Pollen-mediated Gene Flow in Alfalfa : A three-year summary of field research S. Fitzpatrick, P. Reisen, M. McCaslin Forage Genetics International

Introduction: Alfalfa gene flow  In nature, alfalfa is cross-pollinated by bees.  Cross-pollination is required for normal seed production and vigor.  Alfalfa does not out-cross to other species in North America (i.e., no gene flow occurs)  Gene flow between alfalfa populations occurs unless controlled by isolation & management.  Cultivated Feral populations  “Cultivar A” “Cultivar B”

Introduction: Varietal purity  Consistent varietal purity is important for all varieties: both traditional and biotech.  Association of Official Seed Certifying Agencies (AOSCA) has set minimum standard for varietal purity:  1.0 % non-variety off-types maximum in certified seed  0.1% non-variety off-types maximum in foundation seed

Introduction: Varietal purity AOSCA minimum standards for isolation*: Field size: < 5 acres Field size: > 5 acres Foundation Class900 ft600 ft Certified Class165 ft50 ft  Standards are based on gene flow studies in which pest resistance genes were used as pollen markers (Brown, et al., 1986); and some state associations have more stringent standards.

Introduction: Varietal purity  The impact of pollen-mediated gene flow on varietal purity has received new attention with the advent of biotech-derived traits.  Biotech traits may be more readily identified than most non-biotech traits.  Adventitious presence (AP) of approved biotech traits in unintended seed lots is an international, national and commercial commerce issue.

Study Objectives  Expand understanding of alfalfa gene-flow dynamics in commercial seed production settings.  Measure gene flow using a newly available pollen-marker system: cp4-epsps transgene + Roundup Ready® trait  Unique, definitive marker gene  Sensitive, high-capacity greenhouse assay Roundup Ready® is a trademark of Monsanto, LLC

Study Objectives (cont.)  Mimic gene flow between seed fields during commercial seed production  Measure flow at 500 feet to 1 mile  Share data and inform alfalfa community:  ASOCA and NAAIC Regulatory Committee have expressed interest in reassessing seed production standards  Seed company quality assurance program development to meet future needs

: Methods  Location: Canyon Co., Idaho  Agronomic management: typical commercial practices for Pacific Northwest  Pollinator: Leafcutter bees  Plot size:  Source plots were 1 or 1.6 A each (+ pollen marker)  Replicated pollen trap plots were 0.3, 0.7, 1.6 or 2.0 A each (no pollen marker)

NOTE: Special precautions and USDA requirements of the study  Roundup Ready alfalfa is regulated by the USDA under the notification acknowledgement process.  Purpose: Containment of test plants to USDA- authorized conditions  The USDA notification process requires: USDA-APHIS authorities review a detailed application and compliance plan and pre-authorize Applicant must follow USDA and state guidelines for isolation, handling and monitoring FGI and Monsanto operated these studies within USDA rules

Experimental details, Isolation Distance 2000 Study2001 Study2002 Study 0 ftSource (1 A)Source (1.6 A)Source (1.0 A) 500 ft4 reps (0.03 A) ft---2 reps ( A) ft4 reps (0.03 A) ft4 reps (0.03 A)2 reps (1.6 A)2 reps (1.0 A) 2000 ft1 rep (2 A) ft (1/2 mi)--- 2 reps (1.0 A) 3960 ft (3/4 mi)--- 2 reps (1.0 A) 5280 ft (1 mi)--- 2 reps (1.0 A)

2000 Study Design Source Plot (with transgene) Trap plot 2000 ft Wheat Bee domicile North 500 ft 1000 ft 1500 ft Area of all trap plots was 0.03 A, except 2000’ trap was 2.0 A. Source plot was 1.0 A. Fallow 2000 ft

2000 Gene Flow Study (actual scale) 2000 ft 500 ft1500 ft1000 ft

2001 Pollen Flow Trial Source Plot (with RR transgene) Trap Plot 900 ft 1500 ft Bee domicile Wheat

2002 Pollen Flow Trial RR 1 mile 1500’ 1/2 mile 3/4 mile Interplot area was planted with various crops and included roadways.

Methods: Greenhouse assay  Seed (progeny) from traps was collected in a grid pattern  Greenhouse grow-out assay used to detect marker gene:  Seedlings + dominant marker survive and, those without marker are killed  + Results were verified by protein assay + marker no marker

Results Isolation Distance 2000 Study2001 Study2002 Study Mean 500 ft1.39 (1.72) (1.72) 900 ft (0.34) (0.34) 1000 ft0.32 (0.45) (0.45) 1500 ft0.07 (0.17)0.13 (0.17)0.03 (0.06)0.08 (0.13) 2000 ft0.00 (0.05) (0.05) 2640 ft (1/2 mi) (0.02) 3960 ft (3/4 mi) (0.01) 5280 ft (1 mi) (0.01) Seed tested per distance 14,75041,25032,400 Table. Percent observed gene flow and upper bound value of the 99.9% confidence interval (in parentheses)

Results: Gene Flow Decay Curve 99.9% C.I. Values: Y CI = (4x10 6 )(X ), R 2 =0.97

Summary  Spatial isolation methods are effective in controlling pollen-mediated gene flow.  Stringency required for varietal purity (% off- type threshold) may be used to calculate minimum field isolation required.  This data may be used to set reasonable and informed seed field isolation standards for the production of high quality conventional and biotech alfalfa seed products.

Acknowledgements  NAAIC Regulatory Affairs Committee  Monsanto  Glen Rogan, Regulatory Technology Manager  Michael Horak and Todd Pester, Ecological Technology Center  Kirk Remund, SeedCalc3 statistical program  Details of studies are published on- line at: foragegenetics.com ALFALFA