Terminator Seed: The Sequel

8/8/2019 Terminator Seed: The Sequel

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Communiqu

May/June 2007 Issue # 95

Terminator: The SequelDespite the fact that governments re-affirmed and strengthened the United Nations moratorium

on Terminator technology (a.k.a. genetic use restriction technology [GURTs]) in March 2006,public and private sector researchers are developing a new generation of suicide seeds usingchemically induced switches to turn a genetically modified (GM) plants fertility on or off.

Issue: Under the guise of biosafety, the European Unions 3-year Transcontainer Project isinvesting millions of euros in strategies that cannot promise fail-safe containment of transgenesfrom GM crops, but could nonetheless function as Terminator, posing unacceptable threats tofarmers, biodiversity and food sovereignty. Terminator technology genetic seed sterilization was initially developed by the multinational seed/agrochemical industry and the USgovernment to maximize seed industry profits by preventing farmers from re-plantingharvested seed. Researchers are also developing new techniques to excise transgenes from GMplants at a specific time in the plants development, and methods to kill a plant withconditionally lethal genes. This new generation of GURTs will shift the burden of trait controlto the farmer. Under some scenarios, farmers will be obliged to pay for the privilege of restoringseed fertility every year a new form of perpetual monopoly for the seed industry.

Impact: Whether intended or not, new research on molecular containment of transgenes willultimately allow the multinational seed industry to tighten its grasp on proprietary germplasmand restrict the rights of farmers. Industry and governments are already working to overturnthe existing moratorium on Terminator technology at the UN Convention on Biological

Diversity (CBD). In the months leading up to the CBDs 9 th Conference of the Parties (Bonn,Germany 19-30 May 2008), industry will argue that global warming requires urgentintroduction of transgenic crops and trees for biofuels and that Terminator-type technologiesoffer a precautionary, environmental necessity to prevent transgene flow. Ironically, society isbeing asked to foot the bill for a new techno-fix to mitigate the genetic contamination caused bythe biotech industrys defective GM seeds.

Players: Taxpayer-financed research on biological containment of GM crops subsidizes thecorporate agenda. A handful of multinational seed corporations control biotech seeds and theproprietary seed market as a whole has seen unprecedented corporate concentration. In 2006,the worlds top 4 seed companies Monsanto, DuPont, Syngenta and Groupe Limagrain accounted for half (49%) of the proprietary seed market.

Policy: Governments keep trying to find ways to make GM seeds safe and acceptable and theykeep failing. They should stop trying. There is no such thing as a safe and acceptable form ofTerminator. The EU should discontinue funding for research on reversible transgenic sterility,and re-assess funding for other research projects undertaken by Transcontainer. Rather thansupport research on coexistence to bail out the agbiotech industry, the EU should instead fundsustainable agricultural research that benefits farmers and the public. National governmentsshould propose legislation to prohibit field-testing and commercial sale of Terminatortechnologies. Governments meeting at the 9th Conference of the Parties to the Convention onBiological Diversity in Bonn, Germany must strengthen the moratorium on GURTs byrecommending a ban on the technology.


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ETC Group Communiqu, Issue # 95

May/June 2007

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ETC Group Communiqu #95

Terminator: The SequelTable of Contents

Terminator: The Sequel Executive Summary............................................................................1Table of Contents .....................................................................................................................2Introduction..............................................................................................................................31. European Unions Transcontainer Project: Bringing Terminator Back to Life Using ZombieSeed Technology (aka Recoverable Block of Function) .................................................................6

How do Zombie seeds work?..........................................................................................8

How Zombie Technology May Fail ..................................................................................10The Bottom Line on Zombie Technology.........................................................................12

2. The Exorcist: Technologies to delete transgenes from GM plants ..........................................12How Does Exorcist Work?.............................................................................................13How Exorcist May Fail ......................................................................................................14The Bottom Line on Exorcist Technology ........................................................................17

3. Conditional Lethality: Pull-the-Plug Plants..........................................................................17How does Pull the Plug Work?..................................................................................... 18How Pull-the-Plug May Fail .............................................................................................19The Bottom Line on Pull-the-Plug Technology ...............................................................20

Conclusion and Recommendations...................................................................................... 21Appendix: Will Recoverable Block of Function (RBF) work well enough to containtransgenes? .............................................................................................................................23Endnotes .................................................................................................................................26

Boxes and TablesTable 1: Patents on Reversible Genetic Sterility ....................................................................7Box 1: Why Would Farmers Choose to Buy Terminator or Zombie Seeds? .....................10Box 2: Transcontainers Support for Zombie Seed Technology (RBF) is a winningstrategy for corporate seed industry ....................................................................................11Table 2: Patents and Patent Applications on New Methods to Excise Transgenic DNA 16Table 3: Patents and Patent Applications on Conditionally Lethal Plants....................19


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What are GURTs? Genetic Use Restriction

Technology is a broad termthat refers togenetic engineering technologies that use anumber of interactive or interdependentgenes in combination with an environmentalor chemical inducer (such as heat-shock orethanol) to switch on or off the expression ofa plants genetic traits. In the case of varietalGURTs, or V-GURTs, the reproductiveviability of the entire plant is under thecontrol of the company/institution that sellsthe seed. T-GURTs, or trait-specific GURTs,are designed to restrict the use of particular

transgenes (or events), such as those forherbicide tolerance, insect resistance, andother traits. In the official language of theUnited Nations, the term GURT is used torefer to Terminator.

Introduction

Responding to almost a decade ofpopular protest, governments meeting atthe UN Convention on Biological

Diversity in March 2006 (Curitiba, Brazil)re-affirmed and strengthened theexisting moratorium on Terminatortechnology (a.k.a. genetic use restrictiontechnologies GURTs).1 The moratoriumrecommends against the field-testing orcommercialization of seeds that havebeen genetically engineered to producesterile seeds at harvest. Terminatortechnology was initially developed bythe multinationalseed/agrochemical

industry and theUS government toprevent farmersfrom re-plantingharvested seed inorder to maximizeseed industryprofits. Suicideseeds threaten foodsovereigntybecause over 1.4billion peopledepend on farm-saved seed.

Despite the factthat governmentsmeeting in Braziloverwhelmingly supported themoratorium on Terminator, researchersin the public and private sector areexpanding and refining research on anew generation of GURTs. Current

research on biological containment seeksto prevent engineered genetic traits(transgenes) in GM plants fromspreading to non-GM plants and wildrelatives a growing problem for thebiotech industry and for society. (It isalso a major stumbling block inindustrys quest to developpharmaceutical crops and GM trees).However, the same technology that is

being developed to prevent the spread ofpollen and transgenes from GM plantscan also be used to control the plantsreproductive viability and/or preventfarmers from saving and re-plantingharvested seeds. This report examinessome of the new research to developmolecular systems for controllingtransgenes that are simultaneouslyadvancing Terminator 2.0.

In the US and Europe, taxpayer money isbeing used to develop a new suite ofsophisticated molecular technologies tosolve the biotech industrys

contaminationproblem. Society

is being asked tofoot the bill for anew techno-fix tomitigate thegeneticcontaminationcaused by thebiotech industrysdefective GMseeds. Under theguise ofenvironmental

security for GMcrops, industrywill use the newgeneration ofTerminatortechnologies to

tighten its grasp on proprietarygermplasm and biologically restrict therights of farmers to re-plant harvestedseeds. Under some scenarios, farmerswill be obliged to pay for the privilege ofrestoring seed fertility every year.

The first generation of Terminatorpatents (late 1990s) described moleculartechniques that were largely theoreticaland relatively primitive compared tocurrent research on technically advancedGURT technologies. Today, researcherscontinue to develop chemically inducedswitches that turn a plants fertility on


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or off, but they are also developingtechniques to excise transgenes from aGM plant at a specific time in the plantsdevelopment, and methods to kill a plantwith conditionally lethal genes. Thenew generation of GURTs aims to shiftthe burden of trait control to the farmer requiringher/him tobuy a new,proprietaryinducer(most likely achemical) toswitchdesirablegenetic traits including fertility on or

off. The key point is that viability of thecrop would still be controlled by thecorporation that sells the seed.

Biotechs Achilles Heel: Today it isgenerally acknowledged that gene flowfrom some genetically modifiedorganisms (GMOs) to non-GMO plantsand wild relatives has the potential toharm ecosystems or threaten the foodsupply. GM contamination is theAchilles Heel of agricultural

biotechnology. Plants are livingorganisms that operate in a dynamic,evolutionary context they are notmachines and neither industry norgovernment regulators have been able tocontain or control GMOs. The stakes arehigher today becausecommercial firms areconducting open-airfield-tests of GM plantsengineered to producedrugs or industrialchemicals in food andfield crops. As far asthe public and food industry areconcerned, there is zero tolerance forallowing contamination of the humanfood chain with transgenes fromindustrial or pharmaceutical crops.

GMOs can inadvertently contaminate

crops and/or food supplies by two majorroutes.2 Pollen from GM crops can cross-pollinate with related crop plants or wildor weed relatives nearby. GMOcontamination may also occur whentransgenic seeds or plant parts physicallymix with non-GMO crops during the

process ofseedproduction,harvest,storage,transport orprocessing.GMvolunteers

plants that survive in the field from a

previous crop season can also causeunwanted contamination by eitherpollen or seeds.

In the United States, crops not approvedby regulatory authorities for humanconsumption have contaminated fields,or have been discovered in the foodsupply. When unauthorized GMOs arediscovered in export shipments, someimporting governments have deniedthem entry. These incidents have cost

hundreds of millions of dollars inproduct recalls, lost revenues forfarmers, and a giant headache for thebiotech industry which is stillstruggling to convince wary consumersof the benefits of GM foods. For example,

even seven years afterthe StarLink tacodebacle in 2000, thebiotech and foodindustries are stilltesting for the presenceof StarLinkcontamination.

StarLink refers to a GM maize varietysold by Aventis (now owned by Bayer)that entered the food supply although ithad never been authorized for humanconsumption. The total cost for StarLinktesting and product loss has exceeded astaggering $600 million to date.3

According to the GMcontamination register, from1996-2006 there were 146publicly documentedcontamination events involving

42 countries on six continents.1

One enduring lesson from agricultural biotech isthat it is a huge mistake to underestimate biosafetyconcerns. A corollary is that Nature will always finda way; Murphys law implies that no matter howunlikely it seems that genes will flow, they eventuallywill. C. Neal Stewart, Jr., Nature Biotechnology,March 20071


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In the absence of a global monitoringsystem, GeneWatch UK and Greenpeacemaintain a GM Contamination Registerthat records publicly documentedcontamination events.4

Ag biotechs future commercial successdepends on finding a plausible techno-fix to prevent leaky genes from escaping.If governments can be convinced thatbiological containment of GMOs istechnically possible, it will open thefloodgates to new markets for GM crops,and commercial-scale production of GMpharmaceutical plants (plantsengineered to produce drugs), and GMindustrial crops (plants engineered to

produce chemical compounds forindustrial use), as well as GM trees. Anew generation of GM biofuel crops isalso being aggressively promoted as themost efficient route to biofuelproduction, especially in the globalSouth. ETC Group believes that the rushto plant energy crops will shift marginalland away from food production andadversely affect soil, water, biodiversity,land tenure and the livelihoods ofpeasant farmers and indigenous peoples.

Who benefits from publicly financedresearch on biocontainment andTerminator seeds? A handful ofmultinational seed corporations controlthe global biotech seed market. With2006 revenues of $4,028 million,Monsanto the worlds largest seedcompany accounts for one-fifth of theglobal proprietary seed market. The top3 companies Monsanto, DuPont and

Syngenta account for $8,552 million or 44% of the total proprietary seedmarket.5 Why should public funds beused to develop a new techno-fix torescue the Gene Giants defectivetechnology?

Publicly financed research on biologicalcontainment of GM crops subsidizes

corporate Gene Giants. Genetic traitcontrol technology especially geneticswitches to turn fertility on and off willbe promoted as an environmentalsecurity measure. Industry will arguethat its seeds offer an additional level ofbiosafety protection, and it wont be longbefore regulators will require that alltransgenic crops be engineered tocontain fertility switches or otherbiocontainment technologies ostensiblyto prevent promiscuous transgenes fromcontaminating related plants or weedsgrowing nearby.

This report examines three areas ofcurrent research to develop molecular

systems for controlling transgenes(biocontainment) that are simultaneouslyadvancing Terminator technologies.

1) Recoverable block of function(reversible transgenic sterility)2) Gene Excision3) Conditional Lethality Genes

Each section examines how thesetechnologies work and how they mayfail. What are the potential implications

for farmers and food sovereignty,biodiversity and the environment?


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1. European UnionsTranscontainer Project:Bringing Terminator Back toLife Using Zombie Seed

Technology (aka RecoverableBlock of Function)

Brussels Sprout-less? Despite the factthat European consumers haveoverwhelmingly rejected geneticallymodified foods, the EuropeanCommission is supporting a 3-year,5.38 million (US$6.8 million)Transcontainer Project involving 13research partners both public and

private.6

Launched in May 2006, the goalof the Transcontainer Project is todevelop GM crops and trees for Europethat are biologically contained (toprevent the spread of transgenes and thecontamination of conventional or organicplants growing nearby). Transcontainerresearch supports the goal of co-existence the controversial idea thatGM crops and non-GM crops canpeacefully co-exist (or, that acceptablelevels of GM contamination can be

negotiated). In other words, it is apublicly funded initiative to help thebiotechnology industry overcome theEuropean publics rejection of GM foodsand crops.

The Transcontainer Project involvesmultiple research objectives to achievebiological containment.7 These include,1) Chloroplast transformation; 2)Controllable Flowering; and ControllableFertility. Fact sheets describing theresearch in each of these areas areavailable on the Transcontainer website.8

This report does not attempt to providean analysis of all the research objectivesthat fall under the Transcontainerumbrella. Critical analyses of variousmolecular strategies for biocontainmentof transgenes have been analyzed

elsewhere.9 Although several ofTranscontainers research objectivesmerit a strong critique, in this report wefocus on just one of the programmesresearch objectives: To develop a seedlethal transgene containment systembased on Recoverable Block of Function(RBF) in oilseed rape [canola]. Thisresearch is being conducted at theUniversity of Milan, Italy, under thedirection of Professor Martin Kater. TheRBF system was originally developed bya Finnish research group, UniCrop, Ltd.10Kater reports that his group is testing thefirst constructs in oilseed rape, and thathis system is far more promising thanthe Finnish system. The University of

Milan researchers are applying for apatent on their new RBF system.11

Reversible Transgenic Sterility: TheTranscontainer website states that theprojects research on reversible sterilityof GM crops will only partiallyresemble Terminator because its GMplants will include a mechanism to allowfarmers to restore the fertility of thecrops and because its purpose is not torestrict the farmers ability to re-plant

proprietary seed. Reversible transgenicsterility is a method by which theplants fertility can be lost or regained bydesign. Plants are engineered withsterility as the default condition, butsterility can be reversed with theapplication of an external stimulus thatrestores the plants viability. In order forto bring the zombie seed back from thedead, the farmer or breeder must use anexternal stimulus (such as a proprietarychemical) to restore the seeds fertility.

Piet Schenkelaars is a biotech industryconsultant who handles allcommunications for the EUsTranscontainer Project. Given thepolitical sensitivity surroundingTerminator technology, theTranscontainer Project coordinatorsapparently decided it was important to


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hire an information gatekeeper.Responding to questions posed by ETCGroup, Schenkelaar maintains thatreversible sterility is a benefit for farmersbecause it could prevent the spread oftransgenes in the environment, andprevent volunteer plants in the nextgrowing season.12 In the real world,however, corporate Gene Giants will usethis design to transfer the burden of traitcontrol to the farmer who will be obligedeither to buy an external chemicalinducer (likely a proprietary product) torestore the seeds fertility every singlegeneration or buy new seed everyseason.

In another email vetted by Schenkelaars,the Transcontainer researchers in Milanacknowledge that the practicalapplications of reversible sterility may bevery different when it is adopted bycommercial firms: We are an academicgroup who is trying to invent newsystems and give proof of concept.When our system is efficient andbreeding companies would be

interested to use it, they might changethe constructs including anotherinduction system.13

The Transcontainer Project addresses theUN moratorium on field trials and

commercial use of GURTs, but claimsthat the Projects research does not runcounter to the moratorium because GMcrops developed by Transcontainer willonly be tested in laboratories orgreenhouses and they will not be testedin the field or commercialised within thescope of the project. In other words, theProjects after-life is up for (corporate)grabs. The Transcontainer website notesthat the CBD also encourages further

research on the environmental and socio-economic impacts of GURTs a subjectwhich will be examined byTranscontainer researchers. According toSchenkelaars, studies on the socio-economic impact of the RBF system willbe undertaken by Dr. Justus Wesseler atthe Wageningen University in theNetherlands.

Table 1: Patents on Reversible Genetic Sterility

Patent /Application #

Assignee Inventor DatePublished(or

Granted) /

Filed

Description

EP1303628B1 UniCrop,

Ltd.

(Finland)

Kuvshinov, Koivu,

et al.

4 Oct.

2006 / 16

July 2001

DNA construct for controlling

transgene segregation and escape

in a sexually reproducing

transgenic plant comprises a

recoverable block of function

system

WO06005807A1 UniCrop,

Ltd.

Kuvshinov, Koivu,

et al.

19 Jan.

2006 / 5

July 2005

Use of phytoene synthase for

controlling transgene escape

US6849776 UniCrop,Ltd.

Kuvshinov, Koivu,et al.

1 Feb.2005 / 14

July 2000

Molecular control of transgenesegregation and its escape by a

recoverable block of function (RBF)

system

US20050039229A1 UniCrop,

Ltd.

Kuvshinov, Koivu,

et al.

17 Feb.

2005 / 15

July 2004

Double recoverable block of

function

WO0206498A1 UniCrop,

Ltd.

Kuvshinov, Koivu,

et al.

24 Jan.

2002 / 16

July 2001

Molecular control of transgene

segregation and its escape by a

recoverable block of function (RBF)

system


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How do Zombie seeds work?

What is Recoverable Block of Function?Recoverable Block of Function (RBF) tocreate reversible seed lethality is a geneticengineering scheme whereby genes are

put into the crop plant in order to causeseeds to fail to germinate, thuspreventing seeds from passing on theirgenes to the next generation. The purposeof seed lethality in this system, accordingto Transcontainer, is to block thegermination of seeds that bear geneticallyengineered traits (such as herbicidetolerance, insect resistance,pharmaceutical production, etc.). In theRBF system, the gene causing lethality

would be physically linked to thegenetic trait of interest on the samepiece of DNA so that they are inheritedtogether. The gene causing lethality,including its promoters and otherregulatory elements, is called the BlockingConstruct (BC).

Having a BC alone is a dead end, literally.Breeders would be unable to multiplyseeds, and farmers would be unable to re-plant harvested seeds. So another piece of

DNA containing a gene that makes aproduct capable of canceling lethality isalso engineered into the crop. This iscalled the Recovering Construct (RC) and its what makes it possible to bringthe seed back from the dead. The RCcan be turned on when desired, using anenvironmental or chemical trigger, forexample. Some of the RBF systems aretriggered by heat shock, alcoholapplications or antibiotics. Ethanol is thetrigger currently being developed foroilseed rape by Transcontainerresearchers.14 If the RC is not intentionallyturned on, the BC will act alone and seedswill not germinate. Thus the defaultposition for RBF is seed death. A breederor farmer must take a specific action torecover seed viability and the action mustbe repeated in every generation to

maintain seed viability. Although the RBFsystem itself may not be designed withthe intent to restrict seed use per se, itdoes so nevertheless.

Can genes escape through pollen? For

crops such as oilseed rape/canola wherethe seed is the harvested part, robustfertilization is required, so functionalpollen is necessary. Pollen of a seed-lethalRBF variety is capable of normalfertilization of the ovules within the crop,and also of any sexually compatiblevarieties or wild relatives it contacts. Thisis because the lethal factor is designed forproduction at the end of seeddevelopment and/or during germination

and is off in the rest of the plant,including pollen. The RBF genes arepresent, and will be transferred to anyseed fertilized by the RBF pollen. Asthese seeds develop, the BC genes willactivate when the seeds mature andgerminate, and thus there should be nofurther gene escape becausecontaminated seeds will die unlessrescued by activating RC. If it functionsas designed, the RBF system would thusprevent the spread of engineered traits

beyond a single generation. However,that does not rule out the spread ofnegative impacts. For example, if RBFseed cross-pollinates with sexuallycompatible plants growing in aneighboring farmers field, the progeny ofthose plants will fail to germinate whenre-planted. The unsuspecting farmerwould have no way of knowing that apercentage of her/his seeds will be sterileuntil those seeds failed to germinate afterbeing planted. Even if cross-pollinationoccurs infrequently, the negative impactscould be significant. For small farmers, asmall loss may be their profit margin. Forwild plants, a small loss might meanextinction in a marginal habitat, andcould also endanger animals dependingon the plant for food.


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There are many possible configurations ofthe RBF system.15 Some might offer ahigher degree of biological containmentthrough the seed than others. However,all have in common a few characteristicsthat merit public concern.

First, the BC makes something that is

lethal to seeds. In some cases this will bea toxin from a bacterium, fungus or othersource. Will this toxin enter the foodsupply, and how will it be evaluated forhealth effects? The current techniquesand standards for testing GM crops areinadequate to establish unequivocally thesafety of these toxins in food for humans,much less for wild animals that maydepend on the margins of agriculture fortheir survival.

The same concern applies to the RC, if thefarmer or breeder restores the fertility ofhis/her seeds using the necessarystimulus. The seeds will contain theproduct of the recovery gene, and it will

be part of the food supply, so it will alsoneed to be evaluated for safety.

Whether the aim is to restrict seed use ornot, the result of RBF is that the burdenwill be placed on the farmer to apply an

external inducer to restore seed fertility of the right strength, at the right moment

in the growth of the crop and this willhave to be repeated every generation.

Because the seeds of that generation and that generation only would berescued if the RC were activated, thewhole seed-lethality system shouldremain intact if those seeds are planted:The BC would still cause the next batch ofseeds to die, and the RC would still bepresent and could be activated by theinducer again. Although this featuretheoretically functions for the purpose ofbiocontainment, it simultaneouslystrengthens the Terminator properties ofRBF.


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Recoverable block of function is a V-GURT Terminator, where thereproductive viability of the entire plantis under the control of thecompany/institution that sells the seed. Itobliges the farmer to do somethingspecific in order to have fertile seeds toreplant. The Finnish scientist who

pioneered the RBF system in the 1990s,and whose work is being furtherdeveloped by Transcontainer researchersin Milan, classifies his work as a V-GURTtechnology.16 A 2004 report by the USNational Research Council also refers toRBF as a V-GURT.17

Box 1: Why Would Farmers Choose to Buy Terminator or Zombie Seeds?

It may seem strange that farmers would knowingly buy sterile seed when it will meanincreased cost every season farmers will have to buy new seed or buy and apply thechemical required to restore seed viability. The biggest seed companies will dowhatever they can to get farmers on the sterile-seed platform, however, because itenables them to reduce competition and capture a bigger share of the market. GeneGiants will coerce farmers to choose sterile seed by ensuring that the latest technology

(i.e., genetic traits) is available only on that platform. The companies will evenacknowledge as a selling point for sterile seed the very real threat of GMcontamination and the need to contain gene flow. Sterile seed platforms will bepromoted (and in some cases required) as safer, more responsible seed technology. TheGene Giants may keep prices low, initially, as an extra enticement. Once farmers are onthe platform and the competition has been destroyed, companies can start pricing theseed (in the case of Terminator) or pricing the chemical that restores seed viability (inthe case of Zombie) as high as they want. Zombie is a dream scenario for the GeneGiants because it will be cheaper for them to sell farmers an industrial chemical forbringing seeds back to life (rather than pay the seed-multiplication, warehousing anddistribution costs required to sell new seed every planting season). Ironically, the GeneGiants will argue that availability of multiple sterile-seed platforms offers morechoice to farmers!

How Zombie Technology May Fail

There are several potential weaknessesin the RBF system that could affect thefunctioning of the BC or the RC.

EcoNexus, a public interest researchorganization based in the UK, observesthat the use of a V-GURT system forgene containment can only be as good

as its weakest parts, and none of thecomponents tested for any of thepossible V-GURTs systems are 100%reliable or effective.18 In a 2006 briefingsubmitted to the Convention onBiological Diversity, EcoNexus and theFederation of German Scientists providea detailed review of inherentweaknesses in the design and

performance of V-GURTs all of whichapply to RBF technology. Readers whowish to review these issues in moredetail should refer to this document: V-GURTs (Terminator Technology): Design,Reality and Inherent Risks.19

Potential problems withTranscontainers RBF system include,for example:

Insufficient inducer: Activation of therecovery function is likely to be the leastreliable step in the RBF process.Problems include incompletepenetration of the inducer into seedtissues; issues of the inducer changingthe ecology of the crop (ethanol is toxicto many microorganisms, for example);and weather causing delays that could


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result in missing the window foractivating the Recovery Construct (RC).Gene silencing can cause interferencewith the expression of transgenes in aGM plant (especially under stressconditions) and unexpectedly changethe traits or behaviour of the organism.In the case of RBF, for example,silencing of the blocking construct (BC)could disrupt the RBF system and resultin viable seed (allowing transgeneescape).

Segregation of the different geneticcomponents: The GM trait of interestand the RBF genes must remain in closeproximity, on the same strand of DNA,and remain linked during reproduction.

If any of the transgenes involved in theRBF system were to become separatedfrom each other, the system would fail.If segregation occurs, transgenes could

escape and the system failure would bedifficult to detect.

A 2005 paper by the Finnish researcherswho first developed reversibletransgenic sterility provides a glimpse

of the state of the art for RBF, and pointsto many of its potential weaknesses,including gene silencing, mutations andsegregation.20 In order to avoid potentialproblems that could result in seedviability and possible escape oftransgenes, the researchers engineeredthe RBF system in tobacco to containdouble back-up systems. A discussionof this paper, and the technicalchallenges of designing a foolproofcontainment system using RBF, isdiscussed in more detail in theAppendix.

Box 2: Transcontainers Support for Zombie Seed Technology (RBF) is a winning strategy for

corporate seed industry

1. Public funding for Zombie Seed technology subsidizes the corporate research agenda.Transcontainer aims to promote public acceptance of GM crops. It also opens the door to anoxymoronic Terminator a kinder, gentler and environmentally safe suicide seed.

2. The burden of genetic trait control will be transferred to the farmer, who will be obligedeither to buy an external chemical inducer (likely a proprietary product) to restore the seedsfertility every single generation or buy new seed every season. The profit-making appeal ofthe Zombie approach could prove irresistible for the Gene Giants: in the future, farmerscould be encouraged to save harvested seed and rely on the application of a chemical inducer torestore fertility a scenario that would reduce industrys seed multiplication and transportcosts. Ultimately, it would mean lower costs and greater profits for the seed company.

3. By claiming that Zombie seed technology (RBF) means a high degree of environmentalsecurity for GM crops, industry and governments hope to win new markets for biotech including pharma and biofuel crops and GM trees. This is a concern because molecularsystems for controlling transgenes will not be failsafe and may introduce additionalbiosafety hazards. To function as a viable containment system, Zombie seeds must meet thevirtually zero contamination standard to prevent contamination of the food and feed supply(and to prevent contamination of related wild relatives and weeds in the environment).

4. Although the Transcontainer Projects research is limited to European crops, the reality isthat any advances in genetic trait control technology will ultimately be adoptedindiscriminately to further commercial, proprietary goals. If RBF-Terminator systems linkedto seed lethality are commercialized, farmers everywhere (including the global South) willbe obliged to pay for the privilege of restoring seed fertility.


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The Bottom Line on ZombieTechnology

RBF systems linked to seed lethality areclearly V-GURTs because the seeds aregenetically programmed to die during

development or germination. Breedersand farmers can recover viable seeds bytaking some action pre-ordained by thedesign of the RBF system, and thisrecovery action must be undertaken inevery generation to maintain seedviability. The RBF system has manypotential weaknesses because of thecomplexity involved in making both theBlocking Construct and the RecoveryConstruct in a living plant virtually

failsafe.A system that would require farmers torestore fertility of their crops year afteryear undoubtedly seeks to increase seedindustry profits by assuring repeatbusiness (either for the chemical induceror new seeds). Although Transcontainerresearchers in the EU claim that the RBFsystem is not designed to restrict seeduse per se, it does so nevertheless. It isimportant to keep in mind that RBF will

be linked to a proprietary engineeredtrait, which will also be protected bypatents. The act of saving and re-usingRBF seeds will therefore be restricted bylegal as well as biological mechanisms.

The EU Transcontainer Project isadvancing research on a V-GURTsystem. If the project succeeds inconvincing government regulators thatRBF is a viable strategy for containingtransgenes, the researchers will have

developed a Terminator technologyusing public money. If RBF doesnt workprecisely and consistently but ends up inthe field, anyway, the Project will havepaved the way for unprecedented levelsof contamination (involving GM biofuel,pharma and industrial crops), withpotentially disastrous results forbiodiversity, the environment and

human health. For Transcontainerresearchers to divorce themselves fromthis reality is to ignore the real worldimplications of their research and thereality of corporate controlled biotechseeds.

2. The Exorcist: Technologiesto delete transgenes from GM

plants

The biotech industry is increasinglyinterested in gene excision (that is,cutting out or deleting transgenes) as astrategy for both biocontainment andrestricting access to proprietarygermplasm. Recent publications andpatent activity describe methods todelete transgenes from a GM plantduring some stage of its life.

Out, damnd spot! Out, I say! Formore than 15 years, patent applicationshave been filed on methods for excisingtransgenic DNA from plants. Most of theearly work focused on deleting onlythose foreign genes introduced into the

plant for selection purposes. (In theprocess of producing transgenic plants, amarker gene is coupled with a foreigngene that is associated with a desiredtrait, such as insect resistance. Geneticengineers insert the linked genes intoplant cells at the same time. Testing forthe presence of the marker gene revealswhether or not the gene of interest hasalso been transferred successfully.) Oncemarker genes have served their functionduring initial development, they are no

longer needed. Marker genes,particularly antibiotic resistance markergenes, have raised safety concerns,which explains the early motivation toproduce transgenic plants whose markergenes could be excised.21

Footloose and GM-free? Instead oflimiting gene-excision to marker genes,


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recent research is devoted to snipping alltransgenic DNA out of a transgenic plantat some stage in its development beforethe plant flowers and produces pollen,for example, or before it becomes food orwhen expression of the transgene is nolonger desired. As with RBF, research onExorcist technologies reflects the biotechindustrys belated acknowledgementthat co-existence of GM and non-GMcrops has been unworkable. Whileexcising transgenic DNA will not reverseall the effects of the genetic engineering(see below), it is probable that companieswill push to have their products declaredGM Free for the purposes of labelingconsumer products, particularly in

Europe where the public has roundlyrejected GM foods.

Biotech proponents hope that Exorcistcan cleanse GM plants as well as theindustrys image. C. S. Prakash, perhapsthe worlds best-known biotech booster,says of the industrys leaky geneproblems: Most problems raised byscience can be solved by additionalscience itself.22 The game plan is clear:all thats needed to correct a techno-fix

that turned out to be faulty is a new andimproved techno-fix.

Like Zombie methods, Exorcisttechnologies are dual use GURTs: (1)assuming 100% effectiveness, they couldwork as a biocontainment strategy toprevent the escape of transgenes and (2)even at levels of effectivenessconsiderably lower than 100%, theycould work as a biological method toenforce patents (by restricting access toproprietary genes and traits).

Exorcist as a Democratic Terminator?Given the UN moratorium onTerminator, it has become sociallyunacceptable to openly pursue researchon suicide seeds. Exorcist offers aproposed workaround solution to theTerminator taboo. In the opinion of

Ludmila Mlynarova and Jan-Peter Nap,two researchers in the Plant SciencesGroup at Wageningen University in theNetherlands, Exorcist could be aninteresting alternative to Terminator.23They acknowledge that, while farmerscould save Exorcist seeds, in theory, thepractice of seed-saving will be subject tothe national seed laws and intellectualproperty systems.24 They also point outthat with gene-excision technologies,farmers, breeders and seed producers areall faced with additional work tomaintain, develop or test for thetransgene-of-interest in the seed. (In thisway, Exorcist is like RBF.) TheWageningen researchers conclude that

Exorcist could be considered a differentand democratic application ofterminator technology: both producersand growers will have to take additionalsteps to maintain the trait-of-interest,whereas undesired spread isterminated.25 Other researchersdabbling in GM-exorcism put it moreplainly: Excision methods would haveboth technology restriction and biosafetyutilities without seed sterility.26

How Does Exorcist Work?

Exorcist builds on the basic method forexcising marker genes, but a largerpackage of genetic material called agene cassette can be snipped out of theplant. The general process works likethis: Genetic engineers insert the genecassette its helpful to think of thecassette as a group of cars in a toy trainset into the genome of a plant. The

cassette can be made up of lots of cars,including: marker genes, genesassociated with desired traits, a sequenceof DNA that expresses a protein capableof initiating the excision process andpromoters that jumpstart everythingby activating the protein-expressingDNA. The promoter, and therefore thewhole excision process, can be activatedby a chemical stimulus or by a


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developmental stimulus, depending onhow its engineered. The cassette offoreign DNA is flanked on both ends the first and last cars of the toy train bymore foreign DNA, a pair of recognitionsequences (excision sequences). Theydetermine where the excision will takeplace all the genetic material betweenthe two excision sequences will beremoved from the plant, though one ofthe two excision sequences will staybehind. When the chemical ordevelopmental stimulus activates thepromoter, the promoter activates theprotein-expressing DNA. The proteinactivates excision at the sites within theborders of the excision sequences.

Yeah, it does, kind of: Although it maysound silly to transform plants withtransgenes with the aim to remove allincoming DNA, the crucial issue is wherein the life cycle of the crop the transgenesare removed. Ludmila Mlynarova &Jan-Peter Nap, Wageningen University27

In March 2007 researchers at theUniversity of Connecticut (USA)described a GM-gene-deletor system

in Plant Biotechnology Journal that haspiqued interest in gene excisiontechnology.28 The system builds on apatent application the same researchgroup filed in 2001, which describedmethods for removing all (or almost all)of the DNA that was originallyengineered into a plant.29 Their excisionprocess, like the one described above,can be triggered by an externalenvironmental or chemical trigger. It can

also be designed to occur at a particulardevelopmental stage, without the needfor external activation. By timing theexcision to take place beforereproduction, for example, the pollenand seeds would no longer contain theengineered trait, which would not bepassed on. Theoretically, farmers couldsave seeds, and those seeds would notcontain the engineered trait. The

University of Connecticut researchersclaim that they were able to engineertobacco plants that excised thetransgenes from pollen and seeds in allof the progeny seedlings tested morethan 25,000 of them.

How Exorcist May Fail

Gene excision technology is complex andinvolves dozens of elements that need towork in coordination. In its current statethe risk of failure is high. In theUniversity of Connecticut experimentswith GM tobacco discussed above, theexcision was controlled by pollen-and/or seed-specific promoters, ratherthan by promoters that respond toexternal signals. (This method is knownas auto-excision the promoters wereproduced automatically in pollen andseeds, and the transgenic DNA wasexcised in only those parts of the plant.)Thus the plants were not changed intocompletely GM-free plants thoughtheir engineered genes were not passedon via sexual reproduction. The tobaccolines were propagated by cuttings inorder to maintain their engineered traits

in future generations. Although thiscould be useful in some crops that arevegetatively propagated (some fruits,potatoes, and sugarcane, for example),for most applications, plant breederswould need to cross plants without thetraits auto-excising during reproduction.Therefore, external triggers allowingexcision to be activated and deactivatedat will will be necessary. It remains tobe seen whether heat shock, hormones orexternal chemical triggers would be ableto replicate the excision rates claimed inthis study using developmentallyregulated promoters. Whats more, thesetobacco studies were done ingreenhouses under controlledenvironmental conditions. Experiencewith engineered crops over the last tenyears has shown that weather, nutrientlevels and diseases can affect transgene


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expression. Will there be more mistakesin excision in the presence of nutrientdeficiencies, drought or unusualweather? In the age of climate change,linking excision activation to heat shockseems a particularly risky business. Howwill changes in transgene expression beevaluated?

In order to claim transgene containment,the biotech industry acknowledges thatexcision must be failsafe it mustfunction in 100% of the pollen and seedsof the plants. Researchers have estimatedthat just one mistake in one thousandcould be enough to allow escape of thetransgene to wild relatives or

neighboring varieties within as few as 10generations.30 In fact, the researchersconcluded that one mistake in athousand [10-3] might seem small, but itmight not be small enough. There maybe a genuine need for smaller leakageparameters.31 In another recent study byresearchers at Wageningen University inthe Netherlands, success of one excisiontechnique activated during pollendevelopment was measured in samplesranging from 100-17,000 seeds (tobacco

and Arabidopsis thaliana [thale cress]).32The transgene was detected in only0.027% of the seed. While this rate offailure may seem negligible, if the rate isextrapolated to the scale of commercialagriculture, and using canola (ediblerapeseed) seeds as the example, thisfailure rate could amount to nearly 3,000transgenic plants volunteering in thefield per hectare (among the estimated 10

million canola seeds that escape duringharvest).33

The most recent patent application (seeTable 2, below) for excision of transgenicDNA in crops, issued to Pioneer Hi-Bred(DuPont), is similar in concept to themethod developed at the University ofConnecticut, but is based on bringingtogether two different lines of transgenicplants. When the different lines mate, theengineered traits from one line aredesigned to activate engineered traits inthe other line to cause DNA excision in awide variety of configurations. Thus thetrigger for excision is ultimately thecrossing of the two lines. There can also

be a requirement for an additionalexternal trigger as in other GURTs, but itis not necessary. This means that plantbreeders could maintain the transgenicDNA as long as they keep the linesseparated from one another (to avoidactivating the excision process).

But Pioneers invention is not limited toexcision via the crossing of twotransgenic lines. In fact, the patent isquite broad and bold in its claims. The

patent abstract states, By matchingpromoters, responsive to variousinducers, plant tissues or plantdevelopmental states with therecombinase systems [the excisionmachinery], stop fragments andtransgenes, virtually any trait may beexpressed or excised at any plantdevelopment stage or in any plantgeneration.


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Table 2: Patents and Patent Applications on New Methods to Excise Transgenic DNAPatent /

Application #

Assignee Inventor DatesPublished /

FiledDescription

WO0136595A3

See also:

US20060253934A1

Pioneer Hi-Bred

(DuPont)

Yadav,

Narendra S.,

USA

2006-11-09 /

2006-07-21

Methods for conditional

transgene expression and trait

removal in plants virtually any

trait may be expressed or excised

at any plant development stage

or in any plant generation

WO0210415A3

See also:

EP1307570A2

US20020124280A1

University of

Connecticut

Li, Yi et al. 2002-02-07 /

2001-07-27

Methods for the controlled,

automatic excision of

heterologous DNA from

transgenic plants and DNA-

excising gene cassettes for

producing non-transgenic food;

may permit crops produced from

transgenic plants to be co-

mingled with non-transgenic

crops for marketing purposes

US20040143874A1 The Rockefeller

University

Moller,

Simon Geir

2004-07-22 /

2004-01-13

Inducible site-specificrecombination for the activation

and removal of transgenes in

transgenic plants

WO0216609A3 BASF Plant

Science GmbH

Mankin,

Luke

2002-02-28 /

2001-08-27

Self-Excising Polynucleotides and

uses thereof useful for

producing transgenic plants,

removing transgenes from these

plants or crops (e.g. food

commodities), and restricting the

distribution of transgenes within

the environment

WO0216624A1 Institute of

Molecular

Agrobiology,Singapore

Sundaresan,

Venkatesan

et al.

2002-02-28 /

2000-08-25

Reduction of transmission of

transgenes in plants; DNA

construct useful for excisingtransgene from plant at specific

time

WO0229071A3

See also:

US20020078476A1

Maxygen, Inc. Stemmer,

Willem, P. C.

2002-04-11 /

2001-10-05

Methods and compositions

relating to the generation of

partially transgenic organisms

(i.e., a transgenic plant capable

of producing a non-transgenic

agricultural product).

WO02064801A1 Unicrop, Ltd. Kuvshinov,

Koivu, et al.

2002-08-22 /

2002-02-14

Molecular control of transgene

escape by a repressible excision

system

Caveats to Cutting Out DNA:Verification that transgene-escapes arenot occurring is a big problem withExorcist technologies. Since plantsgenerally look the same with andwithout transgenic DNA, a lot ofexpensive and time-consuming testingon a regular basis could be required tocheck whether the excision was workingproperly under real field conditions.

And, of course, in the event that somegenes have escaped, it will be impossibleto recall them.

Jack Heinemann, professor of geneecology at the University of Canterbury,New Zealand, points out that excisingtransgenes at target sites does notentirely reverse the effects of the originaltransgenic gene insertion.34 This makes


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sense, of course: if a human body ispenetrated by a bullet, and the bulletremoved hours later in surgery, thesurgery will not have returned the bodyto its pre-shot state. With Exorcist, one ofthe excision sequences bordering thegene cassette is left behind after thegenetic material in the cassette is excised.If the sequence left behind is lodged inan active gene crucial to the plantshealth, it could act like a mutation in thatgene. In any case, the sequence leftbehind becomes part of the plantschromosome it will be inherited byfuture generations and could causedisruptions further down the line.35 TheUniversity of Connecticut researchers

acknowledge the potential for adverseenvironmental and health effects fromthis non-expressed DNA sequence butthey conclude that these effects shouldbe minimal or relatively easy todetermine.36

Given the thousands of genes ofunknown or incompletely knownfunction in a plant and their nuancedinterrelationships, and the likelihoodthat the piece of DNA that has been left

behind will be randomly inserted in thegenome of the plant, how willenvironmental and health effects bedetermined? Such studies are complexand long-term, and these long-termstudies have yet to be done thoroughlyon the engineered crops currently beinggrown.37

The Bottom Line on ExorcistTechnology

Proponents of Exorcist technology pointout that gene excision technology if itworks as designed wouldnt be aTerminator technology because theprogeny are fertile. Farmers would beable to save harvested seed, and thatseed would no longer contain GM traits.However, even in its current imperfectstate, where the potential for transgene

escape has not been eliminated, itsimportant to remember that Exorcistcould function as an effective, biologicalpatent protection. In practice, thecompany retains control of theengineered traits, by determining whenthe excision takes place and by whatmeans. Depending on the particularmethod of Exorcism, gene-excisionwould either be out of farmers control(i.e., auto-excision) or it would be theirresponsibility (burden) to make sure ithappened: They would have to apply theexternal chemical to stimulate thepromoter and begin the excision process.

Regardless, if the technology fails and

the transgenes are not completelyexcised, it is the farmer who could becharged with infringing the patent onproprietary genes. How would thefarmer prove that he/she is a victim oftechnology failure and not culpable ofpatent infringement or liable forcontamination?

Its unclear how soon Exorcisttechnologies could be commercialized.Many of the schemes envisioned are

quite complex, involving dozens ofelements that need to work incoordination. This will likely take yearsof experimentation, though experiencewith commercial biotech thus far makesclear that the industry doesntnecessarily have to get its product rightto get it to market.

3. Conditional Lethality: Pull-

the-Plug PlantsThe US National Research Councils 2004report, Biological Confinement ofGenetically Engineered Organisms, pointsout that no single bioconfinementtechnique will achieve complete successon its own. The report notes that the useof multiple techniques, with differentstrengths and weaknesses, will decrease


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the probability of failure (because thefailure of one method could be backed-up by the integrity of another).38

In the event that all else fails, companiesare pursuing extreme biocontainmentmethods as an ultimate back-up plan ways to pull the plug on transgenicplants, terminating them and theirtransgenic DNA.

Because methods can fail, a singleconfinement method will not necessarilyprevent transgene escape. US NationalResearch Council, Biological Confinementof Genetically Engineered Organisms, 2004.

How does Pull the Plug Work?Pull-the-Plug plants are engineered sothat the trait of interest and aconditionally lethal gene are juxtaposedand inserted together into the plantsDNA. If the lethal gene is triggered, theplant dies, taking the GM trait downwith it. If its not triggered, the plantlives and can pass on its foreign genes both the gene associated with the trait ofinterest and the lethal gene to the next

generation. The conditionally lethal genecan code for a toxin itself and have apromoter that is triggered by a chemicalor environmental stimulus, or it can codefor an enzyme that transforms anapplied chemical into a toxin. Deadplants dont transfer genes,39 so thismethod should be completely effectiveas a biocontainment strategy for pollen-and seed-mediated gene flow assumingthe trigger works efficiently and is

activated before any gene escape takesplace. The concept of conditionallethality is to create the opposite of anherbicide-tolerant plant an herbicide-susceptible plant, with the herbicideselected to be specific to the engineeredplant (and ideally, not lethal or harmfulto other plants, the environment, peopleor other animals). Pull-the-Plug plantsdiffer from Zombie (RBF) technologybecause the default position is notautomatic death: For Pull-the-Plug plantsto commit suicide, the promoter must betriggered.

The Pull-the-Plug patent issued to DowAgrosciences in the table below40

suggests an unusual twist on the idea ofengineering a plant to self-destruct (ifthat idea werent twisted enough). Theinventors outline a plan to use aconditionally lethal gene to sicken butnot kill plants containing transgenes, asa way to identify visually whichindividual plants contain the genes andwhich do not. If the plant looksdeformed after being given a sub-lethaldose, then it can be assumed the planthas the engineered DNA and can either

be selected to live and rescued bydiscontinuing the trigger, or it can bedestroyed. It may be though thatdiseases could confound theidentification of plants with transgenes:Are the plants looking sickly because ofthe sub-lethal effects of the triggeredgene, or do they have a virus, forexample?


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Table 3: Patents and Patent Applications on Conditionally Lethal PlantsPatent /

Application #

Assignee Inventor Date Published(or Granted) /

FiledDescription

US6753459 Dow

Agrosciences;

National

Research

Council of

Canada

Keller,

Wilfred A.

et al.

2004-06-22 /

2001-06-22

Gene construct comprising a

conditionally lethal gene

functional in a plant cell, useful for

producing a transgenic plant

which can be removed from a

growing environment [and

identified visually]

US20040154054A1

See also:

US6743968

Dellaporta,

Stephen L. et al.

Yale

University

2004-08-05 /

2004-03-17

Genetic construct for the control of

transgenes in transgenic plants

comprises a sex-specific promoter

operatively linked to a suicide

gene that selects against male or

female gametes containing the

suicide gene

EP0658207B1 Bright, Simon, W.

J. et al.

Syngenta

Limited

2002-09-25 /

1993-07-29

Recombinant plant genome

containing gene cascade

requiring a chemical inducer toproduce a mature plant allows

control of expression of plant

genes, and production of non-

viable protectable plants

Why Pull the Plug? Ostensibly, pull-the-plug plants are being developed as astrategy for last-resort biocontainment,which could boost the plausibility of co-existence and the perception that GMplants could be biosafe. The Dow patent

highlights the utility of being able toremove an unwanted plant from agrowing environment. If pharma cropsare rigged with conditionally lethalgenes, for example, soaking a field withthe chemical trigger after harvest wouldpresumably provide added assurancethat no living plants were inadvertentlyleft behind (which, of course, doesntaddress the problem of gene flow whilethe plants were alive).

There is also the more sinister possibilitythat conditionally lethal genes smuggledinto the crops of an enemy populationwould allow the chemical trigger to beused as an anti-crop biological weapon toattack a strategic crop. Is it far-fetched tosuggest that this application of aTerminator technology could be an anti-crop weapon? Unfortunately,

history shows that research on biologicalwarfare against crops is not uncommon.According to Simon Whitby of theUniversity of Bradford (UK), because ofthe potential devastation that can beinflicted on a country by destroying itscrops, all state-supported biologicalweapons programs in the 20th centuryincluded research on anti-cropmeasures.41 While this could beaccomplished quite thoroughly withconventional herbicides, pull-the-plugplants could be introduced unobserved,perhaps during peacetime, allowing thetechnology-holder to exploit the ability tosicken plants rather than kill themoutright.

How Pull-the-Plug May Fail

In the field there are the usual problemsof using chemical triggers: incompletepenetration leading to some plantsescaping, the difficulty of finding achemical trigger that really could beenvironmentally benign when used on acommercial scale and getting the weather


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to cooperate with the treatment schedule.As with other GURT technologies, thereis also the problem of gene silencing andgene mutations (see Zombie technologysection, above); if the lethal gene issilenced or undergoes mutation, it wouldresult in transgene escape.

The Bottom Line on Pull-the-PlugTechnology

Pull the Plug, like Zombie and Exorcist, isalso a dual-use GURT it works asboth a biocontainment strategy admittedly, an extreme one and as apatent protection strategy. Companiescould pull the plug on plants they believeare being grown without the properlicensing agreements. Biotech companies

have previously resorted to draconianmeasures to ferret out farmers suspectedof possible patent infringement (i.e.,hiring private police to investigatepotential violators, establishing toll-freehotlines to encourage farmers to snitch ontheir farm neighbors, etc.). Nowcompanies could threaten a farmersuspected of patent infringement withtriggering the lethal gene or they couldsimply apply a chemical trigger to getpositive or negative confirmation of theirsuspicions. Pulling the plug could be a lotless labor intensive than relying oninformants or taking farmers to court.Conditional lethality also expands thepossibilities for biological warfare against

crops.


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Conclusion andRecommendations

Industry and governments are alreadyworking to overturn the existing

moratorium on Terminator technology atthe UN Convention on BiologicalDiversity. In the months leading up to theCBDs 9th Conference of the Parties (Bonn,Germany 19-30 May 2008), industry willargue that global warming requiresurgent introduction of transgenic cropsand trees for biofuels and thatTerminator-type technologies offer aprecautionary measure andenvironmental necessity to preventtransgene flow. Research on molecularbiocontainment strategies is alreadybeing promoted as a biosafety solutionfor transgenic crops, trees andpharmaceutical-producing plants.42According to its website, the results of theEU-funded Transcontainer Project willcontribute to an informed decisionwhether the [CBD] moratorium should becontinued or modified in the context ofsupporting EU coexistence measures.

Governments and industry are investingmillions of dollars in molecularbiocontainment strategies that cannotpromise fail-safe containment oftransgenes, but could nonethelessfunction as Terminator technologies thatpose unacceptable threats to farmers,biodiversity and food sovereignty. Thesolution to a flawed technology is not anew techno-fix. Ironically, society is beingasked to foot the bill for another new anduntested technology one that is

designed to maximize seed industryprofits in an attempt to reel in thegenetic contamination problem caused bythe very same companies. There is nosuch thing as a safe and acceptable formof Terminator.New research onmolecular containment of transgenes willultimately allow the multinational seedindustry to tighten its grasp on

proprietary germplasm, restrict the rightsof farmers and dictate the conditionsunder which seeds and plants are viable.Under some scenarios, farmers will beobliged to pay for the privilege ofrestoring seed fertility every year a newform of perpetual monopoly for the seedindustry.

ETC Group offers the followingrecommendations:

The technical understanding andpolitical debate surroundingGURTs/Terminator must beexpanded and updated to includetechnical advances in the

technology. With V-GURT orTerminator technologies thereproductive viability or vigor ofthe plant is controlled by thecorporation that sells the seed.New, molecular strategies forbiocontainment are attempting toshift the burden for containingtransgenes to farmers and society.

Governments and civil societymust not succumb to the

technological imperative and theargument that molecularcontainment strategies willprovide a viable solution toprevent the escape of transgenes.Those who are working to resistGM crops must not acceptbiological containment strategiesas a techno-fix for GMcontamination. If GM plants arenot safe, they are unacceptable andshould not be planted.

Public financing should not beused to support Terminatortechnology. The EuropeanCommission should discontinuefunding for research on Zombieseeds (Recoverable Block ofFunction), and re-assess fundingfor other research projects


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undertaken by Transcontainer.Rather than funding research oncoexistence to bail out theagbiotech industry, the EU shouldinstead fund sustainableagricultural research that benefitsfarmers and the public.

Plants engineered to containconditionally lethal genes (pull-the-plug genes) have the potentialto be used as anti-crop weapons, tosicken or kill an enemys crops.Patents on this technology shouldbe revoked and future applicationsdenied on the grounds that they

violate public morality (ordrepublic).

National governments shouldpropose legislation to prohibitfield-testing and commercial sale

of Terminator (V-GURT)technologies.

Governments meeting at the 9thConference of the Parties to theConvention on Biological Diversityin Bonn, Germany must strengthenthe moratorium on GURTs byrecommending a ban on thetechnology.


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Appendix: Will Recoverable Block of Function (RBF) work wellenough to contain transgenes?

Will RBF work well enough to contain transgenes? Many problems will need to beovercome, and although researchers are addressing the points where the technology

might fail, they have a long way to go before this system can be relied upon to keeptransgenes from wandering. The EU-funded Transcontainer Project is using the RBFsystem first developed by Finnish researchers with UniCrop, Ltd. The Transcontainerresearchers have not yet published their work, but claim that it is far more promisingthan the Finnish system. The latest paper from Viktor Kuvshinovs research group(UniCrop) provides the most recent published information on the state of the art forRBF, and points to many of its potential weaknesses.43

In their experiments, tobacco plants are used as a model because they are easy tomanipulate in tissue culture. The goal of the experiments is to engineer tobacco withthe components of RBF to prevent transgenes from escaping via seeds. In particular,

the researchers attempted to engineer the plants in a way that would minimize some ofthe problems they foresaw with the method: gene silencing and mutations resulting inseed viability and thus possible escape of the transgene.

Here is how Kuvshinovs team set up the most important parts of the experiment:

First, the researchers chose the BC which would be responsible for killing seeds if theycontained transgenic DNA, in this case a gene that codes for the toxin barnase.However, genes mutate. If the barnase gene mutated so that the toxin no longer killedthe seeds, the adjacent transgene for a trait of interest could spread via those seeds.Also, plants have ways of recognizing foreign genes and turning them offgenesilencingthat would also allow seeds with transgenes to live. So Kuvshinovs team

decided to reduce these two possible problems by engineering the tobacco plants withtwo genes for barnase that had different DNA sequences but coded for the samebarnase protein, which is possible because there are several codons for each amino acid.Each barnase gene had a unique promoter sequence, as well, so that one was morestrongly expressed during seed development, and the other during germination. Thelikelihood of mutations occurring in two genes and disabling both toxins is much lowerthan for one gene, and the different DNA sequences make silencing less likely. Theyalso arranged these barnase genes in a way that would minimize silencing.

If the tobacco plants behaved as expected with these barnase genes inserted, they wouldgrow normally, make flowers, and then as the seeds developed the barnase gene withthe seed-specific promoter would turn on and the seeds would be non-viable. If forsome reason that gene failed, the second barnase gene programmed to make toxinduring germination would act as a back-up. The result would be seeds that would dieand be unable to pass on transgenic DNA.

If breeders or farmers wanted to get viable seeds they would have to inactivate thebarnase toxin with the RC part of the package. In this case, RC is a gene for barstar, aprotein that inactivates barnase. This gene was fitted with a promoter sequence from aheat shock protein, allowing it to be turned on by treating the tobacco plants with hightemperatures as the seeds were forming. They only used one barstar gene, presumably


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because the researchers were more concerned with biocontainment than with recoveryof viability. Mutations or silencing of the barstar gene would make it difficult to recoverviable seeds, but would not lead to gene escape.

The transgenic trait in the experiment used to determine the success of containment wasa commonly used gene for an enzyme that forms a blue color when a particularsubstrate is added. Tissues containing the transgene can be easily identified by theirblue color.

Putting the pieces together, when they engineer tobacco plants with the two barnasegenes, the barstar gene, and the color trait, they expect to obtain plants that grownormally until seeds form. Then the seeds will either die as they mature or when theygerminate because of barnase, unless the plants are subjected to high temperatures asthe seeds develop. Heat should cause barstar to be made, inactivating the toxin, andthus restoring fertility.

The question is: will this work efficiently enough to kill all of the transgene-containing

seeds for confinement, and will the barstar be able to rescue enough of them to beuseful to farmers and breeders? (Although the purported reason for using the specificarrangements of genes was to discourage mutations and silencing, they did not actuallydetermine if these problems were lessened in this study.)

The authors conclude that yes, it is possible to use this double-BC system to kill seedsefficiently, and that a single RC can rescue enough seeds to be useful. However, a fewaspects of the study deserve comment.

First, only a few of the tobacco plants containing this complex transgenic DNA insertfunctioned as expected. Most of the plants behaved erratically. Many of them werestunted or otherwise looked strange, as if the barnase gene was being expressed outside

of the seed even though the promoters should have kept expression to seeds. Someplants formed seeds that were viable, even without being heat shocked, as if they werenot making enough barnase. Of those that behaved as expected, the levels of barnaseand barstar varied a lot.

This kind of variability in the first plants regenerated from the genetic engineeringprocess is common. Researchers simply sort through the plants for the ones that havethe amount and pattern of gene expression they want, and then grow those to see ifthey maintain the desired expression from generation to generation. However, it isalways possible that the process of genetic engineering can cause changes in the waythe plants grow and develop that are missed in the screening.

Kuvshinov et al. were able to find some tobacco plants containing their transgenicconstructs that did indeed grow normally until seed development, and then all of theirtransgene-containing seeds failed to germinate. Seed sample sizes ranged from 100-1200 seeds, which is not enough to determine if the method works well enough in anagricultural setting where many millions of seeds are formed in a field. They were alsoable to use heat shock to restore fertility, in some cases to 90% or more. However, theydo note that this method of rescuing seeds may not confine genes if the plants areexposed to hot temperatures in the fields.


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To conclude, the researchers did make some progress in proving their concept of RBF,but they have a long way to go before it will function as designed in agriculture. Heatshock will probably not be a good inducer of RC given the normal range oftemperatures in many parts of the world, the barnase/barstar system may not work aswell in large seeds as it does in the very small tobacco seeds, efficiency will have to bevery high to contain genes in agricultural situations where many more seeds areproduced, and their strategy for reducing mutations and silencing has yet to beexperimentally tested for those effects, although they have shown that the large,complex insert can function in the plant. In addition, promoters for BC will have to beoptimized for each species.


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Endnotes:

1 In 2000 the Conference of the Parties to the Convention on Biological Diversity first adoptedthe de facto moratorium on field trials and commercial use of GURTs. The moratorium was re-

affirmed in 2006. The text of the decision is available here:http://www.biodiv.org/decisions/default.aspx?m=COP-8&id=11037&lg=02Genes may transfer by other processes, such as horizontal gene transfer conducted by virusesor parasites (see Heinemann, J.A. and Bungard, R.A. 2005. Horizontal Gene Transfer. In 2nd Ed.Encyclopedia of Molecular Cell Biology and Molecular Medicine (Meyers R.A. ed). Wiley-VCH,p. 223-243).3 According to Jeffrey Barach, Vice-President and Center Director, GMA/Food ProductsAssociation. Comments made during Agrifood Nanotechnology Workshop, Michigan StateUniversity, April 2, 2007.4http://www.gmcontaminationregister.org/ 5 According to seed industry consultants, the Context Network, the value of the globalproprietary seed market was $19.6 billion in 2006. For more information on top 10 seedcompanies, based on 2006 seed revenues:http://www.etcgroup.org/en/materials/publications.html?pub_id=615 6 Transcontainer is a European Commission-funded Specific Targeted Research or InnovationProject (STREP) in the Sixth Framework Programme Priority 5: Food Quality and Safety.Background on Transcontainer: http://www.transcontainer.wur.nl/UK/ 7 Part of Transcontainers work involves development of biocontainment systems that controlfertility. Of the six technical objectives (Fact Sheet: Transcontainer & Controllable Fertility), fiveinvolve male-sterility. Male-sterility reduces contamination by cross-pollination, but viableseeds can pass on genes, so it is not a totally contained system. One of their objectives is tocombine male-sterility with seedless fruits for more complete transgene containment. However,male-sterility alone is invaluable for production of hybrid seeds, and this is one ofTranscontainers goalsto determine the utility of pollen transgene containment technologiesin hybrid breeding systems. Hybrid seed production is, of course, very lucrative for the seed

industry because the seeds command high prices and have to be purchased every season iffarmers want to gain the varietal benefits. However, hybrid seeds are not sterile seeds.8http://www.transcontainer.wur.nl/UK/Fact+sheets/ 9 See for example, Union of Concerned Scientists, A Growing Concern: Protecting the Food Supplyin an Era of Pharmaceutical and Industrial Crops, 2004; US National Research Council of theNational Academies, Biological Confinement of Genetically Engineered Organisms, NationalAcademies Press, 2004, pp. 8-9. See also links on Transcontainer website:http://www.transcontainer.wur.nl/UK/Literature+links/10 http://www.unicrop.fi/control.html11 Email correspondence from Piet Schenkelaars, Schenkelaars Biotechnology Consultancy, toETC Group, March 2, 2007.12 Email correspondence from Piet Schenkelaars to ETC Group, March 2, 2007.13

Ibid.14Ibid.15 See, for example, US Patent 6,849,776 filed July 14, 2000; issued February 1, 2005. Kuvshinov,V. et al., UniCrop Ltd. Molecular control of transgene segregation and its escape by arecoverable block of function (RBF) system.16 Viktor Kuvshinov, Ph.D., is the original developer of RBF strategies for containing transgenes.In an email to ETC Group, 28 February 2007, he writes: RBF should belong to V-GURT.17 The RBF system is briefly described in the section on V-GURTs in Biological Confinement ofGenetically Engineered Organisms, National Research Council, Washington, DC: The NationalAcademies Press, pp. 72-75.


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18 EcoNexus, V-GURTs (Terminator) as a biological containment tool? June 2005.www.econexus.info19 Available on the EcoNexus website: www.econexus.info/pdf/ENx-CBD-GURTs-2006.pdf20 Kuvshinov, V., A. Anisimov, B.M. Yahya, and A. Kanerva, 2005. Double recoverable block offunctiona molecular control of transgene flow with enhanced reliability. Environ. Biosafety Res.

4: 103-112.21 See, for example, patent WO9301283A1 filed by the US Department of Agriculture in 1992 andUS5792924, Biologically safe plant transformation system, issued to the Regents of theUniversity of California in 1998.22 From C. S. Prakash, The Genetically Modified Crop Debate in the Context of AgriculturalEvolution, Plant Physiology, May 2001, Vol. 126, pp. 8-15. The quote continues: For example,appropriate promoters may ensure that pollen will not express genes toxic to beneficial insects,while gene expression strategies, such as sterile pollen, could reduce the risk of gene flow.23 Ludmila Mlynarova & Jan-Peter Nap, Transgenic Plants that Make Non-Transgenic Pollen,ISB News Report, August 2006, on the Internet:http://www.isb.vt.edu/news/2006/news06.aug.htm#aug060324Ibid.25

Ibid.26 Luo, K., H. Duan, D. Zhao, X. Zheng, W. Deng, Y. Chen, C. N. Stewart Jr., R. McAvoy, X.Jiang, Y. Wu, A. He, Y. Pei and Y. Li, 2007. GM-gene-deleter: fused loxP-FRTrecognitionsequences dramatically improve the efficiency of FLP or CRE recombinase on transgeneexcision from pollen and seed of tobacco plants. Plant Biotechnology Journal 5: 263-274.27 Ludmila Mlynarova & Jan-Peter Nap, Transgenic Plants that Make Non-Transgenic Pollen,ISB News Report, August 2006, on the Internet:http://www.isb.vt.edu/news/2006/news06.aug.htm#aug060328 Luo, K., H. Duan, D. Zhao, X. Zheng, W. Deng, Y. Chen, C. N. Stewart Jr., R. McAvoy, X.Jiang, Y. Wu, A. He, Y. Pei and Y. Li, 2007. GM-gene-deleter: fused loxP-FRTrecognitionsequences dramatically improve the efficiency of FLP or CRE recombinase on transgeneexcision from pollen and seed of tobacco plants. Plant Biotechnology Journal 5: 263-274.29

United States Patent 2002/0124280A1, filed July 27, 2001, published September 5, 2002.Methods for the controlled, automatic excision of heterologous DNA from transgenic plants andDNA-excising gene cassettes for use therein. Li, Y. et al, inventors.30 Haygood, H., A.R. Ives, and D.A. Andow, 2004. Population genetics of transgenecontainment. Ecology Letters 7: 213-220.31Ibid.32 Ludmila Mlynarova & Jan-Peter Nap, Transgenic Plants that Make Non-Transgenic Pollen,ISB News Report, August 2006, on the Internet:http://www.isb.vt.edu/news/2006/news06.aug.htm#aug060333 Jack Heinemann, personal communication with ETC Group.34Ibid.35See also, EcoNexus, Genome Scrambling: Myth or Reality? Transformation InducedMutations in Transgenic Crop Plants, Technical Report October 2004. On the Internet:http://www.econexus.info/pdf/ENx-Genome-Scrambling-Report.pdf36 Luo, K., H. Duan, D. Zhao, X. Zheng, W. Deng, Y. Chen, C. N. Stewart Jr., R. McAvoy, X.Jiang, Y. Wu, A. He, Y. Pei and Y. Li, 2007. GM-gene-deleter: fused loxP-FRTrecognitionsequences dramatically improve the efficiency of FLP or CRE recombinase on transgeneexcision from pollen and seed of tobacco plants. Plant Biotechnology Journal 5: 263-274.37 See, for example: William Freese and David Schubert, Safety Testing and Regulation ofGenetically Engineered Foods, Biotechnology & genetic engineering reviews, Volume 21,November 2004. On the Internet:http://www.foe.org/camps/comm/safefood/gefood/testingregbackgrounder.pdf


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38 US National Research Council of the National Academies, Biological Confinement of GeneticallyEngineered Organisms, National Academies Press, 2004, p. 8-9.39 Genes may transfer by other processes, such as horizontal gene transfer conducted by virusesor parasites (see Heinemann, J.A. and Bungard, R.A. 2005. Horizontal Gene Transfer. In 2nd Ed.Encyclopedia of Molecular Cell Biology and Molecular Medicine (Meyers R.A. ed). Wiley-VCH,

p. 223-243).40 United States Patent 6,753,459; filed June 22, 2001; granted June 22, 2004. Transgenic plantsand methods for production thereof. Keller, W.A., S.F. Fabijanski, P.G. Arnison, J.K.Hammerlindl, and S.R. Webb, for National Research Council of Canada; Dow AgrosciencesLLC.41 See http://www.nature.com/nbt/journal/v20/n7/full/nbt0702-656.html42 See, for example: Melissa J. Hills, et al., Genetic Use Restriction Technologies (GURTs):strategies to impede transgene movement, Trends in Plant Science, Vol. 12, No. 4, March 2007.43 Kuvshinov, V., A. Anisimov, B.M. Yahya, and A. Kanerva, 2005. Double recoverable block offunctiona molecular control of transgene flow with enhanced reliability. Environ. Biosafety Res.4: 103-112.

Terminator Seed: The Sequel

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