The scientific risks of GMO
release.
Dr. William H.L Stafford,
Advanced Research Center for
Applied Microbiology,
Department of Biotechnology,
University of the Western Cape.
GMOs and breeding
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We have been modifying our food sources for
thousands of years, selecting for favourable
characteristics. These breeding techniques rely on
fertlization by cross-pollination of the same species
Biotechnology uses the tools of genetic
engineering to modify a plant with any chosen
characteristic. Species barriers can be crossed- we
can take a gene from one organism and place it in
another totally unrelated organism and create
transgenics or GMOs
GMOs
© Strong promoter
© Marker gene
© Desired gene
© Terminator
Marker gene for antibiotic resistance (ampicillin, kanamycin)
Viral Promoter eg. cauliflower mosaic virus (35SCamV)
GMOs (food crops) have been released
-an experiment

Estimated 80 million hectares of
GMOs planted to date. Mainly
cotton, maize, canola, soya.

GMO crops that have
Herbicide resistant genes or
genes for insecticidal toxins
account for more than 93% of
the types of GM crops grown
worldwide..
Scientific Risk Assessments
Absence of Risk = Absence of Science..
Absence of evidence is not evidence of
absence!
 ”No harms reported” is not the same as
”no harms exist”!

Environmental interactions
Outcrossing and horizontal gene transfer
Soil
microbiota
Consumption of GMO
plant: humans, birds,
insects, amphibians,
microbes
Stability and persistence
of transgene product
(eg. Insecticide,
herbicide)
ENVIRONMENTAL RISKS
Random integration into the genome


Despite its importance for safety assessment,
applications submitted to the USDA requesting
permission to commercialise a transgenic line
provide neither the sequence of the genomic DNA
flanking the inserted transgene nor a comparison
with the original genome.
Truncations, rearrangements, tandem repeats at
one or more sites (perhaps reflecting the instability
of the gene constructs) have been reported.
(Collonier, et al. 2003)
T25 maize - LibertylinkTM (Bayer)
Tolerance to herbicide glufosinate
Construct content : truncated bla gene (bla*), pUC cloning vector (pUC), synthetic pat gene (pat),
CaMV 35S promotor and terminator (P35S, T35S).
pUC18
Sequence
expected
Sequence
observed
bla
pat T35S
*
35SCaMV
pUC18
35SCaMV
patT35SpUC18 bla*
Maize DNA
bla*
35SCaMV
(Presence of cloning
vector + the 5 first bp
of bla on the 3’ end )
•
•
Part of pUC cloning vector containing truncated B-lactamase gene (bla)
DNA rearrangement: presence of a second truncated and rearranged P35S on the 5’ end.
•
Insertion site: the 5’ and 3’ ends of the insert show homologies with Huck retrotransposons.
Rearrangements, deletions and
multiple insertions….
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Mon 810 maize YieldGard (Monsanto)
Modified for resistance to lepidopteran insects (butterflies & moths). Company data showed
insert has a P35S driving a CrylAb synthetic gene with terminator T-nos. Analysis revealed
however, that T-nos and part of the 3’ (tail) end of the CrylAb gene have been deleted.
T-nos has been detected elsewhere in the genome. The 5’ (head) end of the insertion
site shows homology to the long terminal repeats (LTR) of the maize alpha Zein gene
cluster.
GTS 40-3-2 Roundup ready soybean (Monsanto)
Modified for tolerance to herbicide glyphosate. Company data showed insert with P35S
driving a composite gene containing the N-terminal chloroplast transit peptide (CPT4)
joined to modified epsps gene with T-nos terminator. Analysis revealed that a 254bp
piece of DNA homologous to the epsps gene and 534bp of unknown DNA have been
joined to the 3’end of the insert. It was not possible to identify the insertion site.
Bt 176 maize (Syngenta)
Modified for tolerance to herbicide glufosinate, male sterility and insect resistance.
Company data showed insert contains P35S driving the bar gene (glufosinate tolerance)
terminated by T35S, followed by the ampicillin resistance (bla) gene plus bacterial
promoter, and plasmid origin of replication, ori. Analysis revealed several fragments, all
containing CaMV 35S promoter, one with P35S joined to T35S, a second with P35S
joined to an unknown sequence, and a third with P35S joined to the bar gene with
the T35S deleted. There were at least three insertion sites.
How do they occur ?
• When transgenic DNA is introduced into the plant
cell a wound-response produces DNA repair
enzymes that use DNA fragments for DNA repair,
resulting in its rearrangement of the plant DNA.
• Twelve representative transgenic rice lines were
analyzed, and found to have several rearrangements
demonstrating transgenic instability. The 35SCamV
promoter was identified as recombination hotspot
(Kohli et al.,1999).
Gene transfer and escape
 Genes
can spread from transgenic plants by
ordinary cross-pollination to nontransgenic
plants of the same or similar species, and
also by horizontal gene transfer to unrelated
species.
 With selection, such elements have increased
penetrance into the environment and cannot
easily be contained or controlled once they
have entered the wider environment!
Hybridisation, Outcrossing and escape:
Gene transfer and escape happens

Herbicide resistant transgenes from GMO plants were 20 times
more likely to escape and spread than the same gene
obtained by mutagenesis. (Bergelson, J. et al. 1998.)

Transgenic DNA introgressed into traditional maize landraces in
Oaxaca, Mexico (Quist D and Chapela IH. 2001). Several cobs
tested positive for the CaMV 35S promoter and sequence
analysis of insertion site by inverse PCR indicated diverse
sequences. Follow-up studies by two Mexican government
laboratories found evidence of the CaMV 35S promoter in 12%
of plants sample from Oaxaca and the adjacent state of Puebla
(Mann 2002).

Transgenic DNA containing the CaMV 35S promoter is
unstable and can randomly insert into the genome.
Contamination
Pollen flow may occur over large distances for some
crops (km for maize, canola; only m for potato).
Transport, storage, and processing of seeds and
crops are also routes for contamination.
 StarLink cry9 corn was approved for animal feed but
not human consumption. It was discovered in a wide
variety of processed foods. Despite a massive recall
of food products and extraordinary efforts to cry9
transgenes still persisted at detectable levels in US
corn supplies 3 years later (USDA 2003b).
Widespread contamination of seedlots
Seed purity has long been an important issue for
agronomists and plant breeders. Test on non-GM
canola seedlots were tested, the majority of tested
seedlots contained at least trace amounts of
genetically engineered herbicide-tolerance traits. In
fact, 97% (32 of 33) of the seedlots tested by Friesen
et al. (2003), and 59% (41 of 70) of the seedlot
stested by Downey and Beckie (2002) had foreign
transgenes present at detectable levels (above
0.01%).
This level of contamination in pedigreed seed is
disturbing since even stringent segregation systems
were not sufficient to deliver pure non-GM canola
seed to farmers in western Canada.

Human error and contamination
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
BT11 maize (Cry1Ab insecticidal toxin under the
35SCaMV promoter and pat gene for resistance to
the herbicide glufosinate, Basta) was approved for
commercial release and grown in the USA (2001)
and later in South Africa (2003).
December 2004, the Syngenta informed the US
government that it had just learned that the Bt11 corn
had been mislabeled and 165 000 tons of Bt10 seed
were grown in the US and the resultant maize was
sold as in the US and abroad.
Although Bt10 is “functionally
equivalent” to Bt11 there are
differences:
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Bt11 is approved for commercial release and human
consumption in, Bt10 is not.
The transgene is inserted at a different position in the plant
DNA.
Bt10 produces only about 1/7th the amount of the insecticidal
protein as Bt11.
More than one Cry1Ab toxin protein produced in Bt11.
The most substantial difference between Bt10 and Bt11 is that
Bt10 contains the bla gene for ampicillin resistance.
Effects on biodiversity

The most obvious effects of cross-pollination already
identified are in creating herbicide-tolerant, or
insecticidal weeds and superweeds and the loss
of locally adapted (‘indigenous’) crop varieties.
Studies with oilseed rape (Brassica napus) have shown
that the Bt gene can be passed on to a wild, weedier
relative (Brassica rapa) (Halfhill, M.D., et al. 2002.).
Horizontal Gene Transfer (HGT)
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Horizontal transfer from the
transgenic plants may spread the
novel genes and gene-constructs to
unrelated species- bactreia, fungi and
viruses in the soil, worms, insects
reptiles, birds, small mammals and
human beings
Horizontal gene transfer has been rare
the billions of years of our evolution,
because of natural species barriers
preventing genetic exchange and
because there are mechanisms which
inactivate or break down foreign DNA.
Horizontal gene transfer of genes from
one species to another may be a major
factor in evolutionary change (Syvanen,
M. 1986)
HGT to soil microbes matters

Microorganisms dominate soil-borne communities, and largely
determine ecosystem functions, such as nutrient cycling and
decomposition. Their direct and indirect interactions with plants
create strong feedback mechanisms, influencing primary production
and vegetation dynamics. They are important in both plant
pathogenicity and protection.

The majority (>99%) of the microbial world is uncultured. Thousands
of bacterial species per gram of soil whose functions are unknown!

Bacteria can take up DNA by several ways- transformation (DNA
uptake), transduction (virus) or conjugation (Hfr mating)
HGT to bacteria can spread antibiotic resistance marker genes to
pathogenic microbes and disrupt ecosystem function.
Factors that increase HGT to bacteria

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Factors affecting the likelihood of uptake of DNA into
soil-borne microbes include: the stability of the
inserted genetic material, the presence of similar
(homologous) sequences in resident microorganisms
the length of time that GM material or DNA remains
intact and the proximity of potential recipient species.
Agents that induce a stress response (J. Beaber et
al., 2003)“SOS response promotes horizontal
dissemination of antibiotic resistance genes,”
Agricultural practices (Continual selection eg.
Herbicide. Ploughing, etc).
HGT to soil bacteria
Horizontal gene transfer (HGT) occurs at very low frequency
(indetectable using some methods)
 Experiments with the the nptII gene in transgenic potato plants
coding for kanamycin resistance, detected HGT to
Pseudomonas stutzeri and Acinetobacter BD413 at a frequency
of 3x10-5 -1x10-4 despite the presence of a more than 106 fold
excess of plant DNA. This dropped to 10-16–10-17 when no
sequence similarity was present (de Vries J., Wackernagel W.,
1998).
HGT to bacteria is most efficient where sequence similarity is
present.
 The bla and npt genes used as antibiotic resistance markers in
some GM crops show sequence similarity to bacterial genes.
 The pat and epsps genes (T25 maize - LibertylinkTM (Bayer)
and GA21 maize (Monsanto) confer resistance to the
glufosinate and glyphosphate herbicides (Basta and Roundup)
also show significant sequence similarity to bacterial genes.

HGT and virus recombination

New and successful variants of viruses do arise naturally by
recombination with a frequency that varies depending on the
virus family (e.g. Chenault and Melcher 1994; Revers et al
1996; Padidam et al 1999). Success of a given variant
depends upon the conditions (selective pressures).

The CamV is a recombination hotspot and therefore subject to
increased HGT. CamV is from pararetroviruses, family that
includes Hepatitis.

Reactivation of dormant viruses and the generation of new
viruses?
Effects of HGT

More important than the frequency of HGT is what
happens to the resulting transgenic microorganisms.
Without positive selection for the new trait, it will
soon be lost and have no further impact on the
system. However, even a low frequency event can
have an important impact if selection is strong.

The novelty of a trait can also influence its potential
impact, as completely novel genes might give rise to
new genetic variants that are not possible within the
normal genetic pool of the system.
Summary of ENVIRONMENTAL RISKS :
 Generation of new bacterial pathogens and the spread of drug
and antibiotic resistance marker genes among pathogens
Reactivation and the generation of new viruses
 Increased resistance to herbicides, leading to super-weed
characteristics
 Position effects with unwanted changes in gene expression and
occurrence of cancers caused by random inserton (insertional
mutagenesis).
 Reduced biodiversity due to GMO selection, outcrossing and
contamination
 Unpredictable effects on genetic evolution and ecosystem
function
High levels of expression of transgene

The CaMv is a strong promoter providing a high level of gene
expression.

All living organisms that interact with the transgenic plant (bacteriabirds and human beings) are exposed to high levels of the
expressed transgene that are new to their physiology. Adverse
immunological or allergic responses can be expected. The GMO
insecticidal toxins such as Bt have been shown to affect beneficial
non-target organisms including lacewings, ladybirds and earthworms
(Birch, A.N.E., et al. 1997, and Marvier, M. 2001.) Bt can persist in
certain soil types for up to 234 days.

There is evidence of insect pests becoming resistant after only a few
years after the transgenic crops were first released since Bt-toxin
genes are expressed continuously at high levels throughout the
growing season.
DNA persistence in the environment

DNA is not completely broken down in the gut. Genes can spread from
ingested transgenic plant material to bacteria in the gut. Antibiotic
resistant marker genes from genetically engineered bacteria can be
transferred to indigenous gut bacteria (Netherwood T, et al. Technical
report on the Food Standards Agency project G010008)

DNA can persist in the soil for years. Transgenes from GMO plants
may be able to spread to soil bacteria, spreading antibiotic resistance
marker genes among the pathogens.
Practically every medical organization that has looked at GM crop safety
has expressed concern, including the American Medical Association,
World Health Organization, UK Royal Society, United Nations Food
and Agriculture Organization, Pasteur Institute, European Food Safety
Authority, and Codex Alimentarius
 The EU has decided to prohibit GMOs with antibiotic resistance genes
after the 31st December 2004 (directive 2001/18EC and Revising
Directive 90/220/CEE
HEALTH RISKS:
Expected and unexpected toxicity
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Transgenic potatoes expressing GNA insecticide (“Snowdrop”,
Galanthus nivalis, lectin) fed to rats resulted in increase in intestinal
mucosal thickness and T-lymphocyte infiltration. (Ewen S. W. B, and
Pusztai, A. 1999)
Monsanto's transgenic soya, has a 26.7% increase in a trypsininhibitor and has been shown to inhibit the growth rate of male rats.
This raises the possibility that transgenic soya is responsible for the
reported recent increase in soya allergy.
Human gene therapy experiments for severe combined
immunodeficiency (SCID) caused by a single non-functional gene
(adenosine deaminase) were halted by the FDA after a second treated
child died of cancer. Molecular analysis showed that the T cells were a
single clone derived from one original cell that has multiplied out of
control. The retroviral vector used – mouse Moloney leukaemia virus –
had inserted into a gene on chromosome 11 causing truncation gene
trucation and oncogenesis.
Requirements for GMO release

Efficient and specific gene targeting to cells

Stable, single insertion of gene at defined site with
no other DNA (viral promoters or antibiotic resistance
genes)
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Normal levels of expression of desired gene
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Proven safety (consumption and the evironment).
X The GMO crops on the market fulfill none of these criteria
Better methods available?
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Site specific recombination using Zn finger protein linked to
integrase-target gene.
Control under endogenous promoters.
Plastid engineering (Daniell et al., 2002)
Other marker genes, such as green fluorescent protein, or
mannose (Joersbo et al., 1998)
Removing the antibiotic resistance genes before the plants are
released for commercial use (Lamtham and Day, 2000; Zuo et
al., 2001), so that these genes can be used during development
and then eliminated from the final product.
Use of introns to prevent expression in bacteria while allowing
plant expression (Libiakova et al., 2001).
A lack of labelling and monitoring
The transgenic genes of GMO crops are covered by patent. Since
these GMO crops can be considered novel inventions
however the food-crop is considered “functionally equivalent”
(mainly un broad nutritional grounds) and no labelling is
required in many countries including as South Africa.
If there are problems……
there is no labelling, no monitoring and ten there will be
difficulties in tracking and establishing liability.
Can we recall the release of a GMO from the environment?
Which is more cost effective- proper risk assessment, monitoring
&labelling or loss of markets (EU) and clean-up cost from
contamination?
Future of GMOs
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It is important to distinguish between contained use
of transgenic organisms and their release to the
environment.
It is vital that GMO crops are proven safe for through
proper independent, long-term feeding trials and
environmental impact assessments
It is essential to monitor GMOs since they have been
released and we need to observe the effects in this
experiment
References
Ho, M.W., Meyer, H. and Cummins, J. (1998). The biotechnology bubble. The Ecologist 28(3), 146-153
 Kohli A.,Griffiths S, Palacios N, Twyman R, Vain P, Laurie D and Christou P. (1999) Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a
recombination hot spot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination" Plant.J. 17,591-601.
 Sengelov G, Kristensen KJ, Sorensen AH, Kroer N, and Sorensen SJ. Effect of genomic location on horizontal transfer of a recombinant gene cassette between Pseudomonas
strains in the rhizosphere and spermosphere of barley seedlings. Current Microbiology 2001, 42, 160-7.
 George A. Kowalchuk, Maaike Bruinsma and Johannes A. van Veen. Assessing responses of soil microorganisms to GM plants TRENDS in Ecology and Evolution Vol.18 No.8
August 2003
 Bergelson, J., Purrington, C.B. and Wichmann, G. (1998). Promiscuity in transgenic plants. Nature 395, 25
 Quist D and Chapela IH. (2001) Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico. Nature, 414, 541-3, 2001
 Syvanen, M . 1986. Cross-species gene transfer: a major factor in evolution? Trends In Genetics pp 1—4
 Hilbeck, A., Baumgartner, M., Fried, P.M. and Bigler, F. (1997). Effects of transgenic Bacillus thuringiensis-corn-fed prey on mortality and development time of immature Chrysoperla
carnea (Neuroptera: Chrysopidae). Environmental Entomology
 Halfhill, M.D., R.J. Millwood, P.L. Raymer, and C.N.Stewart, Jr. 2002. Bt-transgenic oilseed rape hybridization with its weedy relative, Brassica rapa.Environmental Biosafety
Research 1: 19-28.
 Birch, A.N.E., Geoghegan, I.I., Majerus, M.E.N., Hackett, C. and Allen, J. (1997). Interaction between plant resistance genes, pest aphid-population and beneficial aphid predators.
Soft Fruit and Pernial Crops. October, 68-79.
 Hilbeck, A., Baumgartner, M., Fried, P.M. and Bigler, F. (1997). Effects of transgenic Bacillus thuringiensis-corn-fed prey on mortality and development time of immature Chrysoperla
carnea (Neuroptera: Chrysopidae). Environmental Entomology 27, 480-487.
Vaden V.S. and Melcher, U. (1990). Recombination sites in cauliflower mosaic virus DNAs: implications for mechanisms of recombination. Virology 177, 717-26
Halfhill, M.D., R.J. Millwood, P.L. Raymer, and C.N.Stewart, Jr. 2002. Bt-transgenic oilseed rape hybridization with its weedy relative, Brassica rapa.. Environmental Biosafety
Research 1: 19-28.
 Lommel, S.A. and Xiong, Z. (1991). Recombination of a functional red clover necrotic mosaic virus by recombination rescue of the cell-to-cell movement gene expressed in a
transgenic plant. J. Cell Biochem. 15A, 151;
 Greene, A.E. and Allison, R.F. (1994). Recombination between viral RNA and transgenic plant transcripts. Science 263, 1423-5;
 Wintermantel, W.M. and Schoelz, J.E. (1996). Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate
selection pressure. Virology 223, 156-64.
 Sengelov G, Kristensen KJ, Sorensen AH, Kroer N, and Sorensen SJ. (2001) Effect of genomic location on horizontal transfer of a recombinant gene cassette between
Pseudomonas strains in the rhizosphere and spermosphere of barley seedlings. Current Microbiology, 42, 160-7.
Ewen S. W. B, and Pusztai,A. (1999) Effect of diets containing genetically modified potatoes expressing Galanthus nivalis lectin on rat small intestine Lancet 16 October
MacKenzie, D. (1999). Gut reaction. New Scientist 30 Jan., p.4.
 Schubbert, R., Renz, D., Schmitz, B. and Doerfler, W. (1997). Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen and liver via the intestinal wall mucosa and
can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. USA 94, 961-6.
Netherwood T, Martin-Orue SM, O'Donnell AG, Gockling S, Gilbert HJ and Mathers JC. Transgenes in genetically modified Soya survive passage through the small bowel but are
completely degraded in the colon. Technical report on the Food Standards Agency project G010008 "Evaluating the risks associated with using GMOs in human foods"- University of
Newcastle.
Marvier, M. 2001. Ecology of transgenic crops. American Scientist 89: 160-167
Collonier C, Berthier G, Boyer F, Duplan M-N, Fernandez S, Kebdani N, Kobilinsky A, Romanuk M, Bertheau Y. (June 2003) Characterization of commercial GMO inserts: a source of
useful material to study genome fluidity. Poster courtesy of Pr. Gilles-Eric Seralini, Président du Conseil Scientifique du CRII-GEN, www.crii-gen.org
de Vries J, Wackernagel W (1998) Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation. Mol Gen Genet 257:606-613
Koskella, J. and G. Stotzky. 1997. Microbial utilization of free and clay-bound insecticidal toxins from Bacillus thuringiensis and their retention of insecticidal activity
after incubation with microbes. Applied and Environmental Microbiology 63: 3561-3568;
Tapp, H. and G. Stotzky. 1998. Persistence of the insecticidal toxin from Bacillus thuringiensis subsp. kurstaki in soil. Soil
Biology Biochem. 30(4): 471-476.