GENETIC ENGINEERING OF TREE SPECIES: THE CANADIAN EXPERIENCE

Pierre J. Charest

Petawawa National Forestry Institute, Canadian Forest Service, Natural Resources Canada, Chalk River, Ontario, Canada K0J 1J0, (613)589-3090, fax (613)589-2275, pcharest@pnfi.forestry.ca

SUMMARY

The production of transgenic trees is relatively recent and only a few species have been successfully genetically engineered with useful silvicultural traits. Because of the novelty of the application of this technology with trees, very limited information is available on the potential environmental impact of the release of transgenic trees. Several genes involved in important agricultural traits were isolated and used to engineer crop plants. Some of these genes can potentially be used for silvicultural applications and genes involved in traits such as herbicide resistance, insect resistance, and wood quality have been used to genetically engineer trees. Some potential risks to the environment are possible with the commercial release of transgenic trees that are associated with their long life cycle and the presence of wild populations. These risks can be to organisms associated with the transgenic trees or on the species itself. Strategies to minimize risks are available and should be taken into account for environmental impact assessment. In Canada, the commercial release of transgenic trees is covered by the Seeds Act of Agriculture and Agri-Food Canada with potential involvement of other regulatory bodies. The Canadian Forest Service supports the implementation of this regulatory framework by providing technical advice for the issuance of permits and by conducting research on environmental impact and on the basic tree biology (including molecular biology).

Key words: Trees, conifers, silvicultural traits, risks, regulation

INTRODUCTION

Worldwide, genetic engineering of trees is still a new discipline and very little has been done on environmental impact assessment prior to the release of transgenic trees. Most plant studies on environmental releases have targeted crops (Burke et al., 1994; OECD, 1993; Kjellsson and Simonsen, 1994), but this information cannot be directly transposed to trees. This observation is especially true for gymnosperm trees (that include conifers) because all crop plants studied are angiosperms and fundamental biological differences exist between these two classes of plants related to their life cycle.

In Canada, the forest resource is extremely important for the country's economy and significant investment is being made in the new biotechnologies to increase overall managed forest productivity. Emphasis up until now has been on the development of routine techniques for the introduction of genes into tree species but research efforts are moving into the engineering of silvicultural traits such as those involved in wood quality and pest tolerance.

Because of the novelty of transgenic trees in Canada (there were no field tests as of September 1995), the regulatory framework for environmental release is still evolving. At this time, transgenic trees are covered primarily by the Seeds Act under the responsibility of Agriculture and Agri-Food Canada but other acts such as the Canadian Environmental Protection Act (CEPA) are also relevant.

DISCUSSION

The Biology of Tree Species. Tree species have very different biology than most crop plants. This difference is greater with gymnosperm trees than with angiosperm trees because crop plants are also angiosperms. The differences with crop plants common to both classes of trees are associated to their long life cycle and to the fact that they are rarely grown as monocultures. This implies that their interaction with the environment is more pronounced. Furthermore, most commercial trees grown in Canada are also present in natural ecosystems, meaning that gene flow can occur between artificial and natural populations of trees. Additionally, several species can cross-hybridize within the same genus, complicating the potential patterns of gene flow from transgenic trees.

With gymnosperms, differences are related to their position in the evolutionary scale. These plants are considered ancestors of crop plants. Consequently, gene structure and function could be different in this class of plants but studies on gymnosperm genes published until now indicate that most genes are conserved with angiosperms genes showing a high degree of protein and DNA sequence homology. Another difference is that conifers have a complex reproductive cycle (up to 2 years for pines), which has implications related to monitoring and containment of transgenic trees.

Large scale propagation of trees can also be different from crops plants and it is mainly in the time required to obtain enough seed for large scale propagation. This can, however be bypassed by using cuttings (as with poplars) and by using rooted cuttings or tissue culture derived propagules (as with conifer species).

Application of Genetic Engineering to Trees. Tree genetic engineering is dependent upon the availability of tissue culture protocols allowing whole plant regeneration from isolated pieces of tissues and on the availability of DNA transfer methods permitting the modification of the host genome. For the tissue culture protocols, two routes are followed: organogenesis and somatic embryogenesis. Organogenesis is the method of choice for species such as poplar and aspen and consists of plant regeneration through organ formation on an excised piece of plant tissue cultured on the proper tissue culture medium. Somatic embryogenesis is preferred fro conifer species and it is a process mimicking zygotic embryogenesis. Structures similar to zygotic embryos, called somatic embryos, are formed on explants cultured on adequate tissue culture media. These methods allow the rapid multiplication in vitro of any particular genotype that can be submitted to genetic transformation procedures.

For genetic transformation, two methods are used mostly depending on the species to be transformed (Charest and Michel, 1991). The first method makes use of the property of the pathogenic bacterium Agrobacterium tumefaciens to transfer part of its DNA contained on a tumor inducing (Ti) plasmid to infected plant cells. This method is referred to Agrobacterium-mediated transformation and is used with angiosperm trees such as poplars and aspens for production of transgenic trees (Confalonieri et al., 1994; De Block, 1990; Devillard, 1992; Feuillet et al., 1995; Fillatti et al., 1987; Howe et al., 1994; Klopfenstein et al., 1991; Miranda Brasileiro, 1991; 1992; Tsai et al., 1994). The second method is based on brute force to propel microprojectiles coated with DNA into a plant. It is referred to as microprojectile-mediated DNA delivery and it is the only method that has up to now yielded reproducibly transgenic conifers (such as white spruce and black spruce) (Ellis et al., 1993; Charest et al., 1995).

For tree species, the combination of the proper tissue culture protocol with a suitable genetic transformation is essential for recovery of transgenic trees and introduction of genes involved in important silvicultural traits. A list of forest tree species that have been genetically engineered is given in Table 1. As a general trend, gymnosperms are more difficult to engineer than angiosperm trees.

With most of these species, only marker genes to confirm transformation were introduced into the host genome; however, traits such as insect resistance (McCown et al., 1991; Ellis et al., 1993), herbicide resistance (Fillatti et al., 1987; Miranda Brasileiro et al., 1992; Devillard, 1992), bioremediation potential (Stomp et al., 1993), and modification of wood quality (Dwivedi et al., 1994; Feuillet et al., 1995) have been targeted. The genes for these traits were isolated from crop plants and other possibilities are being explored. A brief description and background on potential agricultural traits that are also of silvicultural importance is given below. Several groups now are working on the isolation and characterization of genes from trees that are involved in important silvicultural traits (for a review see Charest and Duchesne, 1995).

Herbicide Resistance. This was achieved by introduction of mutant target enzymes from plants or by introduction of detoxification enzymes from bacteria. Resistances to well known herbicides such as glyphosate (Round up) and chlorsulfuron (Glean) have been achieved (Schulz et al., 1990). Both type of resistance were introduced into transgenic poplar (Fillatti et al., 1987; Miranda Brasileiro et al., 1992; Devillard, 1992).

Insect Pest Tolerance. This has been achieved through the introduction of modified Bacillus thuringiensis-endotoxin genes or of modified proteinase inhibitor genes. Genes effective against lepidopterans, coleopterans and dipterans have been identified. The list is constantly growing and genes against nematodes are also available (Braun et al., 1991; Raman and Altman, 1994). A B. thuringiensis -endotoxin gene encoding resistance to lepidopterans was introduced into poplar and white spruce (McCown et al., 1991; Ellis et al., 1993).

Bacterial Disease Resistance. This was accomplished by using genes from insects that are part of their immune system such as genes encoding for cecropins and apidaecins and introducing them back into plants (Casteels et al., 1989). An alternative strategy is to transfer genes encoding enzymes detoxifying bacterial pathogen toxins (Anzai et al., 1989).

Fungal Disease Resistance. This was done by transferring genes coding for chitinases from bacterium or other plants. Such enzymes have the capacity to degrade chitin which is part of fungal cell wall (Cornelissen and Melchers, 1993).

Viral Disease Resistance. This has been achieved by genetic transformation in plants through three different mechanisms: i. by the introduction of viral genes coding for the virus coat protein mimicking cross protection, ii. by the transfer of an antisense gene to the coat protein of the virus blocking as essential step in virus replication, and iii. by introducing a gene coding for virus satellite RNA that suppresses the symptoms of infection (Fitchen and Beachy, 1993).

Heavy Metal Resistance. This was done by introducing animal genes coding for metallothioneins that have the ability to bind heavy metals such as cadmium and mercury. Alternatively, genes from plants called phytochelatins could be used. This type of engineering is very interesting in view of site remediation with tree species. Furthermore, genetic engineering of plants to enable them to degrade organic compounds for detoxification is now under investigation in species such as poplar (Stomp et al., 1993).

Modified Growth. This was achieved by the transfer of bacterial genes encoding growth regulators. In particular, genes from Agrobacterium were used to modify the ratio of auxin to cytokinin. By doing so, the form and the shape of a plant and its organs can be modified (Klee and Romano, 1994; Kurioka et al., 1992).

Flower Sterility. By introducing chimeric genes specifically turned on in flower tissues and expressing suicide genes such as RNAse genes, flower sterility was achieved. This has been done to engineer male flower sterility (Mariana et al., 1991; DeBlock and Debrouwer, 1993). Another possibility is the modification of expression of homeotic genes controlling flower identity which will render possible the engineering of female flower sterility. For a review on potential for genetic engineering of flower sterility in trees, the reader should consult Strauss et al. (1995).

Frost Tolerance. This was achieved with two different approaches; the introduction of an anti-freeze gene from fish (Georges et al., 1990) and the manipulation of fatty acid metabolism (Murata et al., 1992).

Drought Tolerance. This work has been initiated by looking at plants that have the capacity to survive severe droughts, such as the resurrection plant (Craterostigma plantagineum, Bartels et al., 1992). No success has been obtained yet.

Wood Quality Modification. This is a new avenue that is just being explored. Several genes involved in the lignin biosynthesis pathways of angiosperm and gymnosperm plants have been characterized. Some preliminary genetic engineering work has been done in model plants such as tobacco and in other plants such as poplar (Dwivedi et al., 1994; Kajita et al., 1994; Feuillet et al., 1995).

In Canada, emphasis of tree genetic engineering is on insect pest tolerance, fungal disease tolerance, flower sterility and wood quality modification. Flower sterility is of particular interest because it can be used as a containment measure for gene flow from transgenic trees. Applications of genetic engineering in Canadian forestry will undoubtedly occur first in intensively managed plantations (high-value forestry), in seed orchards and for horticultural species because of the investment required for the production of this elite material.

Potential Risks Associated with the Environmental Release of Transgenic Trees. To insure the benign commercial use of transgenic trees, strong considerations must be given a priori to potential effects of the release of transgenic trees into the environment. As pointed out earlier, information obtained with crop plants is not necessarily applicable to tree species and the two major factors that have to be taken into account are the long life cycle and the presence of wild populations of commercial forest trees. The potential risks that have to be evaluated are: i. development of weediness in transgenic trees (in particular, with poplar and aspen species); ii. gene transfer to wild populations (sometimes called genetic pollution); iii. emergence of new pests when insect resistant transgenic trees are deployed; iv. development of target pests resistant to engineered resistance mechanism in trees; v. deleterious effect on the ecosystem by disturbance of ecological chain; and, vi. genetic erosion of the wild population of trees due to uncontrolled gene flow from the engineered population.

The main difference with short-lived crop plants in evaluating the risks is the potentially more profound effect of long living transgenic trees on the environment (Figure 1). This has direct impact on increasing selective pressures on other organisms associated with transgenic trees. Although the risk evaluation is on the trait that has been genetically engineered, the deployment strategy will be a major factor on the potential environmental impact. Several strategies can be used to minimize or eliminate risks for large scale release of transgenic trees (Raffa, 1989): i. plantation of mosaics of engineered and non-engineered trees; ii. introduction of multiple resistance genes in the case of engineering for pest tolerance; iii. integration with other silvicultural practices; iv. temporal and tissue-specific expression of engineered characters; v. male, female or both flower sterility to contain gene flow to wild populations; and, vi. monitoring of transgenic tree plantations and surrounding forests.

Although almost no directly relevant information is available for tree species, studies on gene flow in natural populations and on basic biology and ecology will contribute significantly to environmental impact assessments of transgenic trees.

The Canadian Regulatory Framework for Trees. Five legislative acts in Canada regulate the release of transgenic trees to the environment; the Seeds Act, the Canadian Environmental Protection Act (CEPA), the Canadian Environmental Assessment Act (CEEA), the Plant Protection Act, and the Pest Control Product Act. The Seeds Act (Agriculture and Agri-Food Canada) is the principal regulatory act and the first point of entry to obtain a permit for field testing or commercial release. This act is overseen by the CEPA that covers all issues related to release of foreign substances in the environment. The Seeds Act complies with all the requirement of CEPA for environmental assessment. The CEEA is applicable only to activities (biotechnology or not) occurring on federal land or funded by the federal government. It insures that an environmental assessment is performed and that there are no potential risks of a given activity before a project is undertaken.

The Plant Protection Act and the Pest Control Product Act are applicable to transgenic trees presenting a potential danger to other plants (weediness) and to transgenic trees genetically engineered for pathogen resistance, respectively.

Future Prospects. For the Canadian Forest Service (Natural Resource Canada) priorities are on the development of a proper regulatory framework that will allow the safe environmental release of transgenic trees including research with model transgenic tree species and microcosms for the study of environmental impacts. Other research comprises the demonstration of the applicability of genetic engineering to trees and to the establishment of a solid knowledge base on gene structure and function in trees, particularly with gymnosperms.

REFERENCES

Anzai, H., K. Yoneyama, I. Yamaguchi. 1989. Transgenic tobacco resistant to a bacterial disease by the detoxification of a pathogenic toxin. Mol. Gen. Genet. 219:492-494.

Bartels, D., C. Hanke, K. Schneider, D. Michel, F. Salamini. 1992. A desiccation-related Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA. Embo. J. 11:2771-2778.

Braun, C.F., J.M. Jilka, C.L. Hemenway, N.E. Turner. 1991. Interactions between plants, pathogens and insects: possibilities for engineering resistance. Curr. Opinion Biotech. 2:193-198.

Burke, T., R. Seidler, H. Smith. 1994. Ecological implications of transgenic plant release. Mol. Ecol. 3:1-89.

Casteels, P., C. Ampe, F. Jacobs, M. Vaeck, P. Tempst. 1989. Apidaecins: antibacterial peptides from honeybees. Embo. J. 8:2387-2391.

Charest, P.J., Y. Devantier, D. Lachance. 1995. Stable genetic transformation of Picea mariana (black spruce) via microprojectile bombardment. In Vitro Cell Dev. Biol. (Plant) Submitted.

Charest, P.J., L.C. Duchesne. 1995. Recent progress in forest biotechnology in Canada. Petawawa National Forestry Institute, Inf. Rep. PI-X-120. 144 pp.

Charest, P.J., M.F. Michel. 1991. Basics of plant genetic engineering and its potential applications to tree species. Petawawa National Forestry Institute, Inf. Rep. PI-X-104. 48 pp.

Chupeau, M.C., V. Pautot, Y. Chupeau. 1994. Recovery of transgenic trees after electroporation of poplar protoplasts. Transgenic Res. 3:13-19.

Confalonieri, M., A. Balestrazzi, S. Bisoffi. 1994. Genetic transformation of Populus nigra by Agrobacterium tumefaciens. Plant Cell Rep. 13:256-261.

Cornelissen, B.J.C., L.S. Melchers. 1993. Strategies for control of fungal diseases with transgenic plants. Plant Physiol. 101:709-712.

De Block, M. 1990. Factors influencing the tissue culture and the Agrobacterium tumefaciens-mediated transformation of hybrid aspen and poplar clones. Plant Physiol. 93:1110-1116.

De Block, M., D. Debrouwer. 1993. Engineered fertility control in transgenic Brassica napus L.: histochemical analysis of anther development. Planta 189:218-225.

Devillard, C. 1992. Transformation in vitro du tremble (Populus tremula x P. alba) par Agrobacterium rhizogenes et regeneration de plantes tolerantes au basta. CR Acad. Sci. Paris 314:(III)291-298.

Dwivedi, U.N., W.H. Campbell, J. Yu, R.S.S. Datla, R.C. Bugos, V.L. Chiang, G.K. Podila. 1994. Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus. Plant Mol. Biol. 26:61-71.

Ellis, D.D., D. McCabe, S. McInnis, R. Ramachandran, D.R. Russell, K.M. Wallace, B.J. Martinell, D.R. Roberts, K.F. Raffa, B.H. Cown. 1993. Stable transformation of Picea glauca by particle acceleration. BioTechnol. 11:84-89.

Feuillet, C., V. Lauvergeat, C. Deswarte, G. Pilate, A. Boudet, J. Grima-Tettenati. 1995. Tissue- and cell-specific expression of a cinnamyl alcohol dehydrogenase promoter in transgenic poplar plants. Plant Mol. Biol. 27:651-667.

Fillatti, J.A., J. Sellmer, B. McCown, B. Haissig, L. Comai. 1987. Agrobacterium mediated transformation and regeneration of Populus. Mol. Gen. Genet. 206:192-199.

Fitchen, J.H., R.N. Beachy. 1993. Genetically engineered protection against viruses in transgenic plants. Ann. Rev. Microbiol. 47:739-763.

Georges, F., M. Saleem, A.J. Cutler. 1990. Design and cloning of a synthetic gene for the flounder antifreeze protein and its expression in plant cells. Gene 91:159-165.

Howes, G.T., B. Goldfarb, S.H. Strauss. 1994. Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants. Plant Cell Tiss. Org. Cult. 36:59-71.

Kajita, S., K. Osakabe, Y. Katayama, S. Kawai, Y. Matsumoto, K. Hata, N. Morohoshi. 1994. Agrobacterium-mediated transformation of poplar using a disarmed binary vector and the overexpression of a specific member of a family of poplar peroxidase genes in transgenic poplar cells. Plant Sci. 103:231-239.

Kjellsson, G., V. Simonsen. 1994. Methods for risk assessment of transgenic plants. I. Competition, establishment and ecosystem effects. Birkhauser Verlag, Boston. 214 pp.

Klee, H.J., C.P. Romano. 1994. The roles of phytohormones in development as studied in transgenic plants. CRC Rev. Plant Sci. 13:311-324.

Klopfenstein, N.B., N.Q. Shi, H.S. McNabb, R.B. Hall, E.R. Hart, R.W. Thornburg. 1991. Transgenic Populus hybrid expresses a wound-inducible potato proteinase inhibitor II-CAT gene fusion. Can. J. For. Res. 21:1321-1328.

Kurioka, Y., Y. Suzuki, H. Kamada, H. Harada. 1992. Promotion of flowering and morphological alterations in Atropa belladona transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid. Plant Cell Rep. 12:1-6.

Leple, J.C., A.C. Miranda Brasileiro, M.F. Michel, F. Delmotte, L. Jouanin. 1992. Transgenic poplars: expression of chimeric genes using four different constructs. Plant Cell Rep. 11:137-141.

Mariana, C., M. De Beuckeleer, J. Truettner, J. Leemans, R.B. Goldberg. 1991. Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347:737-741.

McCown, B.H., D.E. McCabe, D.R. Russell, D.J. Robinson, K.A. Barton, K.F. Raffa. 1991. Stable transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration. Plant Cell Rep. 9:590-594.

McGranahan, G.H., C.A. Leslie, S.L. Uratsu, L.A. Martin, A.M. Dandekar. 1988. Agrobacterium-mediated transformation of walnut somatic embryos and regeneration of transgenic plants. BioTechnol. 6:800-804.

Miranda Brasileiro, A.C., J.C. Leple, J. Muzzin, D. Ounnoughi, M.F. Michel, L. Jouanin. 1991. An alternative approach for gene transfer in trees using wild-type Agrobacterium strains. Plant Mol. Biol. 17:441-452.

Miranda Brasileiro, A.C., C. Tourneur, J.C. Leple, V. Combes, L. Jouanin. 1992. Expression of the mutant Arabidopsis thaliana acetolactate synthase gene confers chlorsulfuron resistance to transgenic poplar plants. Transgenic Res. 1:133-141.

Murata, N., O. Ishizaki-Nishizawa, S. Higashi, H. Hayashi, Y. Tasaka, I. Nishida. 1992. Genetically engineered alteration in the chilling sensitivity of plants. Science 356:710-713.

OECD. 1993. Safety considerations for biotechnology: scale-up of crop plants. Organization for economic co-operation and development, Paris, France. 40pp.

Raffa, K.F. 1989. Genetic engineering of trees to enhance resistance to insects. BioSci. 39:524-534.

Raman, K.V., D.W. Altman. 1994. Biotechnology initiative to achieve plant pest and disease resistance. Crop Protection 13:591-596.

Schulz, A., F. Wengenmayer, H.M. Goodman. 1990. Genetic engineering of herbicide resistance in higher plants. CRC Rev. Plant Sci. 9:1-5.

Stomp, A.M., K.H. Han, S. Wilbert, M.P. Gordon. 1993. Genetic improvement of tree species for remediation of hazardous wastes. In Vitro Cell Dev. Biol. 29P:227-232.

Strauss, S.H., W.H. Rottmann, A.M. Brunner, L.A. Sheppard. 1995. Genetic engineering of reproductive sterility in forest trees. Mol. Breeding 1:5-26.

Tsai, C.H., G.K. Podila, V.L. Chiang. 1994. Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants. Plant Cell Rep. 14:94-97.

Wilde, H.D., R.B. Meagher, S.A. Merkle. 1992. Expression of foreign genes in transgenic yellow-poplar plants. Plant Physiol. 98:114-120.

Figure 1. Interactions between transgenic trees and the environment