Transcript TPS

Technology Protection System (TPS)
Science and Issues
Mel Oliver
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
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
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 Systems
VGURT - Varietal Gene Use restriction Technology
TGURT - Traite Gene Use Restriction Technology
Environmental and Technology Protection
in Cotton
Transgene
Transgene
Pollen carrying LEA-RIP/Barnase
Non TPS
Activated TPS
Transgene
Transgene
Non Germinable Seed carrying LEA-RIP/Barnase
Full System in Planta
Inactive CRE gene
LOX
LOX
Tet-AMV-P35S
RIP CDS
LEA
CRE
P35S(TetO)
Tet represor
Active CRE gene
Tet Induction at
germination
CRE
Excision
LEA
RIP CDS
Nongerminable
Progeny Seed
Oliver, Trolinder, Keim, and Quisenberry 1998
Lea/TetR/RIP R1
Transgenic Ro
Plantlet
Hypocotyl
x
Agrobacterium
Transformation
Multi PCR
TPS
Verified
+
35SOP CRE R1
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-offrame ATG in leader (PCR)
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
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.
Lea-Germination Disruption
Gene Constructs
LOX R
RIP (Del1)
LOX L
gene 7 term
LEA Promoter (4 or 14)
Lea 5’ Leader
Barnase
Att Barnase
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
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
Construction of the Blocked
Germination Disruptor Gene
Lea Promoter + Leader
LOX-L
RIP/Barnase/attBarnase + g7T
LOX-R
Nos -tet repressor-AMV-35S
Construction of 35SOP CRE
Full 35S Promoter
CRE
Nos 3’ term
Region of 35S modified by oligos
to give 3 tet operator sites
Constructs for Tobacco and
Cotton
35SopCRE
Lea4A-- LOXNosTTetR35SLOX -- Del1g7T
Lea4A -- LOXNosTTetR35SLOX -- Barng7T
Lea4A -- LOXNosTTetR35SLOX -- AttBarng7T
Lea14 -- LOXNosTTetR35SLOX -- Del1g7T
Lea14 -- LOXNosTTetR35SLOX -- AttBarng7T
Tobacco Development Scheme
TPS Tobacco
Male
35SoP-Cre
R0
R1
Female
Kan+
Heterozygotes
Kan+
35SoP-Cre
35SoP-Cre
Kan+
Lea Parent
Kan+
35SoP-Cre
35SoP-Cre
Heterozygotes
Homozygotes
Kan+
R3
Lea Parent
Heterozygotes
R2
Kan+
Kan - & +
Heterozygotes
R1
Heterozygotes
R2
Lea Parent
R0
R2
R2
R2-F1
Homozygotes
Kan+
35SoP-Cre
Tet50 and Tet100
R2-F2
Homozygotes
Curently preparing to do
germination tests on MS Plates
Kan+
Kan+
Kan - & +
Lea Parent
Lea Parent
Homozygotes
Heterozygotes
Kan+
R3
Homozygotes
Kan+
Lea Parent
Homozygotes
Progress- 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
Transgenic Tobacco
N O P A J1 L1 N1
+ CRE + His Tag
CRE
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
Tet-repressor Expression
Progeny from 35SopCRE(AX6) x L14AB(E4)
Tet ug/ml
0
Tet ug/ml
50
2
100
10
Tet-repressor Expression
Progeny from 35SopCRE(AK4) x L14AB(E6)
0
Tet ug/ml
Tet ug/ml
50
2
100
10
Progress - Cotton
35SopCRE parents in place - CRE expression
tested
Lea parents - tet repressor expression tested
Both parent lines homozygous
Full System in Planta
LOX
LEA
LOX
Kan Marker
RIP CDS
CRE
Chemical Inducer
at Germination
Excision
LEA
RIP CDS
[
+
Nongerminable
Progeny Seed
CRE
]
(degraded)
Kan Resistance marker lost
USDA-ARS
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
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.
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.
Environmental and Technology Protection
in Cotton
Transgene
Transgene
Pollen carrying LEA-RIP/Barnase
Non TPS
Activated TPS
Transgene
Transgene
Non Germinable Seed carrying LEA-RIP/Barnase
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.
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.
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
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.
Farmers will not pay directly for
TPS.
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.
The competitive advantage for seed
firms will only be gained through
increased breeding of superior
varieties.
This is a distinct advantage of the
farmers.
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
Farmers will continue to have seed
of non-TPS varieties available to
them.
Advantages of TPS
4. Probable prevention of seed sprouting
in the head prior to harvest
Genetic Diversity
There should be no correlation between
TPS and reduced genetic diversity.
Global Perceptions of the TPS
The Reaction of North
American Farmers to the
TPS Technology
Misconceptions
Concerning the
Technology Protection
System (TPS)
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.
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.
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.
MISCONCEPTION:
Farmers will not be able to save seed of nonTPS 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.
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.
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.
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.
MISCONCEPTION:
TPS would be dangerous for the environment.
FACTS:
• TPS is environmentally friendly
• TPS could be an important tool in
preventing the escape of transgenic traits to
wild species through pollen.
Skepticism for "New" Genetics
“We have recently advanced our
knowledge of genetics to a point
where we can manipulate life in a
way never intended by nature -- We
must proceed with the utmost
caution in the application of this
new found knowledge.”
1906 Critique of
Luther Burbank's
Studies on Hybrid Plants