Plant Biotechnology and GMOsLesson 5Genetically Modified OrganismBlue roseA blue rose is a flower of the genus Rosa(family Rosaceae)that presents blue-to-violet pigmentation instead of the more common red,white,or yellow.Blue roses are often portrayed in literature and art as a symbol of love and prosperity to those who seek it,but do not exist in nature as a result of genetic limitations.Blue rose-artificially colouredSince they do not exist in nature as roses lack the specific gene that has the ability to produce a“true blue”color,Blue roses are traditionally created by dyeing white roses.Nominal blue roses have been bred by conventional hybridization methods,but the results,such as Blue Moon are more accurately described as being lilac in color.Blue Moon RosesBlue moon roses are unusually colored roses that were initially introduced to the world in 1965.The roses are not an authentic blue,and resemble more of a pale lilac color instead.They are hybrid tea roses and are part of the Rosa genus.Blue moon roses are characterized by their long stems and are larger than many other varieties of roses,with raised centers.They are highly fragrant with scents that are similar to sweet tea.These roses bloom from summer all the way into autumn.Delphinidin,the primary plant pigment that produces a blue hue but is not found naturally in roses.Rosa Cardinal de Richelieu rose,used for the first genetic engineering experimentsThe Blue Rose was developed by Suntory Flowers Prices were from 2,000 to 3,000 Yen or US$22 to 35 a stemIncreasing crop yieldsTo feed the increasing population we have to increase crop yields.Fertilizers-are compounds to promote growth;usually applied either via the soil,for uptake by plant roots,or by uptake through leaves.Can be organic or inorganicHave caused many problems!Algal blooms pollute lakes near areas of agriculture Increasing crop yieldsAlgalblooms-arelativelyrapidincreaseinthepopulationof(usually)phytoplanktonalgaeinanaquaticsystem.Causesthedeathoffishanddisruptiontothewholeecosystemofthelake.Internationalregulationshasledtoareductionintheoccurrencesoftheseblooms.Chemical pest controlEachyear,30%ofcropsarelosttoinsectsandothercroppests.Theinsectsleavelarva,whichdamagetheplantsfurther.Fungidamageorkillafurther25%ofcropplantseachyear.Anysubstancethatkillsorganismsthatweconsiderundesirableareknownasapesticide.Anidealpesticidewould:KillonlythetargetspeciesHavenoeffectonthenon-targetspeciesAvoidthedevelopmentofresistanceBreakdowntoharmlesscompoundsafterashorttimeChemical pest controlDDTwasfirstdevelopedinthe1930sVeryexpensive,toxictobothharmfulandbeneficialspeciesalike.Over400insectspeciesarenowDDTresistant.Aswithfertilizers,therearerun-offproblems.Affectsthefoodpyramid.PersistintheenvironmentChemical pest controlDDTpersistsinthefoodchain.Itconcentratesinfishandfish-eatingbirds.Interferewithcalciummetabolism,causingathinningintheeggslaidbythebirdsbreakbeforeincubationisfinisheddecreaseinpopulation.AlthoughDDTisnowbanned,itisstillusedinsomepartsoftheworld.Plant BiotechnologyTheuseoflivingcellstomakeproductssuchaspharmaceuticals,foods,andbeveragesTheuseoforganismssuchasbacteriatoprotecttheenvironmentTheuseofDNAsciencefortheproductionofproducts,diagnostics,andresearchPlant Biotechnology“For centuries,humankind has made improvements to crop plants through selective breeding and hybridization the controlled pollination of plants.Plant biotechnology is an extension of this traditional plant breeding with one very important difference plant biotechnology allows for the transfer of a greater variety of genetic information in a more precise,controlled manner.”Genetically modified cropsAllplantcharacteristics,suchassize,texture,andsweetness,aredeterminedonthegeneticlevel.Also:Thehardinessofcropplants.Theirdroughtresistance.Rateofgrowthunderdifferentsoilconditions.Dependenceonfertilizers.Resistancetovariouspestsanddiseases.UsedtodothisbyselectivebreedingWhy would we want to modify an organism?Bettercropyield,especiallyunderharshconditions.HerbicideordiseaseresistanceNutritionorpharmaceuticals,vaccinedeliveryOrnamentalpurposeWhats your opinion on GM food?Genetically modified foodslGenetically modified foods(or GM foods)are foods produced from organisms that have had specific changes introduced into their DNA using the methods of genetic engineering.lThese techniques have allowed for the introduction of new crop traits as well as a far greater control over a foods genetic structure than previously afforded by methods such as selective breeding and mutation breeding.lCommercial sale of genetically modified foods began in 1994,when Calgene first marketed its Flavr Savr delayed ripening tomato.lTo date most genetic modification of foods have primarily focused on cash crops in high demand by farmers such as soybean,corn,canola,and cotton seed oil.lThese have been engineered for resistance to pathogens and herbicides and better nutrient profiles.lGM livestock have also been experimentally developed,although as of November 2013 none are currently on the market.But a GM salmon was awaiting regulatory approval at that time.lThere is broad scientific consensus that food on the market derived from GM crops poses no greater risk to human health than conventional food.lHowever,opponents have objected to GM foods on several grounds,including safety issues,environmental concerns,and economic concerns raised by the fact that GM seeds(and potentially animals)that are food sources are subject to intellectual property rights owned by multinational corporations.Roundup Ready GeneTheglyphosateresistancegeneprotectsfoodplantsagainstthebroad-spectrumherbicideGlyphosate-N-(phosphonomethyl)glycineRoundup,whichefficientlykillsinvasiveweedsinthefield.ThemajoradvantagesoftheRoundupReady”systemincludebetterweedcontrol,reductionofcropinjury,higheryield,andlowerenvironmentalimpactthantraditionalweedcontrolsystems.Notably,fieldstreatedwithRounduprequirelesstilling;thispreservessoilfertilitybylesseningsoilrun-offandoxidation.”Glyphosate-N-(phosphonomethyl)glycineAnaminophosphonicanalogueofthenaturalaminoacidglycine.Itisabsorbedthroughfoliageandtranslocatedtoactivelygrowingpoints.(Meristems!)Modeofactionistoinhibitanenzymeinvolvedinthesynthesisofthearomaticaminoacids:tyrosine,tryptophanphenylalanineGlyphosateGlycineGlyphosate-N-(phosphonomethyl)glycineItdoesthisbyinhibitingtheenzyme5-enolpyruvylshikimate-3-phosphatesynthase(EPSPS),whichcatalyzesthereactionofshikimate-3-phosphate(S3P)andphosphoenolpyruvatetoform5-enolpyruvylshikimate-3-phosphate(ESP).ESPsubsequentlydephosphorylatedtochorismate,anessentialprecursorinplantsforthesearomaticaminoacids.GlyphosateGlycineRoundup Ready GeneGlyphosatefunctionsbyoccupyingthebindingsiteofthephosphoenolpyruvate,mimickinganintermediatestateoftheenzymesubstratescomplex.TheRoundupReady”systemintroducesastablegenealterationwhichpreventsGlyphosatebindingandallowingtheformationoftheessentialaromaticaminoacidsRoundup Ready GeneTheshikimatepathwayisnotpresentinanimals,whichinsteadobtainaromaticaminoacidsfromtheirdiet.GlyphosatehasalsobeenshowntoinhibitotherplantenzymesAlsohasbeenfoundtoaffectanimalenzymes.TheUnitedStatesEnvironmentalProtectionAgencyconsidersglyphosatetoberelativelylowintoxicity,andwithoutcarcinogenicorteratogeniceffectsHowever,somefarmworkershavereportedchemicalburnsandcontactskinburnsEnvironmental degradationWhenglyphosatecomesintocontactwiththesoil,itcanberapidlyboundtosoilparticlesandbeinactivated.Unboundglyphosatecanbedegradedbybacteria.However,glyphosatehasbeenshowntoincreasetheinfectionrateofwheatbyfusariumheadblightinfieldsthathavebeentreatedwithglyphosate.Insoils,half-livesvaryfromaslittleas3daysatasiteinTexasto141daysatasiteinIowa.Inaddition,theglyphosatemetaboliteaminomethylphosphonicacidhasbeenshowntopersistupto2yearsinSwedishforestsoils.Glyphosateabsorptionvariesdependingonthekindofsoil.Insect ResistanceB.thuringiensis(commonly known as Bt)is an insecticidal bacterium,marketed worldwide for control of many important plant pests-mainly caterpillars of the Lepidoptera(butterflies and moths)but also mosquito larvae,and simuliid blackflies that vector river blindness in Africa.Bt products represent about 1%of the total agrochemical market(fungicides,herbicides and insecticides)Genetically modified crops1992-Thefirstcommerciallygrowngeneticallymodifiedfoodcropwasatomato-wasmademoreresistanttorotting,byaddingananti-sensegenewhichinterferedwiththeproductionoftheenzymepolygalacturonase.Theenzymepolygalacturonasebreaksdownpartoftheplantcellwall,whichiswhathappenswhenfruitbeginstorot.Genetically modified cropsNeed to build in a:PromoterStop signal CODING SEQUENCE5 UTRpoly A signalPROMOTERON/OFF SwitchMakes Proteinstop signGenetically modified cropsSotomodifyaplant:NeedtoknowtheDNAsequenceofthegeneofinterestNeedtoputaneasilyidentifiablemakergenenearornexttothegeneofinterestHavetoinsertbothoftheseintotheplantnucleargenomeGoodscreenprocesstofindsuccessfulinsertionCODING SEQUENCEINTRONpoly A signalPROMOTERBuilding the TransgenesPlant Transgenebacterial genesantibiotic markerreplication originPlant Selectable Marker GenePlasmid DNA ConstructON/OFF SwitchMakes Proteinstop signCloning into a PlasmidTheplasmidcarryinggenesforantibioticresistance,andaDNAstrand,whichcontainsthegeneofinterest,arebothcutwiththesamerestrictionendonuclease.TheplasmidisopenedupandthegeneisfreedfromitsparentDNAstrand.Theyhavecomplementarystickyends.TheopenedplasmidandthefreedgenearemixedwithDNAligase,whichreformsthetwopiecesasrecombinantDNA.Cloning into a PlasmidPlasmids+copiesoftheDNAfragmentproducequantitiesofrecombinantDNA.ThisrecombinantDNAstewisallowedtotransformabacterialculture,whichisthenexposedtoantibiotics.AllthecellsexceptthosewhichhavebeenencodedbytheplasmidDNArecombinantarekilled,leavingacellculturecontainingthedesiredrecombinantDNA.So,how do you get the DNA into the Plant?Meristems InjectionsREMEMBER!Thetissueinmostplantsconsistingofundifferentiatedcells(meristematiccells),foundinzonesoftheplantwheregrowthcantakeplace.Meristematiccellsareanalogousinfunctiontostemcellsinanimals,areincompletelyornotdifferentiated,andarecapableofcontinuedcellulardivision.FirstmethodofDNAtransfertoaplant.InjectDNAintothetipcontainingthemostundifferentiatedcellsmorechanceofDNAbeingincorporatedinplantGenomeWorkedabout1in10,000times!Tunica-Corpusmodeloftheapicalmeristem(growingtip).Theepidermal(L1)andsubepidermal(L2)layersformtheouterlayerscalledthetunica.TheinnerL3layeriscalledthecorpus.CellsintheL1andL2layersdivideinasidewaysfashionwhichkeepstheselayersdistinct,whiletheL3layerdividesinamorerandomfashion.Particle BombardmentParticle-GunBombardment1.DNA-orRNA-coatedgold/tungstenparticlesareloadedintothegunandyoupullthetrigger.SelectedDNAstickstosurfaceofmetalpelletsinasaltsolution(CaCl2).Particle Bombardment2.Alowpressureheliumpulsedeliversthecoatedgold/tungstenparticlesintovirtuallyanytargetcellortissue.3.TheparticlescarrytheDNAcellsdonothavetoberemovedfromtissueinordertotransformthecells4.Asthecellsrepairtheirinjuries,theyintegratetheirDNAintotheirgenome,thusallowingforthehostcelltotranscribeandtranslatethetransgene.Particle BombardmentTheDNAsometimeswasincorporatedintothenucleargenomeoftheplantGenehastobeincorporatedintocellsDNAwhereitwillbetranscribedAlsoinsertedgenemustnotbreakupsomeothernecessarygenesequenceParticle BombardmentAgrobacterium tumefaciensOverall processUsesthenaturalinfectionmechanismofaplantpathogenAgrobacterium tumefaciensnaturally infectsthewoundsitesindicotyledonousplantcausingtheformationofthecrowngalltumors.CapabletotransferaparticularDNAsegment(T-DNA)ofthetumor-inducing(Ti)plasmidintothenucleusofinfectedcellswhereitisintegratedfullyintothehostgenomeandtranscribed,causingthecrowngalldisease.SothepathogeninsertsthenewDNAwithgreatsuccess!Overall processThevirregionontheplasmidinsertsDNAbetweentheT-regionintoplantnucleargenomeInsertgeneofinterestandmarkerintheT-regionbyrestrictionenzymesthepathogenwillthen“infect”theplantmaterialWorksfantasticallywellwithalldicotplantspeciestomatoes,potatoes,cucumbers,etcDoesnotworkaswellwithmonocotplantspecies-cornAsAgrobacterium tumefaciensdonotnaturallyinfectmonocotsOverview of the Infection Process1.Agrobacterium tumefacienschromosomalgenes:chvA,chvB,pscArequiredforinitialbindingofthebacteriumtotheplantcellandcodeforpolysaccharideonbacterialcellsurface.2.Virulenceregion(vir)carriedonpTi,butnotinthetransferredregion(T-DNA).GenescodeforproteinsthatpreparetheT-DNAandthebacteriumfortransfer.Ti plasmids and the bacterial chromosome act in concert to transform the plant3.T-DNAencodesgenesforopinesynthesisandfortumorproduction.4.occ(opinecatabolism)genescarriedonthepTiandallowsthebacteriumtoutilizeopinesasnutrient.vir genesopine catabolismpTitrafor transfer to the plantbacterial conjugationAgrobacterium chromosomal DNAchvA chvBpscAoriVT-DNA-inserts into plant genomeAgrobacterium tumafaciens senses Acetosyringone via a 3-component-like system 3 components:ChvE,VirA,VirGPeriplasmicdomainacetosyringoneChvEVirAVirGsugarsTransmitterInhibitorydomainreceiverDNA-binding1.ChvEperiplasmicproteinbindstosugars,arabinose,glucosebindstoVirAperiplasmicdomainamplifiesthesignalPeriplasmicdomainacetosyringoneChvEVirAVirGsugarsTransmitterInhibitorydomainreceiverDNA-binding2.VirA:Receptor kinase1.Membraneproteinfivefunctionaldomains:a)PeriplasmicbindsChvE-sugarcomplexdoesNOTbindacetosyringoneb)Transmembranedomainc)LinkerregionBINDSacetosyringoneNOTEthisisonthecytoplasmicside!PeriplasmicdomainacetosyringoneChvEVirAVirGsugarsTransmitterInhibitorydomainreceiverDNA-binding2.VirA:Receptor kinased)Transmitterdomain(His)auto-phosphorylatesandthentransferstotheresponseregulatorproteinVirGe)InhibitorydomainwillbleedoffthephosphatefromtheHisinthetransmitterdomain(toanAsp)Periplasmic domainacetosyringoneChvEVirAVirGsugarsTransmitterInhibitory domainreceiverDNA-binding3.VirG:Response Regulatora)ReceiverdomainthatisphosphorylatedonanAspresiduebytheHisonthetransmitterdomainofVirAb)ActivatestheDNAbindingdomaintopromotetranscriptionfromVir-boxcontinaingpromotersequences(ontheTiplasmid)PeriplasmicdomainacetosyringoneChvEVirAVirGsugarsTransmitterInhibitorydomainreceiverDNA-bindingAgrobacterium can be used to transfer DNA into plantsAgrobacteria arebiologicalvectorsforintroductionofgenesintoplants.Agrobacteriaattachtoplantcellsurfacesatwoundsites.Theplantreleaseswoundsignalcompounds,suchasacetosyringone.ThesignalbindstovirAontheAgrobacteriummembrane.VirAwithsignalboundactivatesvirG.SummaryActivatedvirGturnsonothervirgenes,includingvir DandE.virDcutsataspecificsiteintheTiplasmid(tumor-inducing),theleftborder.Theleftborderandasimilarsequence,therightborder,delineatetheT-DNA,theDNAthatwillbetransferredfromthebacteriumtotheplantcellSinglestrandedT-DNAisboundbyvirEproductastheDNAunwindsfromthevirDcutsite.Bindingandunwindingstopattherightborder.SummaryTheT-DNAistransferredtotheplantcell,whereitintegratesinnuclearDNA.T-DNAcodesforproteinsthatproducehormonesandopines.Hormonesencouragegrowthofthetransformedplanttissue.Opinesfeedbacteriaacarbonandnitrogensource.SummaryOverview of the Infection ProcessAnd then?.What is the last step?.Tissue cultureThe basics!What is Plant Tissue Culture?Ofallthetermswhichhavebeenappliedtotheprocess,micropropagationisthetermwhichbestconveysthemessageofthetissueculturetechniquemostwidelyinusetoday.Theprefixmicrogenerallyreferstothesmallsizeofthetissuetakenforpropagation,butcouldequallyrefertothesizeoftheplantswhichareproducedasaresult.ReliesontwoplanthormonesAuxinCytokininProtoplast to Plant Callus:Inducedby2,4dichlorphenoxyaceticacid(2,4-D)Unorganized,growingmassofcellsDedifferentiationofexplantLooselyarrangedthinnedwalled,outgrowthsNopredictablesiteoforganizationordifferentiationAuxin(indoleacetic acid)Producedinapicalandrootmeristems,youngleaves,seedsindevelopingfruitscellelongationandexpansionsuppressionoflateralbudgrowthinitiationofadventitiousrootsstimulationofabscission(youngfruits)ordelayofabscissionhormoneimplicatedintropisms(photo-,gravi-)celldivisionfactorstimulatesadventitiousbudformationdelayssenescencepromotessomestagesofrootdevelopmentCytokinin(zeatin,ZR,IPA)Producedinrootmeristems,youngleaves,fruits,seedsOrganogenesisTheformationoforgansfromacallusRuleofthumb:Auxin/cytokinin10:1-100:1inducesroots.1:10-1:100inducesshootsIntermediateratiosaround1:1favorcallusgrowth.Edible VaccinesTransgenic Plants Serving Human Health NeedsWorkslikeanyvaccineAtransgenicplantwithapathogenproteingeneisdevelopedPotato,banana,andtomatoaretargetsHumanseattheplantThebodyproducesantibodiesagainstpathogenproteinHumansare“immunized”againstthepathogenExamples:DiarrheaHepatitisBMeaslesThe End!Any Questions?