Creeping
bentgrass: when
herbicide
resistance goes
wrong
Brigid Meints
CROP 540
11/19/2012
Herbicide resistant weeds
 Weeds
become resistant to herbicides
after repeated use; normal rates of the
herbicide can no longer control the weed
 Transgenic plants engineered to be
herbicide resistant that become weedy
through gene flow
Creeping Bentgrass
(Agrostis stolonifera)
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Specialty grass used for golf course greens, lawn
bowling greens, and lawn tennis
High maintenance grass: requires high levels of
fertilizer, frequent mowing, watering, aerating, and
dethatching
Identification: bright green, fine textured, no auricles,
long and tapered ligule
Perennial growth habit; spreads by stolons to form a
mat or thatch layer above the soil line
Low-growing with a shallow root system
Allotetraploid
Obligate outcrosser: has very small seeds, but can
also propagate asexually
Background
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Glyphosate-resistant creeping bentgrass
created by Monsanto and Scotts: Event
ASR368
In January 2004, Monsanto and Scotts
petitioned the Animal and Plant Health
Inspection Service (APHIS) seeking a
determination of nonregulated status for
creeping bentgrass
APHIS began taking testimony to form an
Environmental Impact Statement
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14 species of Agrostis native to Oregon
Found in agronomic and nonagronomic habitats
Small seeds (6 million per pound) can be dispersed
by wind, water, animals, or mechanical means
Naturally forms interspecific F1 hybrids,
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low in fertility or sterile
but under certain conditions have been shown to
out-compete both parents
Interspecific hybrid: mating of two species,
generally from the same genus. The offspring show
a combination of traits and characteristics from
the two parents. Offspring are often sterile.
Can hybridize with at least 12 other grass species
from Agrostis and Polypogon
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extensive clonal propagation is still possible
Genetics of glyphosate
resistance
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Two components necessary: resistant target
enzyme and sufficient expression of that
enzyme within the transgenic plant
5-Enol-pyruvylshikimate-3-phosphate synthase
(EPSPS) is the target enzyme for the inhibition
of glyphosate in the aromatic amino acid
biosynthetic pathway
Agrobacterium sp. strain CP4 EPSPS was
found to be an exceptional candidate. Does
not have a negative impact on yield, quality,
or nutritional value of the harvested crop
Inherited as a single Mendelian locus
History
 2003:
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162 ha of glyphosate-resistant transgenic creeping
bentgrass tested under APHIS permit in central Oregon
Conducted within a 4453 ha control area established
by the ODA
Located 144 km east of non-transgenic, commercial
bentgrass production
Watrud et al. (2004)
 Majority
of gene flow observed within 2
km (in direction of prominent winds)
 Up to 21 km (sentinel plants) and 14 km
(resident plants)
 Found evidence of gene flow in 75 of 138
(sentinel) and 29 of 69 (resident) A.
stolonifera plants based on seedling
progeny survival after spraying in the
greenhouse
 Sampling
grid
designed to test viable
pollen flow from
transgenic plants
 Sentinel A. stolonifera
plants cultivated in the
Willamette Valley,
transplanted in central
Oregon (first tested for
trait), transported very
carefully
 PCR
primers designed to test for the 1050-bp
segment of the A. stolonifera CP4 EPSPS coding
region. Matched GenBank accession for a CP4
EPSPS construct in glyphosate-resistant soybean
 Maximal
distances
of observed gene
flow. Locations of
sentinel A.
stolonifera, resident
A. stolonifera, and
resident A.
gigantea (A, B, and
C respectively)
 Percent
positive seedling progeny per location for
sentinel A. stolonifera, resident A. stolonifera, and
resident A. gigantea (A, B, and C respectively)
Prevalence and incidence of plants positive
for the transgene and seedling progeny
Reichman et al. (2006)
 Resident
populations of Agrostis species
sampled in nonagronomic habitats
outside of the ODA control zone
 Attempted to discover parentage of
plants positive for CP4 EPSPS, but
Monsanto and Scotts had proprietary
constraints
 Used species-level molecular
phylogenetic analyses
 Of
20,400 plant tissue samples taken,
there were 9 positive plants (0.04%)
 Distributed over 6 of the surveyed
population areas, including the Crooked
River National Grassland
 Not possible to determine which field they
came from, so distances are range
 All near waterways or roadside
 Gene trees showed that all wild
transgenic plants had maternal and
paternal A. stolonifera parents
Why is this crop so
problematic?
 Size,
density, and viability of the pollen
 Wild, sexually compatible species
 Floral synchrony between crop and wild
species
 Large source fields
 Small seed size
History
 2007:
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APHIS completed their investigation into
alleged compliance infractions by The
Scotts Company. Scotts agreed to pay a
civil penalty of $500,000, which is the
maximum penalty allowed by the 2000
Plant Protection Act
The saga continues…
 2012:
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Reports of intergeneric hybridization with
rabbitfoot grass, (Polypogon monspeliensis)
Transgenes confirmed using PCR primers
Produced viable seed, had perennial
growth habit and stolon production
capability
 Intergeneric
hybrid: hybrid between
different genera
Final thoughts
 Careful
consideration of potential gene
flow when introducing transgenic crops
 Consequences associated with that gene
flow
References
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APHIS (2004) Monsanto Co. and The Scotts Co.; Availability of petition for
determination of nonregulated status for genetically engineered
glyphosate-tolerant creeping bentgrass. Federal Register 69, 315-317, January 5,
2004
Heck et al. (2005). Development and Characterization of a CP4 EPSPS-Based,
Glyphosate-Tolerant Corn Event:Crop Sci. 45:329-339 (2005).
Reichman, J. R., Watrud, l. S., Lee, E. H., Burdick, C. A., Bollman, M. A., Storm, M. J., King,
G. A. And Mallory-Smith, C. (2006), Establishment of transgenic herbicide-resistant
creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats. Molecular
Ecology, 15: 4243–4255.
Watrud LS, Lee EH, Fairbrother A et al. (2004) Evidence for landscape level pollenmediated gene flow from genetically modified creeping bentgrass using CP4
EPSPS as a marker. Proceedings of the National Academy of Sciences, USA, 101,
14533–14538.
UC Davis. Statewide Integrated Pest Management: The UC Guide to Healthy Lawns,
Creeping Bentgrass. http://www.ipm.ucdavis.edu/TOOLS/TURF/ TURFSPECIES
creepbent.html
USDA. (2007). Release No. 0350.07. USDA concludes genetically engineered
creeping bentgrass investigation. http://www.usda.gov/wps/portal/usda/
usdahome?contentidonly=true&contentid=2007/11/0350.xml
Zapiola, M. L. And Mallory-Smith, C. A. (2012), Crossing the divide: gene flow
produces intergeneric hybrid in feral transgenic creeping bentgrass
population. Molecular Ecology, 21: 4672–4680.