terminator technology in seeds

1. GeneticGenetic UseUse RestrictionRestriction TechnologiesTechnologies (GURTs)(GURTs)
and their application in seed industryand their application in seed industry
SIDDARAJSIDDARAJ
PGS17AGR7577PGS17AGR7577
Master’s Seminar - IMaster’s Seminar - I
onon
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2. IntroductionIntroduction1
Genesis of GURTsGenesis of GURTs 2
Types of GURTsTypes of GURTs3
General molecular constructionGeneral molecular construction 4
Mechanisms of GURTsMechanisms of GURTs5
State of artState of art 6
Benefits and risksBenefits and risks7
ConclusionConclusion 8
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3. INTRODUCTIOTYPESMOLECULAR
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• Seed being a basic crucial input in agriculture plays a pivotal role in
sustainable agriculture.
• Intensive agriculture requires the use of certified seed. Most growers
cultivate hybrid varieties of maize and other crops that are more uniform
and vigourous than ordinary varieties because of heterosis.
• These advantages are lost when second generation seed is used. To
exploit the benefits of hybrid technology they have to purchase new
seeds every year instead of saving and reuse of seeds. Hence the concept
of terminator technology came into existence.
• Multinational companies who have been unable to clutch farmers back
to recover their returns on investment because of farmers old age
practice of brown bagging and breeding seeds, so they are coming up
with an emerging novel gene protection technology called terminator
technology.
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4. Genetic use restriction technologies are the name given to methods
providing specific genetic switch mechanism that restrict the unauthorized use
of genetic material by hampering reproduction (variety-specific, V-GURT) or
the expression of a trait (trait-specific, T-GURT) in a genetically modified
(GM) plant.
(FAO,
2001)
Synonyms: GURTs/Terminator technology/Suicide seed/Suicide gene
technology/Technology protection system.
GURTs could potentially be applied to all seed-propagated crops.
(Lehmann,1998)
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5. 199
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6. 6

7. Variety-specific
V-GURT
1GURTs
Trait-specific
T-GURT
2GURTs
V-GURT allow breeders to develop plants that grow and form seeds, but the
second generation seed is sterile.
The T-GURT technology has the trait (herbicide tolerance, biofortification, etc.)
controlled by a molecular switch which is activated by a chemical inducer/some
other stimulus, such as heat.
(Visser et al., 2001) 7
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MECHANISMS

8.  It is similar for both T-GURT and V-GURT
1. A repressor gene (the gene switch) that is responsive to an external
stimulus.
2. A recombinase gene (the trait activator gene), the expression of
which is blocked by the repressor.
3. A target gene (toxin coding gene).
 Inducing substance (inducer)
 Mostly of chemical origin
 Biodegradable
 Non-toxic for the ecosystem
 Directly applicable in the field or in seeds
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(Gupta, 1998)

9. Name of motif
sequence
Source Purpose
Tn10 tet repressor Escherichia coli Genetic switch
Toxin coding gene Saponaria officinalis (soapwort)
(Family: Caryophyllaceous)
To encode ribosomal
inactivating protein (RIPs) i.e.,
cytotoxin sporin
Cre recombinase gene P1 bacteriophage Synthesizes molecular scissor
i.e., recombinase which
removes the lox sequence by
site-specific recombination
Lox sequence P1 bacteriophage Prevents expression of toxin
gene in the absence of inducer
substance
Late embryogenesis
abundant (LEA)
promoter
Could be native to host plant or
derived from another organism
Stimulates production of
ribosomal inactivating protein
at the late embryogenesis
development stage of seed
(Source: https://www.ncbi.nlm.nih.gov/)
Table 1. Genes and their sources for GURTs technologyTable 1. Genes and their sources for GURTs technology
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10. For V-GURT, essentially three different restriction mechanisms have been
proposed (Visser et al., 2001).
Terminator gene under the control of LEA promoter linked to the blocking
spacer flanked by lox sequences produces RIPs i.e., saporin toxin.
Recombinase gene encodes a protein Cre (Creates recombination)
recombinase that cuts the lox sites flanking the blocking spacer linked to
the toxic gene.
Cre-Lox recombination system is commonly used to circumvent embryonic
lethality caused by systemic inactivation of many genes.
(Visser et al., 2001)
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11. A Tn10 tet repressor gene produces repressor protein which will bind to
the tet operon linked to recombinase gene thereby preventing expression
of this gene.
The presence of an external inducer prevents binding of the repressor to
the operon.
In the case of U.S. patent number 5,723,765, the chemical inducer is the
antibiotic tetracycline (Jefferson et al., 1999).
Genes under the control of the LEA promoter are only transcribed
during late embryogenesis when the seed accumulates most of its storage
oil and protein and is drying down in preparation for the dormant period
(Hundertmark and Hincha, 2008).
(Visser et al., 2001)
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12. 12
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(Visser et al., 2001)

13. •It is based on a reversed process because it is characterized by the
presence of a gene encoding a disrupter protein permanently active in the
seed, which makes it sterile.
• The gene promoter is under the control of a specific operator sequence.
• A further repressor protein, whose gene is under control of a chemically
inducible promoter, can bind to the operator, inhibiting the expression of
the disrupter gene.
• In the absence of the exogenous chemical inducer, no repressor protein is
expressed hence seeds will get sterile. During the time of selling seeds
chemical inducer should not be applied.
(Visser et al., 2001)
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14. (Visser et al., 2001)
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15. Recoverable block of function (RBF)Recoverable block of function (RBF)
• It is a technology assimilable to ‘second type’ V-GURT, but specifically
designed for gene flow control in transgenic plants, so-called recoverable
block of function (RBF) developed in tobacco by Kuvshinov et al., 2001.
• It consists of a blocking sequence (Barnase) linked to the gene of interest
and a recovery sequence (Barstar), expressed under control of sulfhydryl
endopeptidase (SH-EP) and heat shock (HS) promoters, respectively and
all contained in a single insert.
• The natural expression of the Barnase in embryos and sprouts confers cell
death or prevents sexual reproduction of the transgenic plant.
• The first patent for this technology was granted in 2005 (US 6849776
B1) to the Finnish biotechnology company (Kuvshinov et al., 2005).
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(Visser et al., 2001)
INTRODUCTIOTYPESMOLECULAR
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16. (Visser et al., 2001)
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17. The third strategy is applied to vegetative reproducing species such as
tuber and root crops and ornamental plants where growth is prevented
during the period in which they are stored to increase the ‘shelf life’ of
the product.
• This mechanism patented by Zeneca (Syngenta) in 2001 involves a
permanently active gene able to block the vegetative growth of the plant,
preventing the multiplication of the seeds.
• This default expressed blocking gene can eventually be suppressed by
application of a chemical activating a second gene allowing the plant to
develop.
(Visser et al., 2001)
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INTRODUCTIOTYPESMOLECULAR
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MECHANISMS

18. Mode of action of Saporin, ribosomal inactivating protein inMode of action of Saporin, ribosomal inactivating protein in
cell metabolic pathwayscell metabolic pathways
Saporin toxinSaporin toxin
RIPs display rRNA N-glycosidase activity and depurinate 28s rRNA by
cleaving the bond between adenine and ribose in the exposed loop of the
molecule thus preventing recruitment of translation elongation factors and
subsequent protein synthesis.
(Matthew et al., 2013)
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19. A gene cassette is expressed in the seed and programmed so that
the gene responsible for the production of a toxin/disrupter protein is
instructed to undo a particular plant trait of interest, without killing the
embryo.
Thus, a desirable characteristic may be excised selectively by
applying or withholding chemical application before being sold to
farmers.
There are two mechanisms by which T-GURT work (Visser et al ,
2001)
(Visser et al., 2001)
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20. (Visser et al., 2001) 20
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21. • The gene encoding the trait of interest is kept silent, but it can be
activated by the farmer through the application of a chemical inducer to
the plant or seed.
• In the subsequent fertile generations, the gene is inherited in the inactive
state, so that the chemical must be purchased to exploit the trait of
interest (Shi, 2006).
• The gene of interest can be activated by the farmer by spraying a
‘standing crop’ with an activator only at the occurrence of an
unfavourable event (e.g. pest or disease incidence).
• This focused strategy may help in reducing the build-up of insect
biotypes and virulent pathotypes.
(Visser et al., 2001)
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22. (Visser et al., 2001)
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23. • Intellectual property protection is granted in the form of patents or plant
varietal protection (PVP), also plant breeder’s rights (PBR) and at the
international level by the UPOV (international union for the protection of
new varieties of plants) and by the WTO as trade related aspects of
intellectual property rights (TRIPS) agreement.
• The monitoring of patent right infringement by unauthorized use of seeds
is difficult, time consuming and expensive.
• GURTs are giving a perpetual form of physical protection which would
be an effective mechanism to bypass the intellectual property regulatory
framework.
• The intellectual property protection granted by GURTs has a double target
as it ensures that farmers cannot reuse saved seeds or exploit a valuable
trait without purchasing a chemical.
(Luca Lombardo, 2014)
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24.  To date, there are over 40 granted or
submitted patent families related to
GURTs.
 The holders of the patents include
universities and especially multinational
companies such as Syngenta, Bayer,
Monsanto (D&PL), Ceres, Pioneer and
BASF.
The number of patent applications
started to increase in the late 1990s with
the peak in 2006.
(Luca Lombardo, 2014) 24
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25. Table 2. Comprehensive list of companies with terminatorTable 2. Comprehensive list of companies with terminator
technologytechnology
(RAFI, 2001) 25
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26. Societal group Potential benefits Potential risks Potential costs
Farmers Increased productivity
from improved crop
breeding
Misuse of monopoly
powers by breeders
Reduced seed security
Increased input costs
from seed purchase
Breeders
(especially Private
sectors)
Increases share of
research benefits from
new products
Strict regulations in
response to public
pressure
Increased cost for
access to genetic
resources of other
breeders
Government Reduced investment
requirements in breeding
Fewer enforcement costs
for Plant Variety
Protection (PVP)
Reduction in food
sovereignty
Increased responsibility
for unforeseen effects
and circumstances
Complementary
research and
development
investment
Society Increased agricultural
productivity
Reduced food costs
Reduced genetic
diversity in fields
(reduced sustainability
of agricultural systems)
Reduction in Food
sovereignty
Table 3. Potential benefits and risksTable 3. Potential benefits and risks
(Van Acker et al., 2007) 26
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27. • Hybrids have inbuilt technology protection system as they will show
reduced heterosis and hybrid vigour if saved and reused in second
generation hence farmer has to buy new seeds every year.
• GURTs offers protection biologically which bypass intensive regulatory
frameworks.
• Thus with both systems, farmers are discouraged from saving seed and
both may be used as use restriction technologies (Gupta 1998; Goeschl
and Swanson 2003; Pendleton 2004).
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(Gupta and Lehman, 1998)

28. • In case of hybrid seed production, only recombinase and terminator gene
is utilized.
• One of the parental lines contains the recombinase gene, which becomes
activate only after germination and the other parent contains lethal
(terminator) gene separated from its promoter by a blocking spacer
flanked by lox sequence.
• The recombinase expressed right after germination excises the blocking
spacer bringing the promoter and lethal gene together.
• The seeds harvested from first generation hybrid crops will be normal in
all respects, except that they will not germinate if sown as a seed.
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(Gupta and Lehman, 1998)

29. 29
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(Gupta and Lehman, 1998)

30. Parent 1 Parent 2
Hybrid
F1 - Hybrid seeds given
to farmers for
cultivation
F1 hybrid population
Commercial cultivation
F2 seeds
Sowing of F2
seeds by famers
Seeds will not
germinate
General SchematicGeneral Schematic
representation of terminatorrepresentation of terminator
technology/GURTs in Hybridstechnology/GURTs in Hybrids
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(Gupta and Lehman, 1998)

31. GENERALREVIEW
PAPERS
UNSOLVED
QUESTIONS
CONCLUSION
The main version of the terminator includes a set of three novel genes
inserted into one plant. However, there is another version, which divides
two or three genes on to two plants that are later to be cross-pollinated.
This would secure a much stronger monopoly on the seed market
compared to patents because this technology would ensure that it is
impossible for farmers to save and reuse their once harvested seeds.
(Mukherjee and Senthil Kumar, 2014)
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32. GENERALREVIEW
PAPERS
UNSOLVED
QUESTIONS
CONCLUSION
GURTs developed to secure return on investments through protection of
plant varieties, are among the most controversial and opposed genetic
engineering biotechnological tools as they are perceived as a tool to
force farmers to depend on multinational corporations’ seed monopolies.
In this work, the currently proposed strategies are described and
compared with some of the principal techniques implemented for
preventing transgene flow and/or seed saving.
(Ashok and Arpit, 2017)
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33. GENERALREVIEW
PAPERS
UNSOLVED
QUESTIONS
CONCLUSION
This technology put a ceiling on the illicit use of genetic material either by
hampering reproduction or the expression of a particular trait through an
inducible molecular mechanism, thus approaching to the rescue of long
suffering multinational companies.
The potential hazards of terminator technology includes out-crossing threat,
reduced choice to farmers in procuring seeds and gag on using last year
seeds.
(Yousuf et al., 2017)
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34. GENERALREVIEW
PAPERS
UNSOLVED
QUESTIONS
CONCLUSION
1. Proper segregation of genes because it is important that the three
genes, that is, the toxic protein gene, the recombinase gene and the
repressor gene should segregate together during reproduction
(Daniell, 2002).
2. Gene silencing because the LEA promoter may be subject to
silencing, resulting in malfunction of the system (RIP proteins
would not be produced) and introgression of a GM trait would
become possible (Daniell, 2002).
3. Transgenic pollen can lead to the production of infertile seeds in
adjacent Non-GM fields, causing economic losses for ‘blameless’
farmers (Giovannetti, 2003).
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35. GENERALREVIEW
PAPERS
UNSOLVED
QUESTIONS
CONCLUSION
The issue of GURTs is complex, with different stakeholders
embracing widely opposing views. There are several strong arguments
that have been put forward, both in favour and against the implementation
of these types of technologies.
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36. THANK YOUTHANK YOU
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