Bt-Cotton & Pest Resistant Bt-Toxin

This ppt is made by Basant kumar pradhan, Student of B.Sc. 6th Semester, in Department of botany Guru ghasidas vishwavidyalaya Bilaspur. This presentation mainly focuses on following aspects :-

1. History
2.Introduction of Bt-cotton
3. Why Bt-cotton is produced?
4. What is Bt?
5. What is Bt-cotton?
6. How Bt-cotton is developed?
7. Mode of action of Cry toxin.

1. Pest Resistant; Bt-Cotton
Presented by
Basant Kumar Pradhan
B.Sc. 6
th
Semester
Department of Botany
Guru ghasidasvishwavidyalaya
Bilaspur
Guided by
Miss ShivaliSharma
Assistant Professor
Department of Botany
Guru ghasidasvishwavidyalayaBilaspur

2. ❑History of Bt-Cotton
•FirstreportedfromdiseasedsilkwormfromJapan.
•BerlinercoinedthenameBacillusthuringiensis,isolatedthestrain
fromagrainmillinGermandistrictofThuriengien.
•Firstcommercialpreparationpreparedin1927.
•FirstlargescaleBtbasedproductreleasedforsalein1957bySandoz
Corp.
•ItwasaBtvariety“kurstakistrain”marketedas“Thuricide”.(Spray).
LimitationsofBt-Spray:-
•Low efficacy
•UV-degradable, short field persistence.
•Poor Coverage
Source unknown.. 2

3. www.cicr.org.in
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❑Introduction
•Cottonisoneofthemajorfibercropsofglobalsignificance.
•Itiscultivatedintropicalandsubtropicalregionsofmorethaneightycountriesof
worldoccupyingnearly33mhawithanannualproductionof19to20milliontones
ofbales.
•InIndia,cottonisbeingcultivatedin9.0mha(27.3%)andstandsfirstinacreage
(areaforcottonproduction).
•ThedecisionoftheGeneticEngineeringApprovalcommittee(GEAC)of
GovernmentofIndiaclearingthereleaseofBtcottonforcommercialcultivation
during2002-2003cropseason,isconsideredasoneofthemajormilestonesinthe
historyofcottonimprovementinIndia.
•Incidentally,cottonhappenstobethefirstcroptoreceiveenvironmentclearanceas
GMOinIndianAgriculture,andthushasreceivedmaximumattentionfrom
planners,scientists,socialworkers,media,farmersandgeneralpublic.

4. www.cicr.org.in 4
❑Why Bt-Cotton?
•Today,productivityofIndiancottonsislowestintheworld.In
contrast,themajorcottonproducingcountrieshaveproductivity3to
5timeshigher.
•TherearemanyreasonsoflowproductivityofcottoninIndia.
•Besidesdependencyon70%cottonproductiononvagarieson
monsoon,diverseecologicalandsoilconditions,constantthreat
frompestsanddiseasesisconsideredamajorbiologicalchallengeto
successfulcottonproductivity.
•Amongstthebioticstressfactor,bollwormsarebyfarthemost
seriouspestsofcottonandalternativecontrollingstrategies,
suchasBtcottonisconsideredawelcometechnologicalstep
•Thesuckingpestcomplexcomprisingofaphids,jassids,thripsand
whiteflyarewidespreadandfairlyserious.However,theirdamage
canbeefficientlycontainedbytheexistingpracticesofcultural,
chemical,biologicalandhostresistancemeans.
Challenges

5. •Thebollwormsaremostimportanttissuefeedersandhighlydamaging.Threetypes
ofbollwormsviz.:-
1.Americanbollworm(Helicoverpaarmigera),
2.Pinkbollworm(Pectionphoragossypiella)and
3.Spottedbollworm(Eariasvitella),
Normallyreferredas“Bollwormcomplex”arebyfarthemost
damagingandlossinducingpestsofcotton.Amongstthem,Helicoverpaemergedasa
keypestalloverthecountrycausingashighas80%lossesincotton.
www.cicr.org.in
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Helicoverpaarmigera/ American
cotton bollworm
Pectionphoragossypiella
Eariasvitella
http://www.agroatlas.ru/en/content/pests/Helicoverpa_ar
migera/
https://en.wikipedia.org/wiki/Pink_bollworm apps.lucidcentral.org

6. ❑What is Bt?
•TheBtisashortformofubiquitoussoilbacteriumBacillus
thuringiensis.Thisbacteriumisgrampositiveandsporeformingthat
formsparasporal(Akindofparasitic)crystalsduringstationaryphaseof
itsgrowthcycle.
•Thesynthesizedcrystallineproteinscalled‘endotoxins’arehighlytoxic
tocertaininsects.Theykilltheinsectbyactingontheepitheliumtissues
ofmidgutofcaterpillars.
•Theseproteinoftenappearmicroscopicallyasdistinctlyshapedcrystals
andconstituteabout20-30%ofdryweightofsporulatedcultures.These
proteinsarecharacterizedbytheirinsecticidalactivityandaretherefore
groupedintofourclassesaccordingtotheirspecificityinthedifferent
ordersofinsectsi.e.:-
a)Lepidoptera-specific(CryI),
b)LepidopteraandDiptera-specific(CryII),
c)Coleoptera-specific(CryIII)and
d)Diptera-specific(CryIV).
www.cicr.org.in 6

7. 7
Figure1.
ThreedimensionalstructuresofinsecticidaltoxinsproducedbyBacillusthuringiensisCry1Aa,Cry2Aa,Cry3Aa,Cry3Bb,
Cry4Aa,Cry4BbandCyt2A.
Alejandra Bravo, SarjeetS. Gill, and Mario Soberón. Mode of action of Bacillus thuringiensis Cry and Cyttoxins and
their potential for insect control. Toxicon. 2007 March 15; 49(4): 423–435.

8. •DifferentstrainsofBtproducemorethan25differentbutrelated“Insecticidalcrystal
proteins(ICPs)”.
•Thesearetoxictolarvaeofdifferentinsectsincludingdiseasevectorsandmany
agriculturalpests.
•CottonbollwormsbelongtotheorderLepidopteraandthereforearesensitivetoBt
CryIandCryIIproteins,whicharespecifictothem.
•ThegenebankdatabaseofBacillusGeneticStockCentre(BGSC)havegivenalist
ofCry(Crystal),Cyt(Cytolytic)andVip(Vegetativeinsecticidalprotein)genes
eithersyntheticormodifiedversionsfromB.thuringiensis.
•About22classesofCryincluding126Crygeneshavebeenregisteredalongwitha
Crtgeneand3Vipgenes.ButpopularlyandeffectivelyutilizedareCry1Ac,Cry1Ab
indifferentcrops.
www.cicr.org.in 8

9. •A genotype or individual which is developed by the techniques of
genetic engineering is referred to as transgenic.
•Inotherwords,geneticallyengineeredorganismsarecalled
transgenics.Atransgenicmaybeaplant,ananimaloramicrobe.
•Transgenicplantscontainforeigngeneorgeneticallymodifiedgeneof
thesamespecies.Theforeigngenemaybefromadistantlyrelated
species,closelyrelatedspeciesorunrelatedspeciesorfrommicro-
organismssuchasfungi,bacteriaandviruses.
•Btcottonreferstotransgeniccottonwhichcontainsendotoxinprotein
inducinggenefromsoilbacteriumBacillusthuringiensis.
•Thefirsttransgenicplantwasdevelopedin1983intobaccoinU.S.A.
•Incotton,thefirsttransgenicplantwasdevelopedin1987inU.S.A.
byMonsanto,DeltaandPinecompanies.
www.cicr.org.in 9
❑What is Bt-Cotton?

10. •Lateron,theresearchworkondevelopmentoftransgenicwasintensifiedallover
theglobeandseveraltransgenicplantsweredeveloped.
•Thetransgeniccottonisoftwotypesviz.
(1)Bollguardcotton:Confersresistancetobollworms.
(2)Roundupreadycotton:Resistanttoherbicides.
•TheareaunderherbicideresistanttransgeniccottonisrestictedtoUSA.However,
bollwormresistantBttransgeniccottonhasspreadtoseveralcountries.
www.cicr.org.in 10

11. ❑How Bt-Cotton is developed?
•Thereare5importantstepsforthedevelopmentoftransgenicof
anycrop:-
1)Identificationofeffectivegeneorgenes
2)Genetransfertechnology
3)Regenerationabilityfromprotoplasts,callusortissues
4)Geneexpressionoftheproductsatdesiredlevel
5)Properintegrationofgenessothatcarriedforgenerationsbyusual
meansofreproduction.
•Onceidentificationofbollworminhibitinggeneshasbeen
achieved,molecularbiologistshavestepbystepsolvedthe
problemstoachieveperfecttransgenics.
•Incaseofcotton,Agrobacterium-mediatedgenetransfertechnique
hasbeenessentiallyused.
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12. •Therearefourimportantmethodsofforeigngene(DNA)transferincropplants
(Stewart,1991).viz.
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1)Plasmidmethod,
2)Particlebombardment,
3)DirectDNAuptakeand
4)Micro-injection
Currently,twoDNAdeliverysystemarewidelyusedfordevelopmentoftransgenic
(geneticallyengineered)plantsincotton:-
(1)Agrobacteriummediatedgenetransfer,and
(2)BombardmentofcellswithplasmidDNAcoatedparticles,
•More than 37 transgenic plants have been developed in cotton so far by these two
methods.

13. ❑Agrobacteriummediatedgene
transfer
•ThesoilbornebacteriumAgrobacteriumtumifaciens(termedas
Nature’sGeneticEngineering)isusedfordevelopmentof
transgenicplants.
www.cicr.org.in 13
❖Limitations
•Hostspecificity,
•Somaclonalvariationand
slowgeneration.
❖Advantages
•Firstly,thismethodhassome
controloverthecopynumber
andsiteofintegrationof
transgenewhichisnotpossible
inparticlebombardment
method.
•Secondly,thisisacheaper
method of genetic
transformationthanparticle
bombardmentmethod.
Perlaket.al.(1991)transferred
successfullytheCry1Acgeneto
cottonviaAgrobacteriumwith
CaMVpromoter(Foreigngene
activator)andtheCryprotein
producesbytransgeniccottonwas
foundhighlytoxictobollworms.

14. https://www.slideshare.net/manojsiddartha/bt-crops-ppt 14

15. ❑Particle bombardment method
•IntheparticlebombardmentmethodtheforeignDNAisdeliveredintoplantcells
throughhighvelocitymetalparticles.
•ThismethodhassomeadvantagesovertheAgrobacteriummediatedmethodof
DNAtransfer:
www.cicr.org.in 15
1)Thismethoddoesnotexhibithostspecificity.Hence,itcanbe
effectivelyusedforthedevelopmentoftransgenicplantsin
variousplantspecies.
2)ThismethodistechnicallysimplethanAgrobacteriummediated
DNAtransfermethod.
3)Inthismethod,thereisnoneedofisolatingprotoplast.

16. ❑Mode of action of three-domain
Cry toxins in lepidopteran insects
•ThemodeofactionofCrytoxinshasbeencharacterizedprincipallyin
lepidopteraninsects.
•Asmentionedpreviously,itiswidelyacceptedthattheprimaryactionof
Crytoxinsistolysemidgutepithelialcellsinthetargetinsectby
formingporesintheapicalmicrovillimembraneofthecells.
•Nevertheless,ithasbeenrecentlysuggestedthattoxicitycouldbe
relatedtoG-protein(heterotrimericguaninenucleotide-bindingprotein)-
coupledreceptors(GPCRs)mediatedapoptosisfollowingreceptor
binding.Cryproteinspassfromcrystalinclusionprotoxinsinto
membrane-insertedoligomersthatcauseionleakageandcelllysis.
•Thecrystalinclusionsingestedbysusceptiblelarvaedissolveinthe
alkalineenvironment(pH≈8.5)ofthegut,andthesolubilizedinactive
protoxinsarecleavedbymidgutproteasesyielding60–70kDaprotease
resistantproteins.
16
Alejandra Bravo, SarjeetS. Gill, and Mario Soberón. Mode of action of Bacillus thuringiensis Cry and Cyttoxins and
their potential for insect control. Toxicon. 2007 March 15; 49(4): 423–435.

17. •Toxin activation involves the proteolytic removal of an N-terminal peptide (25–30
amino acids for Cry1 toxins, 58 residues for Cry3A and 49 for Cry2Aa) and
approximately half of the remaining protein from the C-terminus in the case of the
long Cry protoxins.
•The activated toxin then binds to specific receptors on the brush border membrane
of the midgut epithelium columnar cells before inserting into the membrane.
Alejandra Bravo, SarjeetS. Gill, and Mario Soberón. Mode of action of Bacillus thuringiensis Cry and Cyttoxins and
their potential for insect control. Toxicon. 2007 March 15; 49(4): 423–435.
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Figure2.
RelativelengthofCryprotoxinsandpositionof
proteasedigestion.Whiteboxesrepresentthe
protoxinandstripedboxesrepresentthe
activatedtoxin.Solidarrowsshowtheamino-
andcarboxy-terminalcleavagesitesofthe
activatedtoxins.Dotedarrowsshowthe
intramolecularcleavages.CleavageofCry1Aat
residue51resultedinlossofhelixα-1andpre-
poreformation.CleavageofCry4Bresultedin
twofragmentsof18and46kDa,whileCry11A
resultedintwofragmentsof34and32kDa.

18. Schematic representation of the mechanism of action of 3d-Cry toxins in
Lepidoptera at the cellular level, showing the immunolocalization of Cry toxin
during intoxication (Bravo et al., 1992).
LilianaPardo-Lo´pez,MarioSobero´n&AlejandraBravo.Bacillusthuringiensisinsecticidalthree-domainCrytoxins:
modeofaction,insectresistanceandconsequencesforcropProtection.InstitutodeBiotecnologı´a,UniversidadNacional
Auto´nomadeMe´xico,Cuernavaca,Morelos,Mexico. 18

19. •Toxininsertionleadstotheformationoflyticporesinmicrovilliof
apicalmembranes.
•Subsequentlycelllysisanddisruptionofthemidgutepithelium
releasesthecellcontentsprovidingsporesagerminatingmedium
leadingtoaseveresepticemia(Seriousbloodstreaminfection)and
insectdeath.
•OneinterestingfeatureofCrytoxinactivationistheprocessingofthe
N-terminalendofthetoxins.The3-dimensionalstructureofCry2Aa
protoxinshowedthattwoα-helicesoftheNterminalregionocclude
aregionofthetoxininvolvedintheinteractionwiththereceptor.
•Also,itwasfoundthataCry1AcmutantthatretainedtheN-terminus
endaftertrypsintreatmentbindsnonspecificallytoMenducasexta
(Tobaccohornworm)membranesandwasunabletoformporeson
M.sextabrushbordermembranevesicles(BBMV).
•Therefore,processingoftheN-terminalendofCryprotoxinshelpsto
facilitatetoxin-receptorortoxin-membraneinteraction.
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Alejandra Bravo, SarjeetS. Gill, and Mario Soberón. Mode of action of Bacillus thuringiensis Cry and Cyttoxins and
their potential for insect control. Toxicon. 2007 March 15; 49(4): 423–435.

20. Thank You ????????????
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