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

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

• S. Fitzpatrick, P. Reisen, M. McCaslin
• Forage Genetics International
• www.foragegenetics.com

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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

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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

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Introduction Varietal purity
AOSCA minimum standards for isolation AOSCA minimum standards for isolation AOSCA minimum standards for isolation
Field size lt 5 acres Field size gt 5 acres
Foundation Class 900 ft 600 ft
Certified Class 165 ft 50 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.

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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.

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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

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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

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2000-2002 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)

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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

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Experimental details, 2000-2002
Isolation Distance 2000 Study 2001 Study 2002 Study
0 ft Source (1 A) Source (1.6 A) Source (1.0 A)
500 ft 4 reps (0.03 A) --- ---
900 ft --- 2 reps (.7-1.6 A) ---
1000 ft 4 reps (0.03 A) --- ---
1500 ft 4 reps (0.03 A) 2 reps (1.6 A) 2 reps (1.0 A)
2000 ft 1 rep (2 A) --- ---
2640 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)
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2000 Study Design
2000 ft
Fallow
Area of all trap plots was 0.03 A, except 2000
trap was 2.0 A. Source plot was 1.0 A.
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2000 Gene Flow Study(actual scale)
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2001 Pollen Flow Trial
Bee domicile
Source Plot (with RR transgene)
Trap Plot
1500 ft
Wheat
900 ft
Trap Plot
Bee domicile
Trap Plot
Trap Plot
Bee domicile
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2002 Pollen Flow Trial
1 mile
1/2 mile
1500
RR
3/4 mile
Interplot area was planted with various crops and
included roadways.
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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
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Results
Table. Percent observed gene flow and upper
bound value of the 99.9 confidence interval (in
parentheses)
Isolation Distance 2000 Study 2001 Study 2002 Study 2000-2002 Mean
500 ft 1.39 (1.72) -- -- 1.39 (1.72)
900 ft -- 0.28 (0.34) -- 0.28 (0.34)
1000 ft 0.32 (0.45) -- -- 0.32 (0.45)
1500 ft 0.07 (0.17) 0.13 (0.17) 0.03 (0.06) 0.08 (0.13)
2000 ft 0.00 (0.05) -- -- 0.00 (0.05)
2640 ft (1/2 mi) -- -- 0.003 (0.02) 0.003 (0.02)
3960 ft (3/4 mi) -- -- 0.0000 (0.01) 0.0000 (0.01)
5280 ft (1 mi) -- -- 0.0000 (0.01) 0.0000 (0.01)
Seed tested per distance 14,750 41,250 32,400
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Results Gene Flow Decay Curve
99.9 C.I. Values YCI (4x106)(X-2.3673),
R20.97
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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.

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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