Technology Protection System (TPS) Science and Issues

1. Technology Protection System (TPS) Science and Issues Mel Oliver

2. The Need for TPS • To protect the environment from the escape of transgenes to non-crop species • To protect US technology • Stimulate the investment by US companies in plant biotechnology

3. Why USDA-ARS? • Transfer of new technology to the private sector and industry is a responsibility of all Federal research agencies :- Following Laws. • Federal Technology Transfer Act of 1986 • Stevenson-Wydler Act of 1980 • Bahl-Dole Act of 1980 • CRADA -Cooperative Research and Development Agreement ensuring access

4. Benefits of Protection Systems • Protection of the environment from gene escapes into other plant species • Protection of technology provider’s investment against free use of technology • Incentive to companies to develop technologies - new products and new choices for the producer.

5. Genetic Protection Systems VGURT - Varietal Gene Use restriction Technology TGURT - Traite Gene Use Restriction Technology

6. Transgene Transgene Transgene Transgene Environmental and Technology Protection in Cotton Non TPS Pollen carrying LEA-RIP/Barnase Activated TPS Non Germinable Seed carrying LEA-RIP/Barnase

7. LOX LOX Tet-AMV-P35S RIP CDS CRE LEA Full System in Planta Inactive CRE gene P35S(TetO) Tet represor Active CRE gene Tet Induction at germination CRE Excision Non- germinable Progeny Seed LEA RIP CDS Oliver, Trolinder, Keim, and Quisenberry 1998

8. + 35SOP CRE R1 Lea/TetR/RIP R1 Transgenic Ro Plantlet Hypocotyl Agrobacterium Transformation x Multi PCR Verified TPS

9. Germination Disruption Genes • Barnase isolated by PCR from Bacillus amyloliquifaciens DNA • NcoI site engineered at 5’ end • Two forms • unmodified barnase CDS • attenuated barnase - upstream 2 out-of-frame ATG in leader (PCR)

10. Germination Disruption Genes • RIP - plant ribosome inactivating protein • Obtained from Roger Beachy - effective protein synthesis inhibitor in vitro • Two forms • with targeting leader - non effective • without targeting leader - effective • RIP was effective in plants

11. Lea Promoters • Promoters were chosen for time of expression (late in maturation) and apparent lack of expression in leaves in response to ABA • Promoters were Lea 4A and 14A (like). • Isolated by PCR from Coker 312 genomic DNA (4A near identical, 14A 80% similar) • NcoI site engineered at the 3’ end of the promoter for CDS placement.

12. Lea-Germination Disruption Gene Constructs LOX R RIP (Del1) LOX L gene 7 term LEA Promoter (4 or 14) Barnase Lea 5’ Leader Att Barnase

13. Tet Repressor Stuffer Construct tet repressor of Tn10 isolated by PCR from E.coli genomic DNA Full 35S Promoter Tn10 repressor AMV Leader Nos 3’ term

14. LOX Sites • Constructed from oligonucleotides • Cloned and sequenced to confirm • Oriented to negate the insertion of an ATG codon upstream of RIP or Barnase CDS

15. Construction of the Blocked Germination Disruptor Gene Lea Promoter + Leader RIP/Barnase/attBarnase + g7T LOX-L LOX-R Nos -tet repressor-AMV-35S

16. Construction of 35SOP - CRE Full 35S Promoter CRE Nos 3’ term Region of 35S modified by oligos to give 3 tet operator sites

17. Constructs for Tobacco and Cotton • 35SopCRE • Lea4A-- LOXNosTTetR35SLOX --Del1g7T • Lea4A -- LOXNosTTetR35SLOX --Barng7T • Lea4A -- LOXNosTTetR35SLOX --AttBarng7T • Lea14 -- LOXNosTTetR35SLOX --Del1g7T • Lea14 -- LOXNosTTetR35SLOX --AttBarng7T

18. Tobacco Development Scheme

19. Complete TPS in place 35SopCRE parents tested for CRE expression Tet induction and Cre excision of verified Parent lines homozygous Progeny tests complete TPS plants derived from activated seed produce normal seed (appearance, weight and yeild) Seeds from these plants do not germinate (100%) Disruptor genes - LEA4-saporin (RIP) and att-Barnase Progress- Tobacco

20. Cre Expression in Transgenic Tobacco Transgenic Tobacco + - N O P A J1 L1 N1 CRE + His Tag CRE

21. Excision in Tobacco Nos -tet repressor-AMV-35S Lea Promoter + Leader attBarnase + g7 T LOX-R LOX-L Tet activation of CRE - seed treatment LOX In-frame and precise Excision tested in leaf tissue - nested PCR - cloning and sequence

22. Tet-repressor Expression Progeny from 35SopCRE(AX6) x L14AB(E4) 0 2 10 Tet ug/ml 50 100 Tet ug/ml

23. Tet-repressor Expression Progeny from 35SopCRE(AK4) x L14AB(E6) 0 2 10 Tet ug/ml 100 50 Tet ug/ml

24. Progress - Cotton • 35SopCRE parents in place - CRE expression tested • Lea parents - tet repressor expression tested • Both parent lines homozygous

25. LOX LOX CRE RIP CDS Kan Marker LEA LEA RIP CDS CRE Full System in Planta Chemical Inducer at Germination Excision Non- germinable Progeny Seed + ] [ (degraded) Kan Resistance marker lost USDA-ARS

26. Advantages of TPS 1. Biosafety 2. Improved competitive landscape for North American farmers 3. Increased returns to farmers in all areas of the world 4. Probable prevention of seed sprouting in head

27. Advantages of TPS • 1. Biosafety • Prevents the remote possibility of • transgenic genes escaping into the • environment. • a. Volunteer seeds which drop to • the ground will be nonviable.

28. Advantages of TPS 1. Biosafety (cont'd) b. Pollen which could possibly fertilize flowers of wild species near a TPS crop field will produce nonviable seeds.

29. Transgene Transgene Transgene Transgene Environmental and Technology Protection in Cotton Non TPS Pollen carrying LEA-RIP/Barnase Activated TPS Non Germinable Seed carrying LEA-RIP/Barnase

30. Advantages of TPS • 2. Improved competitive landscape for • North American farmers. • TPS will provide a more level playing field for North American farmers as farmers in other countries will also have to pay for improved varieties and transgenic traits.

31. Advantages of TPS 2. Improved competitive landscape for North American farmers. a. Varieties have been pirated out of North America. b. Transgenic traits have been pirated out of North America.

32. Advantages of TPS 3. Increased returns to farmers Because of the possibility of a return on investment in breeding research, many more improved varieties should be available. a. In crops which have not been given optimum breeding attention; - wheat - soybeans - rice

33. Advantages of TPS 3. Increased returns to farmers (cont'd) b. In countries in which breeding research has not been at a level proportionate to their agricultural importance. c. Transgenic traits may be more available to farmers in crops and countries in which they have not been.

34. Farmers will not pay directly for TPS.

35. TPS will be broadly available to both large and small seed firms in the U.S. and in other countries. Therefore, TPS itself will not confer a competitive advantage.

36. The competitive advantage for seed firms will only be gained through increased breeding of superior varieties. This is a distinct advantage of the farmers.

37. Farmers will buy seed carrying TPS only if they receive a return on their investment, such as; a. Improved yields b. Improved quality traits c. More and better pest resistance

38. Farmers will continue to have seed of non-TPS varieties available to them.

39. Advantages of TPS 4. Probable prevention of seed sprouting in the head prior to harvest

40. Genetic Diversity There should be no correlation between TPS and reduced genetic diversity.

41. Global Perceptions of the TPS

42. The Reaction of North American Farmers to the TPS Technology

43. Misconceptions Concerning the Technology Protection System (TPS)

44. MISCONCEPTION: • Seed sterility can spread and wipe out populations and species. • FACTS: • Sterile seeds do not produce plants and • those nonexistent plants cannot produce pollen to spread the sterility trait. • The sterility trait lasts only one generation • and by definition, does not propagate itself.

45. MISCONCEPTION: • Home gardeners will not be able to save seed because of TPS. • FACTS: • Garden vegetable and flower varieties are • not targets for TPS because of the small • markets and acreage and the difficulty and • costs related to converting, through • biotechnology, many different species and • varieties to TPS.

46. MISCONCEPTION: • Gardeners will be prevented from providing viable seeds of heirloom vegetable varieties to others. • FACTS: • Because of the effort and cost, seed • companies will not incorporate TPS into old • heirloom varieties. Seed companies are • more interested in protecting new varieties • carrying technically advanced traits.

47. MISCONCEPTION: • Farmers will not be able to save seed of non-TPS crops in field adjacent to TPS crops. • FACTS: • The large targeted crops of soybean, wheat • and rice are highly self pollinated. • Therefore, the farmers' crop will already be • pollinated by its own pollen before being • pollinated by sterile pollen from TPS plants.

48. MISCONCEPTION: • Farmers will have to use TPS varieties. • FACTS: • Farmers will continue to have the choice of • planting TPS varieties or non-TPS varieties. • They will make their choice based on • whether they get a payback on their • investment in the seed cost each year.

49. MISCONCEPTION: • TPS will put the farmers at a disadvantage. • FACTS: • It should make more and better varieties • available to farmers, in particular varieties • carrying new technologies e.g., transgenic • traits. • New genetics would be available in crops • and geographic areas which, at this time, • are receiving insufficient attention.

50. MISCONCEPTION: • Farmers are against TPS. • FACTS: • Much interest and support for TPS has been • shown by American farmers, both by • individuals and by farmer organizations • such as the Sunflower Association, the • National Cotton Council and the National • Grain Sorghum Producers.