This report has been submitted to the USDA on behalf of ISIS. Please
circulate widely and click onto the link to the USDA docket to register your
U.S. Department of Agriculture Animal and
Plant Health Inspection Service (USDA/APHIS) Biotechnology Regulatory Services
prepared an Environmental Assessment (EA) in response to permit application
(06-250-01r) received from Oregon State University for field-tests of transgenic Populus alba
and Populus hybrids. Comments
on the EA are due by 17 August 2007 at Docket APHIS-2007-0018 http://www.regulations.gov/fdmspublic/component/main.
field tests to be carried out are in relatively small plots on a single 320
acre open site, broadly separated into three areas. Location A includes trials
for reproductive sterility, gibberellin (GA) metabolism, reporter gene constructs
and ‘activation tagging’ mutants, and also includes the clone bank containing
trees that will not be allowed to flower. Two pairs of ramets (vegetative
clones) per event will be planted in a completely randomized design with trees
spaced at a distance of 6 to 10 ft. For certain trials, at least two pairs
of ramets per event will be planted in a completely randomized design with
trees spaced at 10 x 10 ft or 7 x7 ft. Location B will contain trials planted
in two blocks to measure competition and yield of transgenic trees modified
in GA metabolism. Each plot within respective blocks has 25 trees arranged
in a completely randomized design. Location C contains trees engineered with
the lignin modification gene. Two pairs of ramets per event are planted in
a completely randomized design with trees spaced at a 10 X 10 ft distance.
The list of the trees is given
below, followed by a brief description of the risks left out of consideration,
based on which we object to the field releases.
Trees allowed to flower
There are five categories of transgenic
poplars that will be allowed to flower, each category containing multiple
genes and gene constructs.
Genes conferring reproductive sterility
plants engineered with the barstar and barnase genes
derived from the bacterium Bacillus amyloliquifaciens are designed to confer male sterility.
Barnase (a ribonuclease) is expressed only in the tapetum cells of the anther’s
pollen sac during pollen development, resulting in the degradation of host
RNAs and arrest of cell development. This blocks pollen formation and results
in a male sterile plant. As barnase can kill Agrobacterium
(the vector organism used for the transformations) and can inhibit the regeneration
of transgenic plant cells, the barnase gene construct is accompanied by the
barstar gene that produces the specific inhibitor of barnase.
plants are also engineered with the DTA gene derived from the bacterium Corynebacterium
diphtheriae (the causal agent of diphtheria) encodes the A-chain of the diphtheria
toxin. Like the barnase/barstar construct, the preferential expression of
the DTA genes in floral tissues results in reproductive sterility.
reproductive sterility genes for PTLF, PTD, PTAG, PTAP, PMFT, PCENL, PSV,
PFCL, and PAGL24, all from Populus trichocarpa
andgenes for AG and AP1from Arabidopsis thaliana affect flowering and
flower formation, and are designed to reduce or eliminate flowering, pollen
production, or seed formation.
Genes conferring reduced stature/light response
reduced stature include those for GA metabolism: GAI and RGL1 from Arabidopsis
thaliana, PcGA2 OXI1 from Phaseolus
coccineus and PtaGA2 OXI1 from
Populus tremula x alba.
receptor genes for PHYB1 and PHYB2 from the Populus
trichocarpa were inserted to affect light response.
Gene that modify tree chemistry
The sequence for 4CL1 from Populus tremuloides was inserted to alter
lignin levels. This is an antisense copy of the 4-Coumarate CoA ligase (4CL)
gene, which results in a reduction
in the messenger RNA of the target 4CL gene. The 4CL leads to a major branch
point in phenylpropanoid metabolism. The product 4-coumaroyl:CoA
is a precursor for lignin and flavonoids. Lignin, cellulose, and hemicellulose
form the cell walls of xylem, which transports water and supports the tree.
Populus clone 717-1B4 were engineered
with a putative transcriptional regulator gene and a putative AP2 domain-containing
transcription factor, both from Populus tremula x alba.
These trees have been randomly hyperactivated
(activation tagging) with genetic mutations in an attempt to develop “experimental
Other activation tagging mutant constructs
contain only non-coding CaMV 35 S promoter regions and a selectable marker
gene, which are expected to create random mutations via native gene disruption
Some of the noncoding regulatory sequences
introduced as part of the genetic constructs in these poplars were derived
from the plants Nicotiana, Solanum,
Arabidopsis, and the plant pathogens cauliflower
mosaic virus, tobacco mosaic virus, Aspergillus nidulans, and A.
Trees that will not be allowed to flower (maintained in the clone bank)
A few transgenic
clones from earlier work are Populus
species or species hybrids Populus trichocarpa x Populus deltoids
of the sections Aigeiros and Tacamahaca. These will be retained in the cloned
bank and pruned frequently, and will not be allowed to flower; they contain
the Cry3A gene encoding a coleopteran-active insecticidal protein from Bacillus
thuringiensis, the Green fluorescent protein (Gfp) gene from Aequorea
victoria, the nptII gene from Escherichia
coli, the bar gene from Streptomyces
hygroscopicus, and the PTLF gene from Populus trichocarpa in various combinations
Genetic constructions are not adequately explained in the EA
The numerous genetic constructs
listed above will need a small army of trained technicians to do the required
monitoring. The task is well nigh impossible as the EA gives little or no
information on the structure of the transgenic constructs or their functions.
One can only conclude that the USDA/APHIS is rubberstamping the open release
of the largest collection of transgenes and transgenic constructs without
even a pretext at health and environmental risk assessment. We shall attempt
to point out some of the risks below, although that too, is well nigh impossible
on account of the scant information given, but we do our best.
Risks of transgenes not considered
The barnase toxin for cell ablation to control
flowering or pollen production has been used extensively with trees and with
crops. Barnase gene alone driven by a floral promoter tends to have leaky
activity in tissues other than the flowers, thereby reducing the growth of the transgenic tree. However,
when the barnase gene is accompanied by a gene for its natural inhibitor barstar
driven by a promoter giving moderately low level expression, there is enough
barnase activity left to ablate the floral tissue but not the vegetative tissues,
at lease in theory, though this has not been borne out in field trials .
It is clear that barnase in the leaves, stems, and roots of
trees will adversely affect not only the transgenic plant, but also the fauna
and flora of the forest ecosystem. The toxicity
of barnase to mammals is well known and we have pointed that out previously
[2, 3] (Terminator Trees, SiS 26; Chronicle of An Ecological Disaster Foretold,
The diphtheria toxin A chain has been used
as a cell suicide agent in transgenic plants but there does not seem to be
any published study on the safety in animals from eating transgenic plants
modified with the diphtheria a chain gene.
MADS-box genes are a large family of homeotic (regulators for development)
genes that control plant organs; for example, the gene for PTD determines
petal and stamen identity, the gene for PTAG determine stamen and carpel identity
and the gene for PTAP determine flower and perianth identity. These have been
altered by changing their native genetic code [4, 5] and are included in the
transgenic poplars proposed for the field trials. MADS-box transgenes should
not be presumed safe, as they are related to the extensively studied animal
homeotic genes that regulate development  and may well be active in animals.
negative mutants (DNM) are genes altered to produce proteins that interfere
with the function of the wild type gene from which they were derived. Arabidopsis DNM genes AP1 and AG with specifically
altered MADS domain are in the present collection. Also in the collection
are Populus genes for DNM and
PTLF to alter flower initiation, the gene for PCEN to repress flowering and the genes for PAGL24 and
PAGL to promote flowering.
of small RNA molecules that regulate genes by turning off their transcripts. Populus RNAi genes have been used to construct RNAi sterility
clones that have been co-transformed with multiple transgenes [4, 5]. RNAi
constructs raise special concerns as many
such RNAi sequences proved fatal to mice in gene therapy experiments  (Gene Therapy Nightmare for
Reduced stature/light response
Dwarf poplars are presumed to be desirable
because they cannot compete with tall trees should they escape to the wild.
Poplars were engineered with gibberellin (GA) metabolism genes.
are plant growth substances (phytohormones) involved in promotion of stem
elongation, mobilization of food reserves in seeds and other processes. Its
absence results in the dwarfism of some plant varieties. The DELLA proteins
are thought to act as repressors of GA-regulated processes, while GA is thought
to act as a negative regulator of DELLA protein function. Poplars engineered
with GA metabolism genes include the DELLA genes for
GAI and RGL1
from Arabidopsis thaliana, PcGA2
OXI1 from Phaseolus coccineus
and PtaGA2 OXI1 from Populus tremula
x alba. Arabidopsis DELLA proteins RGA and RGL2 jointly
repress petal, stamen and anther development in GA-deficient plants, and this
function is enhanced by RGL1 activity. PcGA2 OXI1 reduces gibberellins in
the plant cells and down regulates GA synthesis. Phytochrome receptor genes
for PHYB1 and PHYB2 from the poplar species
Populus trichocarpa were inserted
to affect light response, and hence the stature of the trees.
Phytochrome is a photoreceptor, a pigment that plants use to detect light.
It is sensitive to light in the red and far-red region of the visible spectrum.
Many flowering plants use it to regulate the time of flowering based on the
length of day and night (photoperiodism) and to set circadian rhythms. It
also regulates other responses including the germination of seeds, elongation
of seedlings, the size, shape and number of leaves, the synthesis of chlorophyll.
PHYB1 and PHYB2 are quantitative trait loci
concerned with bud set and bud flush in Populus.
[7,8,9]. The transgenes in these releases have not been studied
regarding any potential untoward effects,
The 4CL1 gene from Populus tremuloides inserted to alter lignin
levels. Genetic modification of lignin is a key alteration in producing trees
destined for bioenergy production. Low lignin trees are likely to be more
susceptible to pests and to be prone to wind damage because they lack mechanical
strength  (Low Lignin GM Trees
and Forage Crops, SiS 23). Aspen (Populus tremuloides) modified for reduced stem lignin
had normal cellulose content accompanied by reduced lignin content. The transgenic
aspen had reduced root carbon and greatly reduced soil carbon accumulation
compared to unmodified aspen. The trees accumulated 30% less plant carbon
and 70% less new soil carbon than unmodified trees . This makes the transgenic tree highly undesirable
in terms of reducing carbon in the atmosphere, hence defeating the whole purpose
of switching from fossil fuels to biofuels.
Activation tagging is insertional mutagenesis using insertion vectors that
contain strong transcription enhancer to up-regulate a gene near the insertion
site. The insertions appear randomly in the genome, resulting in gain of function
dominant mutations. The activated gene is easily recovered in organism such
as Poplar which has been fully sequenced . AP2 is an activating enhancer
binding protein, a member of a large transcription regulator family in plants.
The AP2 domain is a DNA binding recognition signal. The kind of mutation selection
described above is efficient, but is hardly safe for field test releases, as
it is likely to cause unintended insertional mutagenesis in a range of microorganisms
and animals that interact with the transgenic plants. Insertion mutagenesis
is a major cause of human cancers  (Slipping Through The Regulatory Net: ‘Naked’ and ‘free’ nucleic acids;
ISIS and TWN publication)
This is a potentially a very risky class
of transgenes as they contain numerous regulatory sequences affecting all
aspects of growth and development that have only been discovered within the
past five years  (Subverting the Genetic Text, SiS 24).
Trees that will not be allowed to flower
Trees that will not be allowed to flower
include poplars modified with the Bacilllus
thuringiensis (Bt) Cry 3A gene to control beetles  and those
modified with the PTLF gene controlling flower initiation . Gene flow
leading to transgene dispersal has been studied in hybrid poplars . Mechanical
pruning to prevent flowering seems risky in a large complex array of experimental
trees it seems inevitable that the transgenes will be dispersed.
Horizontal gene transfer especially important for transgenic trees
As for all transgenes, dispersal by horizontal gene transfer is a distinct
possibility, the extensive root system of trees in particular is a hotbed for
horizontal gene transfer and recombination, which is why we have called for
a Moratorium on all
GM Trees and Ban on GM Forest Trees  (SiS 35).
Most of the previous field tests and petitions for non-regulated
status were for plants or animals with single, or a few relatively simple
transgenic constructs involving genes for enzymes and other proteins. The
current application is a major departure in that it contains a plethora of
complex, uncharacterized constructs, and where the main focus of gene modification
is in transcription regulation and worse, non-coding sequences and hyperactivated
insertion mutagenesis. The proposal made no attempt to present the genetic
modifications of the many transgenic lines in a rational and coherent manner,
with diagrams detailing the transgenic constructs in each line being tested
along with an explanation of the function of each gene. The presentation of
the lines is vague, and the many multiple transgene inserts are not clearly
identified. There are so many separate lines being tested in one big 320 acre
site that transgene escape form the site is bound to happen due to human error, and there will be plenty of opportunity
for enhanced horizontal gene transfer and recombination of transgenes in the
extensive root systems of the trees to generate the most exotic new pathogens
out of some of the deadly toxin genes used. At the same time, too little information
is provided to allow the escaped transgenes to be identified by anyone other
than members of the research team proposing the releases. It would be a recipe
for disaster and a travesty of regulation to permit the proposed releases.
This is number 35 of ISIS’ detailed submissions
to US regulatory authorities for environmental releases GMOs.
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Hancock JE, Loya WM,
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CABI Publishing, Cambridge, MA, USA Pp 89-106.
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Science in Society 35