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ISSN:0974-7435Volume10Issue22BioTechnologyAnIndianJournalFULLPAPERBTAIJ,10(22),2014[13887-13893]FrosthardinessaccuracyassessmentbyelectricalimpedancespectroscopyinCatalpaspp.
duringfrosthardeningBaoDi1,JiQian1,YuMeng2,GangZhang1*,Di-YingXiang1,MingGuo31CollegeofHorticulture,AgriculturalUniversityofHebei,Baoding,Hebei071001,(CHINA)2CollegeofLandscapeandTravel,AgriculturalUniversityofHebei,Baoding,Hebei071001,(CHINA)3CatalpaBungeiResearchInstituteofZhoukou,Zhoukou466000,(CHINA)E-mail:zhanggang1201@126.
comABSTRACTThreespeciesofCatalpaspp.
wereusedtomeasuresolublesugarcontentandmembranepermeabilityaswellaselectricalimpedancespectroscopy(EIS)ofstemsduringfrosthardening.
TherelationsbetweenparametersoftheEISandcorrespondingsolublesugarcontentandmembranepermeabilitywereanalyzedrespectively.
ThecomparativelysatisfyingcorrelatedEISparameterswereselectedforestimatingfrosthardinessofstems.
AlsotheconventionalconductivitymethodwasappliedtoestimatefrosthardinessofsteminthreespeciesofCatalpaspp.
.
ThemostoutstandingparameteroftheEISwaschosenbycomparisonofthesetwoestimationfrosthardinessmethods,forthepurposeoftestingtheaccuracyoffrosthardinessassessedbymeansoftheEISmethod.
Thosestudyresultsreveal:1)Duringfrosthardening,solublesugarcontentandmembranepermeabilityincreased;2)ShapesofimpedancespectrawerechangedindifferentspeciesofCatalpaspp.
stem;3)TheEISparametersofspecificlow-frequencyresistancer1andspecificextracellularresistancerehadsignificantpositivecorrelationswithsolublesugarcontentaswellasmembranepermeability.
Amongthemr1rankonthetopforfrosthardinessofstemwithestimationaccuracyof81.
83%.
Inconclusion,theEISdatacanbeusedtoassessfrosthardinessofsteminthreespeciesofCatalpaspp.
withgreaterreliability.
KEYWORDSCatalpaspp.
;Frosthardiness;Accuracy;Electricalimpedancespectroscopy;Solublesugar;Membranepermeability.
13888FrosthardinessaccuracyassessmentbyelectricalimpedancespectroscopyinCatalpaspp.
duringfrosthardeningBTAIJ,10(22)2014INTRODUCTIONCatalpaspp.
isaprecioushigh-qualitytimberspeciesandfamousornamentaltreespeciesbecauseoftheexcellentmaterialqualityandbeautifultreeform[1,2].
Itiscultivatedinsubtropicalregionsofhemispherewithwarmtemperature.
Catalpaspp.
growthandbreedingarelimitedbylowtemperaturemainlyduringwinter,sometimesinearlyspringandlaterfall.
Theselowtemperaturesarecommoninnorthernhemisphere.
InordertoselectthepremiumfrosthardyresourceofCatalpaspp.
,itisnecessarytoestablishfastandobjectivemethodstoevaluatefrosthardiness(FH).
Thereareseveralphysiological,biochemicalandbiophysicalchangesincellsandtissuesduringfrosthardening,aswellasthechangesofFHoftheplants[3-5].
ThecurrentFHassessmentmethodistime-consumingwhichrequiresexpensiveequipment,andaconsiderableamountofmaterial.
TheEISisconsideredasanewfastandeffectivetechniqueforstudyingthestructureoforganicandinorganicmaterial,whichcangetthechangedinformationofphysicochemicalpropertiesofcellswithoutdestroyingit.
RecentlytheEIShasbeendevelopedinmanyfields[6-8].
ThestudiesshowedthattheEISparameterscannotonlyassesstheFHofplanteffectively,butalsocorrelatesignificantlywithFH[9,10].
Intheendoffrosthardening,however,theFHisunderestimatedbytheEISthanbytheothermethods[11,12].
MoststudiesfocusonthecorrelationbetweentheEISparametersandtheFHassessedbyelectrolyteleakage(EL).
However,dataisstillscarceintheaccuracyofFH.
TheaimofthepresentresearchwastostudytheaccuracyofFHassessedbymeansoftheEISmethod.
WeselectthebettercorrelatedEISparametersthroughtheverificationofaccuracy,whichcancomplementandimprovetheEISmethodtoassessFHofplant.
MATERIALSANDMETHODSPlantmaterialThecurrent-yearshootsweresampledinaone-yearprovenancefieldtrialwiththreeCatalpaspp.
atAgriculturalUniversityofHebeiinHebeiprovince(N3850′,E11526′,elevation25m,China).
ThosethreeCatalpaspp.
wereCatalpafargesiif.
duclorxiiofYunnan(N9851′,E2501′,elevation1640m,China),CatalpafargesiiofGansu(N10788′,E3603′,elevation1026m,China)andCatalpaovataofHenan(N11402′,E3283′,elevation88m,China).
Ineachofthethreeprovenances,10graftingplantswereselectedwiththesametreatmentofcontainerandsoil.
Measurementsofthecurrent-yearshootswerecarriedoutfrom20October,2009to20January,2010.
Samplesfrom10graftingplantsofeachprovenanceweretakenatone-monthintervals.
Atotaloffourmeasurementswereused.
Impedanceanalysisofnon-frost-exposedshootsTheelectricalimpedancespectraofthe8shootsfromeachprovenanceateachsamplingdateweremeasuredinlaboratoryimmediatelyaftersampling.
A15-mmsectionwascutfromthemiddleoftheshootsamples.
TheimpedancespectraweremeasuredasdescribedbyRepoetal[9].
ThesampleplacedindirectcontactwiththeelectrodepasteandtheAg/AgClelectrodes(RC1,WPILtd,Sarasota,USA)wassetincontactwiththepaste.
Theimpedancespectrumwasmeasuredat42frequenciesbetween80Hzand1MHz(HP4284ALCRmeter,AgilentTechnologies,USA).
Theinputvoltagelevelofthesinesignalwas100mV(r.
m.
s.
).
Theparametersofthedistributedcircuitmodel(single-DCE)wereestimatedbymeansofacomplexnon-linearleastsquares(CNLS)fittingprogram,whichusesCole-Colemodel.
TheparameterswerefetchedasdescribedbyZhangetal.
[8],whichincludedspecifichigh-frequencyresistancer,specificlow-frequencyresistancerl,specificextracellularresistancere,specificintracellularresistanceri,relaxationtimeanddistributioncoefficientofrelaxationtime.
DeterminationofmemeranepermeabilityA10-mmsectionwascutfromthemiddleoftheshootsamples.
Electrolyteleakage(EL)wasmeasuredasdescribedbyZhangetal.
[8].
Thesampleswereshakenatroomtemperaturefor24hbeforethefirstconductivitymeasurement(C1).
Theconductivitymeasurementofcontrol(deionizedwateronly)wasC01.
Thesampleswerekilledat100℃for20minandshakenforanother24hbeforethesecondconductivitymeasurement(C2),andtheconductivitymeasurementofcontrolisC02.
TherelativeELwasdefinedasequation1:EL=%100022011CCCC(1)DeterminationofsolublesugarconcentrationSolublesugarconcentrationwasdeterminedwithanthronecolorimetricassay.
Determinationofeachtreatmentwasrepeatedfourtimes[13].
MeasurementofFHControlledfreezingtestsTheshootsoffourfreezingtestswererinsedthreetimesbytapwaterandthreetimesbydeionizedwatertoremovesurfacepollutants.
EveryplasticbagwasputintosixbranchesofsamplewithalittledeionizedwaterinordertoavoidBTAIJ,10(22)2014excessivesupercooling.
Onesamplebagoffreezingtemperaturesincludedtemperaturescoolingwas6℃·h-1.
Thesampleswerekept24h.
Immediatelyafterthefreezingtest,themethod,respectively.
TABLE1:ThetemperatureDate20Oct.
20Nov.
20Dec.
20Jan.
FHassessedbyEISandELEIS:Afterfreezingtest,eightshootsfrosthardiness,theEISparameterwasmodeledtemperature:whereyisoneofEISparameters,andAandDdeterminetheasymptotesoftheEL:Afterfreezingtreatment,fourELtest.
Toobtainthefrosthardiness,therelativewithrespecttothetreatmenttemperature:wheredefineasymptotesofthefunction,andBistheSolublesugarconcentrationandmembranepermeabilityThesolublesugarconcentrationoffargesiif.
duclorxiiinJan.
,anddifferedsignificantly(Figure1A).
SugarconcentrationofCatalpafargesiif.
duclorxiiinJan.
Therewereno(P>0.
05).
ThemembranepermeabilityofstemsconcentrationofstemsexceptthatsignificantofCatalpafargesiif.
duclorxiidifferedsignificantlysignificantdifferencebetweenthreespeciesFigure1:Thevariationofsolublesugar(A)andfrosthardening;Catalpafargesiif.
duclorxiiGangZhangetal.
ofeachprovenancewasexposedineachofseventemperaturestemperaturesthatkilledthesamplesandtemperaturesthatcausedkeptatthetargettemperaturefor4handthenmovedinto4thedegreeoffrostdamageintheshootswasquantifiedtemperaturesusedfordeterminingFHinfourcontrolledfreezingtestsTemperature/℃4-3-6-10-15-20-304-6-13-20-25-35-454-8-14-22-30-38-464-8-16-24-34-46-72shootswereusedineachprovenanceandeachfreezingtemperature.
modeledbyalogisticsigmoidfunction(inequation2)withparameters,xistreatmenttemperature,Bisslopeatinflectionpointthefunction.
shootsfromeachfreezingtemperatureforeachprovenancerelativeconductivitywasmodeledbyalogisticsigmoidwhereyandxrefertotheELandtheexposuretemperature,theslopeattheinflectionpointC.
RESULTSmembranepermeabilityofstemsofstemsofCatalpaspp.
wasincreasingduringfrosthardeningsignificantlyamongthreespeciesofCatalpaspp.
betweenCatalpafargesiidifferedsignificantlyfromCatalpaovatainnosignificantdifferencebetweenthreespeciesofCatalpastemsofthreespeciesofCatalpaspp.
followedasimilarsignificantdifferencewasfoundinJan.
(P<0.
05)(Figure1B).
ThesignificantlyfromCatalpaovataandCatalpafargesiiinspeciesofCatalpaspp.
inOct.
,Nov.
andDec.
.
solublesugar(A)andmembranepermeability(B)ofdifferentCatalpaduclorxiiCatalpafargesiiCatalpaovata13889temperatures(TABLE1).
Thenodamage.
Therateof4℃tothawgraduallyforquantifiedbytheEISandtheELsusedfordeterminingFHinfourcontrolledfreezingteststemperature.
Toobtainthewithrespecttothetreatment(2)pointC,Cisfrosthardinessprovenancewereselectedforthefunction(inequation2)temperature,respectively,AandDhardeningexceptCatalpabetweenJan.
andOct.
(P<0.
05)inOct.
andfromCatalpaatalpaspp.
inNov.
andDec.
patternassolublesugarhemembranepermeabilityJan.
,andtherewerenoatalpaspp.
stemsduring13890FrosthardinessaccuracyassessmentbyelectricalimpedancespectroscopyinCatalpaspp.
duringfrosthardeningBTAIJ,10(22)2014EISandEISparameteranalysisofstemsShapesofEISofstemwerechangedindifferentspeciesofCatalpaspp.
anddifferentperiodduringthefrosthardening,whichcoulddemonstratechangingintissuestructureandphysiologicalandbiochemicalchangeincells.
ThespectraofthreespeciesofCatalpaspp.
wereclearlycharacterizedbysinglearc(Figure2).
ThearcofCatalpafargesiif.
duclorxiiwassmallerthanthatofCatalpafargesiiandCatalpaovataduringfrosthardening(Figure2A-D),however,thearcofCatalpafargesiiwasbiggerthanthatofCatalpaovatainNov.
andDec.
(Figure2BandC)andthearcofCatalpaovatawasbiggerthanthatofCatalpafargesiiinOct.
andJan.
(Figure2AandD).
WithfrosthardeningthearcofCatalpafargesiif.
duclorxiibecamebiggerinearlystageandthensmallerlater,themaximumvalueoftoparcwas-10.
24kinOct.
andtheminimumvalueoftoparcwas-18.
55kinJan.
.
Duringfrosthardening,thearcofCatalpafargesiibecamebigger,themaximumvalueoftoparcwas-25.
91kinOct.
andtheminimumvalueoftoparcwas-106.
08kinJan.
,whilethearcofCatalpaovatabecamesmallerinearlystageandthenbiggerlater,themaximumvalueoftoparcwas-29.
80kinNov.
andtheminimumvalueoftoparcwas-124.
76kinJan.
.
AlloftheparametersofCatalpaovataincreasedduringthefrosthardeningexceptdistributioncoefficientofrelaxationtime.
IthadasharpriseinJan.
(Figure3A-E).
InJan.
thevalueofCatalpaovatawashigherthanthatofCatalpafargesiiandCatalpaovatainr,ri,,and,butitwaslowerthanCatalpafargesiiinrlandre(Figure3BandC).
EISparametersvaluesinCatalpafargesiiandCatalpaovatawerehigherthanthoseofCatalpafargesiif.
duclorxiiinallparameters(Figure3A-F),anddifferedsignificantlyinrl,reandinJan.
(P<0.
05).
ThetrendofwassimilarforthreespeciesofCatalpaspp.
,whichwashigherforparametervaluesinNov.
andDec.
andlowerinOct.
andJan.
.
Figure2:ImpedancespectraofstemsofdifferentCatalpaspp.
duringfrosthardening;A:Thedateof20Otc.
,B:Thedateof20Nev.
,C:Thedateof20Dec.
,D:Thedateof20Jan.
);Thespectrawerethepooleddataofeachmonthandcomposedof42differentfrequenciesrangingfrom80Hzto1MHz(fromrighttoleft,respectively);Catalpafargesiif.
duclorxiiCatalpafargesiiCatalpaovataCorrelationbetweenEISparameterandsolublesugarconcentrationaswellasmembranepermeabilitySomeEISparametersandsolublesugarconcentrationaswellasmembranepermeabilityarecorrelated.
Theparametersofr1andrehadsignificantpositivecorrelationswithsolublesugaraswellasmembranepermeability.
Thecoefficientofdetermination(R2)betweenr1andsolublesugarconcentrationaswellasmembranepermeabilitywas0.
621and0.
823,respectively.
Thecoefficientofdetermination(R2)betweenreandsolublesugarconcentrationaswellasmembranepermeabilitywas0.
624and0.
828,respectively(TABLE2).
BTAIJ,10(22)2014GangZhangetal.
13891Figure3:EISparametersofstemsindifferentCatalpaspp.
duringfrosthardening;Catalpafargesiif.
duclorxiiCatalpafargesiiCatalpaovataTABLE2:CorrelationbetweenEISparametersandsolublesugarconcentrationaswellasmembranepermeabilityinstemsofCatalpaspp.
duringfrosthardeningEISparametersSolublesugarconcentrationMembranepermeabilitySpecifichigh-frequencyresistancer0.
4350.
484Specificlow-frequencyresistancerl0.
621*0.
823*Specificextracellularresistancere0.
626*0.
828*Specificintracellularresistanceri0.
4390.
454Relaxationtime0.
3490.
566Distributioncoefficientofrelaxationtime0.
264-0.
105*meansthatcorrelationissignificantatthe0.
05level,andforstem,n=36.
FHassessedbyEISparametersThesugarconcentrationandmembranepermeabilitywerereliablephysicalsignstoassesstheFHofplants.
SoitismorereliableforEISparameterstoassessFHofCatalpaspp.
asaresultofsignificantcorrelationbetweenEISparametersofr1,reandsolublesugarconcentrationaswellasmembranepermeability.
TheFHofeachCatalpaspp.
assessedbyr1weresimilartothatassessedbyreduringfrosthardeningexceptJan.
(TABLE3).
TheFHofeachCatalpaspp.
wassignificantdifferentbetweentwomethodsinJan.
(P<0.
05).
13892FrosthardinessaccuracyassessmentbyelectricalimpedancespectroscopyinCatalpaspp.
duringfrosthardeningBTAIJ,10(22)2014TABLE3:ComparisonofFHinstemsofdifferentCatalpaspp.
assessedbyEISparametersDateCatalpafargesiiCatalpaovataCatalpafargesiif.
duclorxiirlrerlrerlreFH/℃FH/℃FH/℃FH/℃FH/℃FH/℃20Oct.
-10.
368a-10.
359a-11.
383a-12.
051a-9.
848a-9.
806a20Nov.
-19.
781a-19.
715a-22.
104a-22.
078a-31.
655a-34.
306a20Dec.
-22.
173a-22.
426a-38.
006a-38.
393a-21.
331a-21.
637a20Jan.
-24.
728a-12.
247b-28.
298a-16.
217b-27.
451a-29.
776bAccuracytestofFHassessedbyEISTheFHofthreespeciesofCatalpaspp.
assessedbyr1andrewastestedbyEL.
TheresultsshowedthatFHresultsofELcorrelatedwellwithFHresultsassessedbyr1andre.
Thecoefficientofdetermination(R2)ofthreespeciesCatalpaspp.
wasover0.
5,androotmeansquareerror(RMSE)andrelativeerror(RE)werelower.
Theparameterr1wasthebesttoassesstheFHwithR2=0.
80,RMSE=3.
815,RE=18.
17%,andtheassessedaccuracywas81.
83%(TABLE4).
TABLE4:ThefitevaluationindicatorsoftheFHmeasuredvaluesbytherelativeconductivity(x)andEISparameters(y)duringfrosthardeningEISparametersRegressionequationR2RMSERE(%)Specificlow-frequencyresistancerly=0.
9413x-0.
4790.
803.
81518.
17Specificextracellularresistancerey=0.
8473x-1.
14430.
556.
62926.
64DISCUSSIONEIScanprovideinformationforbasicphysicochemicalpropertiesofcells,tissuesandorgans[14].
Inthepresentstudy,duringfrosthardeningthesugarconcentrationandmembranepermeabilityareincreased.
TheEISandEISparametershavecorrespondingchanges,whichcanbeanindicationofchangesinphysicochemicalpropertiesofcells.
Thisisinaccordancewithpreviousstudies[9].
Themembranepermeabilityisincreasedbecauseoflowtemperature,whichleadstoincreaseofelectrolyteexosmoseandconcentrationsofintercellularsubstance.
TheCatalpaspp.
wouldincreasethesugarconcentrationthroughosmoregulationtoenhancetheconcentrationofcellsapandthecapacityofwaterinordertoimprovetheFH,thustheimpedanceoftissuesandorganshaschanged.
Electricpotentialdifferencecanbekeptbyeffectivetransportsystemandalternativeinfiltrationcharacteristicswouldcreatewhencurrentthroughthecellmembrane.
Thus,theEIScharacteristicscanshowthechangeofimpedanceofextracellularresistanceandintracellularresistancebecauseofchangeoftheconcentrationofcellsapandmembranepermeability.
ThesolublesugarconcentrationhadsignificantpositivecorrelationswithFH[15].
TheidentificationofFHbymembranepermeabilityaccordedwiththatoffieldperformance[16,17],sothesolublesugarconcentrationandmembranepermeabilityareoftenusedasidentifyingsignalofFHofplants.
InagreementwithRyyppetal.
[12],thesolublesugarconcentrationandmembranepermeabilityarecloselyrelatedwithFH,andthoseofstemsofCatalpaspp.
aresignificantpositivecorrelationwithr1aswellasre.
ThechangesofEISparametersreflectchangesofthesolublesugarconcentrationandmembranepermeabilityandcanalsoassesstheFHofCatalpaspp.
.
Thesestudiesshowther1isthebestEISparameterforassessmentofFHandhavethebetteraccuracycomparedtootherparameters.
TheFHassessedbyr1isreliabletobeusedinCatalpaspp.
.
Inconclusion,EISisanewapproachtoassesstheFHofplantsandtofiltertheplantresourcewithhighFH.
ThestudyconfirmedtheruleofchangeofthesolublesugarconcentrationaswellasmembranepermeabilityofthreespeciesofCatalpaspp.
andthebestEISparameteronassessmentofFH.
AllofthesestudieswillimprovetheapplicationofEIStechnologyonassessingFH.
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