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INFRAREDCHEMICALIMAGINGOFGERMINATEDWHEAT:

EARLYNONDESTRUCTIVEDETECTIONANDMICROSPECTROSCOPIC

IMAGINGOFKERNELTHINCROSSSECTIONSINSITU

by

HICRANKOC

B.S., Istanbul TechnicalUniversity,2004

ATHESIS

submittedinpartialfulfillmentoftherequirementsforthedegree

MASTEROFSCIENCE

DepartmentofGrainScienceandIndustry
CollegeofAgriculture

KANSASSTATEUNIVERSITY
Manhattan,Kansas

2007

Approved:

_________________
MajorProfessor
DavidL.Wetzel
Abstract

Duringgermination,biochemicalchangesoccurinthewheatkernelbystimulationof
enzymesandhormones,andtheseedreservesaremobilized.Infraredmicrospectroscopyand
imagingenablesalocalizedchemicalinventory,upongermination,tostudytheprocess.Frozen
sectionsofgerminatedwheatmountedontoBaF2 weremappedtoproducefunctionalgroup
imagesforcomparisonwithcorrespondingsectionsofungerminatedkernels.Relativefunctional
grouppopulationsin thescutellumandembryonicaxiswereassessedbeforeandafter
germination.Anaverage23%reductioninlipidtoprotein ratiowasobservedinthescutellum
basedonthecomparisonof53,733spectra.Asaresultof theearly germinationprocess,lipidin
thescutellumwasdepletedtoprovideenergyforthegrowingembryo.
Germinationofthekernelswhileinthefieldbeforeharvestduetohighhumidityis
knownaspreharvestsprouting.Preharvestsproutinghasdetrimentaleffectsontheenduse
qualityofthewheat(sproutdamage)andcauseeconomicloses.Tolerancetopreharvest
sproutingishighlydesirable.Toassistbreedingprogram,anondestructivenearIRchemical
imagingmethodhasbeendevelopedtotestnewlinesforresistancetopreharvestsprouting.The
highersensitivityofsubsurfacechemicalimaging,comparedwithvisualdetection,alpha
amylasedetermination,orviscositytesting,permitsgerminationdetectionatearlystages.A
nearIRchemicalimagingsystemwithanInGaAsfocalplanearray(FPA)detectorinthe1100
nm1700nmrangewasused.Kernelsfromsixdifferentcultivars,includingHRWandHWW
wheat,wereexposedtomoistconditionsfor6,12,24,36,and48hours.Imagesofeach90
kernelgroupwereexaminedkernelsexposedtomoisturefor36hourswerecomparedwith
kernelstreatedfor3hoursasacontrol.Eachkernelwasclassifiedassproutedornotsprouted
withthecriteriaoflog1/Rintensityatselectwavelengthsorselectfactorsofprinciple
componentanalysis(PCA)treatmentofreflectanceintensitydata.Imagingwavelengthrange
wasexpandedbeyond1700nmto2400nmwiththeuseofInSbFPA.Studyforthepotentialfor
unsuperviseddeterminationinnondestructivenearIRimagingwithdetectionwavelengths1200
2400isongoing.Somepreliminaryresultspresentedareencouraging.
TableofContents

ListofFigures........................................................................................................................... vi
ListofTables ............................................................................................................................ ix
Acknowledgements.....................................................................................................................x
Dedication..................................................................................................................................xi
PART1MicrospectroscopicImagingofKernelThinCrossSectionsinSitu:Investigationof
BiochemicalChangesinWheatKernelsduringSproutingbyFTIR....1
1.Introduction....1
1.1. WheatKernel....1
1.1.2.StructureandCompositionoftheWheatKernel.....1
1.1.2.1.Endosperm..........3
1.1.2.2.Bran........3
1.1.2.3.Germ.......................4
1.1.2.4.Brush(Beard)andCrease.......5
1.2.GerminationandPreharvestSproutingofWheat........6
1.2.1.Introduction......6
1.2.2.StagesofGerminationandtheFactorsAffectingtheProcess.....7
1.2.3.BiochemicalChangesduringSprouting......9
1.2.3.1.Enzymesingerminatingwheat...9
1.2.3.1.1.CarbohydrateDegradingEnzymes..10
1.2.3.1.2.ProteinDegradingEnzymes....11
1.2.3.1.3.OtherEnzymesSystems..12
1.2.3.2.ActionofHormonesduringGermination.....12
1.2.3.3.MobilizationoftheStoredReservesandChangesintheKernel.13
1.3.InfraredSpectroscopy.....19
1.4.FourierTransformInfrared(FTIR)Microspectroscopy....21
1.4.1.FourierTransformInstrument........21
1.4.2.Advancesin InfraredMicrospectroscopicInstrumentation.......22
1.4.3.ApplicationsofFTIRMicrospectroscopyonPlantMaterial...24

iii
1.4.4.SynchrotronInfraredMicrospectroscopy......25
2.Experimental....26
2.1.Instrumentation...26
2.1.1.InstrumentationatSynchrotron......26
2.1.2.InstrumentationatKSU.........28
2.1.2.1OpticalsystemandmappingprocedureofSpotlight.31
2.1.2.2.MCTDetector....33
2.2.Samplepreparation.....34
2.3.MicrospectroscopicProcedureandDataProcessing..37
3.ResultsandDiscussion ...43
3.1.ResultsoftheexperimentsperformedatNSLS..43
3.2.ResultsofexperimentsdoneatKSU..49
4.Conclusion.......56
PART2EarlyNondestructiveDetection:NearInfraredChemicalImagingofWheat
GerminationtoAssistPlantBreeding....58
1.Introduction..58
1.1SproutDamage.....58
1.2SproutTolerance..59
1.3DeterminationofSproutDamage....60
1.3.1.Visualassessment(nakedeye)..61
1.3.2.Viscositymethods......61
1.3.3.Enzymaticmethods........62
1.3.4.ImmunologicalFieldTestMethod.63
1.3.5.Othermethods........63
1.4NearIRSpectroscopyandImaging.....64
2.Experimental....67
2.1.Instrumentation...67
2.2.SamplePreparation.....70
2.3.DataAnalysis..72
3.ResultsandDiscussion...75
4.Conclusion...87

iv
References..89
AppendixANearIRAnalysisforSproutDamageTest..96
AppendixBSpectroscopiceffectofdryingandtreatmentoptimization..97

v
ListofFigures

Figure1.1Longitudinalandcrosssectionofawheatkernelshowingdifferentbotanical parts.2
Figure1.2Structure ofthegermofthewheatkernel,longitudinalandtransversesections...5
Figure1.3Distributionofdifferentpartsofthekernel....6
Figure1.4Sequencesoftheeventsinthesprouting8
Figure1.5Thestructureofamylaseandthe(1 4)Dglucosidicbonds...10
Figure1.6Thestructureofamylopectinandthe(1 6)Dglucosidicbonds11
Figure1.7Fluorescephotomicrographsoftransversesectionsofendosperm..13
Figure1.8Schematicmodelofsugartransportfromtheendospermtotheembryo.14
Figure1.9Proteinsynthesisduringgermination...15
Figure1.10Lightphotomicrographofthetransversesectionofthescutellumofadryembryo..16
Figure1.11Lightmicrographsof thescutellumduringgermination....18
Figure1.12DiagramofNSLS...26
Figure1.13ContinumTM infraredmicroscope.27
Figure1.14U2bbeamline..28
Figure1.15SpectrumSpotlightimagingsystem...29
Figure1.16SchematicofIRmicrospectrometerwithasingleelementdetector..30
Figure1.17SchematicofIRmicrospectrometerwithaFPAdetection31
Figure1.18Pathofthevisiblebeamforviewingasampleintransmittance32
Figure1.19Pathoftheinfraredbeamforcollectinganimageintransmittance...33
Figure1.20Thesuccessivesectionsforthefirstsmallslicingangle35
Figure1.21Thesuccessivesectionsobtainedatthelargerangle..35
Figure1.22Thesuccessivesectionsobtainedwiththewidestangle36
Figure1.23Backgroundspectrum.38
Figure1.24Awheatcrosssectionandtotalabsorbanceimage....39
Figure1.25Aspectrumrepresentativeofthescutellumpartofanungerminatedkernel.39
Figure1.26A spectrumshowingbaselineshift.40

vi
Figure1.27Avisibleimageofwheatkernelcrosssectionandtheareamapped.41
Figure1.28Anexampleofbaselinecorrectedpeakareacalculation...41
Figure1.29Thefunctionalgroupmapsforthelipidandcarbohydrate....42
Figure1.30Thefunctionalgroupmapforprotein....43
Figure1.313Dchemicalimagesofthesamewheatkernelcrosssectionshowingtherelative
populationlipid(a),protein(b),andstarch(c).44
Figure1.32Unsproutedkernel3Dchemicalfunctionalgroupmaps..45
Figure1.33Sproutedkernel3Dchemicalfunctionalgroupmaps45
Figure1.34Graphshowingthepopulationofpixelswithhigherlipid/proteinpeakarearatios
withinthescutellumandembryoregion46
Figure1.35Figureaandbhavethreecolumnseachtocompareimagesofsproutedand
unsproutedkernelsandsectionsfromthesekernels..47
Figure1.36Linemapacrossthegermofunsprouted(a)andsproutedkernels(b)..49
Figure1.37Areaswithinthescutellumoutlinedinredonthephotomicrograph.50
Figure1.38Lipidtoproteinpeakareasratioed.50
Figure1.39Photomicrographsof sproutedandunsproutedcrosssectionsofthevarietyTrego..53
Figure1.40PhotomicrographsofsproutedandunsproutedKS2174varietycrosssections54
Figure1.41Photomicrographsof sproutedandunsproutedcrosssectionsofDanby...55
Figure2.1Dormancyandgermination..60
Figure2.2Variouspathwaysofdiffusereflectance..67
Figure2.3SchematicofnearIRimagingsystem..68
Figure2.4Representationof3Dimagedatacubewiththreeaxes70
Figure2.5Anexampleimageofwheatkernelsat2100nmandspectrarepresentingthegermand
theendospermareas...70
Figure2.6Sampleplate.71
Figure2.7Histogramshowingthepixelpopulationofvariousintensitiesat1680nm....73
Figure2.8SpectroscopicimagesandPCAfactorofasinglewheatkernelofsproutedand
unsprouted..75
Figure2.9PCAfactor1imagesof15unsprouted(top)and30sproutedkernels(bottom)..76
Figure2.10PCAfactor4imagesof15unsprouted(top)and30sproutedkernels
(bottom)..77

vii
Figure2.11Germinationresponsesof90kernelsofsixcultivarsat36hr...78
Figure2.12Germinationresponsesof90kernelsofBettyandKS2174tomoistureexposure
times6,12,24,36,and48hr79
Figure2.13Spectraselectedfromgermandendospermsideofthea)Unsproutedsamples,b)
germinatedwetsamples,andc)driedgerminatedsamples...80
Figure2.14EffectofwaterpeakinPCAfactor5loadingofwetsamples...81
Figure2.15Averagespectrafromthedatacubeofstarch...82
Figure2.16Averageof24000spectrafromthedatacubeofgluten.83
Figure2.17Spectraoflipidextractedfromwholewheatandpentosans..83
Figure2.18Meanspectrumofawheatkernel...84
Figure2.19Comparison ofsprouted(36hr)andunsprouted(3hr)kernelsat2310nm...85
Figure2.20Peakheightratio(2310/2060)imageandaverageratiovalues..86
Figure2.21PCAfactor2ofsecondderivativedata..86
FigureB.1Baselinecorrectionof aspectrum97
FigureB.2Baselinecorrected spectraaveragedforcontrol,sproutedwet,sprouteddry97

viii
ListofTables

Table1.1Majordevelopmentsininfraredmicrospectroscopy instrumentation...23
Table1.2Thespectralregionandbaselinepointsusedforparticularpeaks42
Table1.3Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforTrego
controlsections..51
Table1.4Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforTrego
germinatedsections....51
Table1.5Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforKS2174
controlsections..51
Table1.6Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforKS2174
germinatedsections....52
Table1.7Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforDanby
controlsections..52
Table1.8Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforDanby
germinatedsections....52
Table1.9Tabulatednumberofpixelsandthelipid/proteinratioforunsproutedandsprouted
scutelliofthreedifferentwheatcultivars...55
Table2.1Thegrossmean ofthelipid/proteinratioforBetty ..87
TableB.1Thepeakheightcomparisonat1940nmfordifferentdryingmethods...98

ix
Acknowledgements

Firstofall,Iwouldliketothankmymajorprofessor DavidL.Wetzelforhisvaluable
supportandguidanceinmyresearch,educationandexperiences.Iamthankfultohimforthe
exposureheprovidedmeregardingmyacademiccareer Ihadmanyoralpresentationsin
importantconferencesandhadtwopublicationswhichareimportantsuccessforaMSstudent,
andIhadachancetoworkinNationalBrookhavenLaboratory whichisreallyuniquetome.In
additiontothese,IcanneverforgetConnieandDavidWetzelsunlimitedhelpwhilevery far
awayfrommyfamily.Theyalwaystreatedmeasadaughter,thankyouConnieandDr.Wetzel.
WithoutmycommitteememberVirgilSmailnoneoftheseachievementscouldbe
possible.IwouldliketothankDr.Smailforthissupporttoourlabandresearch.Iwantto
conveymythankstoDr. Culberstonforbeingmycommitteemember.
Dearmyparents,sisters,brotherandmyfriends!Ifeelsolucky,youmakemylife
meaningful.ThankyoumyparentsNekariyeandOrhanKoc,mylovelysistersandbestfriends
UmranandKubra,my dearbrotherGokhan.Iwouldliketothankmyfriendsfrommyhome
countryAsli,Aysegul,Sema,SevilandBilginforsharingthelifeincludingallaspects.Iwantto
saythankyoumygoodfriendsMaggie,Sreeram,Val,RamakanthandSibelwhomImetin
Manhattan,theyallbecameapartofmylifebeingsistersandbrotherstome.AndDr.Dogan
andherfamilyalwaysmademefeelmyselfamemberofthemandremindmewarmness,
sincerity,happinessIhadinmyfamily.Thankyou!Hulya,HuseyinandDuruDogan.Mylab
mateEmily,thankyouforbeingapartnerinthelabfortwoyearssharingallchallengesand
goodtime,andexposingmetoyourculture.Ialsothankournewmemberinthelab,Laurenfor
herhelp.

x
Dedication

IdedicatedthisworktomyparentsmymotherNEKARIYEKOCandmyfatherORHANKOC
forteachingmetheimportanceoftwovaluesinthelife,honestyandeducationwhichbuiltmy
characteranddeterminedmywayinthelifeandthankyouforbeingwithme,beingbehindme
allthetimewhereverIam,whateverIamdoing.Eventoughyoudisagreedwithmydecisions
manytimes,andIdidnotlistentoyoumostofthetime,yousupportedmewithoutany
expectations,withoutanylimitations.Youdedicatedyourlifetome,andIhopeIcandogood
thingsforyouandforthecommunity.IamsothankfultoGodthatIamyourdaughter,youare
justperfect.

xi
PART 1MICROSPECTROSCOPICIMAGINGOFKERNEL
THINCROSSSECTIONSINSITU:
INVESTIGATIONOFBIOCHEMICALCHANGESINWHEAT

KERNELSDURINGSPROUTINGBYFTIR

1.Introduction

1.1TheWheatKernel

Wheatisamemberofthegrassfamily,Gramineae,andthegenusTriticumandadryone

seededfruit(1). Thewheatkernel,surroundedbythelemmaandpalea,isproducedinthespikes

atthetopofthestems.Theglumeenclosestheeachspikelet.Thesepartsoftheplantlemma,

palea,andglumearecalledchaff.Thewheatisthreshedbyseparatingthegrainsfromthechaff

mechanically(2).

Wheatsareclassifiedindifferentwaysbasedonfactorssuchaswinterorspringhabitof

growth,timeneededtomature,height,stem,spike,glume,andkernelcharacteristics(endosperm

hardness,color).Therearewhiteorredwheatcultivars.Thepigmentsoftheseedcoatare

primarilyresponsiblefor thedarkcoloroftheredwheat.Moreoverthecolorofthekernelcanbe

affectedbytheendospermtextureandthevitreousnessandtransparencyofthepericarp(3).

Chromosomesaretheprimarycontrolfactorforthecharacteristicsinthewheat.Commonwheat

has21chromosomepairs.Plantbreedersdevelopwheatvarietieswithdesiredcharacteristicsfor

specialpurposes(2).

1.1.2.StructureandCompositionoftheWheatKernel

Thewheatkernelsarefrom810mminlength.Thedorsalsideofthekernelisroundand

attheventralside(oppositethegerm)kernelhasalongitudinalcreaserunningalmostentire

lengthofthekernel(4).Thestructureofthewheatkernelisshowninfigure1.1anditis

1
composedofthreemainpartsincludingtheendosperm,thebranwithvariousprotectiveskinand

thegerm.

Figure1.1Longitudinalandcrosssectionofawheatkernel showingdifferentbotanicalparts
endosperm,branandgerm (reprintedfromref.1).

Beardend

ENDOSPERM

BRAN

GERM

2
1.1.2.1.Endosperm

Endosperm,thefoodbagofthewheatgrain,consistsofstarchyendospermandthe

aleuronelayer.Thealeuoronelayerisbotanicallyoutermostpartoftheendosperm,butduring

millingitisremovedtoformbran.Starchyendospermyieldsflourduringmilling.

Therearethreetypesofcellsinstarchyendospermperipheral,prismatic,andcentral.

Thecellsvaryinsize,shape,andcompositionofthestarchgranulesandproteinsdependingon

theirlocationwithinthekernel.Thecellshavestarchgranulesembeddedinaproteinmatrix,

mostlygluten(storageproteins)(1).Thereislessproteininthecenterwithincreasinglevels

thoroughthebrancoat(3).

Thecellwallsofendospermcontainpentosans,otherhemicellulosesandbetaglucan.

Dependingon thevariety,endospermtexturecanbesoftorhard.Hardwheatendospermstarch

granulesretaintheirshapeduringmilling,whereassoftwheatendospermstarchgranulesfracture

acrossthecells.Thereasonforthisdifferenceisthatstarchgranulesarelesstightlyadheredto

theproteinmatrixinsoftendospermcomparedtohardwheatendosperm(1,2).Inadditionto

variationinhardnessofwheatendospermindividualkernelscandifferinvitreousness.

1.1.2.2.Bran

Thebranconsistsofouterprotectivelayersofthegrainpericarp(outercoating),seed

coat(innercoating)andtheadjacentlayeraleurone.Thepericarpencirclestheseedandconsists

ofseverallayers.Theouterpericarpiscomposedofepidermis,hypodermis,andremnantsof

thinwalledcells(fromtheoutsidetowardthecenterofthekernel)whiletheinnerpericarpis

composedofintermediatecells,crosscells,andtubecells.Thetotalpericarprepresent5%ofthe

kernel andconsistsof6%protein,2%minerals,20%cellulose,0.5% lipid,andtheremaining

partisnonstarchpolysaccharides(1).

3
Underneaththepericarp,thereisinnercoatingoftheseed,seedcoat,whichiscomposed

oftesta(episperm),thepigmentstrandforthecoloredwheats,andthenucellarlayers.The

pericarpandseedcoatareseparatedduringflourmillingtheadjacentaleuronelayeralsoforms

thebranalthoughitisapartofendosperm.Aonecellthickaleuronelayercoversstarch

endospermandgerm.Thecompositionofaleuronelayerincludesminerals,protein,phosphorus

compounds,lipid,andniacin.Inaddition,itisrichinthiamin,riboflavin,andhashighenzyme

activity(1,4).

Allpartsofthebranrepresent14.5%ofthewholewheat.Thedistributionisepidermis

3.9%,crosscelllayers0.9%,testa0.6%,nucellarlayers,andaleurone9.0%(3).

1.1.2.3.Germ

Thegerm,whichaccountsfor2.53.5%ofthekernel,isthereproductiveorgan.Infigure

1.2 thestructureofthegermisshown,itcontainstwomajorpartsembryonicaxisand

scutellum.An epitheliallayerseparatesthewheatgermfromtheendosperm.Duringgermination

theembryonicaxisdevelopsasthebeginningof anewseedandthescutellumprovides

nourishment.Theembryonicaxisiscomposedoftheshoot(plumule)andtheprimaryroot. The

shootisorientedtowardthebrushendofthekernelandiscoveredbythecoleoptileprotective

sheath.Whereas,theprimaryrootisdirectedtowardthebase.Thecoleorhizacoversandprotects

it(3).

Thegermishighinprotein(25%).Theembryonicaxiscontains16%lipidandthe

scutellum 32%.Inadditiontolipidandprotein,ithassimplesugars(18%)mainlysucroseand

raffinose.Itsmineralcontentis5%,anditisalsorichinBvitamins,vitaminE,andenzymes(1,

4).Thegreatestpartofthescutellumisparenchymacellwhichcontainlipidandprotein.

4
Figure1.2Structure ofthegerm ofthewheatkernel,longitudinalandtransversesections
showingdifferentpartsofthegerm,theletterscorrespondingtoeachpartaregiven (reprinted
fromref.2).

U:Scutellum
H:Aleuronecells
N:Epithelium
I:Endosperm
T:Germ
P:Rudimentaryleaves
O:Plumulesheath
M:Compressedemptycellsof
endospermparenchyma
Q:Radicleroot
R:Rootsheath
S:Radiclecap

1.1.2.4.Brush(Beard)andCrease

Thebrushisaclusterofhairslocatedattheoppositeendofthegerm.Thecreaseisan

indentonthecentralsideortheoppositesidefromthedorsalsideofthekernel(2).

5
Thedistributionofdifferentparts ofthekernelisshowninthefigure1.3.Distributionof

theproteinwithinthekernelis72%endosperm,4.5%scutellum,3.5%embryo,15%aleurone

layer,4%pericarp&testa(4).

Figure1.3Distributionofdifferentpartsof thekernel (84%endosperm,13.5%bran,and2.5%


germ)(5).

Scutellum
1.5% Pericarp,4.5%

Embryo1.0% Testa,2.5%

Aleurone,6.5%

StarchyEndosperm
84.0%

1.2.GerminationandPreharvestSproutingofWheat

1.2.1.Introduction

Themature,dormant,airdriedwheatkernelhasa912%moisturecontent,andithaslow

metabolicactivityattheseconditions.Exposuretomoisturetriggersgermination.Thethree

livingtissues(embryo,scutellumandaleuronelayer)playimportantrolesduringgermination.In

thisprocess,thekernelishydratedrapidly,enzymesareactivated,therespirationrateincreases

rapidly,consumptionofreservematerialsisstarted,andprotrusionoftheradiclefromtheseed

coatmayoccurwithin24to48hr.Thisiscalledasgermination.Bytheproductionoftheshoot

androots,anewplantisproducedfromthegrain(5,6).Germinationofthekernelswhilestillin

theearinthefieldbeforeharvestbecauseoftheunfavorableconditionssuchasrainandhigh

6
humidityisknownaspreharvestsprouting.Cerealgrowingregionsoftheworldwherethereis

highprobabilityofrainduringharvestareliabletopreharvestsproutingsuchasEasternEurope,

Chile,Argentina,Brazil,SouthAfrica,Australia,andManitobainCanada.IntheUS, thePacific

Northweststatesareexamplesofplacesthathavesufferedsomeyearsfromthisproblem(7).

Thechangesinchemicalcomponentsofthewheatkernelwiththegerminationprocesshave

detrimentaleffectsontheenduseofthewheatandcauseimportanteconomiclosestothe

farmersbecausethevalueforfermentationislowcomparedtothatformillingandbaking.

Thesedetrimentalchangesarereferredtoaspreharvestsproutdamage(8).Usefulnessofwheat

forbreadmakingpurposesdecreasesrapidlyaftergerminationduetostarchdegradationby

amylase.Preharvestsproutingresultsfromthelackofdormancy.Tolerancetopreharvest

sproutingandembryodormancyarehighlydesirablecharacteristicsthatplantbreedersstudy(9).

1.2.2.StagesofGerminationandtheFactorsAffectingtheProcess

Theeventstakeplaceduringgerminationcanbedividedinthreephasesimbibition,lag

phase,andgermination.Firsteventinthesproutingisthemovementofthewaterthroughthe

grain.Wateruptakeofthekernelisrapidsincethekernelisdry,andtissuesreach3050%

moistureinoneortwodays.Nextseedsenteralagphase,whentheyswellandbecomeheavier,

andmetabolismbecomesactive.Ifalltheenvironmentalconditionsareappropriate,theseed

entersthegerminationphase(10).Duringgermination,theembryotakesinmaterialsfrom

endospermthroughtheepitheliallayerforitsgrowth.Exposuretomoistureactivatesenzymesin

thegerminitiatinggermination.Starchinthevicinityofthegermishydrolyzedintosimplesugar

byamylolyticenzymesandsugarisusedtonourishthegerm.Besidethisenzymaticactivity,

hormonesaffectgerminationbyinfluencingtheformationofamylaseintheendosperm(3).

Duringgermination,thescutellumfunctionsasadigestingandabsorbingorganforthe

7
transferenceofthefoodfromtheendospermtogrowingembryo(4). Sequencesoftheeventsin

thesproutingareshowninfigure1.4.

Figure1.4Sequencesoftheeventsinthesproutingstartingfrommoistureexposure toruptureof
theseedcoat(adoptedfromref.11and12).

RainorMoistureexposureartificially

Movementofthewaterintothegrain

Activationoftheembryo
Fullyimbibedgrain,
beforeembryoemerges.
Productionofgerminativeenzymes
(amylase,proteases)

Mobilizationofstoragematerials
(Proteinandstarch)

Developmentofrootsandshoots

Theembryonicaxisemergesthoroughthe
testa.Thecoleoptileandtheroot(radicle)are
thefirststructurestobeseen

Thereareseveralfactorsaffectinggermination.Theseincludekernelcharacteristicsand

environmentalconditionssuchasmoisture,temperature,andoxygen.Seedviability (abilityto

grow)anditsdormancyareimportantforthegerminationprocess.Theoptimumtemperatureis

8
reportedly2025C(12a).Mares(8) reportedthatanoptimumtemperatureforgerminability was

dependenton dormancylevel oftheindividual varieties. Thestronglydormantvariety achieved

germinationat10C. Thenondormantvariety performedgerminationat20Candthelagperiod

variedwithtemperature.

1.2.3.BiochemicalChangesduringSprouting

Duringthesproutingprocess,therearemanychangesinthemolecularandcellular

biologyofthewheatkernel.Enzymesandhormonesarestimulatedandtheseedreservesare

regulated.Inadditiontotheseenzymaticandhormonalchanges,thecytologicalchangesare

observedinthecells.Thebiochemical,cytologicalandhormonalchangesduringgermination

havebeenstudiedbySwiftandOBrien(13),Fincher(14),BewleyandBlack(15,16,17),Simon

(10),AshfordandGubler(18),CorderandHenry(19),TavenerandLaidman(20,21)andKruger

(22).Inthissection,biochemicalchangesingerminatedwheatassociatedwithdetrimental

effectsonendproductqualitywillbediscussed.

1.2.3.1.Enzymesingerminatingwheat

Enzymesarethemostimportantbiochemicalcomponentsthatformduringgermination

andaffectthequality.Thereisacompartmentalizationofthecomponentsinthekernelwhichis

importantforstoragestabilityofit.Thewheatkernelhasdegradationenzymesandsubstrates,

buttheyareprotectedfromcomingintocontactwhichprovidesthestability (1).Thestarch

degradingenzyme,amylase,isconsideredtobethemainenzyme.Proteolyticenzymescould

alsobeimportant.Thesynthesisandthesecretionoftheenzymesthatcatalyzedegradationof

themacromoleculesinstarchy endospermcontrolthecellularactivityinaleuronelayer(14).The

scutellarepitheliumhasaroleintheproductionandsecretionoftheenzymesaswellas

absorbingtheendospermdegradationproductsandtranslocationofthemtothedeveloping

9
embryo.Enzymesproducedinthealeuronelayerandscutellarepitheliumcanbedividedinto

threegroups:1)enzymesinvolvedinongoingmetabolismwithinthecell,2)enzymesthat

mobilizereservesofscutellarandaleuronecells,and3)enzymessynthesizeddenovoand

secretedinendosperm(14,23).

1.2.3.1.1.CarbohydrateDegradingEnzymes

Thecarbohydratedegradingenzymesingerminatingwheatcanbelistedasand

amylases,debranchingenzymes,cellulases,andglucanases.Amongthesecarbohydrases,

amylasesareknownfortheirharmfuleffectsinbreadmakingprocess.Thisenzymecleavesthe

(14)Dglucosidicbonds(Figure1.5)inthestarchcomponents.Withgermination,this

enzymeformsprogressively,initiallyadjacenttothescutellum,andsubsequentlyadjacenttothe

aleuronelayer.Moreenzymesoccurintheouterlayersofthekernel.Withseveresprouting,

enzymespenetratetheendosperm.CorderandHenry(19)foundthatamylasesactivitywas

increasedoverthefirstfourdaysofgermination.MacGregorandMatsuo(24)observedthatthe

enzymesynthesiswasrelativelyslowupto48hrofgerminationofbarleyinPetridishes.But

after48hr,itincreasedrapidly.

Figure1.5Thestructureofamylaseandthe(14)Dglucosidicbondswhichcombineseach
glucosemoleculesintheamylasemolecule(reprintedfromref.24a).

10
amylaseshydrolyzethe(14)Dglucosidicbondsfromthenonreducingendof

starchandyieldmaltose.Upongermination,the amylaseslevelsincreaseseveralfoldinthe

endosperm.Littleattentionispaidtodebranchingenzymesrelativetopreharvestsprouting.They

cleave(16)Dglucosidicbondsintheamylopectin (Figure1.6)(22,15).CorderandHenry

(19)alsofoundsomeincreaseinothercarbohydratedegradingenzymessuch asglucanase,endo

xylanesse,duringgermination.

Figure1.6Thestructureofamylopectinandthe(16)Dglucosidicbondswhichforms
branching(reprintedfromref.24a).

1.2.3.1.2.ProteinDegradingEnzymes

Proteolyticenzymesareofsecondaryimportancerelativetotheireffectonendproduct

quality.Theseenzymesareverycomplexanddifficulttopurify.Themainenzymesrelatedto

preharvestsproutingareacidendopeptidasesandcarboxypeptides.Theendopeptidasesare

locatedinthepericarp,seedcoat,andaleuronelayeratearlydevelopment,andthendecrease

withmaturation.Theyarealsopresentintheembryoandendospermatmaturitylevels.In

germinatingkernelsthelevelincreasesbythehormonallyinduceddenovosynthesis.They

cleavetheinternalpeptidebondstoobtainsmallerpolypeptides.Carboxypeptideshydrolyze

11
proteinsandpeptidesfromCterminalaminoacidend.Upongermination,theyincreasethree

foldandenzymesinendospermarelocatedlargelyadjacenttoscutellarepitheliumofthekernel

(22,14).

1.2.3.1.3.OtherEnzymesSystems

Therearesomeotherenzymesthatincreaseduringgermination,buttheireffecton

qualityrequireselucidatedviamoreresearch.Lipasesarelocatedmainlyintheshootandfound

toincreaseupongermination.TavenerandLaidman(20,21)reportedthattriglyceridereserves

indifferenttissuesaremobilizedandbrokendownprogressivelyduringgermination.They

foundthatlipaseactivitypresentsintheendospermin12hrofimbitition,andtheincreasein

lipaseactivityisresultedfromthefactorsformtheembryowhicharenotknown.

Inadditiontothelipase,the33foldincreaseinphenoloxidaseswasobserved.Thegray

colorofthebreadcrumbmadefromsproutedwheatresultsfromthisenzymecomplex.The

peroxidesandcatalasesalsoincreaseduringthegerminationtheytakeroleintheoxidative

reactionsaffectingtherheologicalpropertiesofdoughandthepigmentdestruction(22).

1.2.3.2.ActionofHormonesduringGermination

Afterthewateruptake,hormonalsignalsreleasedfromembryocausesynthesisofthe

hydrolyticandotherenzymes.Gibberellic,abscisicacid,auxins,andcytokininsarethe

hormonesthattakeplaceintheseeddevelopmentandgermination(17a).Gibberellicacid(GA)

producedbytheembryodiffusestothealeuronelayerandtriggersthesynthesisandthe

secretionofhydrolyticenzymesandthemobilizationofreservematerials(14,3).AlsoGAis

releasedthroughthescutellumintotheendosperm.Abscisicacidplaysaregulatoryrolein

germination.Cytokininsdonottakeanypartatearlystagesofgerminationbuttheymaybe

importantforcellgrowthinembryo.Auxinaffectsthescutellum(25).

12
1.2.3.3.MobilizationoftheStoredReservesandChangesintheKernel

Mobilizationofthestoredmaterialinthekernelduringgerminationcanbestudiedin

differentways.Usinglightorfluorescentmicroscopyandscanningelectronmicroscopy,

morphologicalchangescanbeinvestigated(Figure1.7). Thisallowsstudyofmacroscopic

changessuchasdamageofstarchgranulesbyamylase.Anotherapproachistheextractionof

biochemicalconstituentsofthekernelatdifferentstagesofthegerminationandtheinvestigation

ofthechemicalchangesofthereserves.Inadditiontothesemethods,modelsystemssuchas

purifiedenzymeandsubstratesystemscanbeused,inwhichsimilarprocessestakeplace.All

methodsprovideinformationonthemobilizationfromdifferentaspects(22).

Figure1.7Fluorescephotomicrographsoftransversesectionsofendospermembeddedin glycol
methacrylate/urea/glutaraldehydeandstainedwithNileBlueAsolution.Note: Starchy
endosperm(end),starch(s),lipiddeposits(bigarrows)andscutellum (sc)partsinthesection
(reprintedfromref.26).

Themajormobilizationofreservesisinitiatedafterradicleelongation,butsome

mobilizationcanoccurbeforecompletionofthegermination.Highmolecularweightmolecules

aremobilizedandconvertedintosmallersimplemoleculesinorderto transfernutrientstothe

growingembryotosupportenergyproducingevents(16).Somemicroscopicstudiesshowedthat

inthesproutedkernelstarchgranulesadjacenttotheembryoweredegradedextensivelyand

proteinbreakdownstartedadjacenttoembryowithin2448hrofgermination.Inthispartofthe

13
kernel,starchgranuleswerefoundtobefreeoftheirproteinmatrix,andbreakdownincreased

progressivelywithincreasinggermination(22,24).

Intheendosperm,starchgranulesarehydrolyzedintoglucosemoleculesbytheenzymes.

Theprocessstartsadjacenttoscutellumandmovesfromtheproximalendtothedistalendofthe

kernelinparalleltothescutellumface,advancingfasteradjacenttothealeuronelayer(14).

Followingthedegradationof starchgranules,carbohydratesincellwallsofendospermarealso

degraded.Thedegradationproducts(glucoseandmaltose)areabsorbedbythescutellum.

Accordingtotheproposedpathway infigure1.8, thedegradationproductsareconvertedinto

sucroseinthescutellumandusedbythegrowingrootandshoot. Noneofthesugaraccumulates

inthescutellum(16,27).

Figure1.8Schematicmodelofsugartransportfromtheendosperm totheembryoin
germinatingwheathighlightingthepotentiallocationof sugartransporters.SPS,Sucphosphate
synthasehexoseP,hexosephosphateandconversionofglucoseintosucrose(reprintedfromref.
27).

Themostof theproteinisstoredmainlyinthealeuronelayerandintheproteinbodiesof

thestarchy endosperm.Thereisaminoramountofproteinreserveinthescutellumand

14
embryonicaxis.Thismaybedegradedintoaminoacidandusedbygrowingembryobeforethe

majorreservesinendospermismobilized(16).Proteolyticactivityproductsinendosperm

includingaminoacids,dipeptides,andsmalloligopeptidesaretransferredtoembryovia

scutellumaftertheyaredegradedintoaminoacids.Thesebuildingblocksareusedforprotein

synthesisofembryogrowth(Figure1.9).

Figure1.9Proteinsynthesisduringgermination showingaminoacidsproducedfromprotein
degradationfunctionasbuildingblocksforproteinsynthesisof thegrowingembryo(reprinted
fromref.25).

Wheatgermdoesnotcontainany nativestarch.Butwhentheseedwaskept24hrunder

moistconditions,starch hadaccumulatedinthegermeventhoughtherewasnochangein

endosperm.Itwassuggestedthatthisstarchmightbeproducedby activityoftheembryo.When

theradicleemergesthroughtheradiclecapandrootsheath,starchgranulesareseeninthecells

15
of thescutellum.Thisisanindicationofthetransferofthestoragematerial fromtheendosperm

tothegerm.Thedissolvedreservematerialistransferredfrom theendospermandreformsas

smallgranulesinthescutellum.Thealeuronecellsdonotshowanychangesuntilstarchis

almostconsumed,thentheircellwallscollapseanddissolve(2,13).

SwiftandOBrien(6,13)studiedthefinestructureofscutellumbeforeandduring

germinationbylightandelectronmicroscopy.Forstudyingthestructurebeforegermination,

kernelsaresoaked3hrbeforefixation.Byaqueousfixationofkernels,therecanbesome

structuralchangesatthemolecularlevel,buttherearenogrossstructuralchangesafter3hrof

soaking.Thescutellumiscomposedofstorageparenchymaandvasculartissue.Indrykernels,

thereisanabundanceofproteinbodiesintheepitheliumandparenchymaofthescutellum.

Proteinandphytinarestoredintheseproteinbodies.Lipidisabundantintheformofsmall

dropletsinthecytoplasmofepitheliumandparenchymacells.Therearenostarchandwatery

vacuolesinthescutellarcells.Inthelightphotomicrographsofatransversestainedsectionofa

drykernelscutellum (Fig.1.10),theabundanceofproteinbodiesisobvious.Proteinbodiesin

theepitheliumaresmallandtheyarelargeinparenchyma.Arrowheadsshowunstainedregions

wherephytatedepositshavebeenlostfromthesection (6).

Figure1.10Lightphotomicrographofthetransversesectionofthescutellum ofadryembryo
(SE:cellofscutellarepithelium,SP:cellofscutellarparenchyma,n:nucleus,pb:proteinbody).
Theabundanceofproteinbodiescanbeseen.Proteinbodiesintheepitheliumaresmalland
largeinparenchyma.Arrowheadsshowunstainedregionswherephytatedepositshavebeenlost
fromthesection (reprintedfromref.6).

16
Theonlychangeinthescutellumafter3hrsoakingistheappearanceanddistributionof

thenuclearchromatin.Inaddition,somemobilizationofstorageproteinsintheepithelium

appears.NougaredeandPilet(1964)suggestthatwateruptakeisveryrapidintheepitheliumand

muchslowerintheparenchymacells(6).

Duringgermination,after6hrexposureverysmallstarchgrainsappearintheepithelium

andparenchymacells(Figure1.11a),andwhenthegerminationcontinues,thesizeandnumber

ofthestarchgrainsincreases.After24hr of germination,therearealargenumberofstarch

granulesintheepitheliumandtheyarebiggerandmoreabundantintheparenchyma(Figure

1.11c).Atthisstageofgermination,proteinbodiesintheepitheliumhavebeendegradedand

smallvacuolesareleft.Breakdownoftheproteinintheparenchymaisslowerandafter24hr

largemassesofpartlyhydrolyzedproteinsareformedbycoalescenceofthebodies.Inthelater

stagesofthegermination,proteinreservesarecompletelydegradedandonlybigvacuolesare

leftinthecells(Figure1.11).

17
Figure1.11Lightmicrographsofthescutellumduringthreestages 6hr(a),12hr(b)and24hr
(c) of germination (reprintedfromref.13).Lightmicrographsofthescutellumduring
germinationshowingdegradationofproteinbodiesintheepithelium.Smallvacuolesareleft
afterdegradation .Intheparenchymalargemassesofpartlyhydrolyzedproteinsareformedby
coalescenceofthebodies(dl:depletedlayerofendosperm,n:nucleus,pb:proteinbody,se:
scutellarepithelium,sp:scutellarparenchyma,v:vacuole,largearrowhead:starchgrainsin
parenchyma,smallarrowheads:starchgrainsinepithelialcells).

a. b.

c.

After24hrgermination,therearesomestructuralchangesinepitheliumcells.They

becomeelongatedandcellwallshavebeenseparatedfromeachother.Alsostarchbreakdown

startsintheendospermadjacenttoepitheliallayer.Inadditiontothesechanges,thereare

changesinorganellesendoplasmicreticulum,mitochondria,nucleus,andplastidsobservedby

electronmicroscopywhichcanbecorrelatedwithmetabolicactivity.

Thereisnodetectablechangeinthelipidbodiesafter12hrsoaking.After24hrthey

becamelesscommonnearthecellmembranesoftheepitheliumcells.Inthelaterstagesof

germination,thereisareductionintheirnumber,howevertheirutilizationisslow.Insome

studies,ithasbeensuggestedthatthestarchinthescutellumisderivedfromthelipidbodies,

18
becausestarchcanbeseeninthescutellumbeforesignificantmobilizationoftheendosperm

reserves(13).

TavenerandLaidman (20)studiedonthetriglyceridemetabolisminthegerminating

wheatkernel.Theyobservedthattriglyceridereservesweredegradedprogressively.Breakdown

startswithin12hrimbibitionintheembryonicaxisandstarchyendosperm.Sincethegermdoes

notcontaincarbohydratereserves,thetriglyceridesmayprovideenergyduringearly

germination.

1.3.InfraredSpectroscopy

Theinfraredradiationincludeswavelengthrange13,00050cm1 or0.75200m. The

entireregionisdividedinthreeparts nearIR0.752.5m(11,0004,000cm1)midIR2.520

m(4,000600 cm1),andfarIR20200m (28). IRspectroscopyhaslongbeenregardedthe

idealtechniqueforqualitativeanalysisandstructuralinformation of organicmaterial.Thesame

istrueforfarIR.ThenearIRregionhasfounduseforquantitativedataanalysis.

Wheninfraredradiationisabsorbedbyaspecies,atomsinmoleculesvibrate.Vibration

frequencyisdependentonthemassesoftheatomsinvolvedandtheforceofholdingtheatoms

together.Onlythesefrequenciesthatcan causetheatominmoleculestovibrateareremoved

fromIRtransmittanceradiation.Thereforetheabsorbancespectrumfrequencyisthe

fundamentalvibrationalfrequency oftheatom.TheIRspectrumisobtainedbythechangeinthe

dipolemomentoftheatomsbymolecularvibrationsandrotations.Information ofthechemical

compoundsisbasedontheabsorptionintensitiesofthevibrationalbands.Thereisalinear

relationbetweentheabsorbanceintensityandtheconcentrationofthemolecules.Relative

amountsofvariouspeaksbasedonabsorbancepeakheightandratiosprovidesemiquantitative

informationinmidIR.

19
ThemidIRregion(4000600cm1)involvingthefundamentalvibrationsandfingerprint

regionhassharpandnarrowpeaksandthepeaksarenotnecessarilyoverlapping.Stretching

vibrationsidentifywhichatomsvibrateandthefingerprintregionrevealstheenvironment.The

lengthofthehydrocarbonchaincanbedeterminedbycomparingtheCH3andCH2 vibrations.

AlsotheunsaturationlevelscanbedetectedbyC=CHorC=Cvibrationalbands.Therelative

populationoflipids,proteins,andcarbohydratescanberevealedbymeasuringtheabsorbanceat

certainfrequenciesforNHtodetermineprotein,OHformoisture,andCHforlipid. Shiftsin

thefrequenciesofthebandsgiveanideaaboutthechemicalenvironmentofinfraredactive

group(29,30). Thereare of twotypesinfraredspectrometerswhichcanscanentirespectrum

dispersiveprism (refraction)orgrating(diffraction)spectrophotometersandinterferometer(FT

IR)spectrometer. FTIRhasbeenwidelyusedmostlyinlast20years.

Oneofthemostimportantadvancesintheinstrumentationofthevibrational

spectroscopyisachievingmicrospectroscopy whenradiantissufficiently conservedtoobtaina

suitableS/Nforspotsizesinafewmicrometers.Inaddition,usageamotorizedstageenables

mappingoftissue.Thisnewcapabilitygivesscientisttheopportunityoflinkingtheanalytical

chemicaltothemorphologyofthematerialinthefieldofview.Followingthese,synchrotron

radiationwasusedforFTIRenablinghighspatialresolution.Chemicalimagingbecame

successfulwiththedevelopmentofcamerasbasedon silicon chargecoupleddevice(CCD)

arrayswhicharesensitivetonearIRandfocalplanearraysindiumgalliumarsenide(InGaAs)

or indiumantominade(InSb),themercurycadmiumtelluride(MCT)detectorssensitivetonear

andmidIRrespectively (31). Theimagingapplicationsdevelopedwiththevibrational

spectroscopyprovidesthesimultaneouscombinationofchemicalandspatialinformation.

Moreover,insomecasesspectroscopicanalyticalinformationcanbeobtainednondestructively.

20
1.4.FourierTransformInfrared(FTIR)Microspectroscopy

Intraditionalmicroscopy,samplepreparationincludesseveralstepssuchasfixationof

thetissue,embeddinginamatrix,andmicrotoming.Histochemicalinformationisobtainedwith

theapplicationofspecificstainsthatselectivelybindtodifferentcomponentsandfromthese

specimensitisverydifficulttogeneratequantitativeinformation.Vibrationalspectroscopy

makesitpossibletoobtainspectraofthemicrotomedsectionsofthetissueswithouttheintrusive

applicationofstainsandchemical reagentslikeinconventionallightandfluorescence

microscopy(29,31).

FTIRmicrospectroscopyandimagingbecamevaluabletechniquestostudythe

molecularchemistryofbiologicalmaterialsby allowingconnectiontomorphology.FTIR

combinesinfraredspectroscopywithlightmicroscopyinordertolocatetheareatobeanalyzed

andthusmolecularinformationisobtainedwithgreatspatialresolutionormicroscopicscale(32,

31). TheFTIRtechniquehasbeenusedtoidentifyanddeterminethemolecularchemistryof

manydifferentmaterialsincludingforensicmaterials,plantandanimaltissues,minerals,

polymerfilmsandpharmaceuticals.

1.4.1.FouirerTransformInstrument

TheFouriertransform instrumentisbasedonaMichelsoninterferometer.Inthissystem,

thebeamisdividedintwopathwayswithabeamsplitter.Twobeamsarecombinedafterpassing

throughthealternatepathwaysandreflectedonthedetector. Thelengthofonepathwayconstant

withafixedpositionedmirrorwhereasintheotherpathwayamovablemirroradjustthelength

asafunctionoftime.Whenacertainfrequencypassesthrough,themovablemirroradjustsin

ordertoobtaintherightdistanceiscombinedwiththesamefrequencyofthefixedpath(zero

pathlengthdifference).Amaximumresponseisobtainedatthatfrequencybyconstructive

21
interferenceinthiscase.Aninterferogramisgeneratedbyholdingthespectralinformationfrom

sampleintimedomainasacosinewave,intensityisrecordedversustimebasedonthespeedof

themovingmirror.Thistimedomainspectrumisconvertedintoafrequencydomainspectrum

withaFouriertransformationfunction (33,29). ThereareimportantadvantagesofFTIRover

dispersiveIRformicrospectroscopy.TheFTIRspectroscopyutilizesaninterferometerinwhich

allfrequenciesoftheradiationpassthoughthesampleandarecollectedbythedetector

simultaneously(Fellgetormultiplexadvantage)withthisopticalscheme.AnimprovedS/Nratio

canbeobtainedinashortamountoftimeandhighS/Nenablesdividingthesignalintomany

channels,thusverygoodspectralresolutionisobtained.Inaddition,itprovideshigherenergy

throughputduetotheabsenceoftheslits(Jacquinotadvantage)(33,28).

1.4.2.AdvancesinInfraredMicrospectroscopicInstrumentation

ThefirstIRmicroscopeattachmenttoanIRspectrometrywasproposedin the1950sby

Coates,Offner,andSiegler(34).Infraredmicrospectroscopybecamemorepracticalby

combiningFTIRspectrometrywithhigh sensitivity detectors(33).ThefirstFTIRmicroscope

wasintroducedin1986,patentedin1989byMesserschmidtandSting.Itwasequippedwith

reflectingopticsanddualmodeimagingplanemasks.TheSpectraTech,IRPLANTM

microspectrometerbecameavailablein1986andhasbeenusedwidely providingbetter optical

efficiencytomaintainS/Nanddualprojectedimageplanemasking.Theconfocaltermhasbeen

previouslyreferredtoasdoubleaperturingbyRobertMesserschmidt,whodesignedthe

SpectraTech IRPLANTM.Thespecimenspectrumbecomescontaminatedwithspectralfeatures

fromtheforeignmaterialoutsideofthetargetspotsizewithsinglemasking.Thiscontamination

iseliminatedwithdualmask,thuspurerspectrumandbetterspatialresolutionisobtained.In

1990,anintegratedsystemconsistingofaninfraredmicroscopeandinterferometerbench was

22
introducedbySpectraTech/IRsTM(35).Numberofthemirrorswaseliminatedfromoptical

path improvingthroughput.Mappingcapabilityisenhancedwithamicroprocessorcontrolled

motorizedstage.

Table1.1 Majordevelopmentsininfraredmicrospectroscopy instrumentation (reprintedfrom


ref.40)
Majordevelopmentsininfraredmicrospectroscopy instrumentation

1949 Firstpaperscombininginfraredspectroscopyandmicroscopy
R.Gore(Science),R.Barer(Nature)
1953
PerkinElmerdevelopedthefirstcommercialmicroscopeforIRspectroscopy
1978 NanometricsintroducedtheNanoSpec20IRmicrospectrometer

1983
BioRadandAnalectintroducedFTIRmicroscopes
1986 SpectraTechinventedtheIRPlanResearchMicroscope

1989
SpectraTechintroducedtheIRsmicrospectrometersystemwithautomatedmapping
1989
SpectraTechinventedtheGrazingAngleObjective
1990
SpectraTechinventedthemicroATRobjective
1994 NIH(LewisandLevin)inventedaninfraredspectralimagingsystemusing
anarraydetector
1998:
SpectraTechinventedtheContinummicroscope(infinitycorrected)
1999
SensIRinventedanATRvideomicroscope
2000 SensIRintroducedanATRvideoFTIRmicroanalysissystem

Oneofthenextstepsinadvancementistheinterfacinganinfraredmicrospectrometerto

synchrotronradiationin1993(37).Thebrightnessofthesynchrotronis1001000timesgreater

thanglobarsourcesprovidingabetterspatialresolutionandhigherS/Nratios. Thedevelopment

ofsingledetectorinstrumentsprogressedsignificantlywiththeintroductionof thefirstinfinity

corrected confocalinfraredmicroscopebyReffnerandVogel(36).Infinitycorrectedmirrors

23
enabledtheplacingofapolorizerprovidingdifferentialinterferencecontrast(DIC) anditalso

extendthepathlength.FTIRwasdevelopedwithaconventionalthermalinfraredlightsource

andsingleinfrareddetector (35). Infraredmicroscopeswerecustomizedforasmalltargetsize

wasachievedwithdual32XSchwarzschildmirrorlensandliquidnitrogen cooledMCTdetector

with a50mx50melement(35).

ImportantprogressinimagingwithFTIRmicrospectroscopy startedwithsubstitutionof

singleelementdetectorwithafocalplanearray(FPA)detectorin1994(38,39).TheFPA

detectorhasanarrayofinfrareddetectorelementsthatprovidescollectionofthespectraof

differentpixelsonthesampleareasimultaneouslybymeasuringtheallfrequencieswithan

interferometer.Initially,ithadaliquidnitrogencooledFPAcamerawithMCTphotovoltaic

detectorsina64x 64arraywithastepscaninterferometer.WiththisinitialFPAsystem,the

spectralqualityofthedataisnotasgoodasasingledetectorFTIRmicrospectrometerdueto

operationat16 cm1 resolutionwithlowS/N.Howeverthisproblemwassolvedwithsome

improvementinstepscan.LaterthequalitywasimprovedwithsomeadvancesinFPAwithrapid

scanarrayinstruments.MCTphotovoltaicopticaldetectorelementswerereplacedby

photoconductivemechanism.FPAinstrumentsand16elementpushbroomlineararray have

beenusedrecently.

1.4.3.ApplicationsofFTIRMicrospectroscopyonPlantMaterial

FTIRmicrospectroscopyhasbeenwidelyusedonplantmaterialandwheat(39,41,42,

43). Inaseriesofpublications,WetzelandcoworkersexaminedbiologicaltissueswithFTIR

microspectroscopy.Theirstudiesstartedwiththefirstmicroscopeaccessoriesandfollowedby

integratedFTIRmicroscope.Inadditiontheirexplorationincludedusingasynchrotronsource

toincreasethespatialresolutionandfinallyFPAimagingsystems.In1987,earlyworkwasdone

24
byWetzel,Messerschmidt,andFulcherbylookingatisolatedcomponentsfromdifferentparts

ofthewheatkernel(44).Thepericarp,seedcoat,aleuronecells,andendospermwereexamined.

Spectrafortheisolatedgluten,starch,lipid,andcellulosewerealsoreported.Followingofthis

studyWetzelandFulcher(45)showedthelocalchemicaldifferenceswithinthesamesection

fromonekernelandcharacterizedeachdifferentbotanicalpart.Thefirstmappingofawheat

kernel insituwasperformedbyWetzelandReffner(46).Themappingwasdonewitha

motorizedstage.Laterthemicrostructureofthewheatkernelwasexaminedwithdifferentmaps

withaprogrammingstageofIRusintegratedFTIR(44).ThefirstFTIRmicrospectroscopic

imagingofwheatsectionbyusingaMCTFPAdetectorwasreportedinliteraturein1999(39).

1.4.4.SynchrotronInfraredMicrospectroscopy

Synchrotroninfraredmicrospectroscopyallowsultraspatialresolution.Therearethree

importantreasonsmakingsynchrotroninfraredmicrospectroscopymoreadvantageousthanthe

techniquesusingaconventionalglobarsource.First,thesynchrotronradiationis1001000times

brighter.Inaddition,itisfreeofthermalnoiseofhotfilamentandthelightemittedishighly

directional.Thereforemaximumradiationfluxonthesmallestpossibleareaprovideshighspatial

resolutionandhighspectralresolutionwithoutexcessivecoaddition(47,48). Inthesynchrotron,

electronsfromanelectronsourcewereacceleratedwithalinearaccelerator.Itproducesan

energyof75millionelectronmilivolts(meV),theseelectronsenteraboosterring,heretheyare

acceleratedmore,andentersavacuumultraviolet(VUV)storagering(Figure1.12).TheVUV

storageringhaseightbendingmagnets,and176radiationports.Whenelectronaccelerates,they

radiateenergyintheformofelectromagneticwaves.Synchrotronlightconsistsofacontinuous

spectrumofelectromagneticradiationrangingfromxraytoinfrared.Infraredbeamfromthe

beamlineportisextractedanddirectedtomicrospectrometer(47).

25
Figure1.12Diagram ofNSLS(A:electronsource,B:linearaccelerator,C:boosteraccelerator
ring,VUV:Vacuumultravioletstorageringwithassociatedbeamlines.Electronsareaccelerated
fromwithalinearacceleratorandproduceanenergy of75millionelectronmilivolts(meV),and
enteraboosterring,andareacceleratedmoreandentervacuumultraviolet(VUV)storagering
(reprintedfromref.29).

2.Experimental

2.1.Instrumentation

FTIRmicrospectroscopywascarriedout on U10bandU2bbeamlinesonthevacuum

ultravioletstorageringattheNationalSynchrotronLightSource(NSLS)ofBrookhaven

NationalLaboratory(BNL)Upton,NY.Initial experimentsandfirstattemptsfor

semiquantitationwereachievedatNSLS. Subsequently,largedatasetswereacquiredfrom three

hardwheatvarietiesandmappedintheKSUMicrobeamMolecularSpectroscopyLaboratory

with thefocalplanearrayinstrument.

2.1.1.InstrumentationatSynchrotron

BeamlineU10bwasequippedwith aContinumTM infraredmicroscope (SpectraTech/

Thermo,MadisonWI)interfacedtoaMagna850FTIRspectrometer(Nicolet/ Thermo,

26
MadisonWI). Schwarzschild32Xobjectiveandcondensermirrorlenseswereusedonadouble

passsinglemaskbeampath toprojecta10mx10mconfocallytargetedspot(Figure1.13). A

50mx 50mliquidnitrogencooledMCTdetectoressentiallymatchedthebeamfromthe

imageplanemaskofthemicroscope. Itallowedsinglerealtimescanningofthespectrumwhile

viewing.WiththeContinumTM infraredfocusisadjustedfromthesinglebeamenergyresponse

atthewavenumberrangeofinterest.Aresolutionof8cm1 wasusedwith16scanscoadded

unlessotherwisestated.

Figure1.13ContinumTM infraredmicroscope.ContinumTM infraredmicroscopeincludes


matchedSchwarzschild32Xobjectiveandcondensermirrorlenseswereusedonadoublepass
singlemaskbeampath,infinitycorrecteddualconfocaloperation (reprintedfromref.35).

BeamlineU2bwasequippedwithaNicPLANTM modelinfraredmicroscope(Nicolet/

Thermo,MadisonWI)interfacedtoaMagna850FTIRspectrometer(Nicolet/ Thermo,

MadisonWI)(Figure1.14). InNicPLANTM microscopeSchwarzschild32Xobjectiveand10X

condenserswereused.Asinglemaskpositionedbeforeobjective,andapinholeimageplane

maskplacedaftercondensertomaintainpurge.Theinfraredfocuswasadjustedtoobtainthe

maximum count(signal)beforespectrawererecorded.Beforedatacollection,visiblefocuswas

27
usedforimagecapture.FordatacollectiononBaF2,infraredfocuswasachievedbasedonthe

intensityofthedetectorresponsepriortoautomatedmappingsequence.Aresolutionof8cm1

wasusedwith16scanscoadded.

Figure1.14U2bbeamline.Beamcomingfromthering(1)entersFTIRspectrometer(2)and
fromthebeamsplitteroftheinterferometeritisdirectedtowardstheIRmicroscope (3),a
computer(4) attachedtothesystem controlsthestage,microscopeandspectrometeroperation.

4
2

2.1.2InstrumentationatKSU

Spotlight300SpectrumOneinstrument(PerkinElmer,Danbury,CT)wasusedinthis

quantitativephaseofthisprojecttomapwheatsectionsof threehardwheatvarietiesin the

transmissionmode.ThisFTimagingmicrospectrometerequippedwitha16element

pushbroomarraywasoperatedwith anominal 6.25mx6.25m pixelsize.8cm1 resolution

wasusedand64scanswerecoadded.Forthebackgroundspectrum,240scanswerecoadded.

Theimagescontaining10003000pixelsweremappedfrom eachsection.TheSpectrum

Spotlightimagingsystem,showninfigure1.15,iscomposedof aninfraredmicroscope, a

SpectrumOneFTIRspectrometerandastagecontrollerthatincludedajoystick

28
Figure1.15SpectrumSpotlightimagingsystem includingaSpectrumOneFTIRspectrometer
connected opticallyinterfacedtoamicroscope, andelectronicsformicroprocessorcontrolof
stage,microscopeandspectrometeroperation (reprintedfromref.49).
Microscope
Computer
Stagecontroller SpectrumOne
Spectrometer

Joystick

ThemicroscopemagnifiesthevisiblelightimageofthesampleandtheMonitorVisible

windowoftheSpotlightsoftwaredisplaystheselectedsampleimageasamosaic.Thedata

collectionandprocessingiscontrolledbytheSpotlightsoftware.Theinfraredmicroscopehasa

motorizedstagewhichiscontrolledeitherbyjoystickortheSpotlightsoftware.Movementof

thismotorizedstageisusedtoselectpointsonthesampletobeanalyzedormapped.Focusing

canbeperformedbymanualadjustmentwhileviewingtheliveimage.Bothinfraredradiation

andvisiblelightaresenttotheremoteaperturebythesamecassegrainmirrorlens(49).

Datacollectioncanbedonein adetectorarray imagemodeorasingledetector point

mode.IRmicrospectrometerwith aFPAisshowninthefigure1.17.Inpointmode,asingle

MCTdetectorrecordsthespectrumofeachspotonthemicroscopestageoneatatime.With

singledetectoraremoteimageoperatingmaskisusedtorestrictthespotsizefromwhicha

spectrumisrecorded.Operatinginimagemode,16MCTdetectorelementsinalineararray

recordspectrasimultaneouslyinparallel.Asthemotorizedstagemoves,mappingaparticular

29
areaofthesampleproducesspectraformanypixelsfromwhichmultiplechemicalimagesmay

begenerated.Thisisdonebyselectingtheareaofinterestby drawingaboxarounditandto

programthescanningoperation.Fromthedataobtained,individualspectracanbeextractedfor

anypixel(x,y)onthesamplearea.Inthisstudy,scutellaoftheeachsection wereanalyzedin

imagemodebyselectingtwoor threerectangularareascontainingbetween10003000pixels

each.Allthespectrawereextractedfromeachofthemapsandusedforfurtherexaminationsand

calculations.

Figure1.16SchematicofIRmicrospectrometerwithasingleelementdetector havingthe
capabilityofconfocaloperationwithaperturesbeforeandaftersample.Thisisageneralized
schematicconsistentwiththeContinumTM andNicPLANTM instrumentsatbeamlinesU10band
U2b(reprintedfromref.50).

30
Figure1.17SchematicofIRmicrospectrometerwithaFPAdetection whichhasanarrayofIR
detectorsenablingwholespectrum scanningoftheentirefieldofviewwithasinglescan
(reprintedfromref.50).

2.1.2.1Opticalsystemand mappingprocedureofSpotlight:

Bothinfraredmicrospectroscopyandvisiblelightopticsexistsinthemicroscope.These

systemsintersectattheaperture.Spotlightenablestoworkintransmittanceandreflectance

operations.Dichroicmirrorsareusedtoreflectinfraredandtransmitthevisiblelight

alternatively.Whenasampleisviewedwiththecamera,aconjugateimageofitisseenatthe

remoteaperture.Bymovingthestagepositionupanddown(Zcontrol)withthejoystick,the

opticalimageisfocused.Viewingasampleintransmittanceisillustrated infigure1.18.

31
Figure1.18Pathofthevisiblebeamforviewingasampleintransmittance.Visiblelightfrom
Lightemittingdiode(LED)illuminator traversesthesampleandissenttotheupperdichroic
mirror(reprintedfromref.49).

Visiblelightfrom Lightemittingdiode(LED)illuminatorisdirectedby thelowerfold

mirrortothelowerdichroicmirrorontothelowercassegrainmirrorlens.Thebeamiscondensed

andfocusedontosamplepositionbylowercassegrain.Afterthelighttraversesthesample,itis

collectedbyuppercassegrainandsentupwardthroughtheimageplanemaskandtheupper

dichroicmirror.

Aftersampleisviewedandanareaisselectedfor analysis,thevisibleimageis

captured.Thepathfollowedby theinfraredbeamfromthebeamsplitterduringmappingis

showninfigure1.19.

32
Figure1.19Pathoftheinfraredbeamforcollectinganimageintransmittance.Infraredradiation
fromthebeamsplitterofthespectrometerisreflectedontothelowerdichroicmirror,attheend
ofthetravelitisfocusedontotheMCTdetector (reprintedfromref.49).

Sinceuppercassegrainisusedforbothvisiblelightandinfraredradiation,focusingthe

samplefromvisibleimagepositionesitcorrectlyforinfraredimaging.Thebeampathin

collectingdatadiffersfromthatforviewing.Infraredradiationfromthebeamsplitterofthe

spectrometerisreflectedontothelowerdichroicmirror thatsendsittothelowercassegrain.The

exitingtheuppercassegrainisfocusedontotheMCTdetectorbythedetectorcassegrain.

IRmagnificationcanbechangedfromlowtohigh(25mto6.25m)by a

microprocessorcontrolledmechanism thatrotates aseriesofmagnifyingmirrorsinto theIR

beam.

2.1.2.2.MCTDetector

Themercurycadmiumtelluride(MCT)detector array intheinfraredmicroscopeis

cooledto77Ktopriortocollectionof data. Thedewariscarefullyfilledwithliquidnitrogen.

Whenfull,thetemperatureofthedetector isreducedandoperatingsensitivityisachieved.The

33
filleddewarkeepstheMCTdetectorcoolattheproperoperatingtemperatureforatleast6hr. At

theendofthistimeperiod,thedewarbeginstoreturntoroomtemperatureandpoweris

switchedofftotheMCT.

2.2.Samplepreparation

Thewheatkernelsweresoakedin distilledwaterat4oCfor68hrinordertosoftenthe

seedsandpreparethemtothecryogenicsectioningwithamicrotome.Keepingatrefrigerator

duringsoakingavoidssproutinginthispreparationstep. Aftersoaking,surfacemoistureofthe

kernelswereremovedwithKimwipesandtheyweremountedonthesampleplate(germside

up)viaembeddinginasupportingmedium TissueTech(OCT)andfrozenat20oCbefore

microtomesectioning.Thethinsections(46m)wereproducedtoobtaingoodandundistorted

absorptionbandsinthetransmissionmode.Thesectionswerethawmountedonto1mmthickx

13mmBariumfluoride(BaF2)disks.Thefrozensectionofthetissuemeltsonthediskduetothe

temperaturedifferenceofthetissueandthedisk.TheBaF2 isnothygroscopicandhasa

transmittancecutoff800cm1.ThetissuesmountedonBaF2 wereanalyzedinthe4000cm1 and

800cm1 rangebyIRmicrospectroscopy.

Optimizingsectioningangle

Somepreliminarysectioningexperimentsweredonetodeterminetheproperanglein

ordertoobtaingoodsectionshavingahighamountofthescutellum.Inthefirstsectioning

experiment,theangleofslicingwasthesmallestanditwasgraduallyincreasedinsuccessive

trials.Thesuccessivelypreparedsectionsforthefirstsmallsectioningangleareshownbelow

(Figure1.20).

34
Figure1.20Thesuccessivesectionsforthefirstsmallslicingangle.Thescutellumareasare
shownintheredboxes.Amountofthescutellumisdecreasinggraduallysuccessivesections
furtherintothekernel.

Theareasshownintheredboxesarewithinthescutellum.Inthefirstsection,the

embryonicaxisisthehighestqualitypartofthesection,intheotherthreesectionsamountofthe

scutellumisdecreasinggraduallyfurtherintothekernel.Itisdifficulttodistinguishbetweenthe

coleorhizaandscutelluminthelastsection.Theselectedareasformappingshouldnotbevery

closetotheembryonicaxisinordertoavoidthecoleorhiza.

Inthesecondsectioningexperiment,ananglelargerthanthefirstwastried.Asseenin

thephotographtheangleismorethan45.

Figure1.21Thesuccessivesectionsobtainedatthelargerangle.Thescutellumareasareshown
intheredboxes.

35
Inthefirstandthelastsections,itisveryhardtomakethedistinctionbetweenthe

coleorhizaandthescutellumtissues,butinthemiddlesectionstheamountandthequalityof the

scutellumtoworkwithisideal.

Inthenextsectioningexperiment,theanglewasthewidestused.Sectioning3:

Figure1.22Thesuccessivesectionsobtainedwiththewidestangle.Notmuchscutellumis
exposedinthesections.

Thisanglewasnotgoodforthepurposeofthisstudybecausenotmuchscutellumis

exposedinthesectionsthatresult.

Thesecondangleusedwaschosenforallsubsequentsectioningduetooptimumthe

scutellumpresentation.ThekernelsofthecultivarsTrego,Danby andKS2174weresectioned

usingthisanglebyadjustingeitherthebladeangleortheseedmountingangle.Thesamples

referredtoascalledascontrolwereunsprouted(0hrgermination)andtheywerenotexposedto

moisture.Priortosectioning,theyweresoakedatrefrigeratortemperatureaspreviouslystated.

Thelaboratorysproutedkernelswereallowedtogerminateforeither24hror36hrbasedon

evidenceoftheseeddormancy.Thesproutedkernelswereexposedtomoistureonmoistblotter

paperinPetridishesandtheyweresectionedimmediatelyaftertheendofthegermination

duration.Ifthesamplescouldnotbesectionedimmediately,thentheywerekeptat80oCfor24

hrtoterminatethegerminationprocess,andsectionedimmediatelyafterthawing.Sections

36
containingbothgermandendospermwithanoptimumscutellumareawereproducedand

analyzedforthesproutedandtheunsproutedkernels.

2.3.MicrospectroscopicProcedureandDataProcessing

ReplicatesofthehardwheatcultivarsHWandJaggerweremappedinarastergridin10

mstepsresultingina10mpixelsizeusingtheContinumTM infraredmicroscopein

preliminaryexperimentswithsectionsfromkernelsthathadbeenanalyzedpreviouslybynear

IRnondestructivesubsurfacepolychromaticcontrastimagingtoreveal thedevelopingembryo.

Foreachpixelinthegridaninfraredspectrum intherangeof 4,000cm1680cm1 was

recorded.ThesamesystemwithaNicPLANTM infraredmicroscopewasusedonanother

synchrotronvisittoperform linemappingfromthegermarea.Thisenabledquantitative

comparisonoftypicalgerminatedvs.ungerminatedpopulationsonthebasisoflipidtoprotein

peakarearatios.WiththeSpotlight,scutellaoftheeachsectionwereanalyzedinimagemodeby

selectingtwoorthreerectangularareascontainingbetween10003000pixelseach.Allthe

spectrawereextractedfromeachofthemapsandusedforfurtherexaminationsandcalculations.

From datacollectedinimagemodefunctionalgroupmapsandpeakarearatiomapswere

produced tocomparethelocalizedspectroscopicdifferencesbetweenofsproutedandunsprouted

kernels(OMNIC,Atlssoftware(ThermoFisherScientific,Madison,Wisconsin),andSpotlight

software(PerkinElmer,Shelton,Connecticut)wereusedtocontroldataacquisition.Malvern's

ISysImagingsoftwarewasusedtoallowfurtherdatatreatment). Initiallytheimportantpeaks

wereselectedandbandassignmentsweremadebasedonthepreviousstudiesonwheatdoneby

Wetzelandcoworkers.Peaksthatareindicatorsofdifferentcompoundssuchaslipid,protein

andstarchinwheatareshown.

37
BackgroundspectrumwereobtainedbymappinganareafreeofsampleonBaF2 diskwas

singlebeamscannedwith8cm1 resolutionand240scanscoaddedtoobtainabackground

spectrum.Thespectrumshowninfigure1.23wasratioedtothesamplespectrumtoproducea

transmissionspectrumfromwhichanabsorbancespectrumwascalculated.WatervaporandCO2

contributionswereremovedwiththiscorrection.

Figure1.23Backgroundspectrum showingrotationalbandsofwatervaporapproximately at
40003800cm1 andC02 at2360cm1
4.45

4.0

3.5

3.0

2.5

E
2.0

1.5

1.0

0.5

0.03
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800.0
cm1

Inthefigurebelow(Figure1.25),aspectrumobtainedfromscutellumareaofan

unsproutedkernelisshown.ThefundamentalvibrationsseeninthespectrumincludeeitherOH

orNHstretchvibrationalbandsat3300cm1.NotethattheOHbandshapeisroundfromstarch

or cellulosechemicalstructures,buttheNHstretchingvibrationproducesasharperpeakandit

isshiftedslightlyfromthe3300cm1 OHabsorptionband(Figure1.24).Thestrongabsorbance

oftheNHstretchinthespectrumindicatesthatthetissueisrichinprotein.CHvibrational

38
bandsat2927 cm1 (CH2 stretch)andat2850cm1 (CH3 stretch)regionarepredominantforthe

lipidcomponents.Asmallpeakatapproximately3015cm1 showsunsaturationofthelipidby

thestretchingvibrationoftheCHattachedtoaC=C.

Figure1.24Awheatcrosssectionandtotalabsorbanceimage.Aspectrumextractedfrom
endospermsideisshowingOHroundpeakat3300cm1.

OH

Figure1.25Aspectrumrepresentativeofthescutellumpartofanungerminatedkernelwith
assignedpeakssuperimposed.
1.240 1650.65
1.20
3291.44
1.15

1.10
2925.27
1.05

1.00

1544.71 1057.97
0.95

0.90

0.85 995.48
2854.51

A 0.80 1398.61
1454.14
0.75 1743.43

0.70 1235.94

0.65

0.60

0.55
2285.09
0.50

0.45
3743.94

0.390
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000
cm1

39
Uponexaminationoftheregion18001500cm1 inthespectrum,firstnotethatthe

strongbandat1740cm1arisesfromthecarbonyl(esterC=Ogroup)whichindicatesthe

presenceoflipid.Thenexttwoabsorptionbandsthataretwoprimaryfeaturesoftheprotein,

amideIandamideIIbandsatapproximately1660cm1 and1550cm1,respectively.AmideI

arisesfromthestretchofC=Oofthepeptidegroupintheprotein.TheamideIIbandismostly

fromtheNHbendingandsecondarilyfromtheeffectofCNstretch.Atapproximately1469

cm1,CHbendingvibrationsoccur.Inbiologicalspecimensthismayrepresentthecarbon

chainsofthelipid.Inthe11001025cm1 regionofthespectrumstrongcarbohydratebandsof

starchandcellulosearefound.Absorptionbandsoccurringat1420 cm1,1370cm1,1335cm1

areindicativeof thecelluloseorhemicelluloseinthesampleandP=Ovibrationsoccursat1235

cm1.

Heterogeneitycanbeexpectedforbiologicalmaterials.Planttissuehasvoidswhich can

increasethescatteringlosses.Alloftheseeffectscontributetobaselineshiftandslope.

Figure1.26showsaspectrumextractedfromamap thathasaseriouslyslopingbaseline.

Figure1.26A spectrumshowingbaselineshiftduetovoidsinthetissue.

40
Assignmentofthepeaksallowsustoknowwhichpeaksareimportantforthesample

beinganalyzed.Inthespectrumofthescutellumpartofthekernelwhichisrichinlipidand

protein,peaksforlipidandproteinarepredominantandsignificantforthisstudy.

Figure1.27Avisibleimageofwheatkernelcrosssectionandtheareamappedinred
rectangularandenlarged.

FunctionalgroupmapsareproducedbyplottingtheintensityofIRabsorptionbandsasa

functionof thex,yposition.Thesemapsshowthedistributionofaparticularconstituentinthe

sectionsandhelpdefinethechemicaldifferenceswithinthespecimen.Functionalgroupmaps

wereobtainedby calculatingthebaselinecorrectedareaunderthepeak.

Figure1.28Anexampleofbaselinecorrectedpeakareacalculation.Leftfigureshowssetting
thespectralrangeswhilerightfigureshowssettingthebaselinepoints.

41
Inthefigure1.28,anexampleofpeakareacalculationisshown.Firstly,thespectral

rangeisdetermined.Next, thebaselinepointsareinserted,andbaselinecorrectedpeakareais

obtained.Inthetable1.2 theintegratedspectralregionandbaselinepointsusedforparticular

peakarepresented.Theseareusedforthecalculationsinthisstudy.

Table1.2Thespectralregionandbaselinepointsusedforparticularpeaks.

Peak (cm1) SpectralRange BaselinePoints(cm1)


Integrated (cm1)
1025 1171890 1171890

1550 15831486 15931486

1650 17131605 17151592

1740 17671715 17731715

2927 29942842 29942817

Whenthebaselinecorrectedpeakareasunderpeaksat1740cm1 and1025cm1 were

calculated,thefunctionalgroupmapsforthelipidandcarbohydrate, respectivelywereobtained.

The1740cm1 functionalgroupmapshowsthelipiddistributionintheareapreviously

displayed.The1025cm1 functionalgroupmapindicatesthatthereislittlecarbohydrateinthe

scutellumarea.

Figure1.29Thefunctional groupmapsforthelipidandcarbohydratewhichareobtainedby
functionalgroupmapsat1740and1025cm1.The1025cm1 functionalgroupmapindicatesthat
thereislittlecarbohydrateinthescutellumarea.

1025cm1
1740cm1

42
AnotherfigurebelowshowstheamideIIproteinpeakareafunctionalgroupmapforthe

samesection.

Figure1.30Thefunctionalgroupmapforproteinat1550 cm1 showingthedistributionof


proteininthescutellumandthespectrumshowingtheareaunderthepeak.

3.ResultsandDiscussion

Fromdatacollectedinimagemodefunctionalgroupmapsandpeakarearatiomapswere

producedtocomparethelocalizedspectroscopicdifferencesbetweenofsproutedandunsprouted

kernels.

3.1.ResultsoftheexperimentsperformedatNSLS

Basedontheassumptionthatthescutellum storehousewasconsumedtoprovide

nourishmentfortheembryoatinitialstagesofgerminationbeforeendospermreservesare

mobilized,thispartofthekernelwasinitiallystudiedspectroscopically.Subsequently the

endosperm,withinthesamesectionsbeingcompared,wasalsoexamined.Changeinthelipid

storehouseinthevicinityoftheembryonicaxiswasthefocusofthisstudy.Biochemicalchanges

wererevealedviathelipid/proteinbandratiofunctionalgroupmapswithinthescutellum

(adjacenttotheembryo)area.

43
Figure1.313Dchemicalimagesofthesamewheatkernelcrosssectionshowingtherelative
populationlipid(a),protein (b),andstarch (c).Thepeakareamapsof1740cm1,1550cm1,and
1025cm1,respectively,resultfrompeaksmarkedinredonthelipid,protein,andcarbohydrate
spectraextractedfromthecorresponding3Dmap.

a. b. c.
c.

Thechemicaldistributionoftheeachbotanicalpartisshownwith3Dfunctionalgroup

mapsinfigure1.31.Chemicalimagesofunsproutedkernelfunctionalgroupsareshownforthe

carbonyl,amideII,andcarbohydratebandsrespectively.(a)highlightslipidinthescutellumand

aleuronecellwalls,(b)showstheproteininscutellumandaleuronecells.Theproteinpopulation

isthehighestintheembryonicaxis.(c) thebaselinecorrectedpeakareaat1025cm1 highlights

thecarbohydrateintheendosperm.From functionalgroupmaps,theproteinrichembryonicaxis

wasclearlydistinguishedfromthelipidrichscutellumandbothofthesebotanicalpartswere

readilydistinguishedfromthecarbohydrateoftheendosperm.

44
Figure1.32Unsproutedkernel3Dchemicalfunctionalgroupmapsofa)thescutellum(lipid)b)
embryonicaxis(protein)c)endosperm(carbohydrate),alsocorrespondingcontrastforeach
botanicalpartfromselectPCAfactormaps.Allimagesarefromthesameimagingdatacube.

1740cm1(carbonyl) 1550cm1(amideII) 1025cm1(carbohydrate)

PCAFactor1 Factor2 Factor3

Figure1.33Sproutedkernel3Dchemicalfunctionalgroupmapsofa)scutellumb)the
developedproteinaceousembryoandc)theendospermnexttotheembryothatisvirtuallyvoid
withrespecttocarbohydrate.NotecontrastalsoincorrespondingPCAfactorimages.

a. b. c.

1740cm1(carbonyl) 1550cm1(amideII) 1025cm1(carbohydrate)

PCAFactor1 Factor2 Factor3


Factor3

Dataforsectionsofsprouted(36hr)andunsprouted(3hr) wheatmappedbymidIR

microspectroscopyareshownin3Dfunctionalgroupmaps(figures1.33and1.32)forlipid

(1740cm1,carbonyl),protein(1550cm1,amideII),andcarbohydrate(1025cm1).Falsecolor

45
contrastforthesesameareaswasobtainedfromthesamerawdataapplyingprinciplecomponent

analysis(PCA)toobtainfactorimagesshown.Figure1.32 isoftheunsproutedkernel.Sprouted

kernelfunctionalgroupmapsforthecarbonyl,amideII,andcarbohydratepeaksareshownin

figure1.33. NotereadilythehighamideIIpopulationintheembryonicaxisof figure1.33bin

comparisontothesamecorrespondingmapintheFigure1.32b oftheunsproutedkernel.This

showsdevelopmentoftheembryoandthebuildupofproteinasitisdeveloping.Theimage

Figure1.33a totheleftofthatembryoexhibitsaridgeoflipidthatisbetweentheembryoand

theendosperm(carbohydrate).The3Dcarbohydratefunctionalgroupmap(figure1.33c) tothe

rightoftheamidemapisadepressioncorrespondingtothelocationoftheembryo.In addition,

mapat1025cm1 indicatesamobilizationinthecarbohydratereserves.Falsecolorimagesinthe

correspondingprinciplecomponentanalysisfactorsof figure1.33showcontrastcorresponding

tothesesametwobotanicalparts.

Figure1.34Graph showingthepopulationofpixelswithhigherlipid/proteinpeakarearatios
withinthescutellumandembryoregion.a)Theungerminatedkernelhasahigherpercentage
(66%)ofpixelswithspectrahavingahigherlipid/proteinpeakarearatiogreaterthan0.6.b)The
kernelsproutedfor36hrhadreducedlipidinthescutellum(41%ofspectrahadapeakarearatio
greaterthan0.6).

Thelipidtoprotein(1740cm1 /1550cm1 )ratioswerecalculatedfromspectraofthe

pixelsextractedfromthescutellumandembryosectionimagesofungerminated(3hr)and

46
sprouted(36hr)kernelsofthesamevariety.Populationsoftheseratiosareshownasgraphs

(Figure1.34)fortheunsprouted(control)andsprouted(36hr)kernels,respectively.Theratio

meanof0.98forpixelsfromtheunsproutedscutellumwascomparedtoameanof0.74for

correspondingpixelsofthesproutedkernel.Theseresultsindicateashiftfromthelipid

populationtoaproteinpopulation.Inthegraph(upper)oftheunsproutedkernel,739of1160

pixelshadabsorbancepeakarearatiosgreaterthan0.6.Thegraph(lower)ofthescutellafrom

sproutedkernelhadonly275of823pixelsbeyondthepeakarearatiosof0.6.Thesedata

representourfirstattemptatsemiquantitationofthechangeinthechemicalbalancebetween

thesetwofunctionalgroups.

Figure1.35(a) and(b)havethreecolumnseachtocompareimagesofsproutedandunsprouted
kernelsandsectionsfromthesekernels.Theleftcolumnscontainthelog1/RnearIRimageof
thatwheatkernel.AdditionalimagesinthesamecolumnsarefromPCAfactorsthatshow
developedembryoincontrast tothebodyofthekernel.InthecentercolumnarethemidIR
microspectroscopicimagesthatshowthecurvatureofthedevelopedembryoandadjacenttissue
outsideofthearcandselectPCAfactorimages.Infigurebthegeometricfeaturesofadeveloped
embryoareabsentinallthreecolumns.

47
Thekernelsectionsshowninfigure1.35wereproducedfromwholekernelsthathad

been testedforgerminationinaseparatestudydesignedtonondestructivelydetectgermination

bysubsurfacepolychromaticcontrast. In figure1.35a of sproutedandb unsproutedkernelsa

columnofnearinfraredwholekernelimagesisshown.Ineachcolumnthetopimageisoflog

1/Rvaluesatasinglewavelengthforeachpixel.Notethat thegermareaontherightendofthe

kernelhasprominentcontrastinfigure1.35a. In figure1.35 b thereisnoevidenceofa

developedembryo.Wholekernelprinciplecomponentfactorimagesofthesproutedkernel

clearlyshowevidenceofadevelopedembryo.Incontrastthereisnosuchprominentfeaturein

thewholekernelnearinfraredimagesoffigure1.35b.Infigure1.35aandbtotherightofthe

wholekernelnearinfraredimagecolumnsareshownanothercolumnofmidIR

microspectroscopicfunctiongroupmapsat1740cm1,1550cm1,and1025cm1.Theseimages

wereobtainedfromsectionsproducedfromthepreviouslynondestructivelyanalyzedwheat

kernels.Thecurvatureshowninthemicrospectroscopicimagesrevealthelineofdemarcation

betweenembryo(lowerright)andthebodyofthekernel(upperleft).Thissamecurvatureis

observedwithselectprinciplecomponentfactormapstotherightoftheactualmidIR

microspectroscopicimages.Incontrast,themidIRmicrospectroscopicfunctionalgroupmaps

andPCAfactormapsoftheunsproutedkerneldonotshowthesamedegreeoforderor

differentiationbetweentheembryoandtheadjacenttissue.

Afterthepreliminarydatashownintheprecedingfigureswasobtained,thefirstattempt

tomeasurethelipidtoproteinpeakarearatioswasdonewithaseriesoflinemapsacrosswithin

thegerm areaattheNational SynchrotronLightSource. Toobtaintherelativelipidpopulation a

numberoflinemapswerecarriedoutwiththeunsproutedandsproutedkernelsrepresenting

48
severalhardwheatvarieties.Figure1.36showsphotomicrographsoftwosectionswiththemap

linessuperimposed.

Figure1.36Linemapacrossthegermofunsprouted(a)andsproutedkernels(b).Average
lipid/proteinvaluesplotted(1.1)aregreaterfortheunsproutedscutellumthantheaverage(0.6)
offirstfiveandlastfiveoneithersideofthesproutedroot.
a. b.

The2927cm1 CH2 stretchingvibration peakareawasusedtorepresentthelipid,andthe

1650cm1 (amideIband)wasusedtorepresenttheprotein.Theratioaveragefrompoints115

alongthelinemapfortheunsproutedsectionwas1.1.Incontrast,forthesproutedkernelofthe

samevariety,thefirstandlastfivepointstakenoneithersideoftheembryohadanaverageratio

of0.6. Theearly imagingevidenceandsubsequentlinemappingareindicativeoftherelative

populationofscutellumlipidinthesproutedkerneltothatoftheunsproutedkernel.Following

theseexperiments,aseriesofquantitativeexperimentswasundertakenwith scanningmaps

including10003000spectra.

3.2.Resultsof thequantitativeexperimentsdoneatKSU

In thephotomicrographofawheatkernelcrosssectionfigure1.37 threerectangles(red)

showtheactualareasofthescutellumofthiskernelfromwhichspectrawerecollected.An

49
artistssketchofawheatkernelsectionisincludedtoshowtheembryoandscutellumwithinthe

germin relationtotheotherbotanical parts.

Figure1.37Areaswithinthescutellumoutlinedinredonthephotomicrographarethose
mapped.Notethecorrespondingbotanicalpartsfromthesketch.

Thethreesectionsforsproutedandthreesectionsforunsproutedkernelsfromeachofthe

varietiesTrego,KS2174,andDanbyweremappedandlipidtoproteinbandratiomapswere

producedby calculatingtheratioof thebaselinecorrectedpeakareasatthe1740cm1 and1550

cm1.Bycomputingthepeakratio(Figure1.38)anyvariationsduetothicknessdifferenceswere

eliminated.

Figure1.38Lipidtoproteinpeakareasratioed.

50
Thenumericalratioswereassembledinanexcelfileandbasicstatisticalcalculations

wereperformed.Thecalculatedresultsarereportedbelow.Maximum,minimumandmean

valueswerecalculated.Alsostandarddeviationsandstandarderrorsforeachgroupwerelisted.

Table1.3Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforTrego
controlsections.
section map area max min mean std se pixels
1 1 1 2.62 0.93 1.56 0.23 0.0078 910
2 1 1.59 0.77 1.13 0.12 0.0027 2331
2 1 1 1.60 0.55 1.11 0.13 0.0030 1863
2 2 1.59 0.63 1.06 0.15 0.0040 1300
3 1 1.53 0.72 1.07 0.11 0.0030 1148
3 2 1.44 0.56 1.04 0.12 0.0040 855
3 1 1 1.30 0.34 0.81 0.13 0.0030 1705
1 2 1.44 0.52 0.92 0.16 0.0060 629
2 1 1.58 0.59 1.01 0.14 0.0030 1716

Table1.4Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforTrego
germinatedsections.
section map area max min mean std se pixels
1 1 1 1.03 0.33 0.81 0.09 0.0030 711
1 2 1.22 0.48 0.87 0.10 0.0020 1512
2 1 1.29 0.49 0.98 0.12 0.0050 529
2 1 1 1.02 0.50 0.74 0.07 0.0018 1552
2 1 1.17 0.45 0.81 0.12 0.0050 500
2 2 1.10 0.53 0.81 0.10 0.0030 896
3 1 1 1.54 0.48 0.84 0.12 0.0050 576

Table1.5Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforKS2171
controlsections.
section map area max min mean std se pixels
1 1 1 1.89 0.62 1.15 0.14 0.0036 1440
1 2 1.61 0.54 1.14 0.18 0.0091 408
2 1 2.1 0.62 1.20 0.18 0.0046 1543
3 1 2.53 0.6 1.13 0.15 0.0039 1452
2 1 1 1.83 0.32 1.03 0.17 0.0028 3366
2 1 1.82 0.36 1.00 0.16 0.0026 3975
3 1 1 1.25 0.65 1.00 0.08 0.0020 1586
2 1 1.32 0.62 1.00 0.09 0.0022 1645
2 2 1.22 0.61 0.95 0.12 0.0039 861

51
Table1.6Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforKS2174
germinatedsections.
section map area max min mean std se pixels
1 1 1 1.59 0.53 0.98 0.14 0.0029 2247
2 1 1.50 0.48 0.92 0.14 0.0033 1632
2 2 1.40 0.43 0.88 0.14 0.0044 936
2 1 1 1.32 0.42 0.86 0.12 0.0024 2448
2 1 1.55 0.46 0.90 0.15 0.0027 2904

Table1.7Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforDanby
control sections.
section map area max min mean std se pixels
1 1 1 1.63 0.74 1.11 0.15 0.0083 345
2 1 1.68 0.79 1.16 0.14 0.0079 314
2 1 1 1.17 0.67 0.94 0.10 0.0053 360
3 1 1 2.09 0.40 0.94 0.22 0.0080 774
2 1 2.51 0.57 1.18 0.30 0.0120 636

Table1.8Maximum,minimum,meanvalues,standarddeviation,andstandarderrorforDanby
germinatedsections.
section map area max min mean std se pixels
1 1 1 1.03 0.43 0.71 0.080 0.0025 1008
2 1 1.10 0.45 0.75 0.095 0.0032 861
2 1 1 1.12 0.45 0.76 0.085 0.0023 1302
3 2 1 1.18 0.40 0.82 0.126 0.0030 1760

52
Figure1.39Photomicrographsof sproutedandunsproutedcrosssectionsofthevariety Trego
withthecorrespondingspectraand3Dmaps. Redboxesindicatetheareamappedinthese
sections.

53
Figure1.40Photomicrographsof sproutedandunsproutedcrosssectionsofthevariety KS2174
withthecorrespondingspectraand3Dmaps.

Figure1.39and1.40showphotomicrographsof sproutedandunsproutedcrosssections

ofthevarietiesTregoandKS2174respectively withthecorrespondingspectraand3Dmaps

images,spectra,andmaps.Notethedifferenceinthemeanratiosbetweentheunsproutedandthe

sproutedsamples.Figure1.41hasphotomicrographimages,spectra,andfunctionalgroupmaps

ofDanbyforsproutedandunsproutedkernel.QuantitativevaluesforthevarietyDanby appear

intable.

54
Figure1.41Photomicrographsof sproutedandunsproutedcrosssectionsofthevarietiesDanby
withthecorrespondingspectraand3Dmaps.

Table1.9Tabulatednumbersofpixelsandthelipid/proteinratioforunsproutedandsprouted
scutelliofthreedifferentwheatcultivars.Resultsshowaveragedspectraldifferencesbetween
21,374sproutedand32,359unsproutedkernels.

Sample pixels max min mean std se


Trego0 12,457 1.63 0.62 1.08 0.14 0.0041
Trego24 6,276 1.20 0.47 0.84 0.10 0.0035

KS21740 17,473 1.82 0.55 1.08 0.15 0.0042


KS217424 10,167 1.47 0.46 0.90 0.14 0.0031

Danby0 2,429 1.82 0.63 1.07 0.18 0.0083


Danby24 4,931 1.11 0.43 0.76 0.09 0.0028

55
Thedatatablesforthreecultivarsprovidequantitationfrom53,733spectraobtainedfrom

31mapscollectedfrom17sections(Table1.9).Eachvariety wasrepresentedbybothsprouted

andunsproutedkernels.Inallcases,itwasobserved thatthelipidtoproteinratioisgreaterthan

1.0intheunsproutedandsignificantlylowerthan1inthesproutedkernelsections.Atotalof

32,359spectrafromthescutellaofunsproutedwheatwerecomparedto21,374spectrafrom

sproutedwheat.Fromallthesenumericaldata,thegrandmeanshoweda23%reductionofthe

lipidtoproteinratiointhescutellumresultingfromthegerminationprocess.

Thereductioninthelipidtoproteinratiocanbecausedbythedepletionofthescutellum

toprovideenergyforthegrowingembryo.Anotherreason,itmaybeduetoincreaseinthe

starchandproteinwhicharetransferredfromendospermforthegrowingembryo.Swiftand

OBrien(1972)foundthatafter24hrlargemassesofpartlyhydrolyzedproteinsareformedby

coalescenceofthebodiesinthescutellum.Therearecontroversystudiesregardingthelipid

consumption.SwiftandOBrien(1972)foundthatafter24hrtheybecamelesscommonnear

thecellmembranesoftheepitheliumcells.Inthelaterstagesofgermination,thereisareduction

intheirnumber,howevertheirutilizationisslow.Insomestudies,ithasbeensuggestedthatthe

starchinthescutellumisderivedfromthelipidbodies.TavenerandLaidman(1972)observed

thattriglyceridereservesweredegradedprogressively.Furtherinvestigationisneededtoexplain

thebiochemicalchangesduringgermination.Otherthanscutellum,embryonicaxisand

endospermofthekernelshouldbestudiedtoexplaintheobservationsbetter.

4.Conclusion
ThegerminatedkernelsselectedfromnondestructiveNearIRimagingexperimentswere

sectionedforsynchrotronMidIRmicrospectroscopy.Midinfraredfunctionalgroupmapsof

sectionshighlightedkernelmorphologicalstructuresinthegermareabasedonthepredominance

56
oftheembryosprotein chemicalcomposition.PCAfactorimagesalsohighlightedtheselect

botanicalpartsincludingthescutellum,embryonicaxis,andendosperm.Sproutedkernelswere

distinguishedfrom unsprouted oneofthesamevariety.Areducedlipidpopulationwasobserved

inthescutellumnexttothedevelopingembryointhe1740cm1 (carbonyl)functionalgroup3D

imageincomparisontothatoftheungerminated(control)kernel.Semiquantitativecomparison

wasmadeon asubsequentsynchrotron linemaps.Thiscomparisonwasbetweenpairsof

sectionsofgerminatedandungerminatedkernelsfromthesamelotrepresentingmultiplewheat

cultivars.Thelipid/amideIratiowascalculatedforspectraof individualpixelsfromlinemaps

acrossthegermarea.Thesedatasupportedpreviousobservationsfrom3Dchemicalimages.

Finally,basedonthepreliminarysynchrotronworkcarefullypreparedsections,representing

threegerminatedcultivarsandtheirungerminatedcontrolswereimagedattheKansasState

University MicrobeamMolecularSpectroscopyLaboratorywithaSpectrumSpotlight300

(PerkinElmer,SheltonCT)witha6.25mx6.25mpixelsize.Fiftythreethousandspectra(32

K unsproutedand21K sprouted)werecompared.Anaverage23%reductioninlipid/protein was

observed.Thesedatawereobtainedfrom 31mapscollectedfrom17sectionsinvolvingsix

differentkernels.Foreachmapthescutellum lipidpopulationofthesproutedkernel was

comparedtothatofacorrespondingunsprouted(control)kernel.Thenumericallipid/protein

ratioswereconsistent.A reductionof scutellumlipidasaresultof theearly germinationprocess

canbecausedbythedepletionofthescutellumtoprovideenergyforthegrowingembryo.

57
PART2 EARLYNONDESTRUCTIVEDETECTION:

NEARINFRAREDCHEMICALIMAGINGOFWHEAT

GERMINATIONTOASSISTPLANTBREEDING

1.Introduction

Germinationof thekernelsintheearbeforeharvestthatmayoccurwhenthereare

unfavorablehumidconditionsisknownaspreharvestsprouting.Cerealgrowingregionsofthe

worldwherethereishighprobabilityofrainduringharvestareliabletopreharvestsprouting

suchasEasternEurope,Chile,Argentina,Brazil,SouthAfrica,Australia,andManitobain

Canada.IntheUS,thePacificNorthweststatesareexamplesofplacesthathavesufferedsome

yearsfromthisproblem(Derera,1989).Thechangesinchemicalcomponentsofthewheat

kernelwiththegerminationprocesshavedetrimentaleffectsontheenduseofthewheatand

causeimportanteconomiclosestothefarmers.Thesedetrimentalchangesarereferredas

preharvestsproutdamage(8).Preharvestsproutingresultsfromthelackofdormancy.Tolerance

topreharvestsproutingandembryodormancyarehighlydesirablecharacteristicsthatplant

breedersstudy(9).

1.1.SproutDamage

Rainfallduringharvestmaycausedifferentdegreesofdamagedependingonvariety,

maturitylevel,thetemperatureandtheexposuretimetounfavorableweatherconditions(8).The

harmfuleffectofthesproutdamagecannotbeeliminated.Interestinwhitewheatisincreasing

incurrentbreedingprogrambecauseitgivesthepossibility ofincreasingflouryieldwithout

affectingthecolor,andofferinghealthbenefitsfromretainedantioxidants.Wheatgrowersare

reluctanttogrowwhitewheatthatissubjecttosprout.Historically,whitewheatwasconsidered

58
highlysusceptibletosproutdamage(25).Currentbreedingprogramsstrivetolimitsprout

susceptibility.

Thepreharvestsproutingdamageaffectsthequalitythecerealbasedproductssuchas

bread,pasta,andbeeradversely.Withtheincreaseofsproutingdoughrheologicalproperties

measuredbyFarinographareweakenedandbreadqualityisdegraded.Becauseoftheexcessive

enzymatichydrolysisofthestarch,waterabsorption,gasretention,doughhandlingandtexture

oftheproductareaffected,doughbecomesstickier(damagedstarchholdslargewater).In

additionchangesinglutenproteinsbyhydrolyticenzymesareeffective(22).Breadprepared

withspoutedwheathaslowerloafvolumeandstickycrumb(51).

1.2.SproutTolerance

Asignificantagronomicobjectiveofthebreedingprogramisimprovingtheresistanceof

anewvarietytopreharvestsprouting.Suchresistancecanoriginatefromthegrainitselforitcan

becausedbythenongrainearcomponents.Resistanceassociatedwithgrainitselfisgenerally

duetothedormancyofthegrain.Dormancyretardsordelaysgerminationoftheseedunder

favorableconditions.Therearetwotypesofdormancyitcanbeexternalwhichisseedcoat

imposedor internalembryorelateddormancy.Seedsmaydevelopwithdifferentlevelsof

primarydormancyatharvestmaturity.Therelationshipbetweendormancyandgerminationis

showninthefigure2.1(52).Secondarydormancywhichpreventsgerminationafterimbibititon

ofwatercanbeinducedbywaterstress,anoxia,orhypoxia(toomuchor toolittleoxygen)or

temperatureextremes(5,7,53).

Whenapplyinggerminationteststheviabilityshouldbeverified.Kernelsthatfailto

germinateforreasonsotherthandormancymaythusbeexcluded(11).Redwheatvarietieshave

historicallybeenmoreresistantthanwhitewheats(54).Andreolietal.(9)reportedthetwomajor

59
geneswhichcontrolthedormancyandstatedthatthereisnomaternaltissueinfluence.Miuraet

al.(55)identifiedchromosomescarryinggenesforseeddormancy.Mares(52)studied

preservationofthedormancyofwheatafterharvest.Itwasshownthatduringthepostharvest

storageofthefreshlyharvestedgraininanairconditionedseedroommaintainedat12C,the

dormancydisappearedin1014daysforthepartiallydormantvarietiesandin2.53monthsfor

thestronglydormantvarieties.Incontrast,whenkernelwasstoredat15C,dormancywas

preservedandgerminationcharacteristicwerethesameasthoseoffreshlyharvestedwheat.

Figure2.1Dormancyandgermination(reprintedfromref.17b)

Thebreedingofdormantvarietiesrequiresthebreakingofdormancybecausetheydonot

germinateifplantedimmediatelyafterharvest.Theapplicationofgibberellicacidandchillingto

4Ccanbeusedtobreakthedormancytobreedwheatswithpreharvestresistance(54).

1.3.DeterminationofSproutDamage

Anobjective,reliable,andreproducibletestisarequirementtoassessthespoutdamage

fordifferentpurposesfromthepointofviewsofgraingrowersandgrain buyers.Unreleasedtest

linesshouldbetestedforthetolerancetopreharvestsprouting.

60
1.3.1.Visualassessment(nakedeye)

Visualinspectionistheonlynondestructivemethodamongsproutdamagetests.Visual

inspectioncanbedoneonthekernels,andbreedersandgraininspectorscountthepercentageof

kernelswhicharevisiblysprouted.Thismethodhaslowsensitivityanddiscriminationpower

comparedtoobjectivemethods(11,56).

1.3.2.Viscositymethods

Theviscositymethodsaredestructiveandappliedtogroundwheatorflour.Inthese

methods,instrumentsareusedtomeasurethickeningthestarchwatersuspensionbyheatingto

itsgelatinizationtemperatureduringmixing.Enzymeactiondecreasestheviscosityofthe

suspension.Thereareuncontrolledsourcesofvariationduetonaturaldifferencesinpasting

propertiesofstarches(57).WaxywheatwithhigheramylopectincontenthaslowFalling

Number(FN)althoughenzymelevelisnotincreased(58).Allthesemethodsaredestructiveand

notapplicabletosinglekernels,theyrequirebulkofsamples.Inaddition,theyarenotsensitive

enough.LowFNintheabsenceofgerminationcanbeobservedduetohighenzymelevelfrom

improperdevelopment,latematurityalphaamylase(25,11,58).

Amylograph:TheAACCapprovedBrabenderamylographmethodismostsensitivetothesprout

damagecomparedtootherviscositytests.Amylographisaviscometerthatmeasuresthe

amylaseeffectonflourpasteviscosityasafunctionoftemperature.Totaltesttimeisbetween

6090min(25,11).

FallingNumber: TheAACCapprovedfallingnumbermethodisdevelopedbyHagberg(1960)

andPerten(1964).Aftermixingofthesuspensionintheviscometerplungerisraisedand

allowedtofalldown.Thetimerequiredtofallacertaindistanceismeasuredinseconds.The

61
fallingnumberforasoundkernelishigh(450550sec),withthesproutdamageand

accompanyingincreaseof amylaselevels,thefallingnumberdecreases(minimum60sec).

RapidViscoAnayzer(RVA):Thismethodwasdevelopedasaquick,simpletestsuitablefor

field.Thismethodgivesresultsequivalenttothefallingnumbertest.Theslurryofmilledgrain

andwaterisheatedandstirred(58).

1.3.3.Enzymaticmethods

DirectAssayofAmylaseactivity

Theviscositymeasurementsshowtheextentofinteractionbetweenamylaseactivity

anditssubstratestarch.Directassaysmeasuretheamylaseactivity onasubstratedependon

theseparationoftheenzymefromthesample.Sinceanaddedsubstratewasused,theresultis

lessdependentonthevariationsofendogenousstarchproperties(58).

LightScattering:Oneofthetechniquesisnephelometry,wheretherateofdecreaseinlight

scattering,nephelos(turbidity)ofalimitdextrinsolutionisrecorded(191GrainAmylase

Analyzer,PerkinElmer).Itincreaseslinearlywiththeamountoftheenzyme.

Colorimetric:Adyelabeledstarchsubstratecanbeusedtodeterminetheenzymeactivity.Dye

moleculesareincorporatedintostarchbycovalentbonding,duringthestarchdigestiondye

releasesintosolutionandcolorismeasuredasanindicationofamylaseactivity(25,11).

FluorescenceMethod:Fluorescentsubstancesareusedtodeterminetheenzymaticreactions.A

fluorochromehasbeenappliedtostudythegerminationofthecerealsbyCarlsbergResearch

Center,Denmark(11).

Otherenzymes:Inadditiontoalphaamylase,theotherenzymesthatareproducedin

germinationcanbeusedtodeterminesproutdamage.

62
1.3.4.ImmunologicalFieldTestMethod

Theenzymelinkedimmunosorbentessay(ELISA)testkittestwasdevelopedinorderto

determinesproutdamagerapidlyinthefield.Itisacolorimetrictestbasedonthedetectionof

amylaseenzymebyspecificantibodies.Inthetest,theextractofthegrainismixedwiththeanti

amylasereagentandacolordevelopingantibody.Thedegreeofthedamageisdeterminedbythe

intensityofthedevelopedcolorin afewminutesbythesandwichingtheextractedenzyme

betweenlabeledandimmobilizedantibodies.Thistestdeterminesthepresenceoftheamylase,

not theactivity(58,59,60).

1.3.5.Othermethods

Density:Donelsonetal.(57)developedadetectionmethodforpreharvestspoutingbyusinga

pregelatinizedstarchsubstrateandcentrifugation.Inthemethod,increasedalphaamylase

activityhydrolysesthepregelatinizedstarchcausingareductionintheweightofcentrifugate.It

isamicrotest,0.3gof sampleand0.2gpregelatinizedstarchareused.Methodismoresensitive

tolowlevelsofamylase.

NearIRAnalysis: Therehavebeenattemptstodestructivelypredictsproutdamagewithnear

infrared(51,61,23,62)correlatedtostandardenzymaticorviscositytests(fordetailssee

appendix)

Note:

Evenintheabsenceofvisiblespoutinginsomecases,theproductionofamylaseduetoearly

stagesofsprouting,maycauseprocessingproblems.Thereforeasensitivetestmethodisneeded.

Untilnow,themethodsfordeterminationofsproutdamagehavebeendestructiveandnot

necessarilysensitive(63).Theonlypreviousnondestructivewayhasbeenvisualassessment

whichcannotdetectthegerminationbefore36hr. Thenondestructiveandsensitivechemical

63
imagingapproachtodetectionofgerminationinintactwheatkernelswasrecentlyintroduced,in

a2006articleentitledChemicalimagingofintactseedswithnearIRfocalplanearrayassists

plantbreeding(63).

1.4.NearIRSpectroscopyandImaging

NearIR(7502500nm)regionofthespectrumarecomposedofcombinationand

overtonesofthefundamentalstretchingandbendingvibrationswhicharenotintenseandsharp

incomparisontomidIRregionwithsharp,strongfundamentalvibrations.Bandsarebroadand

weak,theyoccurathigherfrequenciesthanfundamentalvibrations.Molarabsorptivitiesarelow,

reasonablylinear,andfollowBeerslawthereforenearIRismoresuitableforthequantitative

analysis.ThenearIRtechniqueisrapidandnondestructive.Itisverysuitableforonline

applicationsandhasmanyapplicationsinanumberofindustriesbecausethereisnorequirement

ofsamplepreparation.

Lowabsorptionbandsprovideabetterpenetrationbelowthesurface.NearIRimaging

withdeeperpenetrationofshorterwavelengthsprovidessubsurfacepolychromaticcontrast(35).

NearIRimaginggivesanswerstothequestionsWhat,Howmuch,andWhereby

quantitativeandspatialdistributionidentificationofthechemicalspeciesinasample.NearIR

imagingcanbeappliedinvariousareassuchasnondestructivedeterminationofpharmaceutical

tabletcomponentdistributionandconcentration,determinationof particlesizeanduniformity,

anddistribution ofcomponentsinmixtures.

Atpresent,therearescanninganddiscretewavelengthinstruments.Initially,several

interferencefilterbasedinstrumentwerecommonlyused.Slowscan,gratingmonochromators,

andrapidvibratinggratinginstrumentswerebuilt.Randomwavelengthaccessandrapid

responsephotodiodedetectors(InGaAs)havebeenintroducedandenhancedthedataacquisition

64
speed.Electronicwavelengthswitchinginstrumentsincludeacuostooptictunablefilter(AOTF)

(64,64a),liquidcrystaltunablefilter(LCTF)(65),HadamardTransfer(HTIR)(66),anddiode

arrayspectrographs(67)(67a).

CommonNearIRimagingsystemsarerecentlyavailablein11002400nmregionwith

FPAdetectorsandwithaliquidcrystaltunablefilter(LCTF)oraFTspectrometer.Budevska

(31)reportedstudieson nearIRimaging.VerynearIRimagingstudieswerecarriedoutinthe

siliconphotoconductiveregionof7001100nmbyTaylorandMcClure(1992)withaCCD

cameraandnarrowbandinterferencefilters.Thedistributionofthechlorophyllandmoisturein

plantmaterialwasexamined.Sugiyama(68)accomplishedthevisualizationofsugarcontentof

melonwithaCCDcameraandasingleinterferencebandpassfilter.Nearimagingoftheinsect

detectioninsidewheatkernelwasreportedattwowavelengths(RidgewayandChambers,1998)

byusingwithphotoconductivevidiconcamera.

AliquidcrystaltunablefilterbasedinstrumentwasintroducedbySpectralDimensions/

Malvern(Columbia,MD).ItcanoperatewitheitherInGaASorInSbphotovoltaicdetectors.The

InGaAsoperatedinthe11001700nmwavelengthrangewhileInSboperatesinthe12002400

nmrange.Thistechniquewasappliedtodetectgerminationofwheatkernelsnondestructively

(63),forbacterialidentification(69),andforanalysisofpharmaceutical tablets(70,71).In

addition totheliquidcrystaltunablefilterbasedinstrumentofSpectralDimensions/Malvern

(Columbia,MD), aFTNearIRinstrumentwithfastrecoveryNIRdeuteratedtriglycinesulphate

(FRNIRDTGS)detectorfortransmissionandindiumgalliumarsenideforreflection FPAis

producedbyPerkinElmer(Danbury,CT).TheMicrobeamMolecularSpectroscopyLaboratory

atKSUworkisongoingnearIRinimagingofthecerealsandotherbiologicalmaterials.

65
Bandsin theNearIRregionofthespectrumareoverlappinganditisthereforemore

difficulttointerpretandassignthebandsthaninthemidIRregion.Thevibrationsoflightatoms

withstrongmolecularbondsarepredominantlyseeninthisregion(72).Whentheatomsare

heavyandthebondsareweak,thenthefrequencieswillbelow,andovertonesarenotdetectable

abovebaselineinthenearIR.Becauseofthisreason,mainlybondscontaininghydrogenatoms

attachedtoO,N,andCatomscanbeobserved.InnearIRspectroscopyofthesolids,the

analysisisbasedonmostlydiffusereflectance(73,74).Whenthelightbouncesoffasmooth

polishedsurface,theangleofreflectionwillbeequaltoangleofincident.Thistypeof

reflectanceiscalledasspecularreflectance.Thespecularreflectanceisincreaseswiththe

absorption.Theobjectsinthelifearenotveryflatorpolishedtheyhaveroughsurfacesand

showdiffusereflectance.Theangleofthereflectionisnotequaltoangleofincidentthereisa

changeinthedirection ofthereflection.Whenlightentersthroughasample,itisscattered

withinthebodyandincidentlightinteractswithsampleinseveralmodes.Thesuccessionof

eventsscatterabsorbs,scatterabsorb,scatterabsorbtakesplace,andfinallyaportion ofit

scattersbackfromthebodyofthesample.Thisiscalleddiffuselyreflectedlightanddiffusely

reflectedlightispickedupbythedetector(Figure2.2).

Indiffusereflectancethepathlengthisnotwelldefined.Itchangesinverselywiththe

absorptivityofthesample.InclassicalDRIFTspectroscopy,aKubelkaMunkfunctionisused

themakethespectralresponselinearwithconcentration.f(R)=(1 R)2 /2 R =KC

WhereR issamplereflectanceatinfinitedepth(nonabsorbingmatrix),Kis

proportionalityconstant,Cisconcentration,f(R)ispseudoabsorbance.Thisequationgivesa

reflectioninverselogfunction.Kisbasedontheratioofabsorptioncoefficienttoscattering

coefficient.

66
IfabsorptionisstrongcomparedtoscatteringasitisinthemidIRregion,thena

nonlinearrelationbetweenreflectanceandsampleconcentrationisobtained.Insuchcasethe

specimensaredilutedwithKClandtheKMfunctionisplotted.Whenthediffusereflectance

modeisappliedtonearIRwherethescatteringisverystrongandtheabsorbanceisrelatively

weak,quantitativemeasurementsareobtainedwithoutKMfunction.Inthiscase,empirical

relationshipandmultivariatestatisticseliminatetheneedfordilutionandKMfunctionofferno

benefit.

Figure2.2Variouspathwaysofdiffusereflectance(reprintedfromref.72)

2.Experimental

2.1.Instrumentation

Thedatainthisstudywerecollectedwiththecommerciallyavailablechemicalimaging

system MatrixTMNIRandSapphireNIRofSpectralDimensions/Malvern(Columbia,MD,

USA)whichwasequippedwitha320x256pixelindiumgalliumarsenide(InGaAs)and indium

antominade(InSb)focalplanearray(FPA)detectionsystems,respectively.Withthelensused,

67
eachdetectorelementproducedanominalpixelsizeof38mx38m.Theimagingsystem

includealiquidcrystaltunablefilter(65)forelectronicwavelengthswitchingandappropriate

opticstopreferentiallycapturediffuselyreflectedlightfromeachspecimen (Figure2.3).Kernels

onthepolishedmountingplatewereilluminatedwithfourtungstensourcelampsand

backgroundofspecularlyreflectedradiationwasdirectedawayfromthecollectinglenses.

Opticaldesignoftheinstrumentallowschangingthemagnificationwithdifferentlenses.ISys

chemicalimagingsoftwarepackageversion4.1.1,providedbySpectralDimensions/Malvern,

enabledsimultaneousprocessingofspectraof81,920pixelsinasinglefieldofview.Thesample

mountingplatesweredesignedspecificallyforthenondestructivetestingofmultiplekernelsand

werelocallycustommadeforprepositioningthethirtykernelsinthefieldofviewofthe

commercialimagingsystemused.

Figure2.3SchematicofnearIRimagingsystem

Immediately afterinstrumentstartup,theprogramperformsaninitializationstepduring

whichtheinstrumentwascheckedandfinetunedforthebestqualityandreproducibledata.

Datacollectionwascarriedoutwiththedataacquisitionprogram(SapphireGo)providedwith

68
thesystem.Datawereacquiredinthespectralrange12002400nmat10nmintervalsand16

imageswerecoaddedperwavelength.Thefieldofview(FOV)of46mmx35mmprovidinga

magnificationof125m/pixelwasusedinthisexperimenttoimage20kernelssimultaneously.

Withadifferentlensinthesystem,aFOVof12.8mmx10.2mm,correspondingtoanominalof

40mx40mpixelsize,wasusedinpreliminaryexperimentstoanalyzewithgreaterdetail

twokernelsinthesamefield.Thedatawerecollectedinthediffusereflectionmode.Indiffuse

reflection,theradiationfromthenearIRsourceincidentonthesamplesurface,enterthe

specimenandisdiffuselyreflectedfromthesampleinalldirections.ThelensesoftheFPA

imagingspectrometercollectthescatteredradiation.Aftertheinitializationstep,firstly,darkand

brightbackgrounddatacubeswerecollected,andthensamplesinthefieldofviewwerefocused

andscanned.Adarkdatacubeisobtainedbyscanningahighspecularreflectancematerial(a

metalmirror)andabackgroundcubeisacquiredbyahighdiffusereflectancematerial(an

appropriatesizeofwhiteceramicplatecoveringtheFOVcompletely).Eachdatacubecontains

81,920spectraandisacquiredin45min.Thesedatacubesarethreedimensionalimageswhich

combinesthespectralandthespatialdimensions.Twoaxesrepresenthorizontal(x)andvertical

(y)arespatialdimensionsandthethirdzaxiscontainsthespectraldimension(wavelength)

(Figure2.4).These3Dimagedatacubesenableustoaccessthecompletespectrumofeach

pixelsaswellasspatiallyresolvedintensitiesforallpixelsatselecteddiscretewavelengths.

69
Figure2.4Representationof3Dimagedatacubewiththreeaxes(fromref.75).

Figure2.5Anexampleimageofwheatkernelsat2100nmandspectrarepresentingthegerm
andtheendospermareas.

0.8
Absorbance

0.7

0.6

0.5

1200 1400 1600 1800 2000 2200 2400


Wavelength(nm)

2.2.Samplepreparation

Thepreviousexperimentswereperformedwith theMatrixNIRTM Chemicalimaging

system(SpectralDimensions/Malvern,Columbia,MD)whichhasanInGaAsFPA.Six100g

replicatesofseedofeachvarietyweregerminatedfor3,6,12,24,36,and48hronsoaked

blotterpaperinpetridishes.Thekernelswerefrozenat80Cfor24hrtostopallgermination

processes.Thekernelsweresubsequentlyfreezedried(48hrtoremoveallmoistureandto

stabilizethekernels)untilnoweightchangecouldbedetectedinordertoreducemoisture

70
specificallytoavoidfurthergerminationatambienttemperaturepriortonearIRimaging.The

procedurewasdescribedinourearlierpaper(63). Kernelsweremanuallyplacedonagrid

sampleplateforimaging.Eachplatehada5x6gridofslots(Figure2.6).Asetofthreesuch

plateswasusedtoimage90kernelsofeachlot.

ThefollowingexperimentsweredonewithSapphireNIRofSpectral

Dimensions/Malvern(Columbia,MD,USA)whichwasequippedwithindiumantominade

(InSb)FPA.Wheatkernelsofeachvariety(Danby,Betty,Jagelene,Overley)weregerminated

for3,12,24,and36hronmoistblotterpaper(moistenedwith67mldistilledwater)inPetri

dishes.Aftergermination,thekernelswereremovedfromPetridishessurfacemoisturewas

removedwithpapertowel.Kernelsweremountedontoplatesandthisstepallowedthemtodry

for30min45min.Aftermounting,theywerescanned.Kernelswerefrozenat80Cfor24hrto

stopallgerminationprocesses.Followingterminationofthegerminationprocess,thekernels

weredriedundervacuumat55Cfor3hrtoreducemoisture.Dryingwasdonespecificallyto

avoidfurthergerminationatambienttemperaturepriortonearIRimagingandalsotoavoidthe

maskingeffectofthewaterpeakonthestarchandproteinbands.Fortheseexperiments,20

seedsweremountedoneachplate(Figure2.6).Asetofthreeplateswasusedtoimage60

kernelsofsampleforeach timeperiodofexposuretomoisture.

Figure2.6Sampleplate

2.3Dataanalysis

71
ExperimentswithInGaAsFocalPlaneArray

Reflectancedatawasobtainedfromcollectedrawimagedata(Reflectance=(Sample

Dark)/(BrightDark)). ThereflectancesampledatacubewasconvertedinabsorbanceunitsbyA

=log1/R.Itwasspatiallymaskedtoseparatekernelsfromnonsampleareabyremovingthe

pixelsofradiationspecularlyreflectedoffthepolishedstainlesssteelplate,onwhichseedshad

beenmountedwiththegermup.Fromtheremainingpixels,opticalparameterssuchaslog1/R

images,aswellasprinciplecomponentanalysis(PCA)factorswereselectedtoproducebest

imagecontrast.Themainobjectiveinimagingwastoproducecontrastthatwoulddistinguish

theembryodevelopingwithinthegermofeachindividualkernelfromtheresponseofthesame

factorwithinthebulkofthekernel.AGO/NOGOdeterminationwasmadeforindividual

kernels,andthesummationoftheGOkernelsofthegerminatedgroupindicatedthesensitivity

ofthetest.

NearIRraysinthe1000nm1700nmrangedetectedbytheInGaAsfocalplanearray

penetratedbelowsurfaceofthekerneltoenableprobingofthedevelopingembryoandits

surroundingscutellumwithinthegerm.Visiblelightrayscannotpenetratebelowthesurfaceof

thegermthereforenakedeyescannotseeinternalbiologicaldevelopmentduringtheprocess

beforetheemergenceofasprout.

Fromthemountedseeds,amodelgerminatedkernel(36hrtreatment)andungerminated

(3hr)wasselected.Eachmodelimagewasmaskedspatiallytoremoveadjacentbackground

pixelssotheremainingpixelswouldexclusivelyrepresentdiffusereflectanceoffofthe

individualkernel.Toobtainlog1/R,itwasnecessarytoinverttherawreflectionintensity

valuesandapplyalogfunctiontoachieveanimageofthekernelforeachpixelrepresentedbya

valueoflog1/R.Inthenewdatacube,eachpixelhasanindividualwholerangespectrum.The

72
responsesummationofallthepixelsinthatdatacubeisshownasahistogram,inwhichlog1/R

appearsontheXaxis(Figure2.7).Inordertoremovemostofthebackground,thosepixels

havingalog1/Rofmorethan0.7weredeleted.Theserepresentspecularlyreflectedlightthatis

notdirectedtowardthecamera,butreachesthereasstraylight.Atthelowendofthelog1/R

scale,spuriousresultsoflonepixelsthataredetachedonthehistogramfromthebulkofthe

pixelswerealsoremoved.

Figure2.7Histogramshowingthepixelpopulationofvariousintensitiesat1680nm

Log1/R
Log1/R

Fromthetypicalspectratakenfromthedatacubeoflog1/R,differentwavelengthswere

selectedwhileobservingtheimageinordertooptimizewavelengthchoiceforthemaximum

contrast.InworkingwiththeInGaAsarray,theCHnearIRbandat1680nmwaschosento

representtheregionwhereorganicmaterialshowedamaximumdensityandgavethehighest

contrast.

Processingreflectionintensityrawdatacubeswithoutinversionorproducingalog

functionwasaccomplishedasfollows:aftercroppingthebackgroundpixelssothatthemajor

imagewasofthekernel,ahistogramwasproducedinwhichtheaxisrepresentedreflection

intensity.Inthiscasepixelswithreflectionintensitybelow0.080.10weredeleted,and

73
spurious,highlyintensepixelsseparatedfromthebodyofthehistogramwerealsodeleted.

Principlecomponentanalysis(PCA)wasappliedtoreflectionintensityvaluesoftheresultant

newdatacubewithISysTM software.PCA,imagesofindividualfactorsweredisplayed.Factor1

wasusuallythemostrevealing,however,otherfactors,including3,4,6,etc., showedthe

embryoincontrasttotherestofthebodyofthekernel.Ingeneral,whenjudgingmultivariate

evidenceofgermination,threeimageswereconsidered.Contrastbetweentheintensityof

diffuselyreflectedradiationfromthegermwascomparedtothatfromthebulkofthekernel.

Second,themoreorlessroundshapeindicatinganembryowasexaminedasanintactinternal

feature.Third,thesizeoftheinternalroundportionwasanindicationoftheextentof

developmentofthegrowingembryo.Inallinstances,falsecolorswereusedtoshowcontrast.

Inallcasestheimagingdatacubeof themodelunsproutedkernelandofthemodel

sproutedkernelweretreatedinanidenticalway.Thus,themaximumcontrastbetweenthe

embryoandthesproutedkernelwasachieved.Also,adifferencewasobservedbetweenthe

kernelthatwassproutedandthekernelknowntobeunsproutedbyusingthesameimaging

parameters.Theparametersforthelog1/Rspectroscopicimageandtheparametersusedforthe

PCAfactorswereidenticaltoallowadirectcomparisonbetweenasproutedkernelandan

unsproutedkernel.

ExperimentswithInSbFocalPlaneArray

Inadditiontodataprocessingmentionedinprevioussection,infurtherstudies,some

otherpreprocessingstepswereusedsuchasbaselinecorrection,normalizationorderivativesto

distinctthesproutedkernelfromunsproutedonemoreefficiently.Withpreprocessingsteps,

variationsotherthanchemicalcomponentssuchasphysicaleffectsincludingdifferentseed

morphology,illuminationwereremovedfromthespectralinformation.Furtherdataevaluation

74
wasperformedwith comparisonof PCAfactors,lipid/proteinratios,orwavelengthselective

intensity.

3.ResultsandDiscussion

ExperimentswithInGaAs

Figure2.8showsimageselectedfromrecentarticle(Smailetal.,2006) onchemical

imagingofgerminatedwheat.Theimagesofamodelunsproutedkernelareontheleftcolumn

whileimagesofamodelsproutedkernelappearintherightcolumn.Thefirstimageineach

columnisaspectroscopicresponseinwhicheachpixeldisplaysavalueforlog1/Rat1680nm

wavelength.Followingthespectroscopiclog1/RimageisthefirstfactorofPCAfactormaps

thatalsoshowscontrast.ImagesthatbestrevealedthedevelopingembryowerefromPCA

factors1,3,4and6.FirstfactorinPCArepresents99%varianceinthesinglewheatkernel

image.

Figure2.8SpectroscopicimagesandPCAfactorofasinglewheatkernelofsprouted(right)and
unsprouted(left)indicateembryodevelopmentonthegermside(rightend)ofsproutedkernelby
contrastintheimages.
(a) spectroscopicimage
UNSPROUTEDJaggerwheat SPROUTEDJaggerwheat

(b) PCAfactormaps
Factor 1
1

Alldifferentfactorsindicatethesize,geometry,anddensityofdevelopedembryointhe

sproutedkernel.Noteinthesproutedkernelthatfalsecolorimages(rightcolumn)inamoreor

75
lesscircularshapecontrastareconsideredevidenceoftheexistenceofadevelopingembryo

belowthesurface.

InFigure2.9,kernelimagesofthevarietyTregoareshown.Agroupof15unsprouted

kernelsareonthetopthreerowsandagroupof30kernelsthatsproutedaftermoistureexposure

for36hrareonthebottomsixrows.Noteamongtheimagesofsproutedkernelsthatbothround

shapeandintensityareshownbythefalsecolor,andthatdifferentsizesoftheembryorepresent

differingstagesofdevelopment.

Figure2.9PCAfactor1imagesof15unsprouted(top)and30sproutedkernels(bottom)
representacomparisonbetweenthesproutedkernelsandunsproutedkernels.While30kernels
of30aresproutedin36hrmoistureexposure,germinatedkernelsin3hrmoistureexposureis
lessthan10%.

Factor1

3hr

36hr
36hr

Figure2.10alsoshowsPCAfactor4kernelimagesofthesame15unsproutedkernelsat

thetopandthesame30sproutedkernelsatthebottom.Onbothfigures3and4,twoexamples

ofunsproutedkernelsareshownwithrectangles.Ellipseshighlightsproutedimagesforfactor1

andfactor4.FromtheimagesofthekernelsshowninFigures3and4,itwasdeterminedthat30

76
outof30kernelsweresproutedonthebasisofthePCAfactors1and4.Incontrast,byusingthe

samecriteriaonkernelsexposedtomoisturefor3hr,lessthan10%wereidentifiedasbeing

sprouted.

Figure2.10PCAfactor4imagesof15unsprouted(top)exposedto36hrgerminationand30
sproutedkernels(bottom)exposedto3 hrgerminationshowninfigure2givesthesame
evaluationasfactor1.

Factor4
Factor4

Thegerminationresponseto36hrmoistureexposureisshowninFigure2.11basedona

GO/NOGOclassificationof90kernelsforeachofsixcultivarsbyusingtwoPCAfactors.Note

thattheshadedportionsofthebargraphshowthenumberofsproutedkernelsfromthefieldof

90.Theunshadedportionofthegraphrepresentsthedifferencebetweenthosesproutedandthe

90thatwerepresent.Fortheseresultsnoattemptwasmadetoreexaminethespecimenplatesto

lookfornonviable(dead)kernels.Acorrectionofthesedatabydeletingthenonviableones

wouldincreasethepercentageconsiderably.AmongcultivarsshownonthebargraphinFig.2.9,

inorderlefttoright,Betty,Trego,Danby,Jagger,KS89180,KS2174itisnotedthattheBetty,

77
Trego,Jagger,andKS89180haveahighpercentageofsproutedkernels.Incontrast,Danby

andexperimentallineKS2174showamuchlowerpercentageofgerminatedkernelsoutofthe

90presented.Thisnumericalrevelationofthenonsproutedcharacteristic,evenat36hr,

indicativesthatKS2174isrelativelysproutresistantincomparisonwiththeothercultivars

tested.Thisispreciselythetypeofdatathatcanassistplantbreedersinselectionofsprout

resistancelines.ThenumbersofsproutedkernelsineachoneareBetty 80,Trego85,Danby 70,

Jagger74,KS8918087,KS2174 48.Figure2.11showsthetallyofkernelsclassifiedas

sproutedatthe36hrperiodforeachofthesixcultivarsexamined.

Figure2.11Germinationresponsesof90kernelsofsixcultivarsat36hrarerepresentedasbar
graphs.Amongthesixcultivars,Trego,KS89180,andBettyhavemorethan85%germination.
WhileDanbyandKS2174arerelativelymoreresistanttogermination.

80 85 70 74 87 48
48

Betty Trego Danby Jagger KS89180 KS2174

Figure2.12showsthegerminationresponsetodifferentmoistureexposuretimesfortwo

differentcultivars.Bettyat36hrshowedthat80outof90kernelshadsprouted.Bettyisa

whitewheatthatiscurrentlybeinggrownthroughoutKansas.Notethattheprogressionfrom

48hrexposurewith83kernels,to80at36hr,73at24hr,58at12hr,and52evenat6hr

78
exposure.Incontrasttothosevalues,notetheshadedbarswithamuchlowertallyat48hr of58

downto31outof90at6hr.Betweenthesetwocultivars,thereisabiastowardssprout

resistanceatalltimeperiodsforKS2174,comparedwithBetty.Itisnoteworthythatthesame

numberofsproutedkernels58thatwasproducedwithBettyat12hrrequired48hrforKS2174.

Thus,ittookafourtimeslongerexposuretomoistconditionstoproducethesamegermination

level.ItisthistypeofdramaticdifferencethatshouldbeexploitedtoassistourKansas

AgriculturalExperimentStation plantbreedersproducingnewvarietiesofHardWhiteWinter

wheats. ResearchinnondestructivenearIRimagingisongoingatpresentwithdetection

wavelengths12002400withInSbFPAforthepotentialforunsupervisedGO/NOGO

determination

Figure2.12Germinationresponsesof90kernelsofBettyandKS2174tomoistureexposure
times6,12,24,36,and48hrarerepresentedasbargraphs,openandshaded,respectively.KS
2174ismoreresistanttogerminationandhasapproximately40%lesssproutedkernelsthanthe
Bettyinallmoistureexposuretimes.

79
ExperimentswithInSb

Spectroscopiceffectofdryingandtreatmentoptimization

Wheatkernelswereanalyzedwetimmediatelyaftergerminationanddryafterbeing

freezedriedorovendried.Thisrevealedtheeffectofdryingonthespectra.Withoutdryingthe

waterpeakat1940nmstronglymaskedtheregion20002200nmwhichincludesstarchand

proteinbands.Thiseffectofthewaterpeakismorepredominantonspectraofgerm(Figure

2.13b). Afterdrying(Figure2.13),themaskingeffectwasremoved.

Figure2.13Spectraselectedfromgermandendospermsideofthea)Unsproutedsamples,b)
germinatedwetsamples,andc)driedgerminatedsamples,d)showingaspectrafromeach
sampleinsamepanel.Notethedifferencesofredspectrum(forwetsample)thancontroland
driedspoutedsample.Alsonote,figurebhighlightshugewaterpeaksat1940maskingprotein
andstarchbandsinspectrum.
0hr1freezedrysptrcpylogxCnanmsk.spf(175,97) 0hr1freezedrysptrcpylogxCnanmsk.s pf (160,95)
a. 0.75

0.7
0.75
endo
0.7
0.65
germ
Absorbance
Absorbance

0.65
0.6

0.55 0.6

0.5 0.55

0.45 0.5

0.4 1200 1400 1600 1800 2000 2200 2400


W avelength(nm)
1200 1400 1600 1800 2000 2200 2400
Wavelength(nm)

36hr1f reezedrys ptrcpylogxCnanmsk.spf(163,95)

b. 36hr1freezedrysptrcpylogxCnanmsk.spf(172,94)
0.95

1 0.9
endo
0.85
0.9
germ 0.8
0.8 0.75

0.7 0.7

0.65
0.6
0.6

0.5 0.55
1200 1400 1600 1800 2000 2200 2400
1200 1400 1600 1800 2000 2200 2400 Wavelength(nm)
Wavelength(nm)

80
c. 0.8
36hr1freezedry(afterdrying)sptrcpylogxCnanmsk.spf (165,94)
0.8
36hr1freezedry(af terdrying)sptrcpylogxCnanmsk.spf(147,95)

0.75
germ 0.75 endo
0.7 0.7

Absorbance
0.65 0.65

0.6 0.6

0.55 0.55

0.5 0.5

0.45 0.45

1200 1400 1600 1800 2000 2200 2400


1200 1400 1600 1800 2000 2200 2400
W avelength(nm)
Wavelength(nm)

0hr1freezedry+36hr+36dried(singlekernel)absmask.spf(46,71)

0.75

0.7

0.65
Blue:unsproutedsamples
Absorbance

0.6
Red:germinatedwetsamples
0.55

0.5
Green:driedgerminatedsamples
0.45

0.4
1200 1400 1600 1800 2000 2200 2400
Wavelength(nm)

PCAanalysisisagoodstartingpointindataanalysisprovidingtheinformationhow

variationisdistributedbetweenfactors, whichfactorsareimportantandindicatingthepossible

importantpeakscancausethevariation.ThespatialdistributionofwatercanbeseenfromPCA

factor5(Figure2.14).Itwasconcentratedingermareaofthekernel.

Figure2.14EffectofwaterpeakcanbeseeninPCAfactor5loadingofwetsamplesindicating
averydefinitepeakat1940 nm withahighPCAscore.

IndexedFactorScores(Factor5) 1
0.25
PCALoading
RawSpectrum
0.2
0.8
50
0.15
PCALoading

Absorbance

0.1 0.6
100
Pixels

0.05
150 0.4
0

200 0.05 0.2

0.1
50 100 150 200 250 300 0
Pixels 1200 1400 1600 1800 2000 2200 2400
Wavelength(nm)

81
Whentheeffectofthewaterwasunderstood,differentdryingmethodsincludingfreeze

drying,vacuumovendrying,forceddriftairdryingwerecomparedfrompeakheightsat1940

nmbaselinecorrectedspectra.Itwasobservedthatforcedairdryingwasnoteffective,othertwo

methodsgavesimilarresults.Vacuumovendryingwasselectedbecauseofthesimplicity and

lesstimerequirement(seeAppendixBforthisoptimizationstep).Howeveritwasobservedthat

thereisabigshrinkageinthesizeofthekernelafterdryinginvacuumovenwhilethetexture,

sizewereremainedsameforthefreezedriedsamples.Ovendryingundervacuumdidnotcause

achemicalchangeswithinthekernels,thusitcanbeusedsafely.

Preliminaryexperimentsforthepurposeof understandingbandsin thespectrum

Twomaincomponentsofthewheatarestarchandgluten.Themean spectrumofstarch

andglutenwereshown.Forstarch2100nmischaracteristicwavelengthwhile2050and2180

nm arecharacteristicforprotein.

Figure2.15Averageof18,000spectrafromthedatacubeof starch andassignedpeaks


starchcpylogxCsptrmeanim.spf(1,1) 2320
0.4
1930
0.35 2100
1450 2280
Absorbance

0.3

1570 1700
0.25 1780

0.2

0.15
1200 1400 1600 1800 2000 2200 2400
Wavelength(nm)

82
Figure2.16Averageof24,000spectrafromthedatacubeofgluten
2290
protein2cpylogxCsptrmeanim.spf(1,1)

0.6 2180 23302340


0.55
1980
0.5
2050
0.45 2060
Absorbance

0.4 1700
1500 1730
0.35

0.3

0.25

0.2

1200 1400 1600 1800 2000 2200 2400


Wavelength(nm)

Figure2.17Spectraoflipidextractedfromwholewheatandpentosans(reprintedfromref76.)

InthestudiesbyWetzel(72)andLawand(77),2100nmisassignedforstarch,2180,

2050nm forprotein,2310,2348nm forlipid,2336and2350forcellulose.Inlipidspectrum

(Figure2.17),peaksareat1170,2140,2170nm (CHvibrations).Peaksandassignmentsof

83
wheatandwheatcomponentsweredoneindetailby LawandThachuk(77),thepeaksat1700,

1730,1760 nmisduetoCH2 group,CH1st overtone.2180and2050nm areC=O+AmideIII

combination,NH+amideIIcombinations,respectively.Forstarch2100nmisbyOH

combination,starch andpentosan havepeaksat2280and2320nm(CHcombinations)and1540

and1450nm(OHcombination andOH1st overtone.Forlipid2310and2340nmarefrom

CH2,CHcombination.Alsopeaksat1720and1760nmareduetoCH2 group,CH1st overtone

(76,77). Asseeninthefigure2.18,thespectrumofthewheatisdeterminedlargelybythe

carbohydratecomponents.Starchbandat2100nmisoverlappingwiththeproteinbandsand

maskingthem.

Figure2.18Meanspectrumofawheatkernel indicatingthespectrumofthewheatisdetermined
largelybythecarbohydratecomponents.
0for36(2)truncpylogxCnanmsksptrmeanim.spf(1,1)

0.65

0.6

0.55
Absorbance

0.5

0.45

0.4

0.35

0.3
1200 1400 1600 1800 2000 2200 2400
Wavelength(nm)

Comparisonof sproutedandunsproutedkernelsofvarietyBettybydifferentdata
treatments:

Therearedifferentwaystodiscriminatesproutedkernelfromunsproutedkernel.Inthe

figuresbelowsomeofthemwillbeexplained.Thisdata representanattemptinnondestructive

84
nearIRimagingwithdetectionwavelengths12002400withInSbFPAforthepotentialfor

unsupervisedGO/NOGOdetermination.

Infigure2.19a,embryodevelopmentcanbeobservedbywavelengthdiscriminationat

2310nm butitwasnotverydistinctive.Whendatawerebaselinecorrected,abettercontrastwas

obtainedbetween(Figure2.19b)sproutedandunsproutedkernels.Anddevelopingembryowas

separatedfromrestofthekernelbody.

Figure2.19Comparison ofsprouted(36hr)andunsprouted(3hr)kernelsat2310nm.Image(a)
isabsorbanceintensityat2310nmwhile(b)isbaselinecorrectedintensityat2310nm and(c)
normalizedintensityat2310nm .Leftthreekernelsrepresentunsprouted(3hr)andrightthree
representsprouted(36hr)infigures.

3hrdry 36hrdry
a. b. c. B031d+B361d(absmsk)nrm.spf
10
20 10
20
30 20
40 30
40
40
Pixels

50
Pixels

60 50

Pixels
60
70 60

80 80 70

90 80

100 100 90

110 100

20 40 60 80 100 20 40 60 80 100 110


Pixels Pixels 20 40 60 80 100
Pixels

Inadditiontowavelengthselectivediscrimination,comparisonofpeakheightratioswas

usedfordiscrimination.Infigure2.20peakheight(2310/2060)imagewasobtainedand42

spectraofgermareawasaveragedtofindameanratio.Averageratiovaluesforthegermsideof

eachkernelarerepresented(Figure2.20).Areductioninthelipid/protein ratiowasobserved

withgerminationwhichisconsistentwith theresults reportedinpartonethisthesis.

85
Figure2.20Peakheightratio(2310/2060)imageandaverageratiovaluesforthegermsideof
eachkernel.Notethedifferencesinratiosandreductionwithsprouting.
B031d+B361d(absmsklb3)phrnanmsk.spf

Ratioaverageof 42spectra(pixels)from germ side


20

40 UnsproutedSprouted
Pixels

60
0.42 0.31
80
0.52 0.36
0.49 0.40
100

20 40 60 80 100
Pixels

Ifsecondderivativeisperformed,andPCAisapplied,wecanobtainsimilar

discriminationandFactor2 (Figure2.21)loadingindicatesapeakat2310nm whichcanbethe

sourceofvariation.

Figure2.21PCAfactor2ofsecondderivativedata

Indexe dFactorScor es (Factor 2)


1
0.3
10 PCALoading
RawSpectrum 0.9
20 0.2 0.8
30
0.7
40 0.1
PCALoading

Absorbance

0.6
50
Pixels

0 0.5
60

70 0.4
0.1
80 0.3
90 0.2 0.2
100
0.1
110 0.3
0
20 40 60 80 100 1200 1400 1600 1800 2000 2200 2400
Pixels Wavelength(nm)

Potentialforfutureunsupervisedmultikerneltesting

Withoneoftheproceduresexplainedabove,preliminaryexperimentsweredoneforthe

potential anunsuperviseddiscrimination.Amacrowaswrittentoprocessalltheplatesincluding

20seedsinidenticalwayandrapidly.Inthismacro,firsttheimagewasspatiallytruncatedand

86
convertedinlog1/R(Absorbance)units.Next,itwasspatiallymaskedbetween0.20.75

intensityvaluesinhistogramtoremovepixelsfromnonsamplearea.Aftermasking,theimage

wasbaselinecorrectedwithapolynomialdrawnwithpointsatvalleysof1320,1850,2220and

2380nmandpeakheightratiowascalculatedwithbaselinecorrectedspectra. Next, 4050

pixelsselectedfromgermandendospermpartsofeach20kernelsandtheaverageratiowas

calculatedforthisgroupofpixels(total 9600pixelsforeachcultivar).Thenthegrossmeanof

theseratiosfor20kernelswasobtained(Table2.1).Notethereductionwasobservedforall

kernelsinthisdataset.

Table2.1ThegrossmeanoftheresultsforBetty

Germ area
3hr 0.445
36hr 0.382

Whensimilarprocesswasappliedtonormalizedorbaselinecorrectedpeakintensityat

2310,sameresultcouldbeobtained.Themeanratiofor20kernelsofunsproutedwasfound1.70

whileforthesproutedkernels(36hr)itwasreducedto1.34(normalizeddataintensity).For12

hrsproutedkernelitwasreducedto1.46.

4.Conclusion

FromtheSprouted/Nonsproutedmultivariateclassificationof90moisturetreatedwheat

kernels,itwasevidentthatasinglehardwheatcultivarhadahighersproutingresistancethan

fiveothercultivarsexamined.Thiswasbasedon48of90germinatedaftera36hrtreatment.In

comparisonacompletelygerminated36hrcultivarthathad80of90kernelsclassifiedby

multipleimagedataasgerminated.Excludingthepresenceofnonviable(dead)kernels,the

correctclassificationwouldactuallybehigher.Forboththecultivarwiththehighestgermination

87
andtheonewiththehighestsproutresistanceatintermediatetreatmenttimesof24hr,12hr,and

6hr,differentiationwasevident.Thelessresistantcultivarhadsignificantgerminationevenat

shortexposuretimes.

StudyforthepotentialforunsupervisedGO/NOGOdeterminationinnondestructive

nearIRimagingwithdetectionwavelengths12002400withInSbFPAisongoing.Some

preliminaryresultswerepresentedwhichareencouraging.

88
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AppendixA

NearIRAnalysisforSproutDamageTest

NearIRanalysishasalsobeenusedforpredictionofmechanicalstarchdamagebysome

researchers.Finneyetal.(1988)measuredstarchdamageofgroundwholewheatbyenzymatic

methodandnearIRreflectanceanalysisandcorrelatedthem.Highcorrelations(0.910.94)were

observedbetween twomethods.Inanotherstudy(MorganandWilliams,1995)forthestarch

damageinwheatflour,correlationcoefficientwasfound0.90betweenlaboratoryvaluesand

nearinfraredpredictedvalues.Juhaszetal.(2005)studiedtherelationshipbetweennear

infraredandRVAparametersduringwheatgerminationwithPartialleastsquares(PLS).The

highlinearcorrelation(0.950.98)betweenRVArheologicalparametersandnearIRpredicted

values.MaximumcorrelationwasobservedgelatinizationperiodofRVAmeasurement.Inthis

studyitwassuggestedthatnearIRspectraofdrysampleswereevaluatedmoreeasilythanwet

sampleswhichhadwaterpeaks.Driedsamples(oven120C2hrs)hadhighercorrelation

coefficientscomparedtowetsamples.Juhaszetal.(2007) attemptedtoanalyzegerminated

maizesamplesnondestructivelyimmediatelyaftergerminationwhilewet.Theyobservedthat

correlationispoorinthiscaseduetovariationinspectracausedbywaterandmorphological

characterofseed.Shashikumaretal.(1993)foundafairtogoodcorrelation(0.750.87)between

nearIRandfallingnumberandRVAresults.

96
AppendixB

Spectroscopiceffectofdryingandtreatmentoptimization

Baselinecorrectionwasperformedaspolynomialeithersecondorthirdorderby

selectingvalleysinthespectrumasshowninfigure.

FigureB.1 Baselinecorrectionofaspectrum

0.8

0.75

0.7
Absorbance

0.65

0.6

0.55

0.5

1200 1400 1600 1800 2000 2200 2400


Wavelength(nm)

Theaveragepeakheightat1940nmofminimum100spectraextractedfromgermsideof

thekernelswascalculatedforcontrol,36hrgerminatedkernelandgerminatedanddriedkernel.

Averagepeakheightswerereportedbelow.

FigureB.2 Baselinecorrectedspectraaveragedforcontrol(blue),sproutedwet(red),sprouted
dry(green).Notethewaterpeakinredspectra.

0.35
0.2
0.117 0.3 0.217 0.15
0.080
0.15 0.25
Absorbance

Absorbance

Absorbance

0.2 0.1
0.1
0.15

0.1 0.05
0.05
0.05

0 0 0
0.05

1200 1400 1600 1800 2000 2200 2400 1200 1400 1600 1800 2000 2200 2400 1200 1400 1600 1800 2000 2200 2400
Wavelength(nm) Wavelength(nm) Wavelength(nm)

97
TableB.1 thepeakheightcomparisonat1940nmfordifferentdryingmethods.

Drying Control (0hr) 36hrgerminated 36hrgerminated


wet dry
Freezedrying 0.111 0.177 0.091

Forcedair(tangentair 0.192 0.170


0.126
flow)
Vacuumoven(55C 0.217 0.080
0.117
3hr)

98

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