76Theuseoftheelectrolyteleakagemethodforassessingc

TheuseoftheelectrolyteleakagemethodforassessingcellmembranestabilityasawaterstresstolerancetestindurumwheatMohammedBajji,Jean-MarieKinetandStanleyLutts*LaboratoiredeCytogénétique,UniversitécatholiquedeLouvain,5(bte13)PlaceCroixduSud,B-1348,Louvain-la-Neuve,Belgium;*Authorforcorrespondence(e-mail:[e-mail];phone:[phone];fax:[phone])Received2January2001;acceptedinrevisedform17April2001Keywords:Cellmembraneinjury,Droughttolerance,Electrolyteleakage,Osmoticstress,Polyethyleneglycol,TriticumdurumAbstractThisworkwascarriedouttoadapttheelectrolyteleakagetechniquetodurumwheatandthentoevaluateitsrelevanceintheassessmentofthecellmembranestabilityasamechanismofwaterstresstoleranceinthisspe-cies.Themethodcurrentlyusedisbasedoninvitrodesiccationofleaftissuesbyasolutionofpolyethyleneglycol(PEG)andasubsequentmeasurementofelectrolyteleakageintodeionisedwater.Itconsistsofthreesuc-cessivesteps:(1)awashingtreatmenttoremovesolutesfrombothleafsurfacesandcellsdamagedbycutting;(2)astressperiodduringwhichtheleaftissuesareplungedinaPEG-solutionand(3)arehydrationperiodduringwhichafter-effectsofthestressareevaluated.Duringthewashingperiod,themajorpartofelectrolyteswasremovedwithin15min.Varyingthestressconditionsinfluencedboththepercentandthekineticsofelec-trolyteleakageduringrehydration.Electrolyteleakageexhibitedacharacteristicpatternreflectingtheconditionofcellularmembranes(repairandhardening).Inpractice,werecommenda15-minutewashingtime,a10-hourstressperiodand4hofrehydration.Theextentofthecellmembranedamagenotonlycorrelatedwellwiththegrowthresponsesofwheatseedlingsbelongingtovariouscultivarstowithholdingwaterbutalsowiththerec-ognisedfieldperformancesofthesecultivars.Therelativeproportionofendogenousionslostintheeffusateduringtherehydrationstepmayvarystronglyaccordingtotheelementanalysedandtheprecisenutritionalsta-tusoftheplantshouldthereforebeconsidered.However,anincreaseininorganicionleakagedoesnotfullyexplaintherecordedPEG-inducedincreaseinelectricalconductivity(EC)duringthesubsequentrehydrationstepandorganicionsareprobablyalsoinvolvedinsuchanincrease.IntroductionDroughtresistanceistheresultofvariousmorpholog-ical,physiologicalandbiochemicalcharacteristics.Itsgeneticimprovementincropplantsrequirestheiden-tificationofappropriatedroughtresistancemecha-nismsandparticularlythedevelopmentofsuitablemethodologiesfortheirmeasurementinlargebreed-ingpopulations.Cellmembranesareoneofthefirsttargetsofmanyplantstressesanditisgenerallyacceptedthatthemaintenanceoftheirintegrityandstabilityunderwa-terstressconditionsisamajorcomponentofdroughttoleranceinplants.Thedegreeofcellmembranein-juryinducedbywaterstressmaybeeasilyestimatedthroughmeasurementsofelectrolyteleakagefromthecells.ThemethodisbasedonaninvitrostressofleaftissuesbyaPEGsolutionandasubsequentmeasure-mentofelectrolyteleakageintoanaqueousmedium(SullivanandRoss1979).Ithasanenduringappeal-becauseitrequiresreadilyavailableandinexpensiveequipment,itisnotdestructiveofwholeplants,iseasilyusedonplantmaterialfromavarietyofcul-turalsystemsanditissuitableforanalysinglargenumberofsamples.Suchatechniquehasalsobeenappliedtoquantifydamagestocellmembranesin1PlantGrowthRegulation00:1–10,2001.©2001KluwerAcademicPublishers.PrintedintheNetherlands.XPS53702(GROW)–productelement352339–ICPC/Grafikonvariousabioticstressconditionssuchaslow(Cour-solleetal.2000;Tamura2000;VainolaandRepo2000)andhigh(IsmailandHall1999;Maheswaryetal.1999;SaelimandZwiazek2000)temperatures,airpollution(Gartyetal.2000),saltstress(Chenetal.1999;Sreenivasuluetal.2000),acidconditions(SpencerandKsander1999),heavymetals(DeandMukherjee1996)andeveninresponsetobioticstresses(Adametal.2000;Srirametal.2000).Ithasbeendemonstratedrecentlythatelectrolyteleakagemeasurementsmaybecorrelatedwithseveralphysiologicalandbiochemicalparameterscondition-ingtheplantresponsestoenvironmentalconditionssuchasspectralreflectance(Gartyetal.2000;VainolaandRepo2000),antioxidativeenzymesynthesis(LiuandHuang2000;Sreenivasuluetal.2000),mem-braneacyllipidconcentrations(Laurianoetal.2000),wateruseefficiency(Francaetal.2000;SaelimandZwiazek2000),transverserelaxationtimeofleafwa-ter(Maheswaryetal.1999),stomatalresistance,os-moticpotentialandleafrollingindex(Premachandraetal.1989).Itisthereforenotsurprisingthatelectro-lyteleakagehasbeenrecommendedasavaluablecri-terionforidentificationofstressresistantcultivarsinseveralcropspecies(Leopoldetal.1981;Stevanovicetal.1997).However,despiteitsmanyadvantages,electrolyteleakagewasfoundtobemarkedlyinfluencedbyvar-iousexperimentalparameters,especiallywashingtimeofcollectedsamplesbeforePEGexposure(BlumandEbercon1981;PremachandraandShi-mada1987),intensityanddurationofthePEGtreat-ment(BlumandEbercon1981;Vasquez-Telloetal.1990)anddurationoftherehydrationperiod(Bandur-skaandGniazdowska-Skoczek1995;Bandurskaetal.1997;ZwiazekandBlake1990).Sincethedegreeofsoluteleakagevarieswithspecies(Leopoldetal.1981;Vasquez-Telloetal.1990),acarefulexamina-tionofthetechniquehastobeperformedforeachspeciesstudied.Anotherpointisthatelectrolyteleak-