ETFRN NEWS 34: Biotechnology

Organisations - Institutions - Programmes

• Why Biotechnology for Tropical Trees - A Review of Applications in Tropical Forest Management
• In Vitro Approach for Natural and Induced Biodiversity Conservation of Forest Trees
• Genetic Modification in Forest Trees
• An Integrated Molecular Population Genetic Approach for Forestry Conservation and Management in Thailand
• Isozyme Variation in Natural Populations of Sal (Shorea Robusta) in the Terai Region, Nepal
• Molecular Genetics of Faidherbia Albida
• Assessing Genetic Variability in Leucaena Hybrids
• RAPD Analysis of the Genus Bambusa ‘Sensu lato'
• Genetic Diversity in Natural Populations of Eucalyptus Microtheca
• Assessment of Levels and Dynamics of Intra- Specific Genetic Diversity of Latin American Tropical Trees for Conservation & Sustainable Management
• Germination Syndromes as Tools for Plant Functional Types
• Tissue Culture Pilot Plant Facility for Upscaling and Testing Techno-Commercial Feasibility of Forest Tree Tissue Culture at the National Chemical Laboratory, Pune, India
• Thirty Years of Vegetative Propagation Research on Tropical Trees in Scotland
• Genetics Markers (RAPDS and rep-PCR) for Ecological Studies of the Nitrogen-Fixing Symbiosis
• Mycorrihizal and Rhizobial Inoculations as Tools Legume Tree Conservation and Management in Orinoco Flood Plain, Venezuela
• Symbiotic Interactions between Woody Legumes and Root Nodule Bacteria under Favourable and Adverse Conditions

WHY BIOTECHNOLOGY FOR TROPICAL TREES? – A REVIEW OF APPLICATIONS IN TROPICAL FOREST MANAGEMENT

By Olavi Luukkanen

Forestry differs from agricultural cropping in one fundamental way: management interventions often concern natural or semi-natural ecosystems, and we are faced with the problem of how our work is related to the "natural" genetic processes. Tropical forests are rich in biodiversity and have conservation needs related to specific patterns of reproductive biology in trees. Tree plantations and agroforestry systems, both often based on introduced tree species, have a central role in supplying the desired tropical forest products. These features make forest tree biotechnology applications especially attractive for the tropical zone and for the developing countries.

Plant biotechnology is often mainly associated with sophisticated DNA work related to genome mapping, with or without gene manipulation, or such rapidly advancing techniques as somatic embryogenesis, cryopreservation and artificial seeds. However, it is useful to remember that the history of forestry provides old examples on biotechnology applications in the form of vegetative propagation using stem cuttings, stratification and tree seed handling techniques, manipulation of flowering, and inoculation of trees with root symbionts. Recently, biotechnology has also made rapid advances in relation to wood quality and the underlying genetic, physiological, genetic and environmental factors (cf. Anonymous 2001).

Tree breeding and tree biotechnology are clearly linked. All traditional forms of biotechnology are still relevant, and in this review, the emphasis is on tropical tree biotechnology as related to the production of planting stock for forest regeneration. A more complete text by the author with full references is also available (see the comprehensive report on tropical tree biotechnology published by the International Foundation for Science (IFS, 1998).

Stem cuttings
Rooted stem cuttings are a traditional propagation method used globally mainly for broadleaved tree species but also in some conifers, such as Cryptomeria japonica and Pinus radiata. The largest clonal forestry programme of any forestry species or genus exists in eucalypts. Seedling and epicormic shoot ramets of eucalypts root easily, but mature shoots show an increasingly lower rooting percentage. Up to 100 % rooting can be achieved from basal epicormic shoot material, but problems may arise with species that do not form these shoots easily. Treatment of cut stump surfaces with auxins can induce basal shoots in otherwise recalcitrant eucalypt species. A continuous supply of ramets for stem cutting propagation is achieved from hedged seedlings or grafts; grafting followed by stem excision and epicormic shoot formation ensures juvenile material even when branches of mature trees are used as starting material; this procedure is applicable to most species used in stem cutting propagation. In lesser-known tropical trees, considerable improvement of conventional vegetative propagation has taken place in recent years.

Micropropagation in angiosperm trees
In tropical broadleaved tree species, the practical applications of micropropagation seem to concentrate on tissue culture, while propagation using somatic embryogenesis seems to be more promising in (mostly temperate-zone) conifer propagation. Both groups of techniques require exact protocols for each species, but in several tropical forest tree species practical applications are already available. Forestry-related use of micropropagation for organogenesis has been successfully developed at least in the following genera: Acacia, Bombax, Casuarina, Dalbergia, Eucalyptus, Ficus, Gmelina, Populus, Platanus, Salix, Shorea, Tectona and Terminalia.

Most eucalypts can be vegetatively propagated using traditional stem cutting techniques, but in vitro micropropagation and rooting followed by transfer to soil substrate is gaining popularity because of the prospects of rapid genetic gain by using micropropagation for the production of stools for stem cutting production. Various plant parts have been used as explant material, but the best cultures are generally obtained from cotyledonal or other zygotic embryonal tissues.

Rattans are an example of insufficiently known species that are important in the tropical region but difficult to propagate and grow in plantations. Micropropagation has been suggested as a solution for commercial, agroforestry-type growing of Calamus manan, the most valuable rattan species in Southeast Asia. In bamboos, gregarious flowering and the limited capacity for branch cutting propagation are reasons for introducing micropropagation methods.

The very first woody species in which somatic embryos were produced in 1965 was the tropical sandalwood (Santalum album). Somatic embryogenesis has now been recorded in more than one hundred angiosperm woody species alone. Tropical genera represented among these include Albizzia, Azadirachta, Bambusa, Cassia, Citrus, Cocos, Dendrocalamus, Elaeis, Eucalyptus, Hevea, Mangifera, Phyllostachys and Sinocalamus. The presence of fruit and estate crops and bamboos is conspicuous in this list; the most intensively studied genus is Citrus.

Right selection of the explant seems to be extremely important in somatic embryogenesis, and, commonly, immature embryos or nucellar tissue provide the best starting material. The fact that mature tissues have successfully been used for somatic embryogenesis in many tropical tree families opens up completely new prospects for gene conservation and silviculture based on indigenous tree species.

Field trials must also be used for proper verification of clone performance and especially for checks for somaclonal variation, early maturation and plagiotropism, which otherwise might cause problems. It is essential to include conventionally propagated material as control.

Micropropagation in conifers
Practical applications of micropropagation through organogenesis in conifers have become well established in Pinus radiata plantation management in New Zealand. The reason for not using rooted cuttings has been the fear of poor field performance due to ageing of the clones. This is particularly clear when ortets older than 4-6 years are used. No practical methods for somatic embryogenesis were available at the start of the programme in the late 1970s; therefore, the method chosen was micropropagation from embryonic cotyledons. The in vitro cycles needed include those for shoot formation, elongation and cutting (which can be repeated), callus formation, root induction with auxins, and finally transfer to non-sterile rooting media.

Since 1985, rapid progress has been achieved in micropropagation of conifers using somatic embryogenesis. Basically, there are three different methods for embryogenic culture initiation in conifers: (1) through continuation of natural cleavage polyembryony of explanted immature embryos; (2) through cell division in the epidermal or subepidermal layers of the hypocotyl, cotyledons or needles, and subsequent formation of callus which forms embryo suspensor masses (ESM); or (3) through cell division of small cells within the suspensor system of the explanted immature embryo.

In conifers as a whole, successful results on somatic embryogenesis have been reported in more than 30 species or hybrids. The few tropical species in this list include Agathis australis and Pinus caribaea. In pines, only immature zygotic embryos (often attached to the intact megagametophyte) can easily be used as starting material, which complicates the use of this technology.

For large-scale practical applications of somatic embryogenesis, automatic systems for culturing, delivering and preserving the embryos are being intensively studied and developed. However the production of mature embryos and especially coating them to produce manufactured (artificial, synthetic) seeds (complete with "manufactured megagametophyte") requires further development. Presently, full-scale commercialisation of this technology is prevented by inadequacies both in embryo quality and in the delivery systems. Cryogenic storage was developed early during the studies on somatic embryogenesis of conifers; it allows the preservation of propagation material over the extensive period needed in field testing of the clones.

Genomic techniques
From a forestry viewpoint the most important reason for the development of somatic embryogenesis technology may be its potential for gene transfer and subsequent multiplication of transgenic plants, but the genetic transformation methods used in agricultural plants are less easily applied to trees. As a result, the list of angiosperm species in which successful gene transfer has been reported is conspicuously void of trees.

In conifers, gene transfer to the level of regenerated plants was first reported only in four species: Larix laricina, Picea glauca, P. mariana and Pinus radiata. Much of the potential from gene transfer in conifers remains theoretical. Presently, the major limitation in conifers is the inability to regenerate plants from transformed single cells. It is interesting, howewer, that tropical pines Pinus caribaea, P. oocarpa and P. patula belong to the several conifer species in which protoplasts have been cultured and regenerated.

Once gene transfer has been achieved, genetic selection, using marker traits, such as antibiotic or herbicide resistance, must be carried out so as to separate the transformed cells. Thereafter, plant regeneration is achieved using either organogenesis or somatic embryogenesis. Thus the success of transformation in woody plants also depends of the availability of tissue culture protocols for the species in question. The final result in gene transfer also depends on gene expression as influenced by genetic interaction between DNA sequences and on the stability of the introduced trait. Unwanted escape of transgenes could be prevented by introducing sterility into transformed clones. The final check of gene transfer success is a field trial, but the whole technology is affected by strict regulation and often polemic public discussion.

While genetic transformation (as well as somatic hybridisation) may be of limited practical use for a long time to come, biotechnology in the form of genetic markers already has a significant role in studying and utilizing the intraspecific and interspecific genetic variation in trees. Biochemical and molecular methods allow a precise study of the effects of forestry practices on biodiversity and thus provide tools for the application of criteria and indicators of sustainable forest management.

Virtually all trees can be genetically mapped, and the results have been applied, for instance, to host resistance screening and lignin biosynthesis modification in Pinus taeda. Fusiform rust resistance was found to be under oligogenic and, in some cases, dominant single gene control; since individuals homozygous for the resistance gene can be identified, all seedling progeny of such individuals would be resistant to the pathogen.

Conclusions
Micropropagation does not necessarily require expensive or sophisticated equipment. In fact, micropropagation easily competes with the maintenance of hedged seedlings for rooted cutting propagation -- stacks in a small laboratory can have as good a stock capacity as several hectares of hedges. Laboratory propagation allows year-round production of planting material and may reduce the labour input as compared to stem cutting production; micropropagation can also be combined with in vitro root symbiont inoculation. Micropropagation can and should be combined with the storage of genetic material, so as to allow proper field testing of new breeding material. Instead of cryopreservation, storage can be achieved in modified normal laboratory conditions. The two most important problems associated with micropropagation are maintenance of juvenile material and ensuring sufficient genetic diversity in plantations, using a sufficient number of clones.

Somatic embryogenesis is being rapidly developed for mass propagation of plantation trees. Its advantage is seen in the possibility to quickly produce millions of copies of superior individual trees. This method can be combined with a conventional breeding programme, to replace or complement propagation from stem cuttings or in vitro organogenesis. The available technology in embryogenesis is in most cases too expensive to compete with other methods, but the development of automated production systems may change this situation. Ideally, such systems would cover all steps from culture maintenance and embryo development to the conversion of embryos to autotrophic plants, whereby either direct-seeding of embryos or manufactured seeds could be used.Future research will concentrate on accomplishing all steps in a compact automated system, a bioreactor, and producing mature embryos in liquid medium.

The possibility for cryopreservation of clones in embryo cultures offers an added benefit to the application of somatic embryogenesis. Such storage can be used while field tests are carried out, and mass propagation would be done with the selected superior clones. There is a danger that the selection occurs in the embryogenesis process rather than the expected field performance. Further development of the embryogenesis technology for mature tissues would allow bypassing of cryopreservation during the testing of clones. Field testing is essential in assessing the true value of new planting stock, and it is obvious that wood properties must also be considered in this work.

It must be admitted that the immediate benefits for forestry of sophisticated, high-input techniques such as genetic transformation or somatic hybridisation are not very clear. We should also perhaps be more aware of the danger involved in the perception of "modern" forestry as equal to high-input plantation management. In the tropics, where most forests still are (managed or unmanaged) natural forests, we should not promote intensive tree plantations or any high-input production techniques before studying the possibility to manage the original natural forest, perhaps after improving and rehabilitating it, for optimal production. Plantation performance (taking account of their socio-economic and environmental effects) should be compared with a range of alternative production systems, so as to provide tools for decision making on optimal land-uses.

The following statement, expressed in the introduction chapter of a comprehensive treatise on somatic embryogenesis in woody plants (Jain et al. 1995) tells an old truth about the role of forest management. It emphasises the fact that we have to actively maintain the genetic diversity, using such means as in situ, ex situ and circa situ gene conservation, and also to measure the actual genetic diversity in tree populations in natural and man-made forests when necessary for management decisions. On the other hand, we have to select, multiply and use the suitable genotypes in intensive culture. Technically, this management is fully comparable to growing such estate crops as coffee, oil palm or rubber:

"The challenge to forestry will be to maintain natural biological diversity in forest ecosystems in combination with intensive clonal culture of carefully diversified genotypes selected for the production of wood and other forest products" (Kriebel 1995).

References
Anonymous 2001. Wood, breeding, biotechnology and industrial expectations. Proc. Joint Meeting of EU Funded Projects, the 9th Conifer Biotechnology Working Group,and IUFRO Working Parties 2.04.00 Genetics and 5.01.02 Natural Variations in Wood Quality, Bordeaux, France 11-14June 2001 194 p. Abstracts at website http://www.pierroton.inra.fr/WBB
IFS (International Foundation for Science) 1998. Recent advances in biotechnology for tree conservation and management. Proceedings of an IFS workshop, Florianópolis, Brazil, 15-19 September 1997 (ed. S. Bruns, S. Mantell, C. Trägårdh & A.M. Viana). IFS, Stockholm. [Pp. 203-213 contain a more comprehensive article by the author with full references].
Jain, S.M. et al. 1995, 1999, 2000. Somatic embryogenesis in woody plants. Vol. 1-6. Kluwer.

For further information please contact:
Olavi Luukkanen
Tropical Silviculture Unit
PB 28, FI-00014 University of Helsinki, Finland
Tel.+ 358 9 19158643, Fax + 358 9 19158646
E-mail olavi.luukkanen@helsinki.fi
Website http://honeybee.helsinki.fi/tropic

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IN VITRO APPROACH FOR NATURAL AND INDUCED BIODIVERSITY CONSERVATION OF FOREST TREES

By S. Mohan Jain

The success of any crop improvement program primarily depends on the continuous availability of germplasm with desirable traits. Genetic resources conservation becomes an essential ingredient for breeding and continuous supply of raw material to the industry. Furthermore, large-scale industrialisation, environmental degradation, deforestation and ever-increasing human population growth are placing tremendous pressure on existing resources such as arable land, forests, and natural biodiversity. Deforestation has become a serious problem as a result of forest fires and the use of trees for firewood, timber and paper. In many developing countries, mismanagement and illegal destruction of trees are major serious problems added to deforestation (Jain, 1997). Industrially important trees are being destroyed much faster than the pace of reforestation, resulting in heavy economic looses and destruction of important biodiversity. In some cases, the paper industry is forced to import wood at a much higher price. Reforestation is a slow process often requiring superior planting material, and it is difficult to restore the vegetation of the primary forest. This would increase the demand of improved planting material and high-quality raw material for the industry. Moreover, the loss of natural habitats will narrow down the genetic diversity. Both adverse environmental conditions and industrial expansion may pose danger to the existing germplasm. Therefore, it is justified to induce genetic diversity, for instance, by mutagen treatment and to preserve it using tissue culture (Jain et al, 1998, 2001). Genetic markers are useful to evaluate the genetic diversity and the mechanisms that produce and maintain this diversity. The genetic diversity structure and distribution are important parameters to conserve biodiversity and also to follow the gene flow, especially in transgenic plants.

Tissue culture has facilitated large-scale plant multiplication in woody plants. This has come about with somatic embryogenesis and organogenesis or micropropagation (Jain et al, 1995, 1999, 2000; Ahuja, 1992). Somatic embryogenesis is very effective for clonal propagation, and it has made tremendous progress in some of the commercially important trees belonging to both angiosperms (e.g. bamboo, Eucalyptus spp, Populus spp, Quercus spp, sandalwood, Tilia, Salix, Magnolia, and Betula) and gymnosperms (e.g. Picea, Pinus, Larix, Cycadales, Abies, Sequoia sempervirens, and Pseudotsuga menziesii). Somatic embryogenesis is a process forming embryos from somatic cells without undergoing sexual cycles; it is similar to zygotic embryogenesis. Somatic embryogenic cultures can be multiplied in bioreactors for large-scale production and can readily be cryo-stored for a long period of time without losing plant regeneration capacity and genetic fidelity of regenerated plants. In Silvagen Inc., Canada, clone banks have been initiated for blister-rust-resistant western pine (7 families, 50 genotypes) and high-yielding coastal Douglas-fir (including 12 families and 220 genotypes). For three southern pine species, 400 genotypes and 27 families have been stored (Cyr, 1999). In USA, Weyerhauser Inc. also maintains cryo-storage facilities at several locations for long-term storage of somatic embryogenic cultures of elite forest tree germplasm.

The major disadvantages of somatic embryogenesis are a) high dependence on genotype, b) poor rate of somatic embryo production and c) gradual fluctuation and eventual decline in embryogenic culture potential; finally, since cultures are developed from seeds or seedlings, the material is of unproven genetic value. Somaclonal variation can be prevented by cryo-storage of embryogenic cultures without initial subculture. Another approach for plant multiplication is by organogenesis without going through a callus phase, i.e. shoots can be directly initiated from in vitro-cultured explants such as mature embryos, shoot tips, and adventitious buds, or they can be excised from old trees, as found in Quercus robur, Eucalyptus, Betula, poplars, etc. Micropropagated plants cannot be cryo-stored. However, they can be stored at low temperatures in the cold room. Usually, in vitro shoots or in vitro-rooted plantlets are stored at low light intensity. The major risk with this approach is contamination of the culture material, especially by viruses. This process becomes labour-intensive, since cultures require subcultures on fresh medium at regular intervals that depend on the plant species. Micropropagation is ideally suited for the developing countries because of low maintenance cost.

Molecular marker (AFLPs, microsatellites) and flow cytometer analysis are essential in order to study the genetic fidelity of micropropagated, cryostored, and cold-stored plants. Furthermore, molecular marker analysis would assist in studying the genetic population structure, gene flow, and transgene protection (Jain et al, 2001)

References
Ahuja, M.R.(ed), 1992. Micropropagation of woody plants, Kluwer.
Cyr, D.R.1999. In: Somatic embryogenesis in woody plants. Vol. 4, Kluwer.
Jain, S.M. 1997. In: Plant biotechnology and plant genetic resources for sustainability and productivity. K.N. Watanabe and E. Pehu(eds.). Academic Press.
Jain, S.M. 2001. Euphytica 18: 153-166.
Jain, S.M. et al (eds.), 1995, 1999, 2000. Somatic embryogenesis in woody plants. Vol. 1-6, Kluwer.
Jain, S.M. et al (eds.), 1998. Somaclonal variation and induced mutations for crop improvement, Kluwer.
Jain, S.M. et al (eds.), 2001. Molecular techniques in crop improvement, Kluwer (in press ).

For further information please contact:
S. Mohan Jain, FAO/IAEA Joint Division, International Atomic EnergyAgency, Room A-2206, Box 100, Wagramerstrasse 5 A-1400 Vienna, Austria.
Phone: + 43 1 2600 21623, Fax: + 43 1 26007
Email: S.M.Jain@iaea.org

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GENETIC MODIFICATION IN FOREST TREES

S. Mohan Jain

Recent advances in plant biotechnology have enabled improvement of forest trees with varying success. This is mainly due to differences in longevity, heterozygous nature, life cycles, and the environment in which they grow (Ritter, 1999). Forest trees have long generation cycles, with vegetative phases ranging from one to several decades. Once trees have germinated in nature or transplanted to plantations, they remain anchored in one location where they are exposed to changing environments and other vagaries of nature. Some of these factors may influence their physiology and alter the complex morphogenetic processes. During the long life cycle of trees, many genetic and epigenetic changes are probably important to maintain the within-tree and within-populations genetic diversity, and to assure a long-term survival of individuals and populations. However, it is not surprising that with an increasing world demand for pulp, paper and timber products, along with the growing awareness of the high ecological and social value of indigenous forests, more efforts have been directed using the molecular biology for forest tree improvement (Walter, 1999; Klopfenstein et al, 1997).

Now various techniques are available for gene transfer in forest trees. These include the bacteria Agrobacterium tumefaciens and A. rhizogenes, and the biolistic gun; somatic embryogenic cultures are commonly transformed; and clonal propagagtion of trees can be done with somatic embryogenesis and organogenesis (Jain, 1999; Jain et al, 1995, 1999, 2000; Klopfenstein et al, 1997). In most conifers and other forest trees, there is limited information on the genetic fidelity of somatic seedlings, which is vital for clonal propagation of elite trees (Jain and Ishii, 1998). Several forest tree species have been transformed such as conifers, poplars, sweet gum, eucalypts, elm, and European chestnut. There is a long list of angiosperm and gymnosperm forest trees that have been transformed with marker genes without plant regeneration (Jain and Minocha, 2000). Few tropical or subtropical forest trees have been genetically transformed; they include Japanese persimmon and acacias. The most common trait specific genes introduced in forest tree species are for herbicide resistance, marker genes, rolC lignin, cellulose, insect resistance (Bt, and trypsin proteinase inhibitor), Hepatitis B virus for vaccine, mercuric reductase (merA) for phytoremediation, disease resistance (against Phytophthora cinnamomi and Ophiostoma novoulmi), male sterility, and the glutamine biosynthase gene for efficient nitrogen utilization. World-wide up to the year 1999, a total of 68 field trials with transgenic trees have been approved, out of which 51 are in poplars alone. They include genes for lignin, cellulose, sterility, herbicides, insect resistance and marker genes (Walter, 1999). The long life cycle and extended vegetative phase of forest trees may hamper the monitoring of transgene expression. Transgenic effects may cause abnormality immediately or remain dormant for a long time or even might be lost during the long vegetative phase of the tree species, and therefore it is rather difficult to predict the behavior of transgenes in the future (Jain, 1999; Jain et al 2001).

The major challenges facing introduction of genetically modified forest trees include the stability of transgene expression or silencing effects, containment of transgenes by reproductive options, forest management for reforestation with transgenic trees, genetic engineering and biotechnology specifically for tropical forest trees, and desirable modification of lignin for future sustainable forestry. We will have to come up with technologies to guarantee correct expression of genes over the life span of the tree and for rapid assessment of a high number of genes and promoters using cost effective technology, starting with some model systems, such as genes involved in the reproductive development of Arabidopsis (Walter, 1999). In the case of lignin and cellulose formation, conifer tissue culture systems that provide cells developing lignin and cellulose-containing cell walls, can be used to test a wide range of constraints rapidly and economically. Tree genetic engineering has potential for contributing to a new green revolution, allowing us to grow trees with desired traits and produce high-quality end products. In the final analysis, however, public perception and acceptance of genetic engineering will determine the future of genetically modified trees in forestry.

References (also see references in preceding contribution)
Jain, S.M. 1999. In: Plant biotechnology an in vitro biology in the 21st century. A. Altman, M.Ziv, S. Izhar (eds.). Kluwer. Pp 57-64.
Jain, S.M. and K. Ishii. 1998. In: Recent advances in biotechnology for tree conservation and management, Proceedings of an International Workshop. International Foundation for Science (IFS), S. Burns, S. Mantell, C. Tragardh andA.M. Viana (eds.). Pp 214-231.
Jain, S. M. and S. C. Minocha (eds.) 2000. Molecular biology of woody plants,Vol.1-2. Kluwer Klopfenstein, N.D. et al (eds.). 1997.
Micropropagation, genetic engineering, and molecular biology of Populus. Rocky Mountain Forest and Range Experiment Station, Fort Collins, Colorado, USA
Ritter, E.S. (ed.) 1999. Proceedings of applications of biotechnology to forest trees, Biofor-99, Spain.
Walter, C. 1999. In: Proceedings of applications of biotechnology to forest trees. E.S. Ritter (ed.). Pp331-338.

For further information please contact:
S. Mohan Jain, FAO/IAEA Joint Division, International Atomic Energy Agency, A-2206, Box 100, Wagramerstrasse 5, A-1400 Vienna, Austria.
Phone: + 43 1 2600 21623, Fax: + 43 1 26007
Email: S.M.Jain@iaea.org

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AN INTEGRATED MOLECULAR POPULATION GENETIC APPROACH FOR FOREST TREE CONSERVATION AND MANAGEMENT IN THAILAND

By Suchitra Changtragoon

One of the fundamental requirements for conservation and use of forest tree genetic resources is understanding the biological dynamics of genetic variation within and between species. Considerable variation exists among tree species with respect to the extent of genetic diversity and the way such diversity is organised within and among populations. The extent and the pattern of this diversity are strongly dependent on the amount of genetic polymorphism, pattern of gene flow and mating system.

Molecular genetic marker screening is based on the survey of genetic diversity as revealed by variation at specific gene loci and provides information about the amount and distribution of genetic diversity within and among populations. Furthermore, analysis of gene marker data permits estimation of outcrossing rates and thus monitoring genetic changes caused by factors affecting reproductive biology of a species. Information gained from genetic marker screening is invaluable for identification of populations which are desirable for conserving and breeding purposes and improving forestry practices which inadvertently alter natural gene pools of domesticated species ( Changtragoon and Szmidt, 1997).

Previous research on molecular population genetics of forest trees in Thailand.
The genetic diversity and mating system in some economically important forest tree species in Thailand such as indigenous pines (Pinus merkusii and P. kesiya), neem (Azadirachta indica var. siamensis, A. indica and A. excelsa), Dipterocarpus alatus, teak (Tectona grandis), rattans (Calamus spp.), paper bark tree (Melaleuca cajuputi), Mitragyna brunonis, Pterocarpus macrocarpus, Cycad siamensis have been evaluated by using isoenzyme gene and DNA markers (cf. Changtragoon, 2001; Changtragoon and Finkeldey, 2000).

Among the populations examined, those genetically most diverse within and among populations of the species should merit a high priority for conservation. Since forest trees are long lived compared to annual or crop plants, high genetic diversity and outcrossing rate would guarantee higher possibility of their survival, viability, longevity, and disease and insect resistance for the present and forthcoming generations in a changing environment. Combination of marker-aided population genetic analysis and information about adaptive and quantitative traits as well as forest ecosystems would allow for the development of a comprehensive conservation programme for individual species in each forest type ( Changtragoon, 2001).

Genetic diversity and gene conservation of teak in Thailand.
Fifty-one RAPD (Random Amplified Polymorphic DNA) loci were identified and used to evaluate the genetic diversity in fifteen natural populations of teak in Thailand. Partitioning of genetic variation into within and among population components revealed that about 21 % of the total variation was attributable to differences among populations. The number of polymorphic loci in most of the investigated populations was very high with an average of 72.6%. The average expected heterozygosity was 0.310. Significant differences in allelic frequencies were found for most pairwise comparisons between populations (Changtragoon and Szmidt, 2000). The outcrossing rate ranged between 82-97%. These results suggest that natural populations of T. grandis in Thailand are highly differentiated genetically, implying that multiple sources of materials from at least one population of each province in the northern and central part of Thailand may be required for both in situ and ex situ gene conservation purposes (Changtragoon, 2001).

Ongoing research.
At present, the investigation of genetic diversity and mating system of Rhizophora apiculata, R. mucronata, Azadirachta spp., some Dipterocarpus spp. and bamboo species using molecular markers are on the way.

Acknowledgements.
Some of the research mentioned in this paper was financially supported by the following organisations: Volkswagen Stiftung (VW), International Foundation for Science (IFS), International Plant Genetic Resources Institute (IPGRI), Center for International Forestry Research (CIFOR), Australian Centre for International Agricultural Research (ACIAR), Biodiversity Research and Training Program (BRT), Thailand; and Royal Forest Department, Thailand.

References:
Changtragoon, S. 2001. Forest Genetic Resources of Thailand: Status and Conservation. In: Forest Genetic Resources: Status, Threats and Conservation Strategies (eds. R. Uma Shaanker, K.N. Ganeshaiah & Kamaljit S. Bawa) Oxford & IBH Publishing Co. Pvt. Ltd. New Delhi, pp.141-151.
Changtragoon, S. and Finkeldey. R. 2000. Der Nutzen von Genmarkern fuer die Erhaltung Forstgenetischer Resourcen in Thailand. For.Snow Landsc. Res. 75,1/2: 153- 162.
Changtragoon,S. and Szmidt. A.E. 1997. The evaluation of genetic diversity and resources of tropical forest trees in Thailand by using molecular markers. In: The 6th Annual International Workshop of BIO-REFOR. The University of Queensland, Brisbane, 2-9 December 1997.pp.169-171.
Changtragoon, S. and Szmidt, A.E. 2000. Genetic diversity of teak (Tectona grandis Linn. F.) in Thailand revealed by Random Amplified Polymorphic DNA (RAPD). In: IUFRO Working Party 2.08.01 Tropical Species Breeding and Genetic Resources: Forest Genetics for the Next Millennium.International Conferences Centre, Durban,South Africa. 8-13 October 2000. pp. 82-83.

For further information please contact:
Suchitra Changtragoon, DNA and Isoenzyme Laboratory, Silviculture Research Division, Royal Forest Department, 61 Phaholyothin Rd., Chatuchak, Bangkok 10900, Thailand.
Email: suchitra@mozart.inet.co.th

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ISOZYME VARIATION IN NATURAL POPULATIONS OF SAL (SHOREA ROBUSTA) IN THE TERAI REGION, NEPAL

By Jouni Suoheimo, Chunyang Li, and Olavi Luukkanen

Sal (Shorea robusta) is the most important commercial tree species in Nepal. The species belongs to the Dipterocarpaceae family and also forms extensive forests in the northern states of India. This species has been intensively studied for more than a century, but recent work has still revealed new aspects on its ecology and silvicultural management (Suoheimo, 1999). Until very recent date, only phenotypic traits of S. robusta populations have been studied, and little information exists on the intra-specific variation of S. robusta as revealed by biochemical (isozyme) or molecular (DNA) techniques.

Knowledge of the distribution of genetic variation within and between populations is essential for the conservation of plant genetic resources. The isozyme variation within and among populations has been extensively studied in many woody plants, including tropical species. There is also conclusive evidence showing significant correlation between isozyme variation and quantitative traits. Isozymes as genetic markers are thus of great importance in tropical tree breeding and conservation.

In our study, we investigated the genetic variation of S. robusta in three natural populations in the Terai region, Nepal, using 12 loci from 8 isozyme systems (Suoheimo et al. 1999). The mean number of alleles per locus was 2.16, and 58.3% of the loci were polymorphic (95% criterion for polymorphism). The mean observed and expected heterozygosities ranged from 0.105 to 0.129 with an average of 0.117, and from 0.130 to 0.158 with an average of 0.143, respectively. Only 4.7% of the total genetic diversity was due to differentiation among the populations, and the mean value of genetic distance was 0.018. The results indicated that the majority of the species' genetic variation was found within the studied populations and there was a high genetic similarity among these three natural populations of S. robusta. The sharing of one gene pool among the studied populations suggested a lack of barriers to gene flow.

References
Suoheimo, J. 1999. Natural regeneration of sal (Shorea robusta) in the Terai region, Nepal. Doctoral thesis. Univ. Helsinki Tropical Forestry Reports 19. 134 p.
Suoheimo, J., Li, C. and Luukkanen, O. 1999. Isozyme variation of natural populations of sal (Shorea robusta) in the Terai region, Nepal. Silvae Genetica 48(3-4):199-203.

For further information please contact:
Olavi Luukkanen, Department of Forest Ecology/Tropical Silviculture, Unit P.O.Box 28 (Koetilantie 3)
FIN-00014 University of Helsinki, Finland
Tel: +358 9 19158643, Fax: +358 9 19158646
Email: olavi.luukkanen@helsinki.fi
Website: http://www.honeybee.helsinki.fi/tropic/

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MOLECULAR GENETICS OF FAIDHERBIA ALBIDA (DEL.) A. CHEV.

By: Otto George Dangasuk

Faidherbia albida (Del.) A.Chev. (syn. Acacia albida Del.), a leguminous tree species belonging to the Mimosoideae subfamily, is an important agro-silvo-pastoral species in arid and semi arid lands throughout Africa. Its unique phenology of shedding leaves during the rainy season at the time of higher microbial activities in the soil improves the soil structure, stability and permeability, and provides a microclimate favorable for crops. Retaining the leaves in the dry season, it provides shade and green fodder rich in protein and carbohydrates for livestock. The resulting mulch reduces evaporation thus conserving the available soil moisture. F. albida has a remarkable capacity for recycling nutrients from underground to the surface due to its very deep root system. The tree also stabilizes sand dunes and prevents soil erosion. F. albida does not compete with inter-planted crops for soil nutrients as it enters a period of physiological rest during the crop growing season. It is also used for timber, medicines and food.

In Africa drought and human interference have endangered the genetic resources of this species, prompting its inclusion among the endangered plant species. It has not been domesticated, and it is little used outside its natural range. Plantation projects with this species in Chad and Niger have failed, possibly due to lack of basic knowledge on the amount of genetic diversity and breeding systems.

In our studies, an analysis of genetic variation based on morphological characteristics in twelve and sixteen African provenances of Faidherbia albida in Kenya, although it showed consistent genetic variation across Africa, could not separate the eastern provenances from the southern African ones, probably due to environmental influence and epistatic gene effects (Dangasuk et al. 1997; Dangasuk, 1999). This analysis was not effective in determination of genetic diversity or the phylogenetic relationship among the provenances.

Molecular and biochemical techniques provide a powerful set of tools for the study of plant population genetics. Isozyme analysis in F. albida shows significant deviation from Hardy-Weinberg equilibrium, deficiency in heterozygotes and less differentiation among provenances (Harris et al, 1997; Dangasuk and Gudu, 2000). This could be due to inbreeding; however, a high rate of inbreeding or selfing would have generated considerable genetic differentiation between provenances, which was not detected in the isozyme data. In addition, isozyme results do not allow detailed analysis of genetic structure within provenances of F. albida and hence firm conclusions on phylogenetic relationships among populations cannot be drawn (Dangasuk and Gudu, 2000). Reviews on the levels of variation detected in a range of plant species based on isozyme data reveal that tropical tree species maintain most of their variation within populations (Hamrick, 1993). This suggests that the classical forestry approach, which considers provenance or geographic variation as an accurate predictor of the diversity spectrum within species, may be inappropriate.

For better understanding of population genetics of F. albida, for the purpose of germplasm conservation, there is need to use markers able to show more variations, which would allow the identification of individuals within populations that are genetically different. Restriction fragment length polymorphism (RFLP) is the commonly used DNA marker. RFLP requires large quantities of pure DNA; species-specific DNA probes, and generally uses short-lived radioisotopes in the detection system. Furthermore, RFLP analysis is laborious; making it impractical for many populations based studies. Polymerase chain reaction (PCR) is less technically demanding than RFLPs and requires only a small amount of DNA. In addition, PCR provides flexibility in detecting genetic variation as a variety of primers can be used which are design to reveal particular types of polymorphism (Rafalski and Tingey, 1993). PCR methods include microsatellites, endonuclease restriction of amplified products (ERAP), single–strand conformation polymorphism (SSCPs) and random amplified polymorphic DNA (RAPD).

Unlike other approaches, RAPD analysis requires no prior DNA sequence information and it relies on single short, random oligonucleotides for amplification of unspecified target DNAs. RAPDs therefore represent a PCR-based technology that is immediately applicable to organisms from diverse taxa and, because of the large number of primers available for analysis, potentially provides good overall genome coverage (Williams et al. 1990). Therefore RAPD method capable of detecting individual differences in populations will be used in a study of genetic diversity in sixteen four-year-old F. albida provenances representing the natural distribution range of this species in Africa. The DNA will be nuclear ribosomal DNA, which is well suited for evolutionary and phylogenetic study. The sixteen provenances are currently growing in a randomized complete block design (RCBD) with five replications in the semi arid Baringo District of Kenya.

The long-term objective of the research is the conservation of the genetic resources of F. albida in Africa. The specific objectives are: (1) determination of the phylogenetic relationship among the 16 provenances in order to establish the species center of origin; (2) determination of the extent of genetic diversity in F. albida, using the PCR method of RAPD; and (3) study of individual tree genetic variability using RAPD. Combination of morphological, isozyme and DNA data will then form the basis for conservation, domestication, breeding and improvement of this species in different regions of Africa.

References
Dangasuk, O.G.; Seurei, P. and Gudu, S. 1997. Genetic variation in seed and seedling traits in 12 African provenances of Faidherbia albida (Del.) A.Chev. at Lodwar, Kenya. Agroforestry Systems 37:133-141.
Dangasuk, O.G. 1999. Genetic diversity in Faidherbia albida (Del.) A. Chev. Ph.D. Thesis. Moi University, Eldoret, Kenya.
Dangasuk, O.G. and Gudu, S. 2000. Allozyme variation in 16 natural populations of Faidherbia albida (Del.) A. Chev. Hereditas 133:133-145.
Hamrick, J.L. 1993. Genetic diversity and conservation in tropical forests. Department of Botany and Genetics, University of Georgia, Athens, GA 30602 USA 9pp.
Harris, A.S., Fagg, C.W. and Barnes, R.D. 1997. Isozyme variation in Faidherbia albida (Leguminosae, Mimosoideae). Pl. Sys. Evol. 207: 119-132

For further information please contact:
Otto Dangasuk, Department of Botany, Moi University, P. O. Box 1125, Eldoret, Kenya.
Email: georgedangasuk@yahoo.com

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ASSESSING GENETIC VARIABILITY IN LEUCAENA HYBRIDS: ISOZYMES AS MARKERS

By Maria Teresa Schifino Wittmann and Liane Helena Cardoso de Freitas

Assessing the genetic variability is the first, essential step for plant breeding that indicates what can be done using morphological, physiological, cytological, agronomic and molecular characteristics. Isozymes are good markers, since most of them have simple inheritance, co-dominant expression, complete penetrance and no pleiotropic and epistatic interactions. In addition, they are selectively neutral. The isozyme technique is rather inexpensive as compared to DNA techniques. A research line is being developed by our group with hybrids between the legume trees Leucaena leucocephala and L. diversifolia ssp. diversifolia, aiming to select cold-tolerant plants adapted to the climatic conditions of Rio Grande do Sul (Southern Brazil), to be used as forage specially during the winter and also as an alternative protein bank throughout the year. In this case, isozymes, apart from estimating the population variability and possibly characterising the species, may also provide some markers related to characteristics of agronomic interest . Morphological and phenological variability showed to be expressive (Freitas et al., 1995). Among the three isoenzyme systems (esterase, superoxide dismutase, and malic enzyme) studied, two were polymorphic (EST and SOD), and at least for SOD, species-specific results were detected (Schifino-Wittmann et al.,1996). Twenty selected progenies of these plants are now being analysed. Morphological and physiological studies identified genotypes with good forage production and also cold-tolerance (as measured by leaf retention during winter; Simioni et al. in preparation). Besides EST, SOD and ME, MDH (malate dehydrogenase) was also examined in the selected progenies. Intrafamily variation was found for EST, SOD and MDH. In a further step of assessing the genetic variability, DNA techniques are planned to be used, in order to provide a thorough view of the existing variability at different levels of biological organisation.

For reference publications and further information please contact:
Maria Teresa Schifino Wittman, Departamento de Plantas Forrageiras e Agrometeorologia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul.
Caixa postal 776 91501-970, Porto Alegre, RS Brasil
Email : mtschif@vortex.ufrgs.br

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RAPD ANALYSIS OF THE GENUS BAMBUSA "SENSU LATO"

By Ye Sun, Nianhe Xia and Rushun Lin

Bamboo species of the genus Bambusa are distributed in the subtropical and tropical areas of Asia and the tropical areas of the American continent. There are about 70 species and four subgenera in southern and southwestern China. Subgenus Bambusa includes the armed species and subgenus Leleba accommodates the unarmed species of the genus. Subgenus Lingnania and subgenus Neosinocalamus were established by Chia and Fung when they revised the species of the genus Bambusa in China. At present, molecular markers provide a very powerful tool to study the biodiversity of bamboos. Random amplified polymorphic DNA (RAPD) is widely used in genetic diversity analysis because of its simplicity and low expense. In our research, we employed RAPD molecular markers to study the genetic relationships of species within Bambusa "sensu lato".

Method
Fifteen species were studied in our research, including members of the subgenera Bambusa, Leleba, Lingnania, Dendrocalamopsis and Dendrocalamus. Sixteen primers, selected from sixty primers, were used in amplification. The genetic distances were calculated between each two species by using Nei's genetic distance. The UPGMA (Unweighted Pair Group Method of Arithmetic Average) algorithm was made on the basis of distance matrix.

Results
The results were as follows:

For further information, please contact:
SUN Ye
Center of Systematic and Evolutionary Botany, South China Institute of Botany, Academia Sinica
Guangzhou 510650, P.R. China.
Fax: (++86)(020) 85232831
E-mail: sun-ye@scib.ac.cn

XIA Nianhe
Center of Systematic and Evolutionary Botany, South China Institute of Botany, Academia Sinica
Guangzhou 510650, P.R. China.
Fax: (++86)(020) 85232831
Email: nhxia@scib.ac.cn

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GENETIC DIVERSITY IN NATURAL POPULATIONS OF EUCALYPTUS MICROTHECA

By Chunyang Li and Olavi Luukkanen

Eucalyptus microtheca F. Muell. is characteristically a species of open woodlands with a wide and patchy natural distribution in the arid and semi-arid zones of Australia. Different environmental conditions in its native habitats, such as seasonal change in water availability, have resulted in large intra-specific variation in leaf physiology and growth performance (Li, 1999). E. microtheca has great value as plantation species especially on marginal lands, since it tolerates heat, prolonged drought and calcareous and gypsum soils. In the Sudan, for instance, E. microtheca is the principal plantation tree species. However, there has been relatively little attempt to use biochemical and molecular techniques to determine the genetic relationships among E. microtheca populations. In our studies, isozyme and random amplified polymorphic DNA (RAPD) markers were used to estimate genetic variation in 12 natural populations of Eucalyptus microtheca populations (Li, 1999).

Isozyme markers were analyzed at 13 loci in 9 enzyme systems. The mean number of alleles per locus was 2.25, and 62.2% of the loci were polymorphic (95% criterion for polymorphism). The mean observed and expected heterozygosities were 0.150 and 0.199, respectively. Most of the diversity was located within populations; only 10.36% of the total genetic diversity was due to differentiation among populations. Cluster analysis based on unbiased genetic distance and the UPGMA dendrogram revealed limited genetic distance among populations.

RAPD markers were analysed with 18 primers; the primers yielded a total of 102 polymorphic bands, i.e. an average of 6 polymorphic bands per primer. Molecular markers were used to calculate the similarity coefficients, which were then used for determining genetic distances between the populations. Based on genetic distances, a dendrogram was constructed. Gene diversity values for each population ranged between 0.176 and 0.232 with an average of 0.200. Total gene diversity for this species was 0.240, where 83.3% of the variation was found within populations and 16.7% between populations.

Therefore, according to these isozyme and RAPD analyses, in E. microtheca most of the genetic variation was found within each population and there was high genetic similarity among the natural populations. Levels of genetic diversity were similar to those observed in other Eucalyptus species which also have widespread distributions.

Reference
Li, C. 1999. Drought adaptation and genetic diversity in Eucalyptus microtheca. Doctoral thesis.Univ. Helsinki Tropical Forestry Reports 19. 33 p. + app.

For further information please contact:
Olavi Luukkanen
Department of Forest Ecology/Tropical Silviculture
Unit P.O.Box 28 (Koetilantie 3) FIN-00014 University of Helsinki, Finland
Tel: +358 9 19158643, Fax: +358 9 19158646
Email: olavi.luukkanen@helsinki.fi
Website: http://www.honeybee.helsinki.fi/tropic/

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ASSESSMENT OF LEVELS AND DYNAMICS OF INTRA-SPECIFIC GENETIC DIVERSITY OF LATIN AMERICAN TROPICAL TREES FOR CONSERVATION AND SUSTAINABLE MANAGEMENT

By Andrew Lowe, Eric Bandou, Peter Breyne, Henri Caron, Stephen Cavers, Nathalie Colpaert, Bernd Degen, Rogerio Gribel, Marvin Hernandez, Antoine Kremer, Patrick Labbe, Maristerra Lemes, Rogerio Margis, Marcia Margis-Pinheiro, Marc van Montagu, Carlos Navarro and Julia Wilson

Genetic variation is of paramount importance to the long-term survival of forest ecosystems, since its level and distribution will determine the forests' ability to adapt to changing environmental conditions. Tropical forests are under increasing threat, either directly through exploitation of their products, or indirectly through conversion to other land uses. However, very little is known about the effects of these disturbances on the genetic diversity of forest tree species. It is therefore essential to acquire a good understanding of the level and distribution of diversity in natural populations, before it is possible to predict or measure the impact of any ecosystem disturbance. Little work of this nature has so far been undertaken, and thus our ability to assess the scale of effect of human impacts such as logging, domestication, and fragmentation, is very limited.

The structure of genetic diversity at the population level is determined by factors such as the reproductive biology, and seed and pollen dispersal. The effect of human disturbance on levels of diversity therefore also depends, to some extent, on the disruption it causes to the mating system and gene flow of a target species. Selective logging, for example, may lead to an increase in inbreeding, which results in a reduction of genetic diversity, while forest fragmentation may disrupt pollinator behaviour, and thereby alter patterns of genetic diversity and gene flow.

Approach and methodology
The study we have completed has utilised recent developments in molecular genetics, which have produced many powerful, PCR-based techniques. Chloroplast (PCR-RFLP and sequencing) and nuclear markers (AFLPs and SSRs) were used, as appropriate, to assess levels of genetic diversity, seed flow and pollen flow, by considering the maternal and biparental inheritance of markers.

Activities
The project analysed the level and distribution of diversity in several tree species from different ecosystems in Latin America, and evaluated the effects of human influence upon diversity within these species. As the selected species represent a broad range of model characteristics related to human impacts and life history, this project should enable the identification of key factors likely to modify diversity levels in forest tree populations of importance throughout tropical forest ecosystems.

From the Atlantic coastal forest of Brazil, which has been reduced to about 2% of its original extent and is now internationally recognised as a priority for tropical moist forest conservation, attention focussed on two species, Eugenia uniflora (Myrtaceae) and Anacardium occidentale (Anacardiaceae). In addition, from the Brazilian Amazonian forest, of which about 15 % has been degraded, and which is under ever-increasing pressure, two other species were examined, Swietenia macrophylla (Meliaceae), the ‘big leaf' mahogany, and Ceiba pentandra (Bombacaceae). From French Guyane, species were selected for their contrasting life history traits that are likely to affect levels of genetic diversity, and included the following: Symphonia globulifera (Clusiaceae), Moronobea coccinea, Dicorynia guianensis, Sextonia rubra, and Cecropia sciadophylla (Cecropiaceae). In addition, Tabebuia heterophylla (Bignoniacea) from the Caribbean Islands was investigated.

In Central America, attention focussed on Cedrela odorata (Meliaceae) and Costa Rican populations of Swietenia macrophylla (Meliaceae), Vochysia ferruginea (Vochysiaceae) and Lonchocarpus costaricensis (Leguminoseae), all timber or multipurpose trees of high economic and ecological value.

Conclusions
Molecular markers highlighted important differences in the partitioning of diversity within and among populations. In addition, the role of different breeding systems and colonising strategies in determining diversity was emphasised.

For example, studies indicated that chloroplast DNA variation was generally low, and often showed strong structuring related to population, geographic region and even ancient geological events. However, one species, Symphonia globulifera, showed very high variation and it is thought that cpDNA inheritance in this species may not be strictly maternal, as is usually the case.

In contrast to some other studies, it was difficult to relate the level and distribution of genetic diversity to pollinator or population density. In a study of Symphonia globulifera, it was emphasised that the territorial behaviour of pollinators and flowering patterns could be influential. While bird-pollinated Symphonia had low outcrossing rates, the bat-pollinated Ceiba pentandra showed long pollination distances (~18 km) and highlighted the influence of bat territoriality on genetic diversity.

Results for Swietenia macrophylla (mahogany), from both Central and South America, indicate that this commercially important and heavily logged species is very sensitive to disturbance, and may take generations (100s of years) to recover. Careful management is crucial to prevent populations descending into a downward spiral of genetic resource loss.

These results are only a small subset of those obtained within the project. Overall they highlight the value of molecular approaches for developing conservation and resource management strategies. Thus when challenged by different types of environmental change, species respond differently.

This project has been of enormous benefit to all the partners. The bringing together of high-level molecular expertise with the expert knowledge of distributions and history of populations of tropical tree species has been invaluable. Furthermore, the breadth of the project, which has tackled aspects of the molecular genetics of more than 14 different tropical tree species from a wide range of habitats and with a diversity of breeding systems, has been a tremendous strength. It is hoped that the quality of the collaboration will continue into a third phase of the project, which is about to start, and is be funded under the 5th Framework Programme (GENEO-TROPECO ICA4-2000-20056) of the EU, whom we gratefully acknowledge for their support and encouragement during all phases of this work.

Main Publications
For a complete list of publications, including theses and poster presentations, visit http://www.nbu.ac.uk/inco/referenc.htm

For further information please visit the project website:
http://www.nbu.ac.uk/inco or contact any of the following partners:

Andrew Lowe, Stephen Cavers, Julia Wilson
Centre for Ecology and Hydrology-Edinburgh, Tropical Forests Section, Bush Estate, Penicuik, Midlothian, EH26 0QB, UK.
alowe@ceh.ac.uk, scav@ceh.ac.uk , jwi@ceh.ac.uk,
http://www.nbu.ac.uk/tropical

Carlos Navarro, Marvin Hernandez
Centro Agronómico Tropical de Investigación y Enseñanza (CATIE), Turrialba, 7170, Costa Rica.
cnavarro@computo.catie.ac.cr,
http://www.catie.ac.cr/catie/

Henri Caron, Bernd Degen, Eric Bandou, Patrick Labbe, Antoine Kremer
Institut National de la Recherche Agronomique, Laboratoire de Genetique et d'Amelioration des Arbres Forestières, PO Box 45, Gazinet Cestas, 33611, France.
henri.caron@pierroton.inra.fr, degen.b@kourou.cirad.fr, Antoine.Kremer@zouk.pierroton.inra.fr,
http://www.jouy.inra.fr/

Rogerio Margis, Marcia Margis-Pinheiro
Universidade Federal do Rio de Janeiro, Laboratorio de Genetica Molecular Vegetal, Instituto de Biologia, CCS – Ilha do Fundao Instituto de Biologia, CEP 21944-270, Rio deJaneiro. Brasil.
margisr@ufrj.br, margism@biologia.ufrj.br,
http://www.biologia.ufrj.br/labs/lgmv/

Rogerio Gribel, Maristerra Lemes
Laboratory of Genetics and Reproductive Biology of Plants, Instituto Nacional de Pesquisas da Amazonia, Avenida André, Araújo, 2936, Manaus, AM, Brazil.
rgribel@inpa.gov.br, mlemes@inpa.gov.br,
http://www.inpa.gov.br

Peter Breyne, Nathalie Colpaert, Marc van Montagu
Vlaams Interuniversitair Instituut voor Biotechnologie (VIB) and Institute for Plant Biotechnology for Developing Countries (IPBO), Department of Molecular Genetics, University of Gent, K. L. Ledeganckstraat 35,B-9000 Gent, Belgium.
pebre@gengenp.rug.ac.be, Marc.VanMontagu@rug.ac.be,
http://www.vib.be, http://www.plantgenetics.rug.ac.be/

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GERMINATION SYNDROMES AS TOOLS FOR PLANT FUNCTIONAL TYPES

By Enrique Jurado

Traits associated with seed germination may be of great value for natural resource management if grouped into sensible functional types. For instance knowing the proportions of a given flora that germinate in particular seasons, as well as their germination speed, would help natural ecosystem management both for production purposes and large scale conservation plans associated to climate change. In this article I present some information for the Tamaulipan thornscrub (matorral) in northeastern Mexico from research mainly supported by IFS, CONACYT, and PAICYT.

Seed mass, dispersal syndromes and other plant attributes were investigated for 111 species native to northeastern Mexico. Tests were conducted to investigate whether seeds from Mexico conformed to patterns of seed size and dispersal syndrome spectra found for floras in arid environments from around the world. The distribution of seed mass in the Mexican flora (0.03 mg to 598 mg) was generally similar to that found elsewhere. All of the major seed dispersal syndromes previously found in arid environments were represented in Mexico, although vertebrate dispersal (33 species) was unusually common. There were 31 species with wind-dispersed seeds, 4 ant-dispersed and 43 with no obvious dispersal morphology. Woody species were more likely to have large seeds and herbaceous species were more likely to have small seeds. Woody plants had proportionally more wind-dispersed and less animal-dispersed species than did herbaceous plants. We did not find the expected relationship between time of seed set of vertebrate-dispersed species and the presence of migratory birds or between time of seed set and optimum germination time.

In semiarid environments plants should be selected to germinate at times most suitable for seedling establishment. Germinability and the rate of germination and temperature preference for germination (12°C or 28°C constant) were analyzed in respect to growth form (grass, forb, woody), longevity and seed size. Short-lived species showed a preferential germination at either high or low temperature, whereas long-lived species tended to be season-indifferent. Germinability was >20% for 28 species, < 10% for 17 species, and 21 species did not germinate under any circumstances. Growth form or lifespan did not influence germinability. Woody species germinated rapidly. Germinability and germination rate were positively associated.

Seed germination was investigated for 47 herbaceous and woody species, representative of the Tamaulipan thornscrub flora of northeastern Mexico. More than half of the species had similar germination under spring and autumn conditions, 17 species showed a higher germination percentage under autumn rain conditions. No species had more seeds germinating under spring rain conditions. Germination season response was independent of plant habit, seed size and dispersal syndrome. Sixteen of the species germinated less under the moderate shade ofthornscrub canopy than under direct sunlight, and more than half of the species had similar germination under shade and direct sunlight. Germination in autumn, and away from the root competition of thornscrub, may reflect the importance of soil drought in mortality of young seedlings.

References
Jurado, E., Navar, J., Villalón, M. & Pando M. 2001. Germination associated with season and sunlight for Tamaulipan thornscrub plants in Northeastern Mexico. Journal of Arid Environments (In press).
Jurado, E., Estrada, E. Y Moles, A. 2001. Characterizing plant attributes with particular emphasis on seeds in Tamaulipan thornscrub in semiarid Mexico. Journal of Arid Environments 48:309-321.
Jurado, E., Aguirre, O. Flores, J., Navar, J., Villalon, H. & Wester, D. 2000. Germination in Tamaulipan thornscrub of Northeastern Mexico. Journal of Arid Environments 46:413-424.

For further information please contact:
Enrique Jurado Facultad de Ciencias Forestales, UANL, A.P. 41, Linares, N.L. 67700 México
Email: ejurado@fcf.uanl.mx

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TISSUE CULTURE PILOT PLANT FACILITY FOR UPSCALING AND TESTING TECHNO-COMMERCIAL FEASIBILITY OF FOREST TREE TISSUE CULTURE AT THE NATIONAL CHEMICAL LABORATORY, PUNE, INDIA

By R. S. Nadgauda

A tissue culture pilot plant facility with a production capacity of 1 million plants per year with greenhouse was designed at the National Chemical Laboratory (NCL), Pune, with financial support from the Department of Biotechnology, New Delhi, and commissioned in 1992. This facility has a semiautomated, fully air-conditioned laboratory and a greenhouse, monitored by an advanced computer system. The forest tree species being multiplied in this unit, E. tereticornis, E. camaldulensis (eucalypts), Dendrocalamus strictus (bamboo) and Tectona grandis (teak) were selected on the basis of their economic importance.

The techniques for all these plant species reported earlier in the 1980's were refined considerably. The extensive research and development work carried out was focussed on the following points: Development, refinement and upscaling of protocols by reduction in the number of stages, use of minimal media, rapid multiplication rate and improved survival rates; Methods for successful transportation of plants from greenhouse to the field; Field verificatory trials.

The main features of this project were identification of superior genotypes and cloning of these using tissue culture. Extensive research was carried out to develop the process for mass propagation of the identified genotypes. These were then planted in the field for evaluation. Over 1.5 million plants of all the above-mentioned species were planted all over India in different agroclimatic regions.These trials were undertaken in collaboration with forest departments, agricultural universities and the private sector or organisations.

The field trials were designed as progeny trials, provenance trials and clonal trials with the following objectives:

The growth data collected from different sites and analysed revealed that the tissue culture plants exhibit high uniformity and higher biomass, leading to early rotation and indicating the possibility to use this technology to increase the production per unit area.

Advantages of tissue culture-raised plants over conventional plants.
Field trials were conducted at more than 300 different locations in different states of India and Nepal covering an area of 1500 ha. The field performance of tissue culture-raised plantlets as assessed from different trials was as follows :