NovelTransientAssayforColorExpressioninDetachedAnthuriumSpathesPeterJ.Toves*1,MaureenM.M.Fitch2,XiaolingHe2,RichardA.Criley1,CathieR.Martin3,TeresitaD.Amore1

1UniversityofHawaiiatManoa,Honolulu,Hawaii,USA;2HawaiiAgriculturalResearchCenter,Kunia,Hawaii,USA;3JohnInnesCenter,Norwich,UK

AnthuriumsarethetopgrossingcutflowersintheHawaiifloricultureindustry.DevelopmentofnovelcoloredanthuriumskeepstheHawaiiindustrycompetitiveglobally.

GeneticengineeringwiththegeneFlavonoid3’,5’-hydroxylase(F3’5’H)forexpressionofanthocyaninsinthedelphinidinpathwayisofinteresttocreatenovel,bluecoloredspathes.Thedevelopmentofstablegeneticallyengineeredanthuriumplantsisalengthyprocess.Genetictransformationandselectioncantakeatleastayear.Firstfloweringandvisualizationofgeneexpressiontakesnearlytwoyearsfromplantingout.

Transientexpressionisarapidalternativetostabletransformationforobservinggeneexpression.TransientexpressionistherapidbuttemporaryexpressionofageneshortlyafterDNAdeliverytocellsoftargettissues.Generally,transientexpressioncanbeobserved48hoursafterDNAintroduction.

DeliverysystemsfortransientexpressionincludeparticlebombardmentandAgrobacterium infiltration(i.e.Agroinfiltration).Particlebombardmentisabiolisticsystemforthedeliveryofexogenousgenesintocells.AgroinfiltrationistheinfiltrationofAgrobacteriumtumefacienscarryinggenesofinterestintotargettissues.Previouslyparticlebombardmentofanthuriumspathesresultedinexcessivebrowningofwoundedtissue,makingtransientgeneexpressiondifficulttoassess.

Introduction

Generalizedflavonoidbiosyntheticpathway(AdaptedfromNishiharaandNakatsuka,2011).Thedelphinidinpathwayisofinterestforgeneticengineeringofblueanthurium.F3’5’Histhekeyenzymefordelphinidinsynthesis.

ObjectiveTooptimizeagroinfiltrationasagenetransfersystemfortransientexpressionofthestructuralcolorgeneF3’5’Hinanthuriumspathes.

ConclusionsAgroinfiltrationisapromisinggenedeliverysystemfortransientexpressionofcolorgenesinanthuriumspathes.FurtherexperimentsareneededtovalidatethetransientexpressionofthegeneF3’5’HviaAgrobacterium infiltration.

• Tubeswith2mLofYEBmediumand2μLeachofrifampicin(50mg/mL)andkanamycin(50mg/mL)wereinoculatedwithAgrobacterium andgrownfortwodaysat250rpmonashakerat28°C.

• 5mLofinoculumwastransferredtoaflaskwith50mLYEBand50μLeachofrifampicin(50mg/mL)andkanamycin(50mg/mL),placedonashakerat250rpmand28°C.(FitchandHe,personalcommunication)

• Cellswerecollectedbycentrifugationfor15minat3000gandresuspendedin10mMMES(pH5.5)plus10mMMgSO4foragroinfiltration.

• Bacterialsuspensionwasinjectedintospathesusinga1 mlplasticsyringe.Approximately100 μlofbacterialsuspensionwasinjectedintoeachspot(typically3–4 cm2 ininfiltratedarea),with4to6spotsinasinglespathe.ControlspatheswereinjectedwithinfiltrationbufferwithoutAgrobacterium.

• Spatheswereleftatroomtemperatureunderfluorescentlights.Spatheswereobservedforcolorchangesnearareasofinfiltration.

Experiment1. AssessmentoftransientexpressionofF3’5’Hon‘MarianSeefurth’.Circlesindicateinjectionsiteswithcolorchange.

Experiment3.AgroinfiltrationwithF3’5’H,agroinfiltrationwithoutF3’5’H,andonlyinfiltrationbuffertodetermineifcolorchangeresultsfromAgrobacteriumpathogenicityorF3’5’H.Sixselectionswereevaluated.Resultswerevariableamongselections.

Thisworkwassupported inpartbytheFederalFloricultureGrant,ColorCatalystGrant,andtheUSDANationalInstituteofFoodandAgriculture,HatchProject868HmanagedbytheCollegeofTropicalAgricultureandHumanResources.AgroinfiltratedwithF3’5’H

UH1688

AgroinfiltratedwithF3’5’H

ControlInfiltrationBufferOnly

ControlInfiltrationBufferOnly

UH2010– Bluespotsdevelopedonspathestreatedwithbothagroinfiltrationtreatments

UH2008-- UH2008wastheonlyselectionwithmarkeddifferencesamongtreatments.BrownspotsdevelopedatinjectionsitesforthecontrolagroinfiltrationwithoutF3’5’H,whilebluespotsdevelopedoninjectionsitesonspathesinjectedwithF3’5’H

ControlInfiltrationBufferOnly

ControlInfiltrationBufferOnly

ControlAgroinfiltratedwithoutF3’5’H

ControlAgroinfiltratedwithoutF3’5’H

AgroinfiltratedwithF3’5’H

AgroinfiltratedwithF3’5’H

Marian Seefurth

AgroinfiltratedwithF3’5’HControlInfiltrationBufferOnly

MaterialsandMethods

Results

• TheBacterialsuspensionwasadjustedtoafinalOD600 of0.8

AGROBACTERIUM CULTURE AGROINFILTRATIONofANTHURIUM

Experiment2. Effectofspathedevelopmentalstage(fullyexpandedvs.newlyunfurled)ontransientexpressionofF3’5’H.Circlesindicateinjectionsiteswithcolorchange.