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Identification of escaped transgenic Creeping Bentgrass in Oregon

Information Systems for Biotechnology News Report, USA

Jay R. Reichman and Lidia S. Watrud



Identification of escaped transgenic Creeping Bentgrass in Oregon


When transgenic plants are cultivated near wild species that are
sexually compatible with the crop, gene flow between the crop and wild
plants is possible. A resultant concern is that transgene flow and
transgene introgression within wild populations could have unintended
ecological consequences. In order to begin testing for these potential
effects, it is necessary to locate and monitor wild populations into
which transgenes have escaped. Empirical data on transgene escapes is
just beginning to emerge in the scientific literature, and in the
November, 2006, issue of Molecular Ecology we presented the first
evidence for establishment of transgenic plants in wild populations
within the USA.1 The case involved glyphosate-resistant creeping
bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS (5-
enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium spp.
strain CP4) transgenes that were found in non-agronomic habitats outside
of experimental test plots in central Oregon.

In 2003 under USDA APHIS permit issued to the Scotts Company, flowering
of approximately 162 ha of Roundup Ready creeping bentgrass (event
ASR368) occurred for the first time in Jefferson County Oregon, USA,
within a 4453 ha agricultural bentgrass control area established by the
Oregon Department of Agriculture (Fig. 1).

Our interest in ASR368 as an experimental study system was based on
several factors. Agrostis stolonifera transformed with a CP4 EPSPS
construct (conferring resistance to the herbicide glyphosate) is one of
the first transgenic, perennial, wind-pollinated crops with sexual and
asexual modes of reproduction that is intended to be grown outside of
agricultural fields, i.e., on golf courses. A. stolonifera belongs to a
cosmopolitan genus that includes approximately 200 species worldwide.
There are approximately 34 North American species of Agrostis, 26 of
which are native. Fourteen native and naturalized species are found in
Oregon. Agrostis stolonifera is an obligate outcrossing species and
member of a hybridizing network of at least 12 other grass species from
Agrostis and Polypogon. Of the four species from this complex that grow
wild in central Oregon, A. exarata Trin. (spike bentgrass) is native,
while A. gigantea Roth (redtop), A. stolonifera and P. monspeliensis
(L.) Desf. (annual rabbit's-foot bentgrass) are naturalized.2 CP4 EPSPS
is also a selectable marker that can be used for tracking gene flow from
transgenic cultivars and its potential introgression into wild
compatible populations.

Following the initial ASR368 flowering event, we documented
hybridization of Agrostis plants by viable transgenic pollen as far as
21 km beyond the perimeter of the bentgrass control area in central
Oregon during 2003.3 In that study, seeds were collected from panicles
of sentinel and resident plants that had been placed or were naturally
growing outside the bentgrass control area. Collected seeds were
germinated in a greenhouse and seedlings were sprayed with the herbicide
glyphosate. Survivors of herbicide treatment were tested for production
of CP4 EPSPS protein using a lateral flow test strip, and additional
molecular tests (PCR and sequencing) were run to confirm the presence of
the transgene. Field surveys to assess the establishment of wild
transgenic Agrostis pollen-mediated hybrids were not conducted during
the initial study. However, in a separate survey conducted by Mallory-
Smith et al.,4 numerous CP4 EPSPS positive Agrostis stolonifera plants
were found in agronomic settings inside the bentgrass control area at
locations where either no Agrostis plants were detected in the previous
year, or where they had been removed. The volunteers were presumed to be
GE seed progeny due to their growth in plowed fields of other crops, in
open disturbed spaces, or along irrigation canals near ASR368 fields.
The research recently published in Molecular Ecology expanded on our
previous work and was driven by our interest in determining whether or
not transgenic plants could become established in the environment in non-
agronomic habitats. The paper thus focused on locating and
phylogenetically identifying transgenic Agrostis plants which became
established in wild populations within one year of the initial 2003 test
production season.1

Experimental overview

The objective of our field studies was to locate transgenic plants
established by either crop X wild hybridization or by crop seed
dispersal in wild (native and naturalized) Agrostis populations outside
the control area. Leaf samples from 50 or more plants at each location
were combined and tested for presence of CP4 EPSPS protein using
TraitChek tests (Strategic Diagnostics). Bulk samples that tested
positive were sub-sampled in the field to identify specific CP4 EPSPS
positive plants. In the laboratory, we utilized PCR and sequencing based
approaches to confirm the presence of the transgene in individual
samples. Proprietary constraints by Scotts and Monsanto limited our
access to ASR368 plants and their potential hybrid progeny. We thus used
molecular systematic methods to characterize and identify Agrostis
hybrids. To detect F1 interspecific hybrids among wild transgenics,
their parentage was determined by comparing nuclear ribosomal ITS1-5.8S-
ITS2 (ITS) and maternally inherited chloroplast trnK intron maturase
(matK) DNA sequences.

Locations and habitats of transgenic plants

Fifty-five Agrostis spp. populations were located during field surveys
of publicly accessible lands in the study area. Of the species that are
sexually compatible with the glyphosate-resistant creeping bentgrass
crop, those present in the sampled populations included A. stolonifera,
A. gigantea, and A. exarata. A total of 20,400 plant tissue samples were
collected from these species for analysis with TraitChek kits for the
CP4 EPSPS protein. Approximately 0.04% (9/20,400) of plant tissue
samples tested positive for the protein. The nine positive plants that
were distributed between six of the surveyed populations were identified
as A. stolonifera based on morphology.

Spatial distribution of positive plants was consistent with wind
movement as the primary physical mechanism for transport of both pollen
and seeds from the ASR368 crop fields to resident populations. Seven CP4
EPSPS positive plants were distributed south and southeast of the
control area in the direction of the prevailing winds, while two were
established 0.2 km from the southwestern border. All seven plants to the
south and southeast were found in mesic habitats specifically preferred
by A. stolonifera. Three of these plants were located along banks of
main irrigation canals, three were at a pond, and one was found along
the bank of a small irrigation canal. The two plants found at the
greatest distances outside the control area (3.7 km and 3.8 km to the
southeast, respectively) were located along a canal in the Crooked River
National Grasslands. The two transgenic plants to the southwest were not
associated with a waterway, but were located on a roadside. Resident A.
stolonifera were present within all populations where CP4 EPSPS positive
plants were found, except the two in closest proximity to the control
district. The only Agrostis species located at these two sites was A.
gigantea (Fig. 2).

Molecular transgene detection and species-level parentage determination

For all nine plants that tested positive for CP4 EPSPS protein in the
field, the presence of the engineered construct encoding the protein was
confirmed in the laboratory by PCR and DNA sequencing. Comparisons of
ITS and matK nucleotide variations between the escaped transgenic plants
and various Agrostis reference taxa indicated that both the paternal and
maternal parents of the wild transgenic plants were A. stolonifera.
While our analyses of ITS and matK DNA sequence data have the capacity
to identify recently formed interspecific Agrostis hybrids, none were
present in the nine wild transgenic plants that were found established
in non-agronomic habitats.


Reichman et al. 1 present the first evidence for escape of transgenic
plants from an engineered crop into wild plant populations within the
USA. The distribution and parentage of the wild transgenic plants
suggest that six of the plants established in the wild resulted from
pollen-mediated gene flow to wild A. stolonifera plants and that three
came from dispersed GM seeds. As expected, transgenics were generally
found at sites in the direction of prevailing winds. Evidence for feral
ASR368 comes from three plants established at two sites nearest the
control area where there was an absence of sympatric non-cultivated A.
stolonifera plants. These three plants may have resulted from crop seeds
that were dispersed by various mechanisms; wind, water, wildlife and/or
mechanical means (e.g., vehicles).

Our results demonstrate that even short-term production can result in
transgene flow and transgene establishment within compatible wild
populations at multiple locations that provide suitable habitat. In this
example, transgenic A. stolonifera plants became established up to
several kilometers away from the crop source fields after only a single
growing season, and the escapes were due to movement of both pollen and
crop seeds. When such establishment involves or leads to the formation
of hybrids with either full or partial fertility, then transgene
introgression into wild populations through backcrossing becomes possible.

Because CP4 EPSPS makes plants resistant to glyphosate herbicide,
application or drift of this product could favor continued persistence
of the wild plants that now have this new gene. Plant reproduction and
further spread could cause the herbicide-resistant plants to persist in
wild populations even without additional herbicide use. The long-term
fate and ecological impacts of CP4 EPSPS transgenes within wild Agrostis
populations in central Oregon remain to be determined.

Ruth Martin (U.S. Department of Agriculture Agricultural Research
Service) is thanked for her helpful comments on the manuscript.
Information in this document has been funded wholly by the U.S.
Environmental Protection Agency. It has been subjected to review by the
National Health and Environmental Effects Research Laboratory's Western
Ecology Division and approved for publication. Approval does not signify
that the contents reflect the views of the Agency, nor does mention of
trade names or commercial products constitute endorsement or
recommendation for use.

Figure 1 and 2:

Detailed topographic map on the bentgrass control area and surrounding study sites,

see apr0701-1, apr0701-2

1. Reichman JR, Watrud LS, Lee EH, Burdick CA, Bollman MA, Storm MJ,
King GA and Mallory-Smith C. 2006. Establishment of transgenic herbicide-
resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic
habitats. Molecular Ecology 15(13), 4243-4255
2. MacBryde B. 2005. White Paper: Perspective on Creeping Bentgrass,
Agrostis stolonifera L.,USDA/APHIS/BRS (ver. 12/12/2005) (http://
3. Watrud LS, Lee EH, Fairbrother A, Burdick C, Reichman JR, Bollman M,
Storm M, King G, and Van dewater PK. 2004. Evidence for landscape-level,
pollen-mediated gene flow from genetically modified creeping bentgrass
with CP4 EPSPS as a marker. Proceedings of the National Academy of
Sciences 101(40), 14533-14538
4. Mallory-Smith C, Butler M, Campbell C. 2005. Abstracts of the 2005
Meeting of the Weed Science Society of America, 164.

Jay R. Reichman
US Environmental Protection Agency, Office of Research & Development
National Health & Environmental Effects Research Laboratory
Western Ecology Division
200 SW 35th Street, Corvallis, OR 97333
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Lidia S. Watrud
Research Ecologist / Gene Flow Project Leader
US Environmental Protection Agency, Office of Research & Development
National Health & Environmental Effects Research Laboratory
Western Ecology Division
200 SW 35th Street, Corvallis, OR 97333
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