Pollen-mediated Gene Flow in Alfalfa: A three-year summary of field research

S. Fitzpatrick1, P. Reisen2 and M. McCaslin3

Forage Genetics International, 1West Salem, WI, 2Nampa, ID, and 3Prior Lake, MN

Gene flow between alfalfa populations is a natural occurrence and bee-mediated cross-pollination is necessary for commercial seed production. Alfalfa seed producers use spatial isolation to separate varieties and control bee and pollen flow between fields. The minimum isolation standard for foundation and certified seed fields more than 5 acres is 600 and 50 ft, and for fields 5 acres or less, the standard is 900 and 165 ft, respectively [Association of Official Seed Certifying Agencies (AOSCA), 2003]. One area of interest regarding the application of biotechnology to the improvement of crops is the potential for movement of a transgene into unintended populations such as neighboring seed production fields or feral (non-cultivated) plants via pollen movement. Previous pollen-mediated gene flow information was based on studies that used naturally occurring marker genes (e.g., St. Amand, 2000 and Brown et al., 1986). In this report, we summarize a series of three pollen-mediated gene flow studies that were conducted with the purpose of gaining a more thorough understanding of alfalfa pollen flow dynamics under commercial production and management settings. In each of the three separate studies, 2000, 2001 and 2002, the pollen source plots were planted to Roundup Ready® alfalfa that contained a cp4-epsps transgene marker. Leafcutter bees (Megachile rotundata) were used as pollinators and the gene flow between seed production plots was measured using the transgene as an efficient, high-throughput, pollen marker system. All plants and seed that contained the marker gene were grown and handled in compliance with USDA-APHIS notification guidelines for release and movement of regulated plants.

Table. Isolation distance between trap and source, number of replicates per distance, replicate plot size (acres), trap plot cardinal direction from source and, interplot land cover a,b,c, the mean observed gene flow and the upper bound of true gene flow (i.e., the 99.9% confidence interval upper limit) are given.
Isolation distance /
2000
/
2001 a
/ 2002 a, b / 2000-2002 Mean Observed gene flow (99.9% C.I. upper bound)
0 ft (Source Plot) / Source (1.0 A) / Source (1.6 A) / Source (1 A)
500 ft / Reps 1-4: 0.03 A, N. c / - / - / 1.39% (1.72%)
900 ft / - / Rep 1: 0.7 A, N.
Rep 2: 1.6 A, N.E. / - / 0.28% (0.34%)
1000 ft / Reps 1-4: 0.03 A, N. c / - / - / 0.32% (0.45%)
1500 ft / Reps 1-4: 0.03 A, N. c / Rep 1: 1.6 A, W.
Rep 2: 1.6 A, N.W. / Rep 1: 1 A, S.W.
Rep 2: 1 A, S.E. / 0.08% (0.13%)
2000 ft / Rep 1: 2.0 A, N.W. a / - / - / 0.00% (0.05%)
2640 ft (1/2 mi) / - / - / Rep 1: 1 A, S.W.
Rep 2: 1 A., S.E. / 0.003% (0.02%)
3960 ft (3/4 mi) / - / - / Rep 1: 1 A, S.W.
Rep 2: 1 A, S.E. / 0.0000% (0.01%)
5280 ft (1 mi) / - / - / Rep 1: 1 A, N.W.
Rep 2: 1 A, S.E. / 0.0000% (0.01%)
Mean no. seed tested per trap distance / 14,750 / 41,250 / 60,000
Interplot land cover: a Various crop species typical for the area (e.g., onions, corn, wheat, etc.); b roadways, or, c fallow

Studies were conducted in an irrigated alfalfa seed growing area (Canyon County, Idaho) and were managed according to agronomic management practices typical for the Pacific Northwest Region where leafcutter bees are the introduced pollinator (experimental details are given in the Table). In 2000, all but one of the plots were separated by fallow ground, simulating a “worst-case” interplot management where bees would have no physical or visual barriers to movement. In contrast, all other trap plots during 2000-2002 were spatially isolated by land planted to a variety of crops and/or terrain typical for the region (e.g., wheat, onions, sweet corn, roadways, etc.). In addition to the recommended density of introduced leafcutter bees (2 gal. of loose cell pupae/A), a low number of feral honeybees were observed pollinating alfalfa flowers in the plots. A greenhouse seedling assay was used to measure gene flow between source and trap plots wherein, a sub-sample of seed from each trap plot replicate was evaluated for the incidence of Roundup Ready trait. The upper bound of true gene flow was calculated using the method of Remund et al. (2001). Specifically, the sum of the Roundup Ready marked seed and the total number of seed tested at that distance per location per year were compared, and the 99.9% confidence interval upper limit was calculated and used to estimate the upper bound of true gene flow for that isolation distance.

Results from these studies indicate that pollen-mediated gene flow diminished with increasing distance from the source (Table and Figure). Gene flow among the fallow-surrounded, worst-case management plots was 1.39%, 0.32% and 0.07% at 500, 1000 and 1500 ft, respectively. No gene flow (0.0000%) was detected at 2000 ft (in 2000), however, a single seed of the 30,000 seeds tested (0.003%) carried the trait when isolation was ½ mile (2640 ft) in 2002. Gene flow was not detected at the ¾ or 1 mile isolation distances. Observed gene flow (Yobs) was described by the equation, Yobs= (1x1010)(X-3.6262), R2=0.9391; and, the upper bound of the 99.9% confidence interval for gene flow, YCI was calculated as, YCI= (4x106)(X-2.3673), R2=0.9728.

Findings from these replicated studies demonstrate that alfalfa seed production fields may be effectively insolated from un-desired pollen flow using sufficient spatial isolation and adherence to currently re-commended pollinator manage-ment practices. As an example, the 99.9% confidence interval upper bound for gene flow (YCI) and mean observed gene flow may be calculated at the 900 ft foundation isolation distance as approximately 0.4 and 0.2%, respectively. These values would indicate that the foundation seed field isolation standard would be sufficient to produce seed with 99.6 to 99.8 % varietal purity. The amount of pollen-mediated gene flow in alfalfa—an insect-pollinated crop, as observed in these studies approximates that reported for corn—a wind-pollinated crop. For instance, in a study conducted by Haskell and Dow (1951), outcrossing in corn was 2.33, 0.48 and 0.20% for plants located 410, 984 and 1640 ft (125, 300 and 500 m) from source plants, respectively.

This, and other available gene-flow information may be used by alfalfa seed producers in setting reasonable and informed field isolation standards for the production of high quality conventional and biotech alfalfa seed products. The 2000, 2001 and 2002 study details and data have been posted at:

References:

ASOCA, 2003. Genetic Standards Guide.

Brown, D. E., et al. 1986. Page 123 In: Rept. of the 30th N. Amer. Alfalfa Impr. Conf.

Haskell, G. and Dow, P. 1951. Empire J. of Exp. Agric. (London) 19: 45-50.

Remund, K. M., et al. 2001. Seed Science Research 11: 101-119.

St. Amand, P.C., et al. 2000. Theor. Appl. Genet. 101: 107-114.

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