Evidence for landscape-level, pollen-mediated gene flow from genetically modified creeping bentgrass with CP4 EPSPS as a marker

Abstract

Sampling methods and results of a gene flow study are described that will be of interest to plant scientists, evolutionary biologists, ecologists, and stakeholders assessing the environmental safety of transgenic crops. This study documents gene flow on a landscape level from creeping bentgrass (Agrostis stolonifera L.), one of the first wind-pollinated, perennial, and highly outcrossing transgenic crops being developed for commercial use. Most of the gene flow occurred within 2 km in the direction of prevailing winds. The maximal gene flow distances observed were 21 km and 14 km in sentinel and resident plants, respectively, that were located in primarily nonagronomic habitats. The selectable marker used in these studies was the CP4 EPSPS gene derived from Agrobacterium spp. strain CP4 that encodes 5-enol-pyruvylshikimate-3-phosphate synthase and confers resistance to glyphosate herbicide. Evidence for gene flow to 75 of 138 sentinel plants of A. stolonifera and to 29 of 69 resident Agrostis plants was based on seedling progeny survival after spraying with glyphosate in greenhouse assays and positive TraitChek, PCR, and sequencing results. Additional studies are needed to determine whether introgression will occur and whether it will affect the ecological fitness of progeny or the structure of plant communities in which transgenic progeny may become established.

Fig. 1.

Sampling design to determine gene flow from source fields within the control district to potentially compatible plants outside the control district. A total of 178 sentinel A. stolonifera plants (red circles) were placed outside the control district (6) near accessible public roads spaced 1.6 km apart in the north-south direction and 0.8 km apart in the east-west direction. Given a prevailing wind of 10 km/h from the north or northwest, 76 sentinel plants were located downwind from the control district in a 9.6-km-wide by 3.2-km-deep grid with ≈0.8-km spacing. Remaining sentinel plants were placed at 1.6-km intervals for the next 4.8 km and 3.2-km intervals for the next 6-10 km out to a distance of 16-21 km along six transects corresponding to major highways. In addition to the sentinel plants, 69 compatible resident Agrostis plants (black circles) of A. stolonifera and A. gigantea, plus 10 P. monspeliensis (open circles) located primarily along waterways and in moist soils, were included in the study.

Fig. 2.

Molecular confirmation of the presence of the engineered CP4 EPSPS herbicide-resistance gene. The presence of the CP4 EPSPS gene as verified in a subsample of TraitChek-positive progeny from resident (R1-R5) and sentinel (S1-S5) plants located at various distances from the control district perimeter. All PCR products had the same size and DNA sequence as that amplified from the GM-positive control (A. stolonifera, designated event ASR368). blastn searches (29) revealed that the DNA sequences also matched GenBank accessions AF464188.1, Glycine max CP4 EPSPS (score = 1,271, E = 0.0), and AY125353.1, a synthetic CP4 EPSPS construct. Negative controls included DNA from nonGM (NGM) A. stolonifera, variant Penncross, and a nontemplate control (NTC). +, positive sequence matches; N/A, not applicable.

Fig. 3.

Skewed distribution of GM bentgrass pollen-mediated gene flow to sentinel and resident plants in 2003. Based on the presence and expression of the CP4 EPSPS gene for herbicide resistance, relative frequencies of gene flow among sentinel and resident plant seedling progeny were highest within the first 2 km from the perimeter of the control district and decreased with distance. Arrows depict maximal gene flow distances that were observed. A, B, and C represent locations of sentinel A. stolonifera, resident A. stolonifera, and resident A. gigantea plants, respectively.

Fig. 4.

Prevalence of gene flow based on percent positive seedling progeny of sentinel and resident plants at various distances from the control district perimeter. Kernel smoothing (34) was applied to percent positive seedling progeny (filled circles) of sentinel A. stolonifera plants (A), resident A. stolonifera plants (B), and resident A. gigantea plants (C) to generate spatial maps of the density of percentage positives. Open circles indicate locations where no positive seedling progeny were found. The highest densities of percent positive seedling progeny of sentinel and resident A. stolonifera plants occurred southeast and/or due south of the perimeter of the control district, in the direction of the prevailing winds.