Technology Protection System (TPS)Science and Issues

Mel Oliver

The Need for TPSThe 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

Why USDA-ARS?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

Benefits of Protection Systems

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.

Genetic Protection SystemsGenetic Protection Systems

VGURT - Varietal Gene Use restriction TechnologyTGURT - Traite Gene Use Restriction Technology

Transgene

Activated TPS

Non Germinable Seed carrying LEA-RIP/Barnase

Pollen carrying LEA-RIP/Barnase Non TPS

Environmental and Technology Protection in Cotton

Transgene

Transgene

Transgene

Full System in PlantaFull System in Planta

P35S(TetO)

LOX LOXTet-AMV-P35SLEA RIP

CDS

Tet Induction at germination

LEA RIP CDS

CRE

CRE

Tet represor

Inactive CRE gene

Active CRE gene

Excision

Non-germinableProgeny Seed

Oliver, Trolinder, Keim, and Quisenberry 1998

Hypocotyl

Agrobacterium

Transformation

Transgenic Ro

Plantlet

Multi PCR

Verified

x

Lea/TetR/RIP R1

TPS

35SOP CRE R1+

Germination Disruption GenesGermination 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)

RIP - plant ribosome inactivating protein Obtained from Roger Beachy - effective

protein synthesis inhibitor in vitro Two forms

with targeting leader - non effectivewithout targeting leader - effective

RIP was effective in plants

Germination Disruption GenesGermination Disruption Genes

Lea PromotersLea 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.

Lea-Germination Disruption Gene Constructs

Lea-Germination Disruption Gene Constructs

LEA Promoter (4 or 14)

Lea 5’ Leader

RIP (Del1)

gene 7 termBarnase

Att Barnase

LOX L

LOX R

Tet Repressor Stuffer Construct

Tet Repressor Stuffer Construct

Full 35S Promoter

AMV Leader Nos 3’ term

Tn10 repressor

tet repressor of Tn10 isolated by PCR from E.coli genomic DNA

LOX SitesLOX Sites

Constructed from oligonucleotides Cloned and sequenced to confirm Oriented to negate the insertion of an

ATG codon upstream of RIP or Barnase CDS

Construction of the Blocked Germination Disruptor GeneConstruction of the Blocked Germination Disruptor Gene

Nos -tet repressor-AMV-35S

Lea Promoter + LeaderRIP/Barnase/attBarnase + g7T

LOX-L LOX-R

Construction of 35SOP - CRE

Construction of 35SOP - CRE

Full 35S Promoter

Region of 35S modified by oligosto give 3 tet operator sites

Nos 3’ term

CRE

Constructs for Tobacco and Cotton

Constructs for Tobacco and Cotton

35SopCRE Lea4A-- LOXNosTTetR35SLOX -- Del1g7T Lea4A -- LOXNosTTetR35SLOX -- Barng7T Lea4A -- LOXNosTTetR35SLOX -- AttBarng7T Lea14 -- LOXNosTTetR35SLOX -- Del1g7T Lea14 -- LOXNosTTetR35SLOX -- AttBarng7T

Tobacco Development SchemeTobacco Development Scheme

35SoP-Cre

35SoP-Cre

35SoP-Cre 35SoP-Cre

35SoP-Cre

Lea Parent

Lea Parent

Lea Parent

Lea ParentLea Parent

R0 R0

R1 R1

R2R2

R2 R2

R2-F1

HeterozygotesHeterozygotes

HomozygotesHomozygotesHeterozygotes Heterozygotes

Kan+

Kan+

Kan+

Kan+

Kan+

Kan+

Kan+

Kan+

TPS Tobacco

Kan+Kan+

Male Female

R3R3 35SoP-Cre Lea Parent

Kan+Homozygotes

Tet50 and Tet100

R2-F2

Heterozygotes Heterozygotes

Homozygotes

HomozygotesHomozygotes

Kan - & + Kan - & +

Curently preparing to do

germination tests on MS Plates

Progress- TobaccoProgress- Tobacco

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

Cre Expression in Transgenic Tobacco

Cre Expression in Transgenic Tobacco

-+N P A J1 L1 N1O

Transgenic Tobacco

CRECRE + His Tag

Excision in TobaccoExcision in Tobacco

Nos -tet repressor-AMV-35SLea Promoter + Leader attBarnase + g7 T

Tet activation of CRE - seed treatment

Excision tested in leaf tissue - nested PCR - cloning and sequenceIn-frame and precise

LOX-L LOX-R

LOX

Tet-repressor ExpressionTet-repressor Expression

Progeny from 35SopCRE(AX6) x L14AB(E4)

0 2 10

50 100Tet ug/ml

Tet ug/ml

Tet-repressor ExpressionTet-repressor Expression

Progeny from 35SopCRE(AK4) x L14AB(E6)

0 2 10

50 100Tet ug/ml

Tet ug/ml

Progress - CottonProgress - Cotton

35SopCRE parents in place - CRE expression tested

Lea parents - tet repressor expression tested Both parent lines homozygous

USDA-ARS

Full System in PlantaFull System in PlantaLOX LOX

CREKan MarkerLEA RIP CDS

Chemical Inducerat Germination

LEA RIP CDS CRE

ExcisionNon-germinableProgeny Seed

Kan Resistance marker lost

(degraded)[ ]+

Advantages of TPSAdvantages of TPS1. Biosafety1. Biosafety

2. Improved competitive landscape for2. Improved competitive landscape for North American farmersNorth American farmers3. Increased returns to farmers in all 3. Increased returns to farmers in all areas of the worldareas of the world

4. Probable prevention of seed sprouting4. Probable prevention of seed sprouting in headin head

Advantages of TPSAdvantages of TPS1. Biosafety1. Biosafety

Prevents the remote possibility of Prevents the remote possibility of transgenic genes escaping into thetransgenic genes escaping into the environment.environment.

a. Volunteer seeds which drop toa. Volunteer seeds which drop to the ground will be nonviable.the ground will be nonviable.

Advantages of TPSAdvantages of TPS1. Biosafety 1. Biosafety (cont'd)(cont'd)

b. Pollen which could possibly fertilize b. Pollen which could possibly fertilize flowers of wild species near a TPS flowers of wild species near a TPS crop field will produce nonviable crop field will produce nonviable seeds. seeds.

Transgene

Activated TPS

Non Germinable Seed carrying LEA-RIP/Barnase

Pollen carrying LEA-RIP/Barnase Non TPS

Environmental and Technology Protection in Cotton

Transgene

Transgene

Transgene

Advantages of TPSAdvantages of TPS2. Improved competitive landscape for2. Improved competitive landscape for North American farmers.North American farmers.

TPS will provide a more level playing TPS will provide a more level playing field for North field for North American farmers as American farmers as farmers in other countries will also have farmers in other countries will also have to pay for improved varieties and to pay for improved varieties and transgenic traits.transgenic traits.

Advantages of TPSAdvantages of TPS2. Improved competitive landscape for2. Improved competitive landscape for North American farmers.North American farmers.

a. Varieties have been pirated out ofa. Varieties have been pirated out of North America.North America.

b. Transgenic traits have been b. Transgenic traits have been pirated out of North America.pirated out of North America.

Advantages of TPSAdvantages of TPS3. Increased returns to farmers3. Increased returns to farmers

Because of the possibility of a returnBecause of the possibility of a returnon investment in breeding research,on investment in breeding research,many more improved varieties many more improved varieties should should be available.be available.

a. In crops which have not been givena. In crops which have not been given optimum breeding attention;optimum breeding attention;

- wheat- wheat - soybeans- soybeans - rice - rice

Advantages of TPSAdvantages of TPS3. Increased returns to farmers 3. Increased returns to farmers (cont'd)(cont'd)

b. In countries in which breeding b. In countries in which breeding research has not been at a levelresearch has not been at a level proportionate to their agricultural proportionate to their agricultural importance. importance.c. Transgenic traits may be more c. Transgenic traits may be more available to farmers in crops and available to farmers in crops and countries in which they have not countries in which they have not been.been.

Farmers will not pay directly for Farmers will not pay directly for TPS.TPS.

TPS will be broadly available to TPS will be broadly available to both large and small seed firms in both large and small seed firms in the U.S. and in other countries. the U.S. and in other countries.

Therefore, TPS itself will not confer Therefore, TPS itself will not confer a competitive advantage.a competitive advantage.

The competitive advantage for seed The competitive advantage for seed firms will only be gained through firms will only be gained through increased breeding of superior increased breeding of superior varieties.varieties.

This is a distinct advantage of the This is a distinct advantage of the farmersfarmers..

Farmers will buy seed carrying TPS Farmers will buy seed carrying TPS only if they receive a return on their only if they receive a return on their investment, such as;investment, such as;

a. Improved yieldsa. Improved yields

b. Improved quality traitsb. Improved quality traits

c. More and better pest resistancec. More and better pest resistance

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

Advantages of TPSAdvantages of TPS

4. Probable prevention of seed sprouting 4. Probable prevention of seed sprouting in the head prior to harvestin the head prior to harvest

Genetic DiversityGenetic DiversityThere should be no correlation between There should be no correlation between TPS and reduced genetic diversity.TPS and reduced genetic diversity.

Global Perceptions of the TPSGlobal Perceptions of the TPS

The Reaction of North The Reaction of North American Farmers to the American Farmers to the TPS TechnologyTPS Technology

MisconceptionsMisconceptionsConcerning theConcerning the

Technology ProtectionTechnology ProtectionSystem (TPS)System (TPS)

MISCONCEPTION:MISCONCEPTION:Seed sterility can spread and wipe out Seed sterility can spread and wipe out populations and species.populations and species.

FACTS:FACTS:• Sterile seeds do not produce plants andSterile seeds do not produce plants and

those nonexistent plants cannot produce those nonexistent plants cannot produce pollen to spread the sterility trait.pollen to spread the sterility trait.

• The sterility trait lasts only one generation The sterility trait lasts only one generation and by definition, does not propagate itself.and by definition, does not propagate itself.

MISCONCEPTION:MISCONCEPTION:Home gardeners will not be able to save seed Home gardeners will not be able to save seed because of TPS.because of TPS.

FACTS:FACTS:• Garden vegetable and flower varieties areGarden vegetable and flower varieties are not targets for TPS because of the smallnot targets for TPS because of the small markets and acreage and the difficulty andmarkets and acreage and the difficulty and costs related to converting, throughcosts related to converting, through biotechnology, many different species and biotechnology, many different species and varieties to TPS.varieties to TPS.

MISCONCEPTION:MISCONCEPTION:Gardeners will be prevented from providing Gardeners will be prevented from providing viable seeds of heirloom vegetable varieties to viable seeds of heirloom vegetable varieties to others.others.

FACTS:FACTS:• Because of the effort and cost, seed Because of the effort and cost, seed companies will not incorporate TPS into oldcompanies will not incorporate TPS into old heirloom varieties. Seed companies are heirloom varieties. Seed companies are more interested in protecting new varietiesmore interested in protecting new varieties carrying technically advanced traits.carrying technically advanced traits.

MISCONCEPTION:MISCONCEPTION:Farmers will not be able to save seed of non-Farmers will not be able to save seed of non-TPS crops in field adjacent to TPS crops.TPS crops in field adjacent to TPS crops.

FACTS:FACTS:• The large targeted crops of soybean, wheatThe large targeted crops of soybean, wheat and rice are highly self pollinated. and rice are highly self pollinated. Therefore, the farmers' crop will already beTherefore, the farmers' crop will already be pollinated by its own pollen before beingpollinated by its own pollen before being pollinated by sterile pollen from TPS plants.pollinated by sterile pollen from TPS plants.

MISCONCEPTION:MISCONCEPTION:Farmers will have to use TPS varieties.Farmers will have to use TPS varieties.

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

MISCONCEPTION:MISCONCEPTION:TPS will put the farmers at a disadvantage.TPS will put the farmers at a disadvantage.

FACTS:FACTS:• It should make more and better varietiesIt should make more and better varieties available to farmers, in particular varietiesavailable to farmers, in particular varieties carrying new technologies e.g., transgeniccarrying new technologies e.g., transgenic traits.traits.

• New genetics would be available in cropsNew genetics would be available in crops and geographic areas which, at this time, and geographic areas which, at this time, are receiving insufficient attention.are receiving insufficient attention.

MISCONCEPTION:MISCONCEPTION:Farmers are against TPS.Farmers are against TPS.

FACTS:FACTS:• Much interest and support for TPS has beenMuch interest and support for TPS has been shown by American farmers, both byshown by American farmers, both by individuals and by farmer organizationsindividuals and by farmer organizations such as the Sunflower Association, thesuch as the Sunflower Association, the National Cotton Council and the NationalNational Cotton Council and the National Grain Sorghum Producers.Grain Sorghum Producers.

MISCONCEPTION:MISCONCEPTION:TPS would be dangerous for the environment.TPS would be dangerous for the environment.

FACTS:FACTS:• TPS is environmentally friendlyTPS is environmentally friendly

• TPS could be an important tool in TPS could be an important tool in preventing the escape of transgenic traits topreventing the escape of transgenic traits to wild species through pollen.wild species through pollen.

““We have recently advanced our We have recently advanced our knowledge of genetics to a point knowledge of genetics to a point where we can manipulate life in a where we can manipulate life in a way never intended by nature -- We way never intended by nature -- We must proceed with the utmost must proceed with the utmost caution in the application of this caution in the application of this new found knowledge.”new found knowledge.”

Skepticism for "New" GeneticsSkepticism for "New" Genetics

1906 Critique of 1906 Critique of Luther Burbank'sLuther Burbank's

Studies on Hybrid PlantsStudies on Hybrid Plants