation of an existing gene, typically done with mutagenic chemicals. While these techniques are not effectively in use or even considered important in forestry today, they too point to the fact that large and potentially useful genetic modifications can be made with techniques other than GM technology.
An additional technique now commonly used in both crops and forestry, e.g. in hybrid poplars (Zuffa, Lin and Payne, 1999), is embryo rescuing. This involves the removal of an embryo from a seed that would otherwise not survive on its own and that embryo's cultivation until it becomes a successful "germinant" or seedling. This procedure is typically required when there are differences in basic seed biology between species or parental lines, such as incompatibilities in ovule or embryo development. Although the technique is a rather simple biotechnology, it is important to point out that the plants it produces would not exist without such a rescue effort.
Most of the biotechnologies used in applied forestry today fall into the categories of tissue culture and molecular marker applications. Advances in these two areas of biotechnology have all been logical progressions and refinements of techniques that have been used in plant and tree improvement for decades, if not centuries, to produce clonal lines or varieties.
Most of the popular concern about GM plants, or about the use of any of the other techniques discussed in this article, derives from concern about products grown as food for human consumption. This will rarely be a direct issue in forestry, although careful evaluation will be necessary in the case of multi-use tree species that provide non-wood forest products (NWFPs).
The argument that the genetic changes made in GMOs are "unnatural" has been raised as a concern. It is, however, difficult to say that any particular technique in itself can pose increased biological risk. It is the gene products that may pose a lower or higher risk, irrespective of the technology used to obtain them. To evaluate possible risks fully, in-depth knowledge of the particular genetic transformation is necessary, e.g. the product produced, where the transformation occurred, what "promoter" genes were used and the reliability of the expression over time (Gutierrez, MacIntosh and Green, 1999). This may be particularly true when more than one or two transgenes are incorporated. In such cases there may be requirements for longer field testing, environmental assessments and more caution in deployment (Burdon, 1999). In addition, mutation caused by gene transfer and tissue culture (somaclonal variation) can alter the function of non-target genes in ways that might take some time to detect. These, however, are not genetic issues, but a matter of having appropriate genetic testing and evaluation programmes in place prior to the release and use of GM (or even conventionally bred) trees in managed landscapes.
Herbicide resistance. Herbicide resistance in poplars is probably the best-developed GM technology in forest trees. The first concern with herbicide-resistant GM plants is evidence of the development of resistance in weed populations. The risk may be substantially less in forestry than in agriculture, as herbicides are only used for a short time and with fewer applications during the early period of plantation establishment. Furthermore, total weed control is not necessary in forest tree plantations, so there is less selection pressure for resistance to weeds.
The other concern is cross-breeding with wild populations. While this is possible, it is likely that wild populations that do hybridize with GM trees would have little selective advantage unless adjacent wild populations are treated with herbicide, which is rare in forestry. The main question is then the effect on the genetic structure of local wild populations if cross-breeding does occur (e.g. in in situ conservation areas) and the acceptability of this risk to local resource managers. If this is a concern, reduced flowering or sterility transgenes may be required, along with herbicide resistance in GM trees.
The introduction of herbicide resistance through GM technology is likely to be the most feasible and the most frequently applied genetic modification in trees. Nevertheless, it is only likely for a few well-developed species in certain situations, such as in intensive poplar fibre farms where herbicides are sometimes an approved management tool.
Reduced flowering or sterility. Reduced flowering in forest trees may be desirable in order to steer the products of photosynthesis into wood production, rather than reproductive tissues. However, since such reallocation of resources has not yet been well quantified, the main justification for reduced flowering or sterility development is when it substantially reduces the gene flow to wild adjacent populations of the same species. This may help promote greater acceptance of intensive GM tree plantation forestry adjacent to natural forests. Although research on flowering mechanisms is under way (e.g. Skinner et al., 1999), the stability of sterility-gene expression over time will have to be confirmed in field trials that reflect expected rotation lengths.
Insect resistance. The development of GM crops that are insect resistant is now common, but these crops also create some of the most complex ecological situations that need to be addressed. The first issue of concern is the possible toxicity of the compounds produced in GM insect-resistant plants when they are grown specifically for human consumption or for animal feed as part of associated food chains. Second, there are ecological concerns of cross-breeding with wild relatives and the evolution of resistance in the pest populations, as with herbicide resistance. An additional and serious problem in forestry is that the long generation time of trees allows for many generations of insect populations to challenge a resistance mechanism.
The most developed GM approach for insect resistance in forestry, as in agriculture, has been the use of genes from a natural insect pathogen, Bacillus thuringiensis (Bt). Poplars are again among the tree species in which the technology is most advanced (TGERC, 1999). Research and development of other compounds is under way to reduce reliance on the relatively narrow group of natural Bt toxins (ffrench-Constant and Bowen, 1999). Because of the complex ecological ramifications and public concerns surrounding GM insect-resistant plants, high levels of scientifically sound laboratory and field testing will be required.
Wood property chemistry. It is now technically possible to alter genetically the chemistry of lignin in trees for easier and more environmentally friendly pulping. Genes that are important to the pathway of lignin development in wood have been modified to produce unique wood composition in very young trees (Lapierre et al., 1999). However, genes of relatively large effect on wood chemistry have also been found naturally (e.g. a major recessive gene in loblolly pine [Ralph et al., 1997]), so wood properties can also be modified through conventional selection and breeding. Therefore, many of the same ecological and economic considerations need to be taken into account with or without genetic transformation technology. Two important questions that remain in developing lignin-modified varieties or clones are how economically valuable plantations using such trees would be and whether altered wood may show susceptibilities to environmental stresses. If the answer to the latter is affirmative, it is again likely that reduced flowering or sterility genes would have to be incorporated into material used on a large scale when it is important that there be minimal gene flow to adjacent wild populations (e.g. in in situ conservation areas).
As previously discussed, one of the main ecological concerns with GM plants is the issue of gene exchange with wild populations - an issue already being considered in forestry with regard to seed orchard seed from conventional breeding programmes. In most of the situations for which GM trees will be considered, the plantation species will be an exotic one, so gene exchange would not be a factor; but in cases where it could occur, reduced flowering or sterility is likely to be a basic requirement. Much has been and will continue to be learned from agricultural research on transgene flow from GM crops to their wild relatives. Investigations into these specific questions are under way in forestry (DiFazio et al., 1999).
Although exotic plantation species may have limited risk of cross-breeding per se, the release into the environment of a GMO in most cases poses less of an ecological risk than the introduction of an exotic species. The introduction of an exotic species is a release into the environment of an entirely different genetic constitution, rather than the introduction of a single gene into a native species. The current regulations of the Forest Stewardship Council (FSC, 1999) prohibit the use of GM trees, but suggest that "the use of exotic species shall be carefully controlled and actively monitored to avoid adverse ecological impacts". This recommendation largely runs contrary to the argument made here that the biological risks and economic realities of all forest management options need to be evaluated objectively. On the other hand, a large genetic change made to the overall fitness of a native species and released without adequate consideration of local environmental risks is not appropriate either.
Clearly, potential risks must be balanced with benefits in all types of improved forest trees, considering the deployment schemes that will be used in different locations and at different times. For example, clonal varieties of fruit trees have been used in large continuous orchards for hundreds of years, and this deployment and management approach is considered by most people to be ecologically and economically acceptable, as it presents no unmanageable biotic or abiotic challenges. In plantation forestry for wood production, clonal blocks may be appropriate in some situations, such as poplar plantations, while pure or family-mix planting patterns are likely to be appropriate for most other forestry situations.
In any event, deployment strategies must be designed to minimize the risks of economic losses of the stand as well as future biological losses, such as the development of resistance in pest and disease populations (Roberds and Bishir, 1997). At a minimum, therefore, a few dozen well-tested GM clones will be necessary to meet the typical production and diversity requirements necessary even in intensive forestry programmes.
To help address many of these concerns, a number of countries have developed regulations and restrictions specifying the requirements of confined field testing that are needed before commercial release (OECD, 2000). These requirements, which are necessary to reduce biological and economic risks, will undoubtedly continue to evolve. In addition to national laws and regulations, there are also broader international agreements on biosafety, e.g. the Cartagena Protocol on Biosafety (Convention on Biological Diversity, 2000).
Private investors have taken the lead for most investment in modern biotechnology, and in so doing have also had to manage the associated economic risks. In many situations such investment risks are protected by patents. An obvious concern is, therefore, that access to GM crops and trees will be controlled almost entirely by private corporations. Agreements for the use of techniques or material could be prohibitively expensive, preventing the use of the technology where it would be of value.
In the past, governments were the main investors in breeding and molecular biology research. Their role may have to expand once again in this regard, in order to provide a flow of material and information that can be developed and shared by both private and public institutions (Santos and Lewontin, 1997), as opposed to being controlled largely by private companies. Nevertheless, financial constraints, rather than available technical knowledge, are probably the largest challenge ahead for applying modern biotechnology in less developed countries, although it has been shown, e.g. by the Agricultural Biotechnology Support Project (www.iia.msu.edu/absp/), that arrangements through donations are possible.
Even if financial considerations can be addressed in the research phases of biotechnology, moving the technology from the development stage to operational reality provides a new set of technical challenges with additional expenses (Polonenko, 1999). It is likely that only a few economically important species will warrant such additional investments, and it is on these economic situations and realities that the debate should focus.
As has been alluded to previously, in terms of biotechnology research, one of the most important economic considerations separating forestry from agriculture is that trees require substantially more time for proper evaluation under both laboratory and field conditions. Rapid generation turnover in crop species has allowed genetic modification technology to develop quickly. Although this has been desirable from an economic point of view, the ecological ramifications have been far more difficult to study, as complex experiments must be carried out over several years. Public and government acceptance of GM plants is now probably as dependent on biological risk assessment and risk issue management (Leiss, 1999) as it is on technical developments or economics alone.
For GM trees, there is clearly a need to provide scientifically sound, neutral and intelligible information to the public, so that when and if GM trees become commercially available, informed decisions can be made about their release into the environment. Foresters are generally very aware of the need for public involvement in many other areas of forest management (e.g. chemical applications in forests, game management, felling patterns and plans, allowable annual harvesting levels), so decisions concerning GM trees may simply be an additional issue to be managed in the process.
Research on forest trees is carried out in this tissue culture laboratory at the National Herbarium and Plant Laboratory, Thapathali, Nepal
- G. Allard
This article has sought to address the main issues that should remain relevant to the use of any modern biotechnology in forestry, now and in the future. The following are some summary points and conclusions:
From a genetic perspective, concerns that biotechnology is "unnatural" ignore the dynamic changes in the genetic code that occur within and across species genomes through modification of transposable genes or elements by virus vectors and through mutation. This article has attempted to point out that humans have already had a huge effect on the genetic structure of many plants and animals, using many different technologies which will continue to develop and change over time. However, while those in the forefront of any technology will promote its potential benefits, in the end it will be the economic and regulatory systems of governing bodies at the national and global levels that must evaluate the technology's relevance and appropriateness.
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1 Other terms in use for plants or animals that have been genetically modified through recombinant DNA technology include living modified organisms (LMOs), plants with novel traits (PNTs) and trees with novel traits (TNTs).