BiologicalBiological ConfinementConfinement ofof TransgenicTransgenic PlantsPlants Dr.Dr. HenryHenry DaniellDaniell Pegasus Professor & Trustee Chair, University of Central Florida Technical Founder, Chlorogen Inc. Bioconfinement Methods in Plants -- NRC Report Purpose Method Major Limitations Other Considerations Confine all gene flow Sterile triploids or Few triploid or sterile hybrid Not useful if seed via pollen and seeds interspecific hybrids cases apply or are effective production is desired Use only male or only Not feasible if same species or Not useful if seed female plants that can compatible relatives could production is desired be propagated cross-pollinate with unisexual vegetatively plants; sex expression can be leaky V-GURTs, such as V-GURTs under development V-GURTs should not be original terminator (early); other sterility methods used in food crops if require vegetative propagation growers need to save seeds Reduce spread and V-GURTs with Under development (early) persistence of inducible promoters vegetative that kill vegetative propagules tissues Confine pollen only Male sterility Available for some species, Crop requires other plants could be lost in later as source of pollen if seed generations; transgenic production is desired methods could be more durable Transgene in Under development; not Possible to obtain high chloroplast; feasible for plants with paternal concentrations of desired Maternal inheritance inheritance of chloroplast DNA genetically engineered (most gymnosperms) proteins, but many traits cannot be conferred by chloroplast genes Cleistogamy (closed Under development (early) Results in self-pollination flowers) Apomixis (asexually Under development (early) Hybrid varieties would produced seeds) have high yield and breed true; could become invasive NRC Report, Continued Purpose Method Major Limitations Other Considerations Transgenes Transgenes only in Under development Applicable to grafted absent in rootstocks (early); cannot use scions of certain woody seeds and transgenic traits in species such as grapes, pollen flowers, fruits, seeds fruit trees Transgenes Under development Allows seed production excised before (early); very without spread of reproduction speculative; cannot use transgenes transgenic traits in flowers, fruits, seeds Confine T-GURTs involving Under development Potentially useful; avoids transgenic inducible traits (early); external cues concerns about sterile traits only for transgene plants, but inactive (transgenes expression might not transgenes can still spread can spread) be reliable enough for high efficacy Reduce gene Repressible seed Under development Allows viable seeds to be flow to and lethality (early) produced on same from crop cultivar. Seeds sired on relatives other cultivars or wild relatives would not be viable Cross- Under development incompatibility (early); speculative Chromosome Under development; Applies only to crops that location in possible if relative has are allopolyploids (wheat, allopolyploids nonhomologous cotton, canola) chromosomes; can be leaky Tandem constructs Under development to reduce fitness in (early); requires fitness- crop-wild hybrids reducing trait and their progeny detrimental to wild plants but not crop NRC Report, Continued Purpose Method Major Limitations Other Considerations Phenotypic and fitness Domestication phenotypes Under development; does handicaps to reduce not prevent gene flow need for confinement Auxotrophy (dependence on Under development; does specific nutrients or growing not prevent gene flow conditions) Reduce exposure to Tissue- and organ-specific Promoters available, but Could alleviate the need transgenic promoters that limit expression of greater efficacy needed in for bioconfinement in products in plants transgene many cases; confines some cases transgenic traits but not the transgenes; transgenes can spread Minimize or eliminate Choice of alternative organisms; Economic costs can be Often feasible and highly need for choice not to release in field; choice high, especially if recommended when bioconfinement not to proceed with GEO decision to change appropriate; alternative course is made after choices should be economic investment examined before GEO is developed SeedSeed Sterility:Sterility: TerminatorTerminator TechnologyTechnology • Induces plants to produce non-viable offspring. • Induction can occur by soaking seeds in a solution that induces a promoter. • Uninduced seeds can develop into fertile plants – incomplete induction is a concern. • Better to engineer sterility with an option for restoration of fertility ( FAO 2002). • Weaknesses • The efficacy could be diminished by gene silencing, recombination • Public access to data limited. RecoverableRecoverable BlockBlock ofof FunctionFunction –– KuvstimovKuvstimov etet al,al, 20012001 •• BlockerBlocker __ DNADNA sequencesequence elementelement thatthat interruptsinterrupts aa specificspecific functionfunction resultingresulting inin deathdeath •• RecoveryRecovery –– DNADNA sequencesequence restoresrestores blockedblocked functionfunction activatedactivated byby exogenousexogenous chemicalchemical treatment.treatment. •• BothBoth thethe BlockerBlocker andand RecoveryRecovery sequencessequences areare physicallyphysically linkedlinked toto thethe transgene.transgene. •• StillStill inin earlyearly stagesstages ofof Development.Development. RepressiveRepressive SeedSeed lethallethal ConfinementConfinement • Novel trait is tightly linked to seed lethality in a hemizygous plants (SL/-) • This is crossed with a homozygous plant containing the repressor (R/R). • Seed with SL/R should express the novel trait, produce seeds. • Sexually compatible realtives (-/-) should produce SL/- (non-viable) or R/- (only repressor). • Similar to the terminator technology but facilitates seed production (25% not viable). RepressiveRepressive SeedSeed lethallethal ConfinementConfinement ContinuedContinued…… WeaknessesWeaknesses •• NotNot possiblepossible toto crosscross withwith otherother usefuluseful cultivars.cultivars. •• EarlyEarly stagesstages ofof development.development. •• SiteSite specificspecific integrationintegration ofof transgenestransgenes notnot yetyet achieved.achieved. •• PartialPartial containmentcontainment isis possiblepossible asas longlong asas thethe linkedlinked transgenestransgenes areare locatedlocated onon homologoushomologous chromosomes.chromosomes. •• NearbyNearby relatedrelated cropscrops maymay produceproduce deaddead seeds.seeds. •• IntrogresssionIntrogresssion ofof repressorrepressor genesgenes intointo naturalnatural populations.populations. •• DoesDoes notnot preventprevent seedseed mediatedmediated transgenetransgene escapeescape UnisexualUnisexual plantsplants lackinglacking matesmates • Examples: Holly, kiwi, gingko, avocado, asparagus • Sex specific molecular markers can be use before massive propogation ( Khadka et al, 2002) • May be used in combination with other confinement approaches. Weaknesses – Applies to a narrow range of species Dioecy is known to be quite leaky. Seeds are produced by “male” plants (Poppendieck & Peterson, 1995). CleistogamyCleistogamy (closed(closed flowers)flowers) •• FertilizationFertilization occursoccurs beforebefore flowerflower opensopens •• ObligateObligate cleistogamscleistogams wouldwould bebe effectiveeffective inin preventingpreventing genegene escapeescape viavia pollenpollen WeaknessesWeaknesses:: •• NotNot yetyet availableavailable •• PerpetualPerpetual selfself--fertilizationfertilization couldcould resultresult inin inbreeding,inbreeding, depression.depression. •• SeedSeed mediatedmediated dispersaldispersal notnot preventedprevented ApomixisApomixis (asexually(asexually producedproduced seeds)seeds) • Reproduce asexually by clonally produced seeds. • Progeny are gentetically identical to parent. • Preserves superior genotypes (no need for inbred lines). • Obligate apoximis is extremely rare. • Most apomicts retain low to moderate sexual pollination to stimulate seed formation, in the absence of fertilization. • Apomictic species easily outcompete sexual organisms (e.g. dandelions). Weaknesses • Apomictic GMOs could establish invasive populations. • Not suitable for bioconfinement because even obligate apomicts produce seeds. TransgenesTransgenes absentabsent fromfrom seedsseeds andand PollenPollen Non-transgenic scions on transgenic rootstock • Grapes, citrus, avocados are grown as grafted composites of two genotypes. • Non-transgenic scions could be grafted onto transgenic rootstocks. • Double-grafting (transgenic section sandwiched between non- transgenic) would prevent vegetative propagules. Weaknesses • Not useful for non-woody species. • Applicalble for traits in the root stock. • Not applicable for forest trees (large scale). ExcisionExcision ofof transgenestransgenes beforebefore reproductionreproduction • Trait expressed during vegetative growth. • Does not result in seed sterility. • A chemically induced flower specific promoter drives a recombinase enzyme that excised the transgene (Cre/lox system). Weaknesses • Extremely difficult to guarantee reliability. • Not applicable for traits in seeds. ArtificiallyArtificially inducedinduced transgenestransgenes expressionexpression • Trait is activated by a chemical spray • Example: Salicylic acid for pathogen resistance • An example of T-GURT. Weaknesses • Bioconfinement of expression but not transgene • Early stages of development • Not applicable to traits that require constant expression. CrossCross-- incompatibilityincompatibility