Full text of DOI:10.1007/s11627-000-0027-1

In Vitro Cell. Dev. Biol.—Plant 36:133–136, March–April 2000
᭧ 2000 Society for In Vitro Biology
1054-5476/99 $10.00ϩ0.00
FOREST BIOTECHNOLOGY ’99 —A JOINT MEETING OF THE INTERNATIONAL WOOD BIOTECHNOLOGY
SYMPOSIUM AND THE IUFRO WORKING PARTY FOR MOLECULAR GENETICS OF TREES, 11 –16 JULY,
1999, OXFORD, UK†
D. ELLIS1* and T. STRABALA2
2
1BC Research, Inc., Vancouver, B.C., Canada; Genesis Research and Development Corp. Ltd., Auckland, New Zealand
What better way to keep oneself in perspective when attending the
premier forest biotechnology meeting of 1999 than to walk each day
into a huge glass-roofed edifice, one of the finest examples of neo-
Gothic architecture in England, only to be towered over by the
ancient skeletal remains of an iguanodon, an enormous Cretaceous
dinosaur. Among the other dinosaur skeletons and eggs one had to
pass on the way to the conference room was a display which served
as an inspiration to Lewis Carroll for Alice in Wonderland
containing the most complete remains in existence, a head and
foot, of the now extinct dodo. The Oxford University Museum of
Natural History, site in July 1860 of the infamous Wilberforce and
Huxley debate on creation versus evolution, was also the site of
Forest Biotechnology ’99 almost 140 years later.
The aim of the meeting was to provide a forum to present the most
current advances in the application of molecular biology and
genetics to forest trees. Over 200 scientists from 30 countries
shared information focused on forest tree species covering a wide
range of topics, including in vitro culture, genetic engineering,
molecular analysis of developmental, adaptive and physiological
traits, cell wall biosynthesis and modification, and genomics and
genome mapping. Like the evolution debate of the nineteenth
century, this meeting produced considerable debate and some
external attention because, in addition to the ‘traditional’ scientific
presentations, the meeting played host to an anti-biotechnology
presentation and was also the site of an anti-biotechnology protest
organized by Genetically Engineered Free Forests (GEFF), a group
assembled specifically in response to the meeting. In addition, on
the opening night of the meeting the only field trial of transgenic
trees in the UK, testing trees engineered to suppress lignin
production (CAD antisense), was destroyed.
learn how to grow better wood. He announced that he and a large
group of collaborators were close to final approval for a National
Science Foundation pine genome project with a goal of sequencing
50 000 pine xylem ESTs and making these sequences accessible to
the public. We have since heard that he and his colleagues were
awarded the 3 yr grant. One very exciting element of the project is
that this study will focus on wood formation and include
components for microarrays and mapping.
Numerous advances were reported in tissue culture, principally
in the application of somatic embryogenesis technology. Sara von
Arnold from the Swedish University of Agricultural Sciences in
Uppsala gave an excellent overview of somatic embryogenesis in
conifers and detailed her work with transformation of Norway
spruce (Picea abies). She also highlighted work with lipid transfer
proteins and their regulation in embryo maturation. This presenta-
tion was followed by inspiring talks on progress in somatic
embryogenesis in Eucalyptus globulus (J. Oller, ENCE-DIT,
´
Spain), Theobroma cacao (cocoa) (A. Fontanel, Nestle, France),
Pinus pinaster (A. Ramarosandratana, Laboratoire des Ressources
du Futur, France), Liquidamber styraciflua (S. Merkle, University of
Georgia, USA) and shoot cultures in Tectona grandis (teak) (S.
Widiyanto, Institut Teknologi Bandung, Indonesia). Of special
interest in these talks was the use of floral tissues for the initiation
of embryogenic cultures in Theobroma and Liquidamber and the fact
that in both systems staminate floral parts usually responded better
than other floral organs.
The subject of the meeting then switched to transformation with a
talk on advances with the use of Agrobacterium tumefaciens to
transform conifer tissues, in this case white pine, Pinus strobus (A.
Seguin, Canadian Forest Service, Canada). This work also provided
evidence that SARs decreased the variability of gene expression
among independently transformed lines. Alternative selection
systems for transformed tissue have been a goal for many
laboratories and success with the use of the Multi-Auto Transfor-
mation Vector system (MAT vectors) was reported (H. Ebinuma,
Nippon Paper Industries, Japan). The MAT vectors use an ipt gene
to induce organogenesis. The ipt gene is eventually deleted by the
activation of a co-transformed site-specific recombinase acting on
excision sites that flank the recombinase and ipt genes for removing
the gene. Although this work was first reported in 1997 (Proc. Natl.
Acad. Sci. USA 94:2117–2121), the talk focused on numerous new
extensions of the system such as the use of rolA, B, C and D
fragments and the fact that the MAT systems yield a tenfold
increase in the recovery of transgenic plants relative to NPT in their
hands. Additional reports on transformation included Ulmus spp.
The meeting opened with a keynote address by Ron Sederoff
(North Carolina University, USA) in which he succinctly summed
up the history of forest biotechnology. He reminded us that in the
mid-1980s everyone was saying that they ‘didn’t believe biotech-
nology would work in trees’. By the early 1990s people were saying
that it ‘might work but that it hadn’t yet been proven’. And now, in
1999, everyone says they ‘knew it was a good idea all along’. Ron
went on to further remind us that, despite all the tremendous
progress being made, we still face the same fundamental problems,
in that the world’s forests are still threatened and that we need to
*Author to whom correspondence should be addressed; Email elvis@
silvagen.com
†http://users.ox.ac.uk/ϳdops0022/conference/forest_biotech99_home.html
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ELLIS AND STRABALA
(elm) (K. Gartland, University of Abertay, UK) and an investigation
of methylation patterns of rolC inserts in Populus tremula and how
they may relate to gene silencing (M. Fladung, Institute for Forest
Genetics, Germany).
using an OMT promoter–GUS fusion to show how this gene may be
involved in the differential deposition of lignin in different tissues.
In addition, Boerjan discussed results with the suppression of
caffeoyl-CoA-O-methyltransferase where transformed plants con-
tained brown-stemmed phenotype, similar to CAD suppressed
plants, in addition to increased pulping efficiency. C. Halpin and
co-workers (University of Dundee, UK) are working on methods to
coordinately under- and over-express genes. On the over-expression
side, they are taking advantage of a viral polyprotein cleavage
system to produce polycistronic transcripts of lignin genes which
are then processed by viral proteases. Expression in tobacco has
given good coordinate regulation. On the gene suppression side,
Halpin’s group is experimenting with artificial genes containing
parts of two or three open reading frames from genes encoding
enzymes in the lignin biosynthetic pathway. In addition to
demonstrating that these systems work for the coordinated
expression of two or more genes, their results with the double
antisense inhibition of cinnamyl alcohol dehydrogenase (CAD) with
either O-methyltransferase (OMY) or cinnamyl CoA reductase
(CCR) resulted in male sterile plants. L. Jouanin (INRA, France)
presented the first paper providing evidence that lignin modifica-
tion may be linked to cellulose synthesis as she found a correlation
in plants with decreased OMT and increased cellulose content. She
also found that strong antisense suppression (Ͼ95%) is required
before any phenotypes are observed. J. Grima-Pettenati (Universite
Paul Sabatier, France) concluded the information-packed morning
session with results from tobacco with decreased CCR levels where
an increase in syringyl/guiacyl ratio was observed, as well as the
modification of other cell wall components such as phenolics and
cellulose.
Perhaps the two most significant talks of the meeting were the
back-to-back presentations which provided compelling data that the
lignin biosynthetic pathway proposed 30 yr ago is not the preferred
pathway in plants and, in fact, may not exist as we were all taught.
The pathway had been originally proposed to proceed from
phenylalanine through r-coumarate to ferulate, where it could
either be (1) channelled through coniferaldehyde to coniferyl
alcohol to produce guaiacyl residues or (2) through 5-hydroxy-
ferulate through sinapate, sinapaldehyde and sinapyl alcohol to
produce syringyl residues. Guaiacyl and syringyl subunits are the
two major residues comprising lignin in plants.
C. Chapple (Purdue University, USA) first suggested that syringyl
was not produced through ferulate but rather proceeded from r-
coumarate through r-coumaroyl-CoA and caffeoyl-CoA to feruloyl-
CoA, the precursor for coniferaldehyde. What was crucial regarding
this suggestion was that a gene he previously isolated and
characterized, ferulate 5-hydroxylase (F5H), which catalyzed the
conversion of ferulate to 5-hydroxyferulate, actually has a 1000-fold
lower km value for coniferaldehyde and coniferyl alcohol than it has
for ferulate. This indicates that the pathway for syringyl lignin in
plants likely proceeds through a 5-hydroxylation of coniferaldehyde
or coniferyl alcohol, rather than a 5-hydroxylation of ferulate.
V. Chiang (Michigan Technological University, USA) presented
some elegant organic and biochemical work which corroborated and
complemented the results just reported by Chapple. Again, these
results confirmed that the production of both syringyl and guiacyl
residues proceeds through caffeoyl-CoA to feruloyl and not through
ferulate as was previously proposed. Chiang’s observations
differed from those of Chapple in that Chiang did not observe the
The developmental process of embryogenesis has been investi-
gated by numerous laboratories from
a morphological and
biochemical viewpoint, yet we still lack good markers to aid us in
understanding parameters important for normal embryo maturation
and subsequently healthy plants. J. Cairney (Institute of Paper
Science and Technology, USA) presented one approach to
unravelling the genes involved in the various stages of embryogen-
esis with the use of a differential display to identify genes expressed
at various embryo developmental stages. He reported having a
database of over 500 cDNA clones sequenced and the initiation of
‘transcript profiling’ using DNA microarrays to sort out genes
important for different embryo developmental stages. This was
followed by other exciting talks detailing work on the cloning and
expression of various stress-inducible genes of potential commer-
cial importance. These included an ozone-induced pinosylvin-O-
methyltransferase cDNA from Pinus sylvestris (D. Ernst, GSF-
Institute of Biochemical Plant Pathology, Germany), a polyphenol
oxidase induced by wounding, such as insect feeding, from Populus
spp. (C. P. Constabel, University of Alberta, Canada) and the
characterization of an inducible metallothionin gene from
Casuarina glauca (C. Franche, IRD-GeneTrop, France).
‘There is probably no major biochemical process in plants that is
both so important and so poorly understood at the molecular level as
cellulose synthesis’ (Delmer and Amor, Plant Cell 7:987–1000;
1995). While this statement is still true, considerable progress has
been made in the past five years in our understanding of this
process, as evidenced by the four talks detailing different aspects of
cellulose synthesis. An overview of bacterial and plant cellulose
synthesis was given by D. Ellis (BC Research Inc., Canada) where
the numerous open questions regarding cellulose synthesis were
outlined. He presented work from his laboratory on increasing
cellulose levels by increasing the level of cellulose precursors and
alluded to information that may lead to our ability to modify
cellulose properties, such as microfibril size and angle. This was
followed by the exciting work on the isolation of a cellulose
synthase gene (PtCelA) from aspen and its xylem-specific and
stress-inducible expression (C. P. Joshi, Michigan Technological
University, USA). The expression of cellulose binding domains
(CBD) from cellulase genes was covered by Z. Shani (CBD
Technologies, Israel). This group observed increased plant height
and cellulose fiber length in plants expressing CBD under the
control of the CaMV 35S promoter or a CelA promoter. The session
finished with a talk on the role of cyclic-di-GMP in plant cellulose
synthesis (R. Mayer, Hebrew University of Jerusalem, Israel). The
dependence of cyclic-di-GMP for cellulose synthesis in bacterial
systems has been known for some time. However, whether it is
required for plant cellulose synthesis has previously been unknown.
The evidence presented using the cellulose synthesis inhibitor 2,6-
dichlorobenzonitrile (DCB) certainly suggests a role for cyclic-di-
GMP in plant cellulose synthesis.
Emphasis remained on the cell wall but shifted to the large
volume of work on lignin modification where more progress has
been made in trees than with any other application of genetic
engineering. An excellent overview of work done was given by W.
Boerjan (University of Gent, Belgium) where he presented data

FOREST BIOTECHNOLOGY ’99
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5-hydroxylation of coniferyl alcohol, although the assay conditions
used by the two groups were different. An additional observation
noted by Chiang was that the conversion of ferulate to 5-
hydroxyferulate was noncompetitively inhibited by coniferaldehyde
and the methylation of 5-hydroxyferulate was competitively
inhibited by 5-hydroxyconiferaldehyde.
Collectively, these two independent reports very clearly support
the notion that the pathway to sinapyl alcohol proceeds not through
ferulate, as has been believed for decades, but rather through
coniferaldehyde and coniferyl alcohol. Both have since been
published (Humphries et al., Proc. Natl. Acad. Sci. USA
96:10045–10050; 1999 and Osakabe et al., Proc. Natl. Acad.
Sci. USA 96:8955–8960; 1999). These data also resolve a question
that has been lingering for some time, in that 4-coumarate:CoA
ligase’s (4CL) ability to convert sinapate to sinapoyl-CoA has
always been doubtful. In light of these new data, this portion of the
pathway does not exist, thereby clarifying why this activity has been
difficult to detect. How sinapate esters are formed is now an open
question, as F5H is clearly required for the synthesis of these
compounds.
C.-J. Tsai (Michigan Technological University, USA) presented
work that has since been partially published (Hu et al., Nature
Biotechnol. 17:808–812; 1999) on plants with decreased 4CL
activity where significant increases were observed in plant growth,
including a 7–15% increase in cellulose, height growth, rooting of
cuttings and leaf sizes. M. Gray-Mitsumune (University of British
Columbia, Canada) discussed the use of antisense to suppress b-
glucosidase in spruce to aid our understanding of the role this
enzyme plays in lignin production. Although the plants are still
young, no significant changes in lignin levels have been observed.
J. Dean (University of Georgia, USA; see www.arches.uga.edu/
ϳjeffdean/JDeanUGA.html for pictures of the meeting) discussed
his work with a family of laccase genes in yellow poplar. To date,
his findings are similar to the b-glucosidase work, in that no
phenotype has yet been observed with antisense, whereas ectopic
expression resulted in severe abnormalities in the plants. L. Jouanin
(INRA, France) demonstrated how arabidopsis could be used for the
study of lignin biosynthesis and xylem differentiation with the use of
EST databases and a promoterless GUS fusion for xylem-specific
promoter trapping. A. Kawaoka (Nippon Paper Industries, Japan)
discussed his work on the isolation and characterization of a
tobacco PAL-box binding protein, NTLIM1, and presented evidence
that antisense suppression of this protein greatly reduces lignin
levels by coordinatively down-regulating at least three lignin
biosynthetic genes. The final talk of the day was by C. Grunwald
(University of Hamburg, Germany) where the examination of wood
in 35S-ROLC-transgenic aspen revealed highly abnormal cell wall
characteristics and a dwarfing phenotype which is caused by a
decrease in the number of cell divisions.
consequences of the use of GMOs, and the lack of democratic
process in the deployment of the technology. They see the benefits
of GMOs as short-lived, nonsustainable and not widespread. They
drew a distinction between morally opposed to GMOs and therefore
not needing sound scientific rationale for their opposition. For the
most part, this was an interesting segment of the meeting as neither
party had spoken to the other in an organized nonconfrontational
setting previously. Discussions continued for over 2 h the following
day between The Soil Association and a small group of the
scientists. These discussions did not drastically change the thinking
or operations of either party, but they did open up an understanding
that all of us involved hope in the future will foster less animosity
and greater cooperation to ensure that systems are put in place to
study and respond to concerns about the use of genetic engineering
in forestry.
S. DiFazio (Oregon State University, USA) followed The Soil
Association with results from his studies on the risks of outcrossing
of transgenic poplar. This is truly pioneering work in trees and is
focused directly at providing experimental tools to assess and look
at risk management to limit the spread of transgenic plants into the
environment. His studies included measurements on pollen flow,
setting up numerous field plots to look at regeneration of non-
engineered poplar to assess the rate at which regeneration happens,
and data collection to make a model to predict spread by pollen or
seed. Results on outcrossing indicate that most outcrossing
occurred within 10 m of the parent tree but 74% of the outcrossed
pollen came from beyond 1 km so that, clearly, the potential for
transgenic pollen spread is high in poplar. S. Maury (IBMP du
CNRS, France) then presented work with transgenic tobacco with
reduced lignin showing decreased resistance to tobacco mosaic
virus, as well as a decreased induction of cell wall phenolics
thought to be involved in a plant’s defence after wounding, although
this later phenomenon is not related to viral resistance.
An overview of several programs, both ongoing and proposed, was
presented starting with a UK Forestry program on releasing wild
cherry varieties (N. Hammatt, Horticulture Research International,
UK). D. Duncan (Monsanto, USA) discussed the proposed joint
venture between Monsanto, International Paper, Westvaco and
Fletcher Challenge Forests. The joint venture hopes to be a magnet
for forestry biotechnology worldwide and would focus on traits such
as herbicide resistance, improved growth rates and improved fiber
quality. J. Charity (Forest Research, New Zealand) then discussed a
program for the transformation of conifers as well as the
characterization and isolation of genes involved in reproduction.
S. Strauss (Oregon State University, USA) presented an overview of
the research being conducted by the Tree Genetic Engineering Co-
operative (TGERC) involving transgene flow, Bt and RoundUp
Ready field-grown poplars and the control of flowering in poplar.
With the spread of transgenes being a contentious issue, it was no
surprise that there were a number of talks investigating reproduc-
tion and reproductive structures in trees. O. Nilsson (Swedish
University of Agricultural Sciences, Sweden) continued the
discussion on flowering with his research investigating the
competence to respond to a flowering signal. His work suggested
that although a flowering signal such as LFY or AP1 was present,
some other factors must be in place for the plant to respond to these
homeotic signals and he discussed several arabidopsis mutants
which confirmed this hypothesis. This certainly helps explain why
numerous researchers have not had success in inducing early
The following day of the meeting was one of contrast as the
deployment of transgenic trees was discussed whilst an active
protest (complete with drums and someone dressed up in a
Frankenstein costume) against the use of genetically engineered
trees was going on outside the University Museum. The day began
with talks by representatives of The Soil Association (P. Holden and
M. Wenban-Smith; www.SoilAssociation.Org), an organization in
support of organic farming and completely opposed to the use of
GMOs. The concerns they raised included the impact on
biodiversity, the lack of containment, unknown and unforeseen

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flowering with the ectopic expression of these flowering genes. A.
Brunner (Oregon State University, USA) presented very clear data
on the expression patterns and characterization of poplar AG and
AP1 homologues. J. Skinner (Oregon State University, USA)
discussed work using a poplar DEF homologue, PTD, promoter to
control a cytotoxin gene in poplar. Co-transforming a 35S-LFY-
responding poplar genotype (early flowering) with this cytotoxic
construct, they have obtained preliminary evidence that suggests
flowering may be disrupted, although the experiments are still in
the very early stages. T. Sopanen (University of Joensuu, Finland)
presented work on the isolation of arabidopsis floral homologues
from birch and the induction of flowers in arabidopsis by expression
of a birch AP1 homologue.
R. Rutledge (Canadian Forest Service, Canada) continued the
discussion initiated by Nilsson on the competence to respond to a
flowering signal. He presented evidence that although a spruce AG
homolog and arabidopsis LFY have dramatic effects on flowering
when ectopically expressed in arabidopsis, identical constructs in
spruce have no effect on the formation of reproductive structures. A.
Collins (University of Oxford, UK), described work on phase change
in eucalyptus and the isolation of an apparent TFL/CEN ortholog
from eucalyptus which complements an arabidopsis TFL mutant.
Research involving the evolution of seed plants via analysis of
protein and carbohydrates in gymnosperm ovular secretions and
their role as barriers to pollination was discussed by P. von Aderkas
(University of Victoria, Canada). The final presentation on
reproduction dealt with somatic embryogenesis of Bambusa edulis
and the unusual phenomenon of a proliferating spikelet (spikelet-
produce-spikelet) culture in the presence of thidiazuron (W. Chang,
Academia Sinica, Taiwan).
Prior to the meeting, IUFRO had begun the process of having
each of its working parties draft a position statement on GMOs and
plantation forestry. The Working Party on Molecular Genetics was
further advanced with this process than other Working Parties, with
S. Strauss (Oregon State University, USA) having prepared a draft
position statement which was presented at the meeting and was the
subject of considerable debate. While many participants felt some
statement should be made in response to the public protests at the
meeting it was decided by a majority of the participants that the
position statement as written could not go forward for one reason or
another. The position statement has since been extensively revised
and circulated via email to all participants, where it was approved
by a majority response. The final position statement can be found at
www.fsl.orst.edu/tgerc/iufro pos-statm.htm.
The molecular analysis of tree genomes, although lagging behind
that of herbaceous species, is still being carried out by several
groups. I. Allona (ETSI Montes Ciudad Universitaria, Spain)
presented work on the sequencing of 1097 ESTs from Pinus taeda
compression wood. This study suggested that there are several
unique sequences in pine xylem that are not found in arabidopsis.
B. Sundberg (Swedish University of Agricultural Sciences, Sweden)
has taken a different approach and asked if arabidopsis could be
used to study wood formation. He presented data showing secondary
xylem formation in arabidopsis and differential effects on secondary
xylem formation with mutations in different ethylene receptor
genes. Sundberg ended with a brief introduction to the Swedish
poplar EST program where nearly 30 000 ESTs have been
sequenced. T. Strabala (Genesis Research and Development Corp.
Ltd., New Zealand) discussed what is probably the most ambitious
sequencing project in trees to date, the EST sequencing of
Eucalyptus grandis and Pinus radiata with over 100 000 ESTs
from each species, making this project comparable to that of
Drosophila. Numerous important variables involved in managing
such a project were discussed, including the fact that despite the
large number of sequences already done they are still obtaining
novel sequences at a frequency of one in every five. To date, this
project has sequenced ESTs from over 30 different libraries
including tissue cultured cells.
D. Neale (UDSA-Forest Service, USA) discussed the initiation of
an international collaboration, the Conifer Comparative Genomics
Project, in which the map of loblolly pine containing nearly 1000
genetic markers is being used as a basis to construct comparative
maps for all important pine species. To date, they have found good
conservation of gene order on chromosomes among pine species. T.
Kondo (Forest Tree Breeding Center, Japan) reported on progress
with markers in Pinus thunbergii to use in the selection of
resistance to pine needle gall midge. A. Seguin (Canadian Forest
Service, Canada) discussed recent results on the study of wound-,
salicylic acid- and jasmonic acid-induced 14-3-3 protein genes
from white spruce and hybrid poplar. C. Plomion (INRA, France)
discussed the use of the proteome for protein quantitative locus
analysis in Pinus pinaster and the generation of a proteomics
database. V. Storme (University of Gent, Belgium) discussed the
generation of three AFLP maps of Populus deltoides, P. trichocarpa
and P. nigra and the alignment of these maps using microsatellites.
These data have begun to be used to clone disease-resistant genes
in poplar. G. Moran (CSIRO Forest and Forest Products, Australia)
has been using RFLP markers and a three-generation pedigree to
map QTLs linked to frost tolerance, trunk diameter, height and leaf
area in Eucalyptus nitens. D. Prat (INRA, France), utilizing AFLP,
RAPD and ISSR mapping, has obtained 67 QTL markers with
various traits of interest in larch.
The final section of the meeting dealt with the application of
molecular genetics and biotechnology in conservation and with
novel species. M. Morgante (DuPont, USA) has used a microsatellite
approach to map Norway spruce grown in several central European
countries, giving ϳ2100 cM coverage of a 2840 cM genome.
Morgante and his collaborators have found a novel transposable
element in spruce which they call Alisei. They have developed
markers from this element’s LTRs and have begun to use it in an
AFLP-based approach using an LTR primer and EcoRI adapter
primers from digested genomic DNA. Five papers (missed by these
authors) followed, detailing further genomic characterization
through mapping of conifers, oak, wild cherry and the Honduran
species Swietenia humilis, as well as cryopreservation of
Eucalyptus.