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[SANET-MG] GM oilseed rape GT73



September 15, 2004

Prof. Joe Cummins

“Genetically-modified oilseed rape GT73: information on the transgenes”

The European Union (EU) will soon vote on the acceptance of genetically
modified (GM) oilseed rape the background information provided here may
provide a resource that is useful in evaluating the hazards of the crop.

Oilseed rape (Brassica napus) is grown as a commercial crop in 50
countries,with a combined harvest of over 40 million metric tonnes. The
major producers of rapeseed in 2000 were China, Canada, India, Germany,
France, Australia, and the United Kingdom. Canola is a genetic variation
of B. napus with low levels of the natural rapeseed toxins glucosinolate
and erucic acid. Canola is grown for its seed, which represents a major
source of edible vegetable oil and pressed cake from oil extraction is
also used in livestock feeds (1). Oilseed rape is called Canola in North
America because the commercial oil producing varieties were developed in
Saskatchewan, Canada.

Monsanto canola GT73 was released commercially in 1995 in Canada (2) and
the same strain ,designated RT73, was released commercially in the
United States in 1999(3). Japan approved release of GT73 in 1995 (1) and
Australia in 2003(1). approval of the releases was based on ,
essentially, the same data sets in all of the countries.

Canola strain GT73 (RT73) involves the herbicide glyphosate resistance
phenotype. Two primary genes were used , first the The EPSPS gene codes
for the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The
EPSPS gene put into GT73 was isolated from the common soil bacterium
Agrobacterium tumefaciens and is a glyphosate tolerant form of EPSPS.
The EPSPS enzyme is part of an important

biochemical pathway called the shikimate pathway, which is involved in the

production of aromatic amino acids. When conventional canola plants are
treated with glyphosate, the plants cannot produce the aromatic amino
acids but the enzyme from the transgene is insensitive to glyphosate.
The canola line GT73 contains a second gene that codes for a modified
version of glyphosate oxidase (GOX) enzyme . The gox gene inserted into
GT73 was isolated from the bacterium Ochrobactrum anthropi.. Glyphosate
oxidase (GOX) enzyme accelerates the normal breakdown of the herbicide
glyphosate into two compounds, aminomethylphosphonic acid (AMPA) and
glyoxylate.(1,2,3,4) In the absence of GOX unacceptable levels of the
herbicide may accumulate in canola cake used in animal feed.

The two primary genes were introduced into Canola from a plasmid using
the bacterium , Agrobacterium tumefaciens,. Only the primary genes and
the sequences necessary for their activity in the plant cell were
inserted into the canola cells while sequences from the plasmid such as
the plasmid origin of replication and a gene for streptomycin resistance
were lost from the commercial strain. The EPSPS and gox genes were each
driven by the 35S promoters from a modified figwort mosaic virus. The
shikimate pathway is located in the chloroplast so chloroplast transit
signal peptides sequences from the ribulose biphosphate carboxylase and
EPSPS of Arabidopsis targeted the primary genes to the chloroplast.
Finally, the sequences for the two primary genes were ended with the
copies of the 3’ (terminal) end of the pea rbcS E9 gene that included
the poly adenylation signal and the transcription termination signal .
The gene complexes were inserted in a stable and Mendelian fashion but
the exact site of their insertion was not reported (3).

Few of the regulatory documents released on Canola GT73 (RT73) dealt
with extensive alterations in the genetic codes of the primary genes in
canola GT73 but all of them acknowledged that the codes were alter to
enhance production of the bacterial gene products in the plant. The
United States Food and Drug Administration consultation on canola GT73
provided a somewhat fuller description of the alterations in the
bacterial DNA (5) while the a patent for the EPSPS used in canola GT73
provided an extensive description of the code alteration (6). Native
transgenes from bacteria or humans do no function very well in crop
plants because gene expression is influenced by codon bias geared to
optimum gene expression within plants, mammals or bacteria for that
reason the genetic code is altered by genetic engineers to achieve
optimum gene expression. The alterations need not extensively alter the
amino acid sequence in proteins because the code provides alternative
letters in the third position of the codon. However, the optimized genes
used in modified crops are mainly synthetic approximations of the real
bacterial gene (7). The synthetic genes are very different from the
genes that evolved in bacteria and for that reason their characteristic
recombination and mutation deserves special attention that has been
ignored by regulators.

Even though the native bacterial genes were altered in DNA sequence the
proteins tested for mammalian toxicity and environmental safety were not
isolated from canola GT73 but from the native bacterial genes which
produced the enzymes by fermentation. The bacterial surrogate enzymes
were assumed to be identical to the enzymes produced in canola GT73 by
cursory observations using gel electrophoresis , N terminal analysis and
enzyme activity even though the presence of four anomalous amino acids
were noted in the bacterial GOX (4). It cannot be concluded that the
safety tests were meaningful, or even effective, because there has been
no labeling of the food and feed produced from the modified crops.
Safety tests should be conducted on the real thing not the bacterial
surrogates.

The regulatory reviews leading to commercialization of canola NK73
without exception discounted the rapid pollution of transgenic crops by
wind spread pollen or by seed dispersal by animals or vehicles. There is
clear and growing evidence that widespread deployment of GM canola will
lead to widespread contamination of conventional crops . A 2003 report
showed that 95% of certified seed stock in western Canada were polluted
to detectable levels with glyphosate tolerance genes and 52% exceeded
the allowable contamination of certified seed (8). The widespread
deployment of GM canola for a variety of herbicides is leading to
pyramiding of the genes for herbicide tolerance creating crops that turn
into ferile weeds that are difficult to eradicate (9).

Before canola GT73 is approved it is essential that the real gene
products not their bacterial surrogates be tested. The synthetic genes
used in this crop should be studied extensively regarding their
stability, mutability and recombination properties. The DNA sequences of
these genes were never exposed to normal evolution so their behavior
warrants unique scrutiny. The tendency of canola transgenes to pollute
conventional crops should not be ignored.

References

1.agbios Data base product description MON-00073-7 (GT73,RT73) pp1-3
http://www.agbios.com/dbase.php?action=ShowProd&data=GT73%2C+RT73
<http://www.agbios.com/dbase.php?action=ShowProd&data=GT73%2C+RT73>

2.Canadian Food Inspection Agency Plant Biosafety Office Decision
Document DD95-02:Determination of of environmental safety of Monsanto
Canada Inc.’s Roundup Herbicide tolerant Brassica napus canola line GT73
1995 pp1-10 http://www.inspection.gc.ca/english/plaveg/bio/dde.shtml

3.Animal and Plant Health Inspection Service USDA Docket no. 98-089-2
Monsanto co. determination of Nonregulated status for canola genetically
engineered for glyphosate tolerance 1999 pp1-33
http://www.aphis.usda.gov/brs/aphisdocs2/98_21601p_com.pdf

4. ANZFA Australia New Zealand Food Authority Draft risk analysis report
application A363 Food produced from glyphosate tolerant canola line GT73
2002 pp 1-73 http://www.agbios.com/docroot/decdocs/01-290-009.pdf

5.US Food and Drug Administration Biotechnology Consultation Note to the
File BNF No.000020 Monsanto’s glyphosate tolerant canola line GT73 1995
pp1-4 http://www.cfsan.fda.gov/~rdb/bnfm020.html
<http://www.cfsan.fda.gov/%7Erdb/bnfm020.html>

6. Eicholtz,D,Gasser,D. and Kishore,G.
Glyphosate-tolerant-5-enopyruvyl-3-phosphoshikimate synthetase
1999United States patent 5,866,775

7.Cummins,J. Synthetic genes in food crops 2004 ISIS Press Release

http://www.i-sis.org.uk/

8.Cummins,J. Transgenic contamination of certified seed stocks 2003

ISIS report http://www.i-sis.org.uk/

9.Ho,M. What lurks behind triple herbicide tolerant oilseed rape?” 2002
ISIS report

http://www.i-sis.org.uk/

.

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