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Improved Methods for In-vitro Plant Regeneration

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Improved Methods for In-vitro Plant Regeneration

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AWARD:$10,000USD|DEADLINE:4/20/15|ACTIVESOLVERS:242|POSTED:2/16/15

Source:InnoCentiveChallengeID:9933678Type:Ideation
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Detailed Description & Requirements

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Background
Agrobacteriumtumefaciensmediatedtransformationofsugarbeetsislimitedbythekeyrequirementthatexplants
(typicallyleafcuttings)areabletoproduceregenerabletissueswhenculturedinvitro.Thevastmajorityofplant
tissuesandgenotypesdonotrespondtoinvitrocultureortransformationtechniques,aphenomenonknownas
recalcitrance.Theinabilityofplanttissuestoregenerateinvitrolimitsadvancesinplantscienceandbiotechnology.
Therefore,morerobustmethodstoregeneratewholetransgenicplantsfromtissueculturedmaterialare
desired.

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Summary

CurrentMethods:
Transgenicsugarbeetplantsaretypicallyproducedusingamethodtotransformshootbasemeristematictissue,
originallydescribedinLindsey&Gallois(1990).Inthismethod,shootbasetissueslicesareisolatedandthen
inoculatedwithAgrobacteriumtumefaciens(hereafterAgrobacterium),whichisabletointegratethedesiredTDNA
intotheplantgenome.SincetheTDNAconfersresistancetoanherbicideorantibiotic,thetransformedcellsare
selectedbyincubatingtheshootbaseslicesinamediumcontainingoneofthesecompounds.Duringtheselection
step,theresistantplanttissuesdeveloptransgenicshoots,whilethenontransgenictissuesdie.However,themethod
hassomedrawbacksinregardtothequalityoftheproducedtransgenicplants(e.g.chimerism)aswellwiththe
overallefficiencyingettingtransgeniclines.
Interestingly,thestomatalguardcellsfromleafderivedprotoplastculturesofsugarbeetshowedanexceptionalability
toregenerateplants.Despitethegreatpotentialofthatdiscovery,thegenerationoftransgenicplantsbyusingthat
systemistediousandtheefficiencyisstilllow(about2%)(Halletal.,1996).Morerecently,alternativeapproaches
usingalsoregenerationsystemsfromsingletransformedcellsaredescribedintheliterature(Kishchenkoetal.,2005
Kagamietal.,2015).Briefly,thesemethodsincludethegenerationofunorganizedcellmasses(callus)whicharise
frompiecesofplantorgans.Inthiscase,Agrobacteriumtransformsindividualcellseitherintheplantorganbeforethe
establishmentofthecalluscultureorinthecallus.Nextthetransgeniccellsareselectedasdescribedabove,and
developmentallyreprogrammedtoregenerateaplantorgan,usuallyashoot.However,thesemethodssufferfromlow
efficiencyinplantregenerationandextremegenotypedependency.

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Challenge Data

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Solvers

Eventhoughsomegenotypesshowthepotentialtoregeneratewholeplants,thesuccessrateisoftenlowandvery
variablebetweenindependentexperiments.Thereasonsforthesevariationsarecompletelyunknown.
Weareconfidentthatweareabletoachievehighratesoftransferoftransgenes(throughtheuseofafluorescent
marker).However,duetothelackofregenerationcapabilitythesetransgeniccellsarenotabletodevelopintowhole
plants.
Wehavetestedanumberofmodificationsofdifferentparametersforthosemethodsdescribedintheliterature.Those
modificationsincludehormonetypesandconcentrations,enhancersofcallusproductionandplantregeneration,
diversetissuetypesandgenotypes,variousAgrobacteriumstrains,gellingagents,differentconditionsforgrowingthe
plantsinvitro,etc.However,therateofsuccess(regenerationofaplantinvitrofromeitheranexplantoracallus)is
stillverylowand,moreimportantly,alltheseparametersandmethodsareverygenotypedependent.

Submissions

Solver Map

References:
LindseyK.andGalloisP.,1990.TransformationofSugarbeet(Betavulgaris)byAgrobacteriumtumefaciens.JExp
Bot41,529536.
HallR.D.,RiksenBruinsmaT.,WeyensG.J.,RosquinI.J.,DenysP.N.,EvansI.J.,LathouwersJ.E.,LefbvreM.P.,
DunwellJ.M.,vanTunenA.,KrensF.A.,1996.Ahighefficiencytechniqueforthegenerationoftransgenicsugar
beetsfromstomatalguardcells.NatureBiotech14,11331138
KishchenkoE.M.,KomarnitskiiI.K.,andKuchukN.V.,2005.Productionoftransgenicsugarbeet(BetavulgarisL.)
plantsresistanttophosphinothricin.CellBiolInt29,1519.
KagamiH.,KurataM.,MatsuhiraH.,TaguchiK.,MikamiT.,TamagakeH.,andKuboT.,2015.SugarBeet(Beta
vulgarisL.).KanWang(ed.),AgrobateriumProtocols:Volume1,MethodsinMolecularBiology,vol.1223,335347.
TheChallenge
Takingalloftheseintoaccount,theSeekerislookingforimprovedregenerationmethodsfromexplantsculturedin
vitrodefiningregenerationastheprocesstoobtainaplantthroughorganogenesisorsomaticembryogenesis.The
methodsoranswersshouldimprovetheregenerationofstabletransformedcellsinrecalcitrantplantspecies(Sugar
beets)ortissues.Thesemethodsshouldberobustandreliable,meaningthattheresultscanbereproducedand
producetransgenicplantswithadesiredefficiencyexceeding10%(calculatedastheamountofindependent
transgeniceventsobtainedperprimaryexplant,whichmeansthatin10outof100isolatedexplants,the
Agrobacteriumhassuccessfullyinfectedaplantcellthatwilldevelopintoatransgenicplant).Inordertoallowthe
furtheruseofthesetransgenicevents,itisnecessarytotransferthetransgeneintothenextgeneration.Itisimportant
tonotethattheregeneratedplantsmustbefertile.
Weareparticularlyinterestedinsolutionswhichcouldinvolvetheearlydetectionofthetissueorgenotypeamenable
fortransformationandregeneration,thereprogrammingofanonregenerabletissuetoaregenerableone,the
enrichmentofatissueinregenerablecells,etc.

Information for Academics

IfyouareaUSUniversityorCollege
Professor,student(graduatestudent
orundergraduate),oryouworkata
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ClickHereforImportantInfo.

Additional Information

Theproposedmethodshouldmeetthefollowingrequirements:
1.

RegenerationoffertilesugarbeetplantsfromasinglecelloriginwhichisamenabletoAgrobacterium
tumefaciensmediatedtransformationmethods

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2.

Efficiencyexceeding10%fortheregenerationofplants(calculatedasthepercentageofexplantswhich
canbeculturedinvitrosuccessfullytoafullplantaftertransformation).

3.

Themethodshouldbeapplicabletomultiplegenotypesinthebestcaseitwouldbegenotype
independent.

Challenge Attachments
Therearenoattachmentsforthis
Challenge.

Project Criteria
Thesubmittedproposalshouldincludethefollowing:
1.

AdetaileddescriptionoftheproposedplantregenerationprotocolrelevanttotheSolution.

2.

Ifapplicable,adetailedplanorguidelineontestsorexperimentswhicharenecessarytovalidatethe
solution.

3.

Referencetoliterature(commercial,patent,orpeerreviewed)tosupportyourclaims.

4.

Examplesofwherethesolutionisbeingconsideredtobeutilizedorhasbeenutilizedbefore.

5.

Lengthofproposalnotlimitedaslongasitincludesexecutivesummarynotexceeding500words.

Aprotocolforplantregenerationisarecipeinvolvingdifferentexplants,mediacomponentsandcultureconditions.
Wewouldliketoavoidproposalsthatonlyincludethetestingofjustdifferentphytohormonesconcentrations(e.g.use
0.25mg/lofthephytohormone6BenzyladenineforcallusinductionasdescribedinKagamietal.,2015),media
componentcombinations,genotypesandtissuetypes.ThepreferenceforthisChallengeistolookfor(an)outofthe
boxanswerstotheproblemofsugarbeettransformation,meaning,ideastodiminishrecalcitrance.
Inthebestcase,theSolverhasfoundinanotherrecalcitrantplantspeciessomewaystoimprovetheshoot
regenerationfromtransformedcellsandhasgoodargumentstobelievethatthisdiscoverycouldbealsoapplicableto
sugarbeet.
SubmittedproposalsalongwithallrelevantsupportingdatashouldincludetheinformationdescribedintheDetailed
DescriptionoftheChallenge.
SubmittedproposalsshouldnotincludeanypersonalidentifyinginformationtheSolversdonotwanttomakepublic,
oranyinformationtheSolversmayconsiderastheirIntellectualPropertytheydonotwanttoshare.
ThisisanIdeationChallenge,whichhasthefollowinguniquefeatures:
Thereisaguaranteedaward.Theawardswillbepaidtothebestsubmission(s)assolelydeterminedbythe
Seeker.Thetotalpayoutwillbe$10,000,withatleastoneawardbeingnosmallerthan$5,000andnoaward
beingsmallerthan$2,500.
TheSolversarenotrequiredtotransferexclusiveintellectualpropertyrightstotheSeeker.Rather,by
submittingaproposal,theSolversgrantstotheSeekeraroyaltyfree,perpetual,andnonexclusivelicense
touseanyinformationincludedinthisproposal.
AftertheChallengedeadline,theSeekerwillcompletethereviewprocessandmakeadecisionwithregardstothe
WinningSolution(s).AllSolversthatsubmittedaproposalwillbenotifiedonthestatusoftheirsubmissionshowever,
nodetailedevaluationofindividualsubmissionswillbeprovided.

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