With assistance and inputs from Jean Marc Bouvet, Jean Marc Gion, Yves Prin, Daniel Verhaegen and Philippe Vigneron and Antoine Galiana (for the genetic modification section)
“Biotechnology provides important tools for the sustainable development of agriculture, fisheries and forestry and can be of significant help in meeting the food needs of a growing and increasingly urbanized population,” reads an FAO press release dated 15 March 200012. The field of modern biotechnology is indeed often considered as one of the fields of scientific research in which the most rapid advances have been made in recent years.
Several elements can explain the growing interest of forest scientists, conservationists and tree growers in modern biotechnologies. They include the unique roles and functions that trees, major structural constituents of forest ecosystems have, their special biological characteristics, and their importance in the provision of environmental, social and economic goods and services. Special features of interest to scientists and geneticists include the low level of domestication of forest trees and their rich genetic diversity; their long life cycles, long generation times and late sexual maturity; their spatial requirements; the multiplicity of species and the low degree of heritability of traits of interest, linked to weak juvenile–adult correlations and the importance of genotype–environment interactions. Application of biotechnologies in forests has been seen as a unique opportunity for obtaining new information on the extent, patterns and functioning of tree genetic diversity; and for providing new tree varieties and reproductive materials adapted to changing environmental, social and economic environments (Fenning and Gershenzon 2002).
Specific developments in biotechnology in the forestry sector have been addressed in a large number of conferences, meetings, publications, electronic fora and Internet web pages13. Owing to this abundant literature, this report, commissioned from CIRAD-Forêt by FAO in December 2003, will not describe the types and classifications of forest biotechnology in detail. It aims instead to fill a gap in global data and statistics on research in and applications of biotechnology for forest trees. Given the scientific and technical potential created by an increasingly accurate knowledge of forest tree species genomic structure, it is important to have an overall picture of the current status of forest biotechnology developments, together with trends and future prospects. The objective of this document is thus to review and summarize research, and the suitability and practical use of biotechnology in the forestry sector, and to provide tentative global analyses.
For the purpose of the study, a simple data set has been developed. The data set gathers major biotechnology activity (i.e. a given technology developed or used in a given country, on a given tree species or variety, by a given laboratory team, for a given purpose [Appendix 2.7.1]).
Data originated from (i) systematic searches in CAB Abstracts and associated global scientific databases, (ii) searches on the Internet (including sites of private companies, governmental and non-governmental institutions and linked references, and (iii) personal enquiries, observations and communications. Most significant publications, including those produced by major laboratories and teams, have been included in the data set. The study was mainly conducted between February and September 2004.
The data set included basic fields such as country, type of biotechnology, information source, reference or Internet site, stage of development, species or genera involved and, whenever available, year when the activity was conducted. These fields were considered to be a minimum set of requirements for entering a biotechnology activity in the database. The reference period covers approximately the last 10 years, although more than 75 percent of the data were from between 2000 and 2004. Internet references, however, could not always be dated.
The data set is in no way comprehensive, and some of its limitations reflect the difficulties of such information gathering. The study revealed language limitations (international databases cover only a fraction of the literature in Chinese and Russian, for example). International databases also tend to reflect past research activities, and only a small share of on-going research work. An additional flaw of the data set is related to private (corporate) research and commercial applications, for which public-domain information is generally scarce. Despite its drawbacks, the data set provides a sample (of unknown global representativeness) of materials available in the public domain. No attempt has been made to gather classified information. In total, data on 2 716 activities were collected, and their analysis supports the present report. Data, statistics and conclusions presented in this report should therefore be considered with caution and as general indicators.
A quick review of the literature shows that various classification systems have been used to categorize modern biotechnologies used in forestry14. In some countries, the term biotechnology refers to genetic modification exclusively. Yanchuk (2001) proposed three main categories: (i) biotechnology employing molecular markers, (ii) biotechnology aimed at enhancing plant propagation and large-scale production of uniform plant material, and (iii) biotechnology for modifying the genome of forest tree species. In the present report, the broad categories include:
1. The characterization of genetic diversity of forestry species, including diversity structure studies, gene flow and human impacts on forest stands: characterization, population genetics and diversity studies.
2. The functional and applied aspects of genetic investigation: genetic mapping, marker-assisted selection (MAS) and genomics (collectively referred to as MMG).
3. Research and applications in vegetative propagation: micropropagation (in the broad sense).
4. The alteration of genomes by insertion of genes: genetic modification.
Individual data (biotechnology entries) were categorized according to the above classification once they had been entered into the data set.
The report presents detailed analyses of the data sets for the categories 1–4 above, and provides separate summaries for biotechnologies excluding genetic modification, and genetic modification technologies.
The information compiled in the data set of biotechnology activities excluding genetic modification represents 2 196 references (or 81 percent of all activities reported). Activities were reported in 76 countries, broken down by regions as follows: 39 percent of the activities were reported in Europe, 24 percent in Asia, 23 percent in North America, 6 percent in Oceania, 5 percent in South America, 3 percent in Africa and less than one percent in the Near East (Figure 2.1.1A). Both developed countries (24 countries, representing 68 percent of biotechnology activities) and developing countries and countries in transition (52 countries, or 32 percent of activities) were represented (Figure 2.1.1B). Developing countries and countries in transition were mainly represented by India (27 percent of these countries’ activities), China (17 percent), Brazil (7 percent), South Africa (5 percent) and Malaysia
(4 percent). Three countries (India, China and Brazil) accounted for 52 percent of all biotechnology work reported in developing countries and countries in transition.
Species surveyed belonged to 142 botanical genera. Sixty-two percent of the information collected in the database regarded research carried out on less than six genera including Pinus (20 percent of biotechnology activities excluding genetic modification), Eucalyptus
(11 percent), Picea (9 percent), Populus (9 percent), Quercus (7 percent) and Acacia
(6 percent) (Figure 2.1.2). Just four genera (Pinus, Eucalyptus, Picea and Populus) account for almost half of the compiled biotechnology activities excluding genetic modification.
Work was found to be relatively evenly spread between the three main categories of biotechnology categories apart from genetic modification: characterization of tree species genetic diversity represented 32 percent of biotechnology activities, MMG 26 percent, and micropropagation 42 percent (Figure 2.1.3A). Differences were more marked when tree genera were considered (Figure 2.1.3B). The forestry sector appears to have rapidly adopted markers developed for agricultural crops (Figure 2.1.4). Isozymes and random amplified polymorphic DNAs (RAPDs) have been widely used for genetic diversity description although the present trend seems to favour microsatellites (nuclear and chloroplast) and amplified fragment length polymorphisms (AFLPs). Driven by research on genomics, expressed genome banks (ESTs [expressed sequence tags]) are being widely developed.
The majority of the work reported is still mainly at the experimental stage in the laboratory. Genetic diversity characterization has less than one percent of its reported activities in the field, MMG 2.5 percent and micropropagation 5 percent (Figure 2.1.5). Field tests are still mainly geared to supporting laboratory research. These results possibly reflect the origin of the information in the data set. While research activities in the public sector are relatively easy to collect, especially through international research storage databases, information on commercial applications is generally restricted and incomplete.
Commercial applications of micropropagation are, however, generating increasing interest. The potential is huge although, up to now, only several thousand hectares seem to have been established globally using micropropagated materials.
In South America, particularly in Brazil, some companies are reported to be integrating micropropagation into the clonal propagation process: micropropagation is used to ‘store’ clones in mother blocks (gene banks) in the laboratory as potential sources of responsive nursery stock plants for large-scale mass propagation. The use of rooted cuttings has allowed the propagation costs to be lowered significantly.
Great expectations have been raised about the possible contribution of biotechnology to tree selection and breeding, and its commercial applications. Genomics and proteomics should greatly help breeders in tree selection, in particular in the identification of traits of interests. However, it remains difficult to predict when new forest tree varieties selected with biotechnology tools will become available on the market. Although genetic diversity characterization started some 30 years ago, very limited large-scale commercial application has yet been reported in forest tree genetic resources conservation and management.
Figure 2.1.1A. Distribution of reported forest biotechnology activities (excluding genetic modification) by world region
Figure 2.1.1B. Distribution of reported forest biotechnology activities (percent of activities in the data set, excluding genetic modification) by country (for the 15 countries most represented, making up 77 percent of the data set of entries excluding genetic modification)
Figure 2.1.2. Distribution of reported forestry biotechnology activities, excluding genetic modification, by genus
Figure 2.1.3A. Distribution of biotechnology activities, excluding genetic modification, by broad category (genetic diversity characterization; mapping, marker-assisted selection and genomics [MMG]; and micropropagation)
Figure 2.1.3B. Distribution of biotechnology activities, excluding genetic modification, (genetic diversity characterization; mapping, marker-assisted selection and genomics [MMG]; and micropropagation) by number of tree genera
Figure 2.1.4. Distribution of molecular markers used in forest biotechnology activities, excluding genetic modification
Figure 2.1.5. Distribution of reported forest biotechnology activities, excluding genetic modification, by category and applications (laboratory studies, field trials and commercial deployments)1
1MMG: mapping, marker-assisted selection and genomics.
Genetic diversity is sometimes considered to be the invisible dimension of biological diversity. The present structure of genetic diversity is the result of the evolutionary history of the species exposed to natural selection pressures in variable environmental conditions. Natural selection at the local level is an evolutionary force opposed to gene flow. The combination of the two forces creates a powerful mechanism for maintaining within-species diversity. The use of molecular descriptors (markers) of the genome has allowed the measurement of genetic variation between genotypes and within/between populations, as well as the effectiveness of seed and pollen dispersal.
Studies carried out on forest tree genetic structure and on reproductive regimes use RAPD and AFLP markers, as well as nuclear markers and microsatellites, to increase understanding of the relationship between genome variations and genetic diversity. Molecular markers, especially neutral markers, provide an important tool for studying the structure of populations. However, experience shows that the resulting information on diversity, for example in ecology or taxonomic studies, is not always a good indicator of the patterns of variation in traits subject to selection pressures. A complementary approach is often required, consisting of studying the genetic diversity in specific regions of the genome. For example, SNPs (single nucleotide polymorphisms) are used to study the impact of evolutionary forces on the variability of allelic forms of a given gene, and their phenotypic expression.
Work reported on tropical trees often involves studies of the impacts of tree harvesting and fragmentation on species’ reproductive patterns and changes in genetic diversity at the population level (Chase et al. 1996; Dawson et al. 1997; Dick 2001; Dutech et al. 2002; White et al. 2002). In temperate species, several studies focused on gene flow in natural populations (Quercus, Cedrus) and the evolution of genetic diversity under forest management (Streiff 1998; Fady et al. 2003). Genetic diversity descriptions are numerous and diversified in terms of species and methodologies, and are mainly aimed at verifying some theoretical aspects, or at providing elements for the elaboration of conservation strategies (Petit et al. 1998; Newton et al 1999). Applications of results at operational level have been infrequently reported as yet, except in the case of rosewood (Aniba rosaeodora) and sandalwood (Santalum) (J. M. Bouvet, personal communication).
On-going studies on gene flow and, more specifically, on pollen flow, are based on the use of molecular markers, mainly microsatellites (Dow and Ashley 1998; Streiff 1998; Chaix 2003) and on paternity analysis (Gerber et al. 2000, 2003). Objectives include:
• estimating and studying the evolution of genetic variability over time;
• assessing effective population sizes;
• studying biological mechanisms of reproduction, either in natural populations or in improved populations (in selection programmes);
• studying pollen pollution in tree seed production areas (seed stands or seed orchards) or in the context of GM trees.
Data set analysis
Most of the activities compiled in the data set have the overall objective of conserving and managing forest genetic resources in a sustainable way, and providing a better understanding of genetic diversity and evolution through the study of gene flow. This includes analyses of the consequences of human practices on the evolution of forest tree genetic diversity, as well as development and validation of theoretical models of population genetics.
Work has been reported from all regions of the world (Figure 2.1.6A) including Europe
(44 percent), followed by North America (22 percent), Asia (19 percent), South America
(6 percent), Oceania (6 percent) Africa (3 percent) and the Near East (less than one percent). When one considers the origin of the species being studied, the situation shows broadly similar patterns (Figure 2.1.6B).
At least 99 genera are being worked on: Pinus (19 percent), Quercus (12 percent), Eucalyptus (8 percent), Acacia (7 percent) and Picea and Populus (6 percent) are the most frequently cited genera (Figure 2.1.7). Reported objectives of genetic diversity characterization studies (Figure 2.1.8) include: evaluation of genetic diversity (57 percent), gene flow studies
(15 percent), fingerprinting (12 percent), and conservation (6 percent).
Figure 2.1.6A. Distribution of forest genetic diversity characterization activities by region where the work is/was carried out
Figure 2.1.6B. Distribution of forest genetic diversity characterization activities by region of origin of the species studied; ‘unknown’ relates to undocumented sources, or to hybrid species, varieties and clones
Figure 2.1.7. Distribution of genetic diversity studies by forest tree genus
Figure 2.1.8. Main objectives of reported studies on forest tree genetic diversity
For a long time, forest tree seed has been collected from natural populations and, less frequently and only recently, from artificial stands (including planted stands or seed orchards). Since genetic information was lacking, most collections were based on phenotypic selection. At the beginning of the twentieth century, more attention started to be given to seed origin; numerous arboreta were established around the world and international seed exchange, documented to a greater or lesser extent, increased significantly.
Later, growing awareness of the importance of intraspecific variation led to a more systematic exploration of the natural distribution of species of proven or perceived interest. With the assistance of national and international organizations, comprehensive explorations were made in the 1960s and 1970s for several genera and species including Tectona grandis, Gmelina arborea, Eucalyptus, Pinus and Acacia. Seeds collected were used in provenance tests, which in turn provided basic information and new materials for seed production areas or tree seed orchards. Nowadays, biotechnology tools allow more in-depth exploration of within-species genetic diversity, through the study of genomic variability.
Use of biotechnology in tree breeding and selection
Marker-assisted selection (MAS) has given further impetus to tree breeding and selection. Molecular markers are genetically linked to a given allele on a given locus and can therefore be used to predict the presence of the allele with great accuracy. The first markers developed (RAPDs, RFLPs [restriction fragment length polymorphisms] AFLPs and microsatellites) were neutral in the sense that their function, if they had any, was unknown.
They have provided a way of estimating genetic diversity and allowed comparison between individuals in different growth and development conditions. They have also been used in profiling to tag and then single out individual trees.
Co-segregation of RAPD and AFLP type markers has allowed the construction of genetic maps. Genetic maps can be used to assess the degree of evolutionary relatedness of a number of species and identify the zones where gene variations are statistically linked to the variability of quantitative traits. These zones are called QTLs (quantitative trait loci). QTL research focuses on the genetic architecture of traits of interest. However, QTL research results are not easily transferred to other tree populations: the QTLs identified so far are closely linked to the populations sampled. The use of QTLs in tree selection is thus increasingly questioned.
For the above reasons, the use of candidate genes became necessary for applying MAS to forest trees. The candidate gene approach was made possible by the development of gene search methods (AFLP, EST banks and SSR, cDNA), transcriptomics and proteomics. In order to use those genes in selection, their molecular variability must be linked to the variation of the targeted trait of interest. This requires associating appropriate field experiments with genetic mapping.
Proteomic analysis offers an important field of basic research that many research teams are willing to develop. Proteomic studies can find applications in such fields as biotic and abiotic stress response, effects of genetic mutations, gene expression regulation, QTL validation and genetic variability (Plomion 2000; Pilate et al. 2002).
Information technology is an important asset since work on biotechnology requires the handling of massive catalogues of genes, transcripts and proteins. The use of bioinformatics, in particular, to transfer results from one given (model) species to other species, in variability studies, fingerprinting or tree breeding, is particularly valuable.
Mapping, MAS and genomics (MMG) studies are found in all regions of the world except the Near East (Figure 2.1.9). Europe and North America represent 43 percent and 34 percent, respectively, of the total MMG activities, followed by Asia (11 percent), Oceania (8 percent), South America (2 percent) and Africa (2 percent). The work documented in Africa (South Africa and Congo) and South America (Brazil) relates to Eucalyptus almost exclusively.
MMG activities are being currently applied to approximately 40 genera (Figure 2.1.10) among which Pinus (32 percent), Populus (18 percent), Eucalyptus (12 percent) and Picea
(9 percent) are the most studied.
Objectives of the research work (Figure 2.1.11) include wood properties improvement in relation to lignin composition (57 percent), abiotic resistance for cold or drought stress
(20 percent), genetic diversity (8 percent), growth rate (6 percent), flowering (5 percent) and phytosanitary aspects of biotic resistance (4 percent). The work is being pursued mainly on expressed gene products (53 percent), genetic map construction (27 percent), gene candidate research (12 percent) and QTLs (6 percent). More recent proteomics research constitutes
2 percent of the activities documented (Figure 2.1.12).
Figure 2.1.9A. Distribution of research related to mapping, marker-assisted selection and genomics (MMG) in forest tree species by region where the work is being carried out
Figure 2.1.9B. Distribution of research related to mapping, marker-assisted selection and genomics (MMG) in forest tree species by region of origin of the species studied
Figure 2.1.10. Distribution of mapping, marker-assisted selection and genomics (MMG) activities, by genus
Figure 2.1.11. Distribution of the main traits targeted in marker-assisted selection studies
Figure 2.1.12. Methodological approaches associated with mapping, marker-assisted selection and genomics (MMG) in forestry
The construction of genetic maps is a prerequisite for MAS. Genetic mapping is currently being developed for more than 25 forest species with the main objective of making maps denser by positioning a larger number of markers. Genera and species include:
• Eucalyptus (Grattapaglia & Sederoff 1994; Verhaegen & Plomion 1996; Byrne et al. 1995; Marques et al. 1998; Brondani & Grattapaglia 1999; Gion et al. 2000; Myburg et al. 2003), including E. camaldulensis, E. globulus, E. grandis, E. nitens, E. tereticornis and E. urophylla;
• Populus (Cervera et al. 2001; Yin et al. 2002) in particular P. deltoides, P. nigra and P. trichocarpa;
• Picea (Plomion 2000) including: P. abies, P. glauca and P. mariana;
• Pinus (Kubisiak et al. 1995; Plomion 2000) including P. brutia, P. edulis, P. elliottii, P. palustris P. pinaster, P. radiata, P. strobus, P. sylvestris and P. thunbergii;
• other species (Plomion 2000; Butcher et al. 2002; www.pierroton.inra.fr/genetics/Quercus): Acacia mangium, Cryptomeria japonica, Larix leptolepis [=L. kaempferi], Pseudotsuga menziesii, Quercus robur and Taxus brevifolia.
Several plant functional genomics programmes have undertaken linkage map construction, and significant developments in this area have been made with some forest trees. The search for linkages between coding regions and traits linked to wood quality has allowed some gene candidates to be identified (especially in lignin synthesis pathways) although they are not yet used in routine selection programmes. There are noteworthy EST bank development programmes underway for several species in several genera, including Pinus, Populus, Eucalyptus and Betula. The gene candidate approach has been used in a dozen species of Pinus (Plomion et al. 2000; Whetten et al. 2001; Brown et al. 2003; Garnier-Géré et al. 2003; Le Dantec et al. 2003; McMillan 2004), at least five Eucalyptus spp. including hybrids (Gion et al. 2000; Thamarus et al. 2002; De Melis et al. 1998; Plomion et al. 2003; Kirst et al. 2004; Ranik et al. 2004) and in three Picea spp. (McDougall 2000).
More recently, research work has been undertaken on other traits such as water stress tolerance (Dubos and Plomion 2003; Dubos et al. 2003). Proteomic studies have been initiated on wood properties for a limited number of species: Pinus pinaster, Eucalyptus gunnii, Populus spp. and Picea sitchensis.
In vitro vegetative propagation or micropropagation is aimed at cloning superior individuals or at ‘bulk’ (in mixture) propagating new genotypes with high genetic potential but available in limited quantities (such as materials obtained by controlled pollination).
Tree species can be micropropagated by microcuttings or by somatic embryogenesis.
Micropropagation by microcuttings consists of mass producing vegetative copies of desired genotypes by either axillary or adventitious budding. In the latter case, differentiated cells, usually from superficial tissues, must undergo a de-differentiating process before new shoot formation is initiated. Ultimately, production of independent and self-sufficient individuals is completely dependent on the de novo formation of an adventitious root system.
Somatic embryogenesis, or production of embryos from somatic cells, is in fact a cloning technique, as opposed to zygotic embryogenesis in which germinal cells give rise to seedlings that are all genetically different. The process of somatic embryogenesis derives usually from callus formation induced by applying cytokinic or auxinic exogenous growth regulators to very juvenile plant tissues. In the most favourable situations, some undifferentiated cells of these calli can evolve into somatic embryos characterized, similarly to zygotic embryos, by a shoot–root bipolar structure. This basically distinguishes somatic embryogenesis from microcuttings consisting first of a shoot from which an adventitous root must subsequently develop.
The majority of GM trees is likely to be used in the form of clonal materials and will need to be vegetatively propagated. In the case of conifers, somatic embryogenesis, especially when derived from a single cell, seems the most suitable regeneration and propagation technique. In broad-leaved species, vegetative propagation of GM materials is likely to use a combination of micropropagation and rooted stem cuttings, at least in the beginning.
Database analysis
Asia accounts for 38 percent of documented activities in forest tree micropropagation, followed by Europe (33 percent), North America (16 percent), South America (7 percent), Africa (3 percent), Oceania (2 percent) and the Near East (one percent) (Figure 2.1.13A). Sixty-four countries active in this field have been identified. A closer look shows that species from Asia (27 percent) and Europe (21 percent) predominate (Figure 2.1.13B), which may suggest that the activities in Asia are concentrated on indigenous species, while Eucalyptus spp. are micropropagated in all continents. Species originating from South America and Africa are almost absent from the data set. Micropropagation activities seem to take place mainly in countries with significant tree planting programmes.
Most micropropagation research activities are at a very advanced stage. Germplasm and protocols are likely to be available for large-scale deployment soon, if not already operational, for species and genera including:
• Anogeissus in India (Saxena and Dhawan 2001);
• Acacia mangium and A. mangium × A. auriculiformis in Malaysia (Galiana et al. 2003), Indonesia and Vietnam (O. Monteuuis, personal observation);
• Eucalyptus in Vietnam, India (O. Monteuuis, personal observation), Australia (Watt et al. 2003) and South America (Levis W. Handley, personal communication);
• Pinus in Canada and New Zealand (Lelu and Thompson 2000), and Pinus taeda in the USA (Levis W. Handley, personal communication);
• Tectona grandis in Brazil, Thailand and Indonesia (O. Monteuuis personal observation) and Malaysia (Goh et al. in press).
Micropropagation techniques have been tested on at least 82 forest tree genera (many of them of proven or potential interest in forest plantations). Research work has been devoted to Pinus (13 percent), Picea (13 percent), Eucalyptus (11 percent), Acacia (7 percent), Quercus
(6 percent), and Populus, Larix and Tectona grandis (4 percent each) (Figure 2.1.14). The first five genera account for 50 percent of all activities carried out in forest tree micropropagation. Somatic embryogenesis accounts for 65 percent of activities in micropropagation followed by cell/tissue culture (17 percent), micropropagation by microcuttings in in vitro conditions (13 percent), cryopreservation/conservation (4 percent) and embryo rescue (one percent) (Figure 2.1.15).
Figure 2.1.13A. Distribution of micropropagation activities by region of activity
Figure 2.1.13B. Distribution of micropropagation activities by region of origin of the species studied
Figure 2.1.14. Distribution of micropropagation activities by genus
Figure 2.1.15. Categories of biotechnologies used in forest tree micropropagation
Micropropagation by microcuttings is carried out on more than twenty species including:
• Populus alba, P. deltoides, P. tremula and Populus hybrids in Germany and India (Cornu 1994), Spain (Bueno et al. 2003) and Lithuania (Kuusiene 2002);
• Eucalyptus camaldulensis, E. globulus, E. grandis, E. nitens, E. tereticornis and E. urophylla in South Africa, Spain and Portugal (Watt et al. 2003), India (Watt et al. 2003; Nadgauda in press), Vietnam and Thailand (O. Monteuuis personal observation), and Australia (Bandyopadhyay et al. 1999);
• Acacia mangium, A. melanoxylon, A. mangium × A. auriculiformis in Malaysia and South Africa (Galiana et al. 2003; Monteuuis et al. 2003; Quoirin 2003);
• Tectona grandis in India (Bonga and Von Aderkas 1992; Nicodemus et al. 2001; Nadgauda in press), Vietnam, Brazil and Indonesia (O. Monteuuis personal observation), Thailand (Kjaer et al. 2000), Costa Rica (Schmincke 2000), Malaysia (Monteuuis & Goh 1999; Goh & Monteuuis 2001; Goh et al. in press) and Australia (Monteuuis, personal observation) .;
• Larix in Canada and France (Charest 1996; Lelu and Thompson 2000);
• Pinus radiata, P. taeda and P. pinaster in France (Dumas and Monteuuis 1991; Monteuuis and Dumas 1992), New Zealand15 and the United States (Rahman et al. 2003);
• Pseudotsuga menziesii in the United States (Ritchie et al. 1994);
• Sequoia sempervirens and Sequoiadendron giganteum in France (Bonga and Von Aderkas 1992; O. Monteuuis personal observation) and Germany (www.cnr.berkeley.edu/~jleblanc/www/Redwood/rdwd-Micropro.html);
• Anogeissus latifolia and A. pendula in India (Saxena and Dhawan 2001);
• Betula pendula in Norway (Saebo et al. 1995) and Finland (Cornu 1994);
• Paulownia fortunei in Australia (O. Monteuuis personal observation);
• Platanus acerifolia in China (Liu and Bao 2003);
• other species include Gmelina arborea, Artocarpus chaplasha, A. heterophyllus, Azadirachta indica and Elaeocarpus robustus in Bangladesh (Sarker et al. 1997; Roy et al. 1998).
The advantages of somatic embryogenesis in comparison with micropropagation by microcuttings, especially with regard to multiplication rate and genetic modification applications, explain the major research investments in the technique. However, there are still serious obstacles to large-scale operational application of somatic embryogenesis to forest trees, for example:
• Only some species and, within these species, only some genotypes can produce somatic embryos.
• Success has been obtained, with few exceptions, mainly with juvenile tissues coming for instance from immature zygotic embryos.
• There are risks that somaclonal variation may decrease the value of the genotypes produced by somatic embryogenesis, resulting in a considerable waste of time, material and money. True-to-typeness, particularly, may remain a problem for certain genotypes, and efforts are still needed for optimizing this technique to make it more reliable, especially when using mature selected genotypes.
Although activities on somatic embryogenesis have been reported in all regions of the world, it seems that countries with conifer planting programmes show a stronger interest in this micropropagation technique:
• Larix in Canada (and France) (Charest 1996; Lelu and Thompson 2000);
• Picea abies, P. glauca, P. mariana and P. sitchensis in Canada, France and Ireland (Thorpe 1995; Charest 1996; Park et al. 1998; Lelu and Thompson 2000; Park 2002; Sutton 2002; Lelu-Walter and Harvengt 2004);
• Pinus banksiana, P. patula, P. radiata, P. strobus and P. taeda in Canada (Park 2002), South Africa (Jones 2002), New Zealand (Lelu and Thompson 2000), the United States (Jones 2002; Sutton 2002) and Chile (Lelu-Walter and Harvengt 2004);
• Pseudotsuga menziesii in the United States and Canada (Lelu and Thompson 2000, Sutton 2002);
• Eucalyptus globulus (Pinto et al. 2002), E. grandis and E. dunnii (Watt et al. 1999);
• Gmelina arborea, Artocarpus chaplasha, A. heterophyllus, Azadirachta indica and Elaeocarpus robustus in Bangladesh (Sarker et al. 1997; Roy et al. 1998).
Investments in vegetatively micropropagated species of commercial interest have resulted in successful large-scale applications in several countries in Europe, North America, Asia and Oceania. Unfortunately, there is a lack of basic information on areas planted with forest vitroplants.
The first regeneration of a genetically modified (GM) forest tree was achieved in 1986 in Populus. Since then, the genus has become a model for genetic modification and related tree biotechnology studies. The first attempt to genetically modify a conifer (Larix) was reported in 1991 (Huang et al. 1991). Introducing targeted genes into the genome of a forest tree is a way to obtain GM plants. It is also a basic research tool for a better understanding of gene functioning in woody plants.
Review of objectives and methodologies
Transformation methods consist of inserting into the genome of the host plant a mutated gene or a gene from another organism either by microinjection/projection (direct transformation), or through a vector such as Agrobacterium tumefaciens (indirect transformation).
Most successful work on genetic modification of forest trees species genomes so far has been obtained by using juvenile material, often from explants produced from early germinations tissues which have much higher regeneration capacities than older material. Successful genome modification reports of adult selected plant material are very rare, except in poplars precisely because of its greater capacity to regenerate.
Traits subject to genetic modification are discussed below.
A first method has consisted in introducing a mutated version of the gene encoding the enzyme target for various herbicides: glyphosate for Populus alba × P. grandidentata, P. trichocarpa × P. deltoides, Eucalyptus grandis, Larix decidua and Pinus radiata (the herbicide blocks the synthesis of tryptophan, tyrosine and phenylalanine), or chlorosulfuron for Populus tremula and Pinus radiata (the herbicide blocks the synthesis of leucine, isoleucine and valine).
A second and more frequently used strategy consists of introducing a microbial gene encoding an enzyme for the detoxification of the herbicide, and has been applied to Populus alba, P. alba × P. tremula, P. tremula × P. alba, P. trichocarpa × P. deltoides and Eucalyptus camaldulensis (Ho et al. 1998; Moralejo et al. 1998) and Pinus radiata and Picea abies (Bishop-Hurley et al. 2001).
The first strategy consists of using microprojectiles to insert a gene encoding an endotoxin which binds to the receptors in the intestine of Lepidoptera, Coleoptera and Diptera, lysing the organ and killing the insect. This was done in Populus alba × P. grandidentata, P. tremula × P. tremuloides and Picea glauca. GM Populus trees and Pinus radiata expressing the Bacillus thuringiensis endotoxin ‘Bt’ have been obtained.
A second transformation method is based on the introduction of a gene coding for a protease inhibitor that modifies insect digestion, causing the death of the pest. Studies used potato gene pin2, a protease inhibitor introduced into P. alba × P. grandidentata through A. tumefaciens (Klopfenstein et al. 1991), and the gene of a rice protease inhibitor introduced into P. tremula × P. tremuloides (Heuchelin et al. 1997).
A third approach focuses on simultaneous modification of two genes for enhanced resistance to insects. This was achieved in Liquidambar styraciflua, combining a peroxidase anionic enzyme gene involved in cell growth and wall development with a ‘Bt’ gene (Sullivan and Lagrimini 1993).
In order to produce sterile trees and prevent possible dispersal of transgenic pollen in the environment, an approach based on genetic ablation has been tested on poplar (Skinner et al. 2000). This technique, which consists of expressing a cytotoxic gene under the control of a very specific poplar floral promoter, resulted in more than 90 percent of transformed lines lacking floral structures. Other approaches, based on the suppression of key flowering genes, are being tested.
Quantitative and qualitative modification of lignin
Modification of lignin composition or content is being actively pursued because of the expected financial gains from pulp processing improvements. Lignins, which enhance cell wall mechanical properties and hardness, are difficult to process and are a significant limitation in processing wood into paper pulp by chemical treatment. Genetic transformation to modify lignin characteristics is a key research feature on species used in the paper industry. The aim is to regulate the activity of key enzymes involved in the lignin biosynthesis pathway (Lapierre et al. 1999; Jouanin et al. 2000; Li et al. 2003). Active on-going research targets the effects of lignin biosynthesis in Populus on soil carbon transformation and storage16.
Other traits that have been considered for genetic modification include: resistance to diseases (Mayer 2004; www.genewatch.org/) in Populus, Betula pendula, Alnus and Juglans.; bioremediation in Populus; enhanced growth through higher efficiency of nitrogen assimilation in Picea and Populus (Gallardo et al. 1999), and modification of gibberellin synthesis in Populus.
Genetic modification activities represent 520 entries in the data set, or 19 percent of all biotechnology activities recorded worldwide. Research is reported in 35 countries and on
29 tree genera. North America is the lead region, accounting for 48 percent of genetic modification activities on forest tree species, followed by Europe (32 percent), Asia
(14 percent), Oceania (5 percent) and Africa and South America (less than one percent each) (Figure 2.1.16), while no activity is reported in the Near East. Genetic modification research at the country level is led by the United States (219 entries in the data set, or 42 percent of the world’s recorded activities on GM forest trees) (Figure 2.1.17). Studies carried out by, or in cooperation with, agencies or companies from developed countries have been identified in Brazil, Chile, China, Indonesia, South Africa and Uruguay.
Populus accounts for 47 percent (Figure 2.1.18) of data set entries, followed by Pinus
(19 percent), Eucalyptus (7 percent) and Liquidambar and Picea (5 percent each). The pedigree of a number of hybrid poplars used in China has not been ascertained. Several countries have already established GM tree field trials.
Interestingly, while the first GM forest trees were developed some fifteen years ago, about three-quarters of the total number of species recorded in the dataset have been genetically transformed during the past five years.
The main studies underpinning genetic modification (Figure 2.1.19) target gene expression (21 percent), in vitro regeneration tissue culture (18 percent), herbicide resistance
(13 percent), biotic resistance (12 percent) and lignin properties (9 percent).
Figure 2.1.16. Distribution of data set entries on genetic modification by region of the world
Figure 2.1.17. Distribution of reported forest tree genetic modification research activities by country (only those with more than five such activities in the data set are included)
Figure 2.1.18. Distribution of reported forest tree genetic modification research activities by genus
Figure 2.1.19. Main reported objectives of research activities in forest tree genetic modification
In vitro regeneration of transformed plants is still a technical limitation for many species and genotypes. Genetically transformed lines have been regenerated for Populus, Eucalyptus, Pinus (including P. taeda17) and Picea. Technical processes involved in regeneration are not always well controlled or even studied; especially in mature tissues where the situation is the most critical. No GM tree is reported to be commercialized in international databases to date (Mayer 2004; www.agbios.com).
Insertion of genes controlling traits of interest in a given forest species is limited by insufficient knowledge of the molecular control of these traits. Most of the traits important in wood production, such as growth rate, adaptability, and stem and wood quality, are under polygenic control, and therefore depend on more than a single gene. Biosafety regulations governing GM trees are often a concern due to the long period of time necessary to monitor environmental effects (including gene stability, pollen and seed dispersal, and impact on other elements of the ecosystem). Most existing regulations are both stringent and involve high development costs compared to annual crops. Collaborative genomics programmes such as those carried out on Populus, Pinus and Eucalyptus, with the on-going characterization of genes of interest (including those coding for flowering, and lignin and cellulose biosynthesis), and the identification of specific promoters of transgenic expression, are expected to boost advances in genetic modification of forest trees. Studies to assess the stability of GM tree growth under abiotic stress, and their environmental impact, have been initiated.
Unlike work carried out on crop or farm animal species, the domestication of forest trees is, with few exceptions, very recent. However, within a few decades, knowledge transfer from agriculture to forestry has been so fast that genetic knowledge of a few tree model genera, such as poplar (Populus), pine (Pinus) and Eucalyptus, is about to become as important, in terms of complexity and quality, as the genetic knowledge of the main crop species. Modern biotechnologies have potentially outstanding applications in the forestry sector, mainly because of the potential genetic gains they could confer. Expected gains for tree breeders include new genetic pools and significant reduction in tree selection time.
Owing to technical difficulties, high costs, and time needed to evaluate forest species, tree breeding programmes have long been limited to the mass selection of the best individuals. Between the 1970s and 1990s, development of horticultural techniques such as grafting, propagation by cuttings and controlled pollination, as well as information technology development, contributed to the implementation of sophisticated and efficient tree improvement activities. Long-term conventional tree breeding programmes and large-scale clonal propagation techniques have resulted in, and will continue to provide, significant productivity gains, especially for fast-growing species such as Eucalyptus and Populus.
While the objectives of tree breeding are clearly defined and address end-users’ (tree growers’) requirements (in terms of yield, wood quality, and biotic and abiotic resistance), the situation in biodiversity conservation is less clear. Several issues (including knowledge of species biology, ecosystem conservation, restoration of endangered species, and decision-making tools for ecosystem or forest management) are common to biological diversity and forestry.
However, in spite of a considerable number of studies carried out on hundreds of forest tree species, there are still very few reported biotechnology applications. Many countries are currently engaged in biotechnology-supported forest biodiversity conservation programmes. Nonetheless, there are only a few reported direct impacts at policy or technical levels.
The impact of biotechnology on genetic studies has allowed the use of cutting-edge methodologies with forest tree species in spite of the late domestication process compared with crop species (Sedjo 2003). The sequencing of the Populus genome has significant implications for forestry (Campbell et al. 2003). Eucalyptus gene sequencing, which was reported during a IUFRO (International Union of Forest Research Organizations) Tree Biotechnology meeting in 2003, has since been pursued by the Eucalypt Genome Initiative18, the Eucalyptus Genome Sequencing Project Consortium19 and the Brazilian project Genolyptus. The Dendrome Project20 is a collection of forest tree genome databases and other forest genetic information resources, with a special emphasis on conifers and Pinus. It is in genome sequencing that research seems currently most active.
The development of large-scale vegetative propagation techniques, essentially based on micropropagation or somatic embryogenesis, makes it theoretically possible to deploy superior planting materials rapidly and effectively. This is already the case for some coniferous and broad-leaved tree species including Eucalyptus and teak (Tectona grandis). Micropropagation techniques are also necessary to regenerate GM plants.
Genetic modification has been applied to forest trees mainly during the last two decades, at the experimental stage in the laboratory, to alter various traits such as herbicide, metal, salt, or insect resistance or cold tolerance. The rationale for producing trees with reduced lignin content for the pulp and paper industry is generally based on the expected environmental and economic benefits (Christensen et al. 2000). Equally if not more important, genetic modification is often considered as a tool to improve knowledge of tree biology and functioning, with significant potential applications; for example, for studying cell wall properties and wood formation. Since genes regulating secondary tissue and lignin formation in the annual plant Arabidopsis thaliana were identified (Goujon and Jouanin 2003), research on the genetic basis of wood quality in forest trees has significantly increased (mainly in Eucalyptus, Pinus and Populus).
With the exception of micropropagation, for which some applications are reported, most public domain information relates to research activities. Field applications appear extremely limited, according to the literature. Nevertheless, and in spite of unclear economic rationale, the use of biotechnology in forestry research has increased and provided new knowledge on tree biology and functioning. It also appears that forest biotechnology is increasingly funded by the public sector in academic research, while the private sector seems to focus investments more on specific traits of commercial interest such as wood quality.
Commercial deployment of new biotechnology-based forest tree varieties of a limited number of taxa (Eucalyptus, Pinus taeda, P. radiata, P. pinaster, Populus) can be expected in the near future. The impact of biotechnology applications on global wood supply may, however, take longer to materialize (not before 2020, according to Seppälä 2003). In this context, genetic modification appears to be the most significant and controversial sector of forest biotechnology. It is noteworthy that in some countries the terms ‘biotechnology’ and ‘genetic modification’ are used as synonymously, adding to the confusion.
During the last decade, several collaborative groups or consortia have been created by public or private partners for applying specific forest biotechnologies in a number of countries, including:
• ArborGen, created in 1999, with the aim of enabling research on genetic modification, has been working on herbicide resistance, growth and fibre quality for paper pulp in Eucalyptus, Pinus radiata, Populus, Pinus taeda and Liquidambar sp.
• GenForSA, created in 1999 with the main objective of modifying and testing disease resistance of Pinus radiata, while improving wood quality and formation (Owusu 1999).
• Monfori Nusantra Indonesia, created in 1996, has been involved in mass production of tissue cultures of Tectona grandis, Acacia and Eucalyptus for field trial establishment and commercialization.
• The Oregon State University’s Tree Genetic Engineering Research Cooperative (TGERC) has carried out research on the use of GM poplar in plantations.
• The Poplar Molecular Genetics Cooperative (PMGC) has been established with the aim of improving knowledge of genetic and molecular mechanisms responsible for productivity and quality trait variations in hybrid poplars.
• The North Carolina State University Forest Biotechnology Industrial Research Consortium (FORBIRC) mission is to integrate genome technology, metabolic engineering, traditional tree breeding and wood and paper science into a research organization directed to the creation of superior wood as a raw material and as a product.
The interest induced by the first genetic modifications of tree species at the end of the 1990s has since declined. Regulatory frameworks have been established in many countries for GM organism (GMO) field experiment or commercial deployment, with increased testing costs. In a number of countries, hostile reaction from the public or/and environmental groups has also limited deployment of GM trees. Some authors have questioned the profitability or return on investment of MAS techniques, at least in the short term and the medium term (Robinson 1999).
In Europe, forestry research priorities now focus on stronger synergies between biotechnology and conventional selection and breeding programmes mainly for wood improvement. The aim is to fill the gap between forest trees and agricultural crops in terms of the level of domestication. As a result, tree improvement tends to use genomic and proteomic approaches for achieving trait selection more rapidly. This is done by taking advantage of the advances obtained in model plants such as Arabidopsis (Kirst et al. 2003), and of the considerable increase of information exchange resulting from the latest bioinformatics progress. More and more effort is devoted to research aimed at better understanding biotic and abiotic stress responses. Significant investments have been made in genomics research on Populus and Pinus within the framework of the Poplar Genomics Initiative and the Pine Mapping Project.
In the tropics, wood production in volume per hectare is significantly higher than in the temperate zone. Several large-scale companies have recently invested in tree planting in tropical and subtropical areas. Some paper companies tend to fund their own research activities and keep them confidential. More emphasis seems to be put in genomics and proteomics, and particularly sequencing, thanks to developments on Arabidopsis thaliana, Oryza and Populus.
At the same time, there is increasing information coming from genome banks on wood quality, growth and stress resistance. Good examples of EST development include Eucalyptus in the framework of the Genolyptus project in Brazil, Populus21, Pinus taeda22, and P. pinaster and Quercus23, although applications for practical field selection are very unlikely to be operational for at least a decade.
Some predict that the next few years are more likely to see significant field applications of forest genomics research than of genetic transformation research, whose application is seriously hindered by strong environmental opposition and stringent regulations. Developments in agricultural crops are very meaningful is this respect. Genomics and proteomics outcomes are expected to boost conventional tree breeding and improve the efficiency of existing programmes.
The production costs of a new forest tree variety using conventional selection (well-known species, with a short juvenile phase) is roughly estimated at about US$400 000 and requires 15 to 20 years to be developed (Fenning and Gershenzon 2002). The calculation does not include the investment required for the selection of the suitable species. On the other hand, genomics, supported by bioinformatics in the framework of MAS, requires substantial equipment and running budget (as an indication, a supportive biotechnological platform costs several million dollars). In turn, such biotechnologies are expected to enable breeders to access more accurate and numerous traits of interest for greater improvements, while reducing the new variety production costs thanks to bioinformatics.
As regards developing countries and countries with economies in transition, there are few references available on their involvement in forestry biotechnology. The limited literature mainly refers to micropropagation in Vietnam, Malaysia, Indonesia and India. Malaysia has a reported strong oil palm molecular biology programme, including genetic modification.
However, some emerging countries with advanced financial, institutional and human capacities (including Brazil, India24, and China) have made significant breakthroughs in advanced forest biotechnology. Brazil has been actively involved in research on Eucalyptus through the Genolyptus project that constitutes at the moment the cutting edge in research on the genus worldwide. China is very active in poplar genetic modification. It can be reasonably assumed that after a period of time, results from research in technically advanced countries will benefit an increasing number of developing countries and countries in transition.
African countries, with the exception of Kenya and South Africa, are far less advanced in forest biotechnology. However, notable development of agricultural crop biotechnology in countries like Nigeria, Uganda, Côte d’Ivoire, Morocco and Algeria (particularly through AAB25) and even in the forestry sector (ISAAA in Kenya26), constitute positive indicators suggesting that the situation is improving. Adoption of well-targeted forest biotechnology, possibly for drought-resistance, seems a crucial issue for the future of such activities.
Micropropagation has for the past several years been the most striking example of forest biotechnology applications, at least in terms of numbers of plants produced and acclimatized. Applications have been reported mainly in Asian countries, although there is still a lack of reliable information as far as operational field planting is concerned.
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Data analysed in this study were stored in a simple Excel data set. The data set gathers major biotechnology entries (i.e. a given technology developed or used in a given country, by a given laboratory team, on a given species or variety, for a given purpose). Since a single activity can be reported and published many times in different journals, a significant amount of time has been devoted to singling out the activities and selecting the latest (or most comprehensive) reference (= source of information, or activity, or entry, or record).
Collection dates: February to April 2004 with revisions in September 2004.
Methodology: *Systematic searches on CAB Abstracts from 1994 to 2004 using combinations of key words (including forest trees, MAS, micropropagation, proteomics, genomic, EST, lignin, transformation, GMO). Examples: forest trees × genetic diversity, forest trees × micropropagation, proteomics × eucalyptus.
*Research on Google using the key words indicated above. A search in Google provided 632 000 references to ‘forest biotechnology’ in November 2004 (all languages).
*Team research by topic, on the Internet, through personal contacts, and through reviews of literature. Topics included:
• Micropropagation
• Genetic transformation
• Genetic improvement, genomics, etc.
• Structuration of genetic diversity
• Mycorrhizae, Rhizobium
*Review of the proceedings of the latest international conferences
Confidentiality: *Only public information was gathered, including personal communications and observations.
The following databases and international data sets have been used through CABI: AGRICOLA, AGRIS, ASFA, CAB Abstracts, Econlit, FSTA, TROPAG & RURAL, and ULRICH International Periodicals.
Data structure: In total, 2 716 records have been collected. Of these, 2 644 records were directly related to forest trees, while 72 records addressed forest trees indirectly: studies on mycorrhizae (Frankia and Rhizobium) accounted for 36 records, fruit trees and ornamental trees 30 records, and bamboos and rattans six records.
Information on crop trees has not been searched systematically, and the data set is not representative of activities taking place on domesticated trees. However, a number of genera that comprise wild populations and cultivated varieties (including Ficus, Malus, Morus, Pyrus, Prunus) have been included, when insufficient information was available from the source of information to ascertain the degree of domestication of the germplasm on which activities were carried out. These classification uncertainties represent approximately one percent of the data set.
A number of forest tree species cannot be categorized in specific regions of provenance. This is the case for most hybrid Populus clones, including euramerican poplars (Populus × P. euramericana) and many Asian varieties.
Eurasian species (including Pinus sylvestris, Populus nigra …) have been categorized as of European origin.
http://biotech.jrc.it
http://dendrome.ucdavis.edu
http://elonmerkki.net
http://fabinet.up.ac.za
http://mediaforest.net
http://ogm.agriculture.gouv.fr/
http://webdoc.sub.gwdg.de
http://web.ahc.umn.edu/biodata/
www.agbios.com
www.agnet.org
www.americanlands.org
www.aphis.usda.gov/
www.arborea.ulaval.ca
www.arborgen.com/
www.beckerunderwood.com
www.biorize.com
www.biospace.com
www.cellfor.com
www.checkbiotech.org
www.cirad.fr
www.cnr.berkeley.edu
www.csiro.au
www.ctnbio.gov.br
www.edinburgh.ceh.ac.uk
www.efi.fi
www.ermanz.govt.nz
www.etfrn.org
www.fao.org
www.forestresearch.co.nz
www.fsc.org
www.fungi.com
www.genet-info.org
www.genewatch.org
www.greenpeace.org
www.grolife.com
www.ipgri.cgiar.org
www.isb.vt.edu
www.itto.or.jp
www.iufro.org
www.ncl-india.org
www.nf-2000.org
www.nrcan-rcan.gc.ca
www.phcmexico.com.mx
www.philombios.com
www.pierroton.inra.fr
www.premiertech.com
www.rbge.org.uk
www.rti.org
www.strategis.ic.gc.ca
www.ucl.ac.be
www.wrm.org.uy
Africa: Cameroon, Congo, Côte d’Ivoire, Ethiopia, Ghana, Kenya, Madagascar, Senegal, South Africa, United Republic of Tanzania, Uganda
South America: Argentina, Brazil, Chile, Costa Rica, Cuba, Guyana, Mexico, Uruguay, Venezuela (Bolivarian Republic of).
North America: Canada, United States of America
Asia: Bangladesh, China (including the province of Taiwan), India, Indonesia, Japan, Republic of Korea, Malaysia, Myanmar, Nepal, Pakistan, Philippines, Singapore, Thailand, Viet Nam
Near East: Egypt, Iran (Islamic Republic of), Kuwait, Saudi Arabia, Tunisia, United Arab Emirates
Europe: Austria, Belarus, Belgium, Bulgaria, Croatia, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Israel, Italy, Latvia, Lithuania, Luxembourg, Netherlands, Norway, Poland, Portugal, Romania, Russian Federation, Serbia and Montenegro, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey, United Kingdom.
Oceania: Australia, New Zealand.
12 See: www.fao.org/WAICENT/OIS/PRESS_NE/PRESSENG/2000/pren0017.htm or www.fao.org/biotech.
13 See, in particular, Appendix 2.7.2 for Internet sites.
14 See: www.fao.org/biotech/sector5.asp.
15 See: www.forestresearch.co.nz.
16 See: www2.ncsu.edu/unity/lockers/project/forestbiotech/news.html
17 Source: APHIS; www.aphis.usda.gov/
18 See: www.up.ac.za/academic/fabi/eucgenomics/EGI/.
19 See: http://forests.esalq.usp.br/.
20 See: http://dendrome.ucdavis.edu/Gen_res.htm.
21 See: http://Poppel.fysbot.umu.se, http://mycor.nancy.inra.fr/PoplarDB.html.
22 See: http://cbc.umn.edu, http://dendrome.ucdavis.edu.
23 See: www.pierroton.inra.fr.
24 See: http://dbtindia.nic.in/ebc/ptc.htm.
25 See: www.aab.org.dz.
26 See: www.isaaa.org/Projects/Africa/trees.htm.
27 Note: the distribution of countries and territories by regions of the world is in line with the FAO regional classification. See:
www.fao.org/countryprofiles/physical_presence.asp?lang=en.
