Large number of flowers and tertiary branches, and higher reproductive success increase yields under salt stress in chickpea

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European

Journal

of

Agronomy

j our na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / e j a

Large

number

of

flowers

and

tertiary

branches,

and

higher

reproductive

success

increase

yields

under

salt

stress

in

chickpea

Vincent

Vadez

a,∗

,

M.

Rashmi

a

,

K.

Sindhu

a

,

Mithila

Muralidharan

a

,

R.

Pushpavalli

a

,

Neil

C.

Turner

b,c

,

L.

Krishnamurthy

a

,

Pooran

M.

Gaur

a

,

Timothy

D.

Colmer

b,c,d

aInternationalCropsResearchInstitutefortheSemi-AridTropics(ICRISAT),Patancheru502324,AndhraPradesh,India

bCentreforLegumesinMediterraneanAgriculture,TheUniversityofWesternAustralia,35StirlingHighway,Crawley,WA6009,Australia cTheUWAInstituteofAgriculture,TheUniversityofWesternAustralia,35StirlingHighway,Crawley,WA6009,Australia

dSchoolofPlantBiology,TheUniversityofWesternAustralia,35StirlingHighway,Crawley,WA6009,Australia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28May2011

Receivedinrevisedform19March2012 Accepted19March2012 Keywords: Salinitystress Salttolerance Reproductivebiology Transpiration Flowerabortion Podabortion Cicerarietinum

a

b

s

t

r

a

c

t

Salinityisamajorproblemworldwideandimprovingsalttoleranceofchickpea(CicerarietinumL.)will allowexpansionofproductiontomoremarginalareas.Plantreproductionsuffersundersaltstressin chickpea,butitremainsunclearwhichprocessismostaffectedandwhattraitsdiscriminatetolerant fromsensitivelines.Threepotexperimentswerecarriedouttocomparetheeffectsofsaltapplication (17gNaClkg−1Alfisol)atsowing(SS)andatthestartofflowering(SF)ongrowth,canopytranspiration,

plantarchitecture,andflower,podandseeddevelopment(timing,numbers,mass,abortion).Sixpairs oftolerant/sensitivelineswithsimilarfloweringtimeswithineachpair,butdifferentamongthepairs, wereused.ShootbiomasswassimilarintolerantandsensitivelinesintheSSandSFtreatments,whereas theseedyielddecreasedmoreunderSSandSFtreatmentsinthesensitivelines.Theflower,podandseed numberswithinallpairswashigherinthetolerantthaninthesensitivelinesinthenon-salinecontrols, butthedifferencesinnumbersofseedsandpodsfurtherincreasedinboththeSSandSFtreatments. Bycontrast,neitherthedurationoffloweringorpodding,northepercentageofflowerorpodabortion, discriminatedtolerantfromsensitivelines.Innon-salinecontrolsthenumbersofprimarybrancheswas 100%higheracrossthesensitivelines,whereasthenumberoftertiarybrancheswas8-foldhigheracross tolerantlines.Therelativetranspirationofthetolerantlinesinthesalttreatmentswasabovethatforthe sensitivelinesinthreepairsoftolerant/sensitivelines,butdidnotdifferwithintwopairs.Ourresults demonstratethatconstitutivetraits,i.e.numbersofflowersandtertiarybranches,andadaptivetraits, i.e.highnumberofseedsundersaltstress,arebothcriticalaspectsofsalinitytoleranceinchickpea.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Salinity is a major and increasing problem worldwide that needstobeaddressedinordertomaintainagricultural produc-tion.Geneticapproachestoimprovecroptoleranceofsalinity(i.e. breeding) will be important, and especially since management optionsrequirealargeinvestmentthatpoorfarmersareunlikely toimplement.Chickpeaisgrowninvariousregionschallengedby increasingsoilsalinity(Flowersetal.,2010).Thereexistsgenetic variationforsalinitytolerancewhichcanbeusedtobreedsuperior varieties(Vadezet al.,2007;Krishnamurthyetal.,2011). How-ever,breedingwouldbemademoreefficientbyfocusingonthose traitsthatarecritical,butstillrelativelyunknown,forthesalinity toleranceofchickpea(Flowersetal.,2010).

∗ Correspondingauthor.Tel.:+914030713643;fax:+914030713074. E-mailaddress:v.vadez@cgiar.org(V.Vadez).

Althoughsalinityaffectsshootgrowth,itseffecton reproduc-tiveprocesses is relativelymore severein chickpea. Genotypic tolerances,basedonseedyieldobtainedundersalineconditions, wererelated moretomaintainingalargenumber ofseedsand lesstomaintainingahighbiomassproductionrelativetoa non-salinecontrol(Dhingra andVarghese,1993;Vadezet al.,2007; Krishnamurthyetal.,2011).Fromtheearlydevelopmentofflower meristemsuntil the development of seedsin thepods, abiotic stresses can affect a number of processes. Abiotic stresses are knowntoaffectmeiosisduringgameteproductionandmale steril-ityappearstobemorecommonthanfemalegametesterility(Saini, 1997).Flowerproductionwasdecreasedunderdroughtin chick-pea(Nayyaretal.,2005;Fangetal.,2010),orunderheatstressin groundnut(VaraPrasadetal.,2000).Flowerabortionwasanother causeforyielddecreaseunderdroughtinastudythatshowedthat cultivatedchickpeaabortedalargernumberofflowersthanwild germplasm(Nayyaretal.,2005), orinchickpea exposedtocold whereplantsproducedflowersbutfailedtosetpods(Clarkeand 1161-0301/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved.

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Siddique,2004;Srinivasanetal.,1998).Podabortionalsowasthe keylimitationtoseedyieldinchickpeaexposedtodroughtstress (Behboudianetal.,2001;Leportetal.,1999,2006;Fangetal.,2010). Finally,thedurationandrateofseedfillingcanalsoexplainyield variationsunderdroughtstressinchickpea(Daviesetal.,1999). So,anumberofprocessesduringthereproductivephasecanbe affectedbyabioticstresses.Thereis,unfortunately,limited knowl-edgeonwhich oftheseprocessesaremostaffectedinchickpea exposedtosalinityandwhethertolerantandsensitivelinesdiffer insensitivityofoneorseveraloftheseprocessestodetermineseed yieldinsalinesoils.

Carbohydratesupplycouldbealimitation.Reproductive struc-turesare quitedemandingfor carbohydratesandthesupply of sucrosetothedevelopingembryoswasshowntobecriticalto res-cueembryosofwaterstressedplantswherephotosynthesiswas inhibited(Zinselmeieretal.,1999).So,reproductivefailureunder saltstresscouldberelatedtodecreasedtranspirationrelativeto unstressedplants,wheretranspirationis asurrogatefor photo-synthesis/carbonsupplytothedevelopingembryos.Recentdata (Vadezetal.,2011)alsosuggestthatinearlychickpealines,where floweringissimultaneouswithsustainedshootgrowth,thehigh yieldinglineswerethose havingbothreproductivesuccessand sustainedshootgrowthundersalinestress.Thesustainedgrowth undersalinestresscouldleadtoincreasedbranchingand toan increasednumberofreproductivenodesandflowers.So,the ques-tionremainswhethershootgrowthandbranchingcouldleadto morereproductivestructures,especiallyinearlydurationlines.

Theoverallobjectiveofthisworkwastopinpointtraitsthat dis-tinguishtolerantandsensitivelines,withaparticularfocusonplant architectureandreproductivebiology.Theworkwasperformed withsixpairsoftolerant/sensitivelinesofchickpeainwhich flow-eringtimewassimilarwithineach pair.Afirstobjectivewasto compareeffectsofsaltstressapplicationatsowingandflowering onbiomassandyield,withthehypothesisthatsalteffectswouldbe thesameinthesetwotypesoftreatmentsifreproductionwasthe mostsensitiveplantprocesstosalinityinchickpea.Asecond objec-tivewastoassesstheeffectofsaltonphenologicaldevelopment (flowering/poddingduration)andgrowthpatterns(rooting,shoot branching).Athirdobjectivewastoinvestigatethedirecteffects ofsalttreatmentonreproductivestructures(flowernumberand abortion,podnumberandabortion,seednumberandsize).Thelast objectivewastoinvestigatehowsalinityaffectsplanttranspiration duringreproduction.

2. Materialandmethods

2.1. Growthconditionsandtreatments

Chickpea(CicerarietinumL.)wasgrownundersalineand non-salineconditionsin20cmdiameterpotsfilledwith4kgofVertisol soil(VerticInceptisol)collectedfromtheICRISATfarm,mixedwith farm manureat a rateof 50:1 (soil:manure, w/w), autoclaved, sieved and sun dried.The soil [pH 8.1, CEC/clay ratio=0.8 and anelectricalconductivity=0.10dSm−1insaturatedpasteextract (ECe)(ElSwaify etal.,1985)]wasfertilizedwithdi-ammonium phosphateandmuriateofpotash,atarateof0.3gand0.2gperkg soil,wellmixedwiththesoilbeforefillingthepots.Soilwas inoc-ulatedwithstandardchickpearhizobiuminoculumatthetimeof sowing.

ThreeexperimentswerecarriedoutbetweenNovemberand MarchatICRISATheadquarters(Patancheru, AP,India,Latitude: 17◦3153N,Longitude:78◦1554E),twooutdoors(Experiments 1and 2)and onein a greenhouse(Experiment3).The average maximumtemperaturesrangedbetween25.3and36.8◦Cand min-imumtemperaturesbetween8.4and22.0◦Coutdoors.Theaverage

maximumtemperaturesrangedbetween29.7and32.6◦Cand min-imumtemperaturesbetween15.4and16.1◦Cinthegreenhouse. Fourseedswereplantedineachpot.Thesewerethinnedtotwo plantsperpotat3weeksaftersowing.

Threetreatments wereused: a non-salinecontrol(C),a salt treatmentapplied at thetime of sowing (SS), and a salt treat-mentappliedatthebeginningofflowering(SF),thereforeapplied atdifferentdatesdependingongenotype.Thetwosalttreatment swereequivalentandcorrespondedtoasaltapplicationin the irrigationwaterinsufficientquantitytowettheVertisoltofield capacity(1Lper4kgpot)andresultintheequivalentof80mM NaClinthesolution(1.17gNaClkg−1 soil).Thesaltwasapplied insplitapplications.InSS,halfthedosewasappliedatsowingby wettingthesoilwith1Lof40mMNaClsolution,whilethe sec-onddosewasapplied1weekaftersowingbyadding400mLof 100mMNaCl.InSF,halfthedosewasappliedwhenallplantsofa givenpairoflineshadstartedflowering,byflushingthepotswith 1Lof40mMNaCl,andthenthefollowingdayflushingagainwith 1Lof80mMNaCl.Ateachtime,thenon-salinecontrolpotswere alsoflushedwith1Lofwatercontainingnosalt.LinesICC1431and ICC6263mistakenlyreceivedanadditionalLof40mMNaCl solu-tionintheSFtreatment,likelyexplainingtheirhighershoot,pod andseedmassdecreasethantheotherlines.Therefore,upto flow-eringtime,theplantsoftheSFtreatmentandtheCtreatmentwere treatedthesameway.AftersaltapplicationintheSSandSF treat-ments,potswerewateredwithtapwatercontainingnosignificant amountofNaCl,andmaintainedclosetofieldcapacity(determined gravimetrically)toavoidanincreaseinsaltconcentrationinthesoil solution,butalsotoavoidleachingofthesalt.

2.2. Plantmaterialsanddetailsofexperiments

Experiment1(Exp.1)wascarriedouttocomparetheplant archi-tecture,rooting,andtimingofpod/seedproduction,thenumberof flowersandflowerabortion,alongwiththeseedyield/pod/seed numberandshootdrymass,andtoassesstheeffectofsaltstress ontherateoftranspirationatthetimeofflowering(SF).Primary brancheswerethoseproducedonthemainstem,whilesecondary and tertiarybrancheswerethose producedontheprimaryand secondarybranches,respectively.Exp.1wasconductedoutdoors andusedfivepairsoflinesthatwereclassifiedassalttolerantor salt sensitivebased onseedyieldin salineconditionsin a pre-viousevaluation(Krishnamurthyetal.,2011):ICC1431/ICC6263 (tolerant/sensitiveineachcase);JG11/ICCV2;ICC9942/ICC15802; ICC3512/ICC13283;ICC7819/ICC7571.Thesefivepairsoflineshad similarfloweringtimewithinpair,i.e.37DAS,49–51DAS,49–51 DAS,49DAS,and49DAS,respectively.Thiswasimportantbecause previousreportshowedhighertolerancetosalinityinearly dura-tionlines(Vadezetal.,2007).Threetreatmentswereused(C,SS, SF),eachwitheightreplicatepotspergenotype.Fourreplicatepots perlineandtreatmentwereharvestedat30daysaftertreatment applicationintheSFtreatmentforassessingrootingandbranching, whereastheotherfourreplicateswerekeptuntilmaturity.

Experiment 2 (Exp.2) was carried out to confirm the measurements of Exp.1, and contained an extra pair of tol-erant/sensitive lines, with the objective to compare the yield reduction and flower/pod/seed number and abortionin theSS and SF treatments. In this experiment, no plants were grown in non-saline soil. Exp.2 was conducted outdoors and used six pairs of tolerant/sensitive lines (flowering time in paren-thesis): ICC1431/ICC6263 (46–44 DAS); JG11/ICCV2 (35 DAS); ICC9942/ICC15802 (45–44 DAS); ICC3512/ICC6877 (46 DAS); ICC7819/ICC7571 (46 DAS); ICC5845/ICC13283 (46 DAS). Four replicatepotsperlinewereusedineach ofthetwotreatments (SS,SF).

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Experiment 3 (Exp.3) was carried out to follow the dura-tionoffloweringandpoddingundersaltstress,andtocompare flower/pod/seed number and abortion in salt-treated/control plants. Exp.3 was conducted in a greenhouse and used three pairs of tolerant/sensitive lines (flowering time in paren-thesis): ICC1431/ICC6263 (44 DAS); JG11/ICCV2 (34 DAS); ICC9942/ICC15802(44DAS).Threetreatmentswereused(C,SS, SF),eachwithfourreplicatepotsperline.

2.3. Flowerandpodtagging

InExp.1,reproductivesuccesswasassessedindifferentweeks aftersaltapplicationbytaggingflowersappearingduringanentire weekwithathreadofaparticularcolour,adifferentcolourbeing usedforeachweeksothatatharvestflowerproductionaswell aspodandseedproductioncouldbefollowedonaweeklybasis. InExp.2 a similarprinciple wasfollowed forflower tagging. In Exp.3,proceduresfollowedcloselyLeportetal.(2006),andeach flowerwastaggedatthetimeofappearanceandthedatewritten onthetag.Subsequentlythedateofpodappearance,ifany,was recordedonthetag.Thereforethestartsandendsoffloweringand poddingweredocumentedforeachplant,andforallthree treat-ments(C,SS,SF).Thistaggingprocedurealsoprovidedthetotal numberofflowersproducedbyeachplant(totalnumberoftags perplant),thenumberofpodsperplant(numberoftagswitha dateofpodding),thenumberoffertilepods(numberofseedsper podandplant,countedatharvest),andenabledcalculationofthe percentageabortionofflowersandpods.

2.4. Canopytranspiration

FourdaysbeforesaltapplicationintheSFtreatmentofExp.1,a uniformandthicklayerofplasticbeadswasappliedtothesurface ofeachpotofeachtreatment.Previousworkhadshownthatthe beadsreducedsoilevaporationbyabout90%sothatweighingof potscouldbeusedasameasureofplanttranspiration(Ratnakumar etal.,2009).Eightreplicatepotsineachtreatmentandlinewere weighedeveryalternatedayinthemorningfrom4dayspriorto until4weeksaftertheapplicationofsaltandthemassrecorded. Transpirationratewascalculatedfromthepotmassdifferences andamountsofwateradded(potmassdaynminuspotmassday n+2pluswateraddeddayn).Saltapplicationwasmadeatnight andtranspirationpriortosaltapplicationwasmadebyweighing thepotslateintheafternoon.AfterflushingtheSFandCpotswith theirrespectivetreatments,potswerelettodrainovernightandpot masstakenagaininthemorning.Thisallowedthemeasurementof planttranspirationbeforeandaftertheapplicationofsalt.Plants werethenre-wateredto90%field(i.e.pot)capacity,toensurethat nowaterstressoccurredandthattherewasnodrainage.

Transpirationratios(TR)werecalculatedforeachdayby divid-ingplanttranspirationinSFbythemeanofthetranspirationinC foreachline.Thennormalizedtranspirationratios(NTR)were cal-culatedbydividingindividualTRvaluebythemeanofTRvalues beforetreatmentswereapplied,forindividualpots.

2.5. Harvestprocedures

Whenthe plants reachedmaturity, primary, secondary and tertiarybrancheswerecounted.Thetwoplantsperpotwere pro-cessedseparatelyandthedatesoffloweringandpoddingonthe tagsandthenumberoftagsperplantwererecorded.Podswere separated,weighedafterdryinginaforcedairovenat70◦Cfor2 daysandcounted.Podswerecrushedandseedswereseparated, weighed,andcounted.Eachplantwasovendriedseparately at 60◦Cfor48handweighedforshootdrymass.

InExp.1,asetofplants(4replicatepotsineach oftheC,SS andSFtreatments)wasalsoharvestedat30daysafterSF treat-mentapplicationtoassessrootbiomassandbranchingpatterns.At harvestinExp.1,theECeofthesoilwasmeasuredinallpots fol-lowingstandardprocedures(Krishnamurthyetal.,2011);soilECe averaged0.97dSm−1intheSStreatmentand1.34dSm−1intheSF treatment,thereforethesalinitywasslightlymoresevereintheSF treatment.

2.6. Statisticalanalyses

Theexperimentaldesignswereeachacompletelyrandomized blockwithtreatmentasthemainfactorandlinesasthesub-factor; witheightreplicatesinExp.1andfourreplicatesinExp.2andExp.3. InExp.1,eightreplicatesperlineandtreatmentwereusedtoassess thetranspirationresponsetosaltapplicationintheSFtreatment andcomparedtotheCtreatment.Thenfourofthesereplicateswere usedfortheharvestat30daysafterSFapplicationandtheother fourreplicates werekept untilmaturity. One-way ANOVA was usedformeancomparisonswithintreatments.Two-wayANOVA wasusedtocompareC,SSandSFtreatmentsandtheirinteraction effectsontheparametersmeasured.

3. Results

3.1. Comparisonofsaltapplicationatdifferentstages

Theshootdrymassdecreasedbyonaverageabout45%when saltwasappliedatthetimeofsowing(SS-Exp.1).However,lines variedinthedegreeofreductioninshootdrymassfrom25to80% (Table1).Bycontrast,withsaltappliedatflowering(SF)theshoot masswasreducedonaveragebyabout20%.Therealso,linesvaried intherangeofbiomassreduction,withearlydurationlinesJG11 andICCV2showingthelargestdecreaseof45and33%,respectively. IntheCtreatment,theaverageshootdrymassoftolerantand sen-sitivelinesweresimilar,andthereductionunderSSwassimilar fortolerantandsensitivelines.Under SF,theshootdrymassof thetolerantlineswasreducedbutnotthatofthesensitivelines (Table1).

ThepodmasswasonlyslightlydecreasedintheSStreatment (about13%).However,linesvariedinthereductionofpodmass reduction.Sensitivelineshadanaverage24%decreaseinpodmass underSS,whereaspodmassoftolerantlinesdidnotdifferfromthe control(Table1).ResultswereverysimilarintheSFtreatment:no significantdecreaseinpodmassoverall,variationacrosslinesin thatpercentagedecrease,andanaveragedecreaseinthesensitive lines(21%),whereasthepodmassofthetolerantlinesunderSFwas notdifferentfromtheCtreatment(Table1).Inaddition,themean podmassofeachgroupoflineswassimilarunderSSandSF treat-ment.Nevertheless,thepercentagechanges foreach lineunder SFandSSwerepoorlycorrelated,indicatedalsobyahighly sig-nificantgenotype-by-treatmentinteraction(Table1).Thisshowed thatsomelinessufferedtheSSstressmorethantheSFstress(e.g. ICC13283),whereasotherssufferedtheSFstressmorethantheSS stress(inparticularearlylinesJG11andICCV2).

Resultsfortheseedmasswereverysimilartothose onpod massfortheSStreatment.Thedecreaseinseedmassinthe sensi-tivelinesunderbothSSandSFtreatmentswasabout25%,whereas theseedmasswasunchangedcomparedtotheCtreatmentin tol-erantlines.The percentagechanges alsosignificantlyvariedfor lines.Forinstance,thepodmassoftolerantJG11decreased15% underSF,whereastheseedmassincreased35%.Conversely,the podmassofsensitiveICC13283decreased4%underSS,whereasthe seedmassdecreased30%(Table1).Thetwo-wayANOVArevealeda

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Table1

Shoot(excludingpods),podandseedmass(gperplant)atmaturityinfivepairs(Exp.1)oftolerant(T)/sensitive(S)linesexposedtonon-salinecontrolconditions(C),saline conditionsappliedfromthetimeofsowing(SS),andsalineconditionsappliedfromthetimeofflowering(SF).Dataaremeansoffourreplicatepots(twoplantsperpot,data expressedperplant).Two-wayANOVA(F-value)wasusedtocompareline(G)treatment(T)andgenotype-by-treatmenteffects(G×T).Leastsignificantdifferencesforeach oftheG,T,andG× Teffectsareprovided.

Shootmass(g) Podmass(g) Seedmass(g)

C SS SF C SS SF C SS SF ICC1431(T) 13.72 2.68 11.05 6.81 4.71 6.80 5.18 3.74 5.15 ICC6263(S) 12.09 3.32 12.52 9.77 3.48 7.48 6.29 2.39 4.26 JG11(T) 7.24 4.12 3.89 3.89 5.26 3.30 3.17 4.41 4.41 ICCV2(S) 6.56 2.60 4.40 4.44 3.12 2.11 3.07 2.35 2.35 ICC9942(T) 10.49 6.63 11.18 10.05 9.54 7.08 8.14 7.64 5.33 ICC15802(S) 12.84 7.88 12.20 9.61 7.58 6.16 6.84 5.58 4.16 ICC3512(T) 10.45 7.79 10.81 8.92 9.33 11.88 6.71 6.98 8.97 ICC13283(S) 14.51 12.89 18.59 7.74 3.22 7.40 5.48 2.00 3.91 ICC7819(T) 22.57 13.35 15.24 6.11 7.24 9.18 4.36 4.90 5.70 ICC7571(S) 17.19 10.49 13.77 5.03 7.77 5.67 3.72 5.41 3.52 LSD 4.60** 2.01** 3.01** 2.68** 2.50** 1.97** 2.11** 1.97** 2.03** Meantolerant 12.89 6.91 10.43 7.15 7.22 7.65 5.51 5.53 5.91 Meansensitive 12.64 7.44 12.29 7.32 5.03 5.76 5.08 3.55 3.64 Geffect 35.90** 20.31** 15.40** Teffect 62.11** 4.75** 3.47* G×Teffect 3.38** 5.19** 3.39** LSDG 1.87 1.31 1.10 LSDT 1.02 0.71 0.60 LSDG×T 3.24 2.26 1.91 ns,non-significant. *SignificantatP<0.05. **SignificantatP<0.01.

predominantgenotype(G)effectforpodandseedmass,although thegenotype-by-treatmentinteractionwasalsohighlysignificant. InExp.2theresultsweresimilartothosefromExp.1,in partic-ularthefactthattheseedmasswassimilarinSSandSFtreatments forbothgroupsoftolerantandsensitivelines.However,the aver-ageshootdrymassofthetolerantandsensitivelinesweresimilar underSSandSF.Therealso,theseedmasswasabout60%higher in thetolerant(7.4gper plant) than inthe sensitive(4.3gper plant)linesacrossbothSSandSFtreatment(Table2).Alsoinboth Exp.1andExp.2,theseedmassunderSSwithineachpairof toler-ant/sensitivelineswashigherinthetolerantthaninthesensitive lines,whichconfirmedearlierassessmentsontheselines(Vadez et al.,2007; Krishnamurthy et al., 2011). Thetwo-way ANOVA revealedamajorgenotype effectonboth shootandseedmass, whereasthegenotype-by-treatmentinteractionwasnotsignificant forseedmass(Table2).

3.2. Phenologicaldevelopment

Thelengthofthefloweringperiod,measuredinExp.3,wasabout 4dayslongerinthetolerantthaninthesensitivelinesintheC treat-ment(33.0daysversus28.6days)(Table3).UnderSS,thisperiod wasreducedby17and15daysinthetolerantandsensitivelines. UnderSF,thelengthofthefloweringperiodwasalsosignificantly reducedcomparedtotheCtreatmentbyabout8daysinboththe tolerantandthesensitivelines.Thelengthofthepoddingperiod wasalsoslightlylongerinthetolerantthaninthesensitivelinesin theCtreatment(30.6daysversus26.0days).UnderSS,thelengthof poddingwasshortenedby16and15daysinthetolerantand sen-sitivelines.UnderSFitwasreducedby9and8daysinthetolerant andsensitivelines.So,thereappearedtobenomajorassociation betweensensitivitytosaltstressandthelengthoffloweringor podding.

3.3. Plantmorphologyanddevelopment

Thenumbersofprimarybrancheswerehigherinthesensitive thaninthetolerantlinesunderthenon-salineCtreatment,except

inJG11/ICCV2andICC7819/ICC7571(Table4).TheSStreatment significantlydecreased thenumberofprimarybranchesby40% inthetolerantlinesand20%in thesensitivelines,whereas the SFtreatmentdidnotaffectthenumberofprimarybranches.The numberofsecondarybrancheswassimilarinthetolerantand sen-sitivelinesintheCandSStreatments.TheSStreatment,butnot theSFtreatment,decreasedsecondarybranchingby45%and25%in

Table2

Shoot,and seed mass(gplant−1)at maturity insix pairs(Exp.2)of tolerant (T)/sensitive(S)linesexposedtosalineconditionsappliedfromthetimeofsowing (SS)andsalineconditionsappliedatthetimefromflowering(SF).Dataaremeansof fourreplicatepots.Two-wayANOVAwasusedtocomparegenotype(G)treatment (T)andgenotype-by-treatmenteffects(G×T).Leastsignificantdifferencesforeach oftheG,T,andG×Teffectsareprovided.

Shootmass Seedmass

SS SF SS SF ICC1431(T) 14.57 10.57 9.14 7.56 ICC6263(S) 12.06 8.71 6.04 3.67 JG11(T) 9.86 10.97 7.72 9.73 ICCV2(S) 5.89 4.84 2.44 4.38 ICC9942(T) 15.79 11.76 7.70 7.15 ICC15802(S) 12.06 15.60 5.68 5.79 ICC3512(T) 13.20 11.65 6.66 7.16 ICC13283(S) 15.57 15.73 2.88 2.93 ICC7819(T) 14.33 16.04 8.83 6.08 ICC7571(S) 18.86 15.47 5.32 5.81 ICC5845(T) 10.55 14.13 6.22 4.54 ICC6877(S) 17.15 20.86 4.24 3.13 LSD 5.30** 2.94** 1.68** 1.69** Averagetolerant 13.05 12.52 7.71 7.04 Averagesensitive 13.60 13.54 4.43 4.28 Geffect 9.94** 11.87** Teffect 0.65ns 1.56ns G×Teffect 1.97* 0.77ns LSDG 3.11 2.26 LSDT ns ns LSDG×T 4.40 ns ns,non-significant. *SignificantatP<0.05. **SignificantatP<0.01.

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Table3

Lengthofthefloweringandpoddingperiod(days),totalnumberofflowersperplant,andpercentageflowerabortion,totalnumberofpods,seeds,andemptypodsper plant,percentageofpodabortion,inthreepairs(Exp.3)oftolerant(T)/sensitive(S)linesexposedtonon-salinecontrolconditions(C),salineconditionsappliedfromthe timeofsowing(SS)andfromthetimeofflowering(SF).Dataaremeansoffivereplicatepots.Two-wayANOVAwasusedtocomparegenotype(G)treatment(T)and genotype-by-treatmenteffects(G×T).LeastsignificantdifferencesforeachoftheG,T,andG×Teffectsareprovided.

Lengthofflowering Lengthofpodding Numberofflowers Flowerabortionpercentage

C SS SF C SS SF C SS SF C SS SF ICC1431(T) 22.5 15.1 25.1 19.1 12.1 22.7 51.7 20.5 67.2 32 31 35 ICC6263(S) 30.6 16.3 27.9 26.5 14.4 25.0 36.0 14.9 52.7 28 32 36 JG11(T) 40.8 17.3 28.4 36.9 14.6 21.5 58.2 21.0 37.3 40 26 39 ICCV2(S) 17.8 8.5 9.2 16.9 7.6 10.0 13.1 4.9 7.6 08 10 8 ICC9942(T) 35.6 14.6 19.7 35.7 14.5 17.7 73.3 15.4 48.3 37 21 37 ICC15802(S) 37.4 16.3 25.0 34.5 12.1 19.4 34.2 12.6 38.1 25 25 35 LSD 10.6** 5.2** 6.0** 11.2** 4.7* 8.5* 20.9** 4.0** 13.8** 14** 12** 12** Averagetolerant 33.0 15.7 24.4 30.6 13.7 20.6 61.1 19.0 50.9 36 27 37 Averagesensitive 28.6 13.7 20.7 26.0 11.4 18.1 27.8 10.8 32.8 20 20 26 Geffect 16.5** 7.86** 26.6** 15.3** Teffect 61.7** 44.5** 74.3** 4.93** G×Teffect 3.07** 2.72** 6.57** 1.23ns LSDG 4.1 4.74 7.6 0.07 LSDT 2.89 3.35 5.4 0.05 LSDG×T 7.1 8.21 13.2 ns

Numberofpods Numberofseeds Numberofemptypods Podabortionpercentage

C SS SF C SS SF C SS SF C SS SF ICC1431(T) 33.0 13.9 44.2 25.7 12.6 36.6 7.5 1.4 7.6 22 10 18 ICC6263(S) 26.6 9.9 33.0 21.6 7.6 29.4 5.5 1.6 3.5 21 15 11 JG11(T) 33.2 15.1 22.9 27.8 12.6 19.2 5.0 2.5 3.9 15 17 18 ICCV2(S) 12.0 4.4 7.0 10.9 3.8 6.0 1.5 0.6 1.0 17 14 12 ICC9942(T) 46.0 12.1 31.4 32.4 10.3 26.6 13.0 1.9 4.9 31 19 23 ICC15802(S) 25.2 9.7 26.3 18.2 8.2 21.3 6.8 1.5 5.2 25 23 28 LSD 13.0** 2.5** 11.0** 12.9* 2.5** 10.2** 5.7* ns 2.9** ns ns ns Averagetolerant 37.4 13.7 32.8 28.6 11.8 27.5 8.5 1.9 5.5 22 20 15 Averagesensitive 21.3 8.0 22.1 16.9 6.5 18.9 4.6 1.2 3.2 21 17 17 Geffect 19.4** 12.9** 7.12** 1.93ns Teffect 61.0** 39.8** 26.2** 1.30ns G×Teffect 4.51** 2.84** 2.95** 0.50ns LSDG 5.22 5.06 2.0 ns LSDT 3.69 3.58 1.4 ns LSDG×T 9.05 8.77 3.4 ns ns,non-significant. * SignificantatP<0.05. ** SignificantatP<0.01. Table4

Totalnumberofprimary,secondaryandtertiarybranchesperplantandrootdrymass(gperplant)infivepairs(Exp.1)oftolerant/sensitivelinesexposedtonon-salinecontrol conditions(C),salineconditionsappliedfromthetimeofsowing(SS),andsalineconditionsappliedfromthetimeofflowering(SF).Dataaremeansoffourreplicatepots. Two-wayANOVAwasusedtocomparetreatmentandtreatment-by-genotypeeffectusingonlytheSSandSFtreatment.Two-wayANOVAwasusedtocomparegenotype (G)treatment(T)andgenotype-by-treatmenteffects(G×T).LeastsignificantdifferencesforeachoftheG,T,andG×Teffectsareprovided.

Primarybranches Secondarybranches Tertiarybranches Rootdrymass

C SS SF C SS SF C SS SF C SS SF ICC1431(T) 5.7 2.0 5.5 32.0 4.3 23.2 31.0 0.7 35.2 4.6 0.7 4.2 ICC6263(S) 19.2 12.2 15.0 43.2 3.2 25.7 11.7 0.0 2.5 5.5 1.0 3.6 JG11(T) 11.5 12.0 15.0 17.0 14.2 24.0 4.5 0.2 0.5 2.4 1.3 1.9 ICCV2(S) 10.7 7.2 8.5 6.2 6.5 7.2 0.0 0.0 0.0 2.0 1.1 1.2 ICC9942(T) 6.7 5.7 5.2 25.7 17.2 26.0 40.2 22.2 45.5 4.1 2.4 4.0 ICC15802(S) 15.2 15.2 14.7 39.7 40.7 30.7 11.5 3.7 4.0 5.7 2.8 4.1 ICC3512(T) 16.0 6.0 17.0 35.0 19.5 33.7 12.7 8.7 12.2 4.4 3.7 3.8 ICC13283(S) 26.2 15.2 25.2 45.5 26.5 35.2 1.5 0.2 2.2 7.4 5.1 7.5 ICC7819(T) 16.2 6.5 12.2 62.2 34.7 37.2 28.7 12.0 26.5 7.0 4.1 6.5 ICC7571(S) 15.5 19.5 20.0 47.5 45.0 48.7 12.0 8.2 10.2 6.1 3.5 4.5 LSD 5.8** 4.6** 4.8** 10.5** 13.2** 7.6** 12.4** 6.1** 7.9** 1.2** 1.4** 1.4** Averagetolerant 10.0 6.4 10.7 27.4 13.8 26.7 22.1 8.0 23.4 3.9 2.0 3.5 Averagesensitive 17.9 12.5 15.9 33.7 19.2 24.7 6.19 1.0 2.2 5.1 2.5 4.1 Geffect 22.2** 21.1** 43.9** 23.9** Teffect 22.4** 20.2** 57.8** 52.9** G×Teffect 3.16** 3.12** 11.7** 2.97** LSDG 3.3 7.5 4.8 0.9 LSDT 1.5 3.4 2.2 0.4 LSDG×T 4.6 10.7 6.8 1.4 ** SignificantatP<0.01.

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Table5

Numberofflowers,percentageofflowerabortion,numberofpodsandseedsperplant,seednumberperpod,infivepairs(Exp.1)oftolerant/sensitivelinesexposedto non-salinecontrolconditions(C),salineconditionsappliedfromthetimeofsowing(SS),andsalineconditionsappliedfromthetimeofflowering(SF).Dataaremeansof fourreplicatepots.Two-wayANOVAwasusedtocomparegenotype(G)treatment(T)andgenotype-by-treatmenteffects(G×T).Leastsignificantdifferencesforeachofthe G,T,andG× Teffectsareprovided.

Numberofflowers Flowerabortion Numberofpods Numberofseeds Seednumberpod−1

C SS SF C SS SF C SS SF C SS SF C SS SF ICC1431(T) 52.0 35.0 51.8 76 76 83 39.7 27.0 43.0 33.3 30.7 36.0 0.86 1.16 0.84 ICC6263(S) 45.8 18.5 40.5 77 72 91 35.5 13.8 37.0 25.5 10.5 20.0 0.72 0.79 0.55 JG11(T) 26.8 27.8 25.3 68 88 86 18.3 24.5 22.0 14.8 22.5 14.0 0.83 0.92 0.63 ICCV2(S) 26.3 14.3 14.0 86 97 91 22.0 13.8 12.8 16.5 11.8 8.0 0.73 0.95 0.63 ICC9942(T) 70.8 66.5 52.5 73 88 86 51.3 58.8 45.0 62.8 65.3 40.8 1.23 1.12 0.92 ICC15802(S) 51.3 42.0 47.0 78 86 86 40.0 35.8 40.5 34.0 24.8 19.0 0.85 0.71 0.48 ICC3512(T) 61.3 70.5 80.8 78 91 93 48.0 64.0 75.3 36.3 42.5 61.3 0.76 0.67 0.81 ICC13283(S) 33.0 29.0 55.7 80 59 70 26.3 18.3 39.3 22.0 10.5 17.7 0.84 0.53 0.46 ICC7819(T) 53.0 71.3 64.3 56 71 90 29.5 50.5 57.5 17.0 23.3 28.0 0.58 0.47 0.49 ICC7571(S) 30.7 51.5 52.5 72 73 75 22.0 37.5 40.0 18.0 24.3 18.5 0.81 0.64 0.45 LSD 8.9** 4.6** 3.3** 15* 14** 10** 10.8** 12.5** 11.3** 9.3** 11.5** 9.9** 0.22** 0.29** 0.19** Averagetolerant 52.7 54.2 54.9 70 83 88 37.33 44.95 48.5 32.82 36.8 36.0 0.85 0.87 0.74 Averagesensitive 37.4 31.0 41.9 79 77 83 29.17 23.80 33.9 23.20 16.3 16.6 0.79 0.72 0.51 Geffect 13.9** 7.18** 40.0** 52.8** 12.9** Teffect 19.9** 16.87** 11.3** 0.70ns 15.9** G× Teffect 3.90** 2.88* 5.4** 5.57** 1.9* LSDG 3.3 0.07 6.4 5.7 0.13 LSDT 1.8 0.04 3.5 ns 0.07 LSDG×T 5.7 0.12 11.1 9.8 0.23 ns,non-significant. *SignificantatP<0.05. **SignificantatP<0.01.

thetolerantandsensitivelines.Thetraitofplantarchitecturethat discriminatedtolerantfromsensitivelinesthemostwasthe num-beroftertiarybranchesundernon-salineCconditions,whichwas morethan300%higherinthetolerantlines(22.1perplant)that inthesensitivelines(6.2perplant).TheSStreatmentdecreased thenumberoftertiarybranchesdramatically,by65%and90%in thetolerantandsensitivelines,whereastheSFtreatment signifi-cantlydecreasedthenumberoftertiarybranchesinthesensitive linesonly(Table4).

Therootdrymasswashigherinthesensitivelinesthaninthe tolerantlinesundernon-salineCconditions(Table4).TheSS treat-mentdramaticallyreducedtherootdrymass,byabout50%inboth tolerantandsensitivelinessothatthedifferencesinrootdrymass werenolongersignificantbetweentolerantand sensitivelines. UndertheSFtreatment,therootdrymassdidnotdecreaseinthe tolerantlines,butsignificantlydecreasedinthesensitivelinesso thatthedifferencesinrootdrymasswerenolongersignificant betweentolerantandsensitivelines(Table4).

3.4. Flowerproductionandabortion

Themostremarkabledifferencewasinthegreaternumberof flowersproducedunderthenon-salineCtreatmentinthe toler-antlines(52.7flowers)about40%higherthaninthesensitivelines (37.4flowers).Undersalineconditionsthenumberofflowerswas 75%higherinthetolerantthaninsensitivelinesintheSS treat-mentand30%higherinthetolerantthaninsensitivelinesinthe SFtreatment(Table5).InExp.2,thenumberofflowersinthe tol-erantlineswasabout100%higherthaninthesensitivelinesin boththeSSandSFtreatments(Table6).Eachofthesixpairsof tol-erant/sensitivelinestestedshowedahigherflowernumberinthe tolerantthaninthesensitiveline,varyingfrom55%to170%higher. SimilarresultswerefoundinExp.3wherethetolerantlineshada highernumberofflowers(61.1flowersperplant)thaninthe sen-sitivelines(27.8flowersperplant)(Exp.3–Table3).Despitethe variationinthetotalnumberofflowerswithineachpairof toler-ant/sensitivelines,thetrendwasclearwithineachpair.UnderSS

andSF,thenumberofflowersofthetolerantalsoremained75%and 55%higherthaninthesensitivelines,withthetrendbeingfollowed withineachpair.Interestingly,ICC1431andICC6263produced sig-nificantlymoreflowersundertheSFthanundertheCtreatment. So,aclearandconfirmedtrendacrosstheexperimentsandlines wasforalargernumberofflowersinthetolerantlines,regardless oftreatment,shownbyamajorgenotypiceffectonflowernumber, despitealsoahighlysignificantgenotype-by-treatmentinteraction (Tables3and5).

InExp.1flowerabortionintheCtreatmentwascomparablein thetolerantandsensitivelines(70%and79%)andsimilarresults werefoundintheSS(83%versus77%)andSF(88%versus83%) treatments(Table5).TheseresultswereconfirmedinExp.2by sim-ilarflowerabortioninthetolerantand sensitivelinesin theSS (about56%)andtheSF(about52%)treatments(Table6).InExp.3, theflowerabortionwasonaverageslightlyhigher(36%)inthe tol-erantthaninthesensitivelines(20%)underCtreatment.Thelower flowerabortionpercentageinExp.3thaninExp.1couldberelated tothefactthatExp.3wascarriedoutintheglasshouseunderlower evaporativedemandandpossiblyamildereffectofsaltstress.The flowerabortiondecreasedonlyslightlyundertheSStreatment(26% and22%)andwassimilarinthetolerantandsensitivelines.This percentageremainedrelativelysimilartotheCtreatmentunderSF (37%and26%),andwasnotsignificantlydifferentinthetolerant andsensitivelines(Table3).

3.5. Podandseednumber,seednumberperpod,andseedsize InExp.1,thenumberofpodswas28%,43%and89%higherin thetolerantthaninthesensitivelinesfortheC,SSandSF treat-ments,respectively.Thenumberofseedswas41%,125%,and116% higherinthetolerantthaninthesensitivelinesfortheC,SSand SFtreatments,respectively(Table5).Seedmassandseednumber werecloselyrelatedinExp.1(R2=0.87forSS;R2=0.77forSF).So, podandseednumberweregenerallyhigherinthetolerantthan inthesensitivelines,regardlessoftreatment.Nevertheless,there wasahighlysignificantgenotype-by-treatmentinteractioneffect,

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Table6

Numberofflowers,podsandseedsperplant,seednumberperpod,andpercentageofflowerabortioninsixpairs(Exp.2)oftolerant(T)/sensitive(S)linesexposedtosaline conditionsappliedfromthetimeofsowing(SS)andsalineconditionsappliedfromthetimeofflowering(SF).Dataaremeansoffourreplicatepots.Two-wayANOVAwas usedtocomparegenotype(G)treatment(T)andgenotype-by-treatmenteffects(G×T).LeastsignificantdifferencesforeachoftheG,T,andG×Teffectsareprovided.

Numberofflowers Flowerabortionpercentage Numberofpods Numberofseeds Seednumberperpod

SS SF SS SF SS SF SS SF SS SF ICC1431(T) 117.7 125.1 41 51 68.4 58.3 76.0 63.3 1.11 1.10 ICC6263(S) 77.5 80.1 50 60 38.0 31.7 26.9 29.4 0.71 0.93 JG11(T) 106.6 125.9 48 53 48.0 54.1 42.1 48.9 0.87 0.91 ICCV2(S) 28.4 55.0 16 52 19.0 25.4 16.4 24.3 0.88 0.99 ICC9942(T) 110.6 97.4 36 32 70.4 64.9 75.2 54.6 1.09 0.87 ICC15802(S) 73.4 54.7 57 51 31.0 25.9 25.7 23.3 0.83 0.91 ICC3512(T) 154.0 124.1 64 63 52.6 46.1 48.6 44.4 0.92 0.98 ICC13283(S) 54.4 45.7 71 63 16.6 17.0 13.9 17.3 0.91 1.12 ICC7819(T) 129.7 96.0 60 65 52.2 33.1 41.7 38.3 0.79 1.25 ICC7571(S) 67.0 54.7 56 43 30.3 27.3 24.4 25.0 0.84 0.92 ICC5845(T) 188.0 160.1 66 72 58.4 45.6 64.3 46.3 1.10 1.04 ICC6877(S) 85.9 69.6 69 71 27.0 18.4 23.0 15.0 0.83 0.82 LSD 25.6** 23.6** 24** 16** 12.2** 12.0** 12.0** 11.4** 0.15** ns Averagetolerant 134.4 121.4 52 56 58.3 50.4 58.0 49.3 0.98 1.03 Averagesensitive 64.4 60.0 53 57 27.0 24.3 21.6 22.4 0.84 0.95 Geffect 18.2** 3.70 20.89** 24.98** 2.99** Teffect 0.99ns 0.10ns 7.34** 4.86* 0.64ns G×Teffect 1.00ns 0.75ns 0.86ns 0.60ns 0.30ns LSDG 26.3 0.18 14.1 14.5 0.21 LSDT – – – – – LSDG×T ns ns ns ns ns ns,non-significant. * SignificantatP<0.05. ** SignificantatP<0.01.

sothatdifferenceswerelargerbetweentolerantandsensitivelines underSSandSFconditionsthanintheCtreatment.InExp.1,the seednumberperpodinthetolerantandsensitivelineswassimilar intheCandtheSStreatment(0.87versus0.72),buthigherinthe tolerantthaninthesensitivelinesintheSFtreatment(0.74versus 0.51)(Table5).

ResultswereconfirmedinExp.2wherethenumberofpodswas 116%and107%higherinthetolerantthaninthesensitivelinesfor theSSandSFtreatmentrespectively.Similarly,thenumberofseeds was168%and120%higherinthetolerantthaninthesensitivelines fortheSSandSFtreatment,respectively(Table6).Thenumberof seedsperpodinExp.2wasnotsignificantlydifferentinthetolerant andsensitivelines(0.98versus0.84intheSStreatment;1.03versus 0.95intheSFtreatment)(Table6).

InExp.3thenumberofpodswasalso75%,71%and48%higher inthetolerantthaninthesensitivelinesfortheC,SS,andSF treat-mentrespectively.Similarly,theseednumberwas69%,82%,45% higherinthetolerantthaninthesensitivelinesfortheC,SS,andSF treatment,respectively(Table3).Thetrendwassimilarwithineach pairofsensitive/tolerantlines.Podabortionwassimilarinthe tol-erantandsensitivelinesinthenon-salinecontroltreatment(22% and21%),remainedunchangedintheSS(20%and17%)andinthe SF(15%and17%)treatments.Therefore,flowerandpodabortion didnotdiscriminatethetolerantfromthesensitivelines(Table3), butthenumberofseedsundersalttreatmentdid.

Theseednumberwasalsofollowedweekbyweekafter flower-ing.Underalltreatments,thenumberofseedsproducedperplant perweekdecreasedovertime.UnderCconditions,theseed num-berper plantdidnot differbetweenthetolerant andsensitive linesinanyofthefourindividualweeksfollowingtheinitiation offlowering.Bycontrast,theseednumberfromweek1and2was significantlyhigherinthetolerantthaninthesensitivelinesunder boththeSSandSFtreatments(Fig.1A).Theseresultssuggested thatreproductionsufferedtheeffectofsaltapplicationmostlyin the2weeksfollowingthebeginningofflowering.Theseedsizeof thesensitivelineswassimilarinthetolerantandthesensitivelines inalltreatments.

3.6. Canopytranspiration

Thenormalizedtranspirationratioremainedabove1.0inall tolerantlines,exceptatlatestagesaftertreatmentinICC3512and

16 20 nt-1 T-C 8 12 16 number plan T-C S-C T-SF S-SF TSS 0 4 1 2 3 4 Seed n T-SS S-SS 1 2 3 4 1 2 3 4 1 2 3 4

Weeks after flowering

32 16 24 ed weight T-C S-C T-SF SSF 0 8 100-see S-SF T-SS S-SS

B

A

Weeks after flowering

Fig.1.Seednumber(perplant)(A)and100-seedmass(g)(B)producedduring weeks1,2,3,and4afterinitiationoffloweringintolerantlines(T,closedsymbols andsolidlines)andsensitivelines(S,opensymbolsanddashedlines)exposedto non-salinecontrolconditions(C,circles),salineconditionsappliedfromthetimeof sowing(SS,triangles),andsalineconditionsappliedfromthetimeofflowering(SF, squares).Dataarethemeandataforeachlinewithineachofthetolerant/sensitive lines(n=5).BarsrepresentLSD(0.05%).

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1.00 1.20 1.40 1.60

*

*

*

*

1.00 0.80 1.20 1.40

*

*

0.00 0.20 0.40 0.60 0.80 4 7 11 14 18 21 25 29 32 35 0.00 0.20 0.40 0.60 4 7 11 14 18 21 25 29 32 35 4 7 11 14 18 21 25 29 32 35

Normalized Transpiration Ratio

Normalized Transpiration Ratio

Normalized Transpiration Ratio

Normalized Transpiration Ratio

Normalized Transpiration Ratio

Days since transpiration measurement started

4 7 11 14 18 21 25 29 32 35

Days since transpiration measurement started

Days since transpiration measurement started

1.20 1.40 1.60

*

*

*

*

1.00 1.20

A

D

0.20 0.40 0.60 0.80 1.00 0 0.20 0.40 0.60 0.80 0.00 35 32 29 25 21 18 14 11 7 4

Days since transpiration measurement started

Days since transpiration measurement started

0.00 35 32 29 25 21 18 14 11 7 4 1.20

*

B

E

0.20 0.40 0.60 0.80 1.00

Tolerant

Sensitive

0.00 35 32 29 25 21 18 14 11 7 4

C

Fig.2. Normalizedtranspirationratio(NTR)infivepairsoftolerant/sensitivelinesICC1431/ICC6263(A),JG11/ICCV2(B),ICC9942/ICC15802(C),ICC3512/ICC13283(D), ICC7819/ICC7571(E)exposedtosalineconditionsfromthetimeofflowering(SF).Tolerantlinesarewithclosedsymbolsandsolidlines.Sensitivelinesarewithopen symbolsanddashedlines.Thearrowsindicatethetimeoffloweringandthereforethetimeofsaltapplication.Dataaremeansof8replicatepots.BarsrepresentLSD(0.05%).

ICC7819(Fig.2).TheNTRwascloseto1.0inmostsensitivelinesand wasevenbelow1.0inICC13283(Fig.2).Inthefirst,secondandthird pairoftolerant/sensitivegenotypes,theNTRofthetolerantwas abovetheNTRofthesensitivelines,fromabout10days(Fig.2A), 20days(Fig.2B),or15days(Fig.2C)aftertreatment.Inthefourth pair,theNTRwasalsooccasionallyhigherinthetolerancethanin thesensitiveline(Fig.2D).Inthefifthpair,therewasnosignificant differenceintheNTRatanystage(Fig.2E).

4. Discussion

Thetreatmentappliedatflowering(SF)appearedtoreducethe seedyieldandrelatedcomponentsbyasimilarmagnitudetothe treatmentappliedatthetimeofsowing.Nevertheless,therewas somedegreeofinteractionwiththelinesindicatingthatsomewere moresensitivetoeitheroneoftimesofimposingsalinitystresses. Amongthefloweringcharacteristics,neitherthedurationof flow-eringandpodding,northepercentageofflowerandpodabortion, discriminatedthetolerantfromthesensitivelines.Rather,itwas thenumberofflowersproducedundernon-salineconditionsthat discriminated tolerantfrom sensitivelines in each of thepairs tested.Higherflowernumbersresultedinhighernumbersofpods

andseedsperplantinthetolerantthaninthesensitivelinesunder non-salineconditions.Undernon-salineconditions,tolerantlines alsohadhighernumberoftertiarybranches.Insalinetreatments (bothSSandSF)thedifferencesbetweentolerantandsensitivelines inflowernumbersandthereforepodandseednumberswereeven greater.Seednumberperpodwasnotdifferentbetweenthe toler-antandsensitivelinesinanytreatment.Therelativetranspiration aftertreatmentimpositionwasslightlyhigherinthetolerantthan inthesensitivelinesinthreepairsoftolerant/sensitivelines. 4.1. Timingofsalinitystress:SFandSStreatmenteffects– genotypeinteraction

Theresultsshowedthattheeffectofsaltapplicationfromthe timeoffloweringcausedasimilareffectontheyieldcomponents asthesalt treatmentfromthetimeof sowing.By contrast,the shootmasswasmoreaffectedbythetreatmentappliedfrom sow-ingthanthetreatmentappliedfromthetimeofflowering.These resultsindicatedthat theeffectof saltstressoperated predom-inantlythroughaneffectonreproduction,sincewelldeveloped plantstreatedwithsalinityfromfloweringhadsimilaryield reduc-tionstothosetreatedwithsalinityfromthetimeofsowing,despite

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theirlowershoot biomass(Table1).This isin agreementwith previousworkthatpointstoreproductivebiologyasthemost salt-sensitivephaseinchickpea(DhingraandVarghese,1993;Katerji etal.,2005;Vadezetal.,2007;Krishnamurthyetal.,2011;Samineni etal.,2011).However,therewasasignificantgenotypeby treat-mentinteraction.ForinstanceearlydurationlinesJG11andICCV2 alsoshowedbiomassreductioninthesalttreatmentappliedatthe timeoffloweringandsointhecaseoftheearlylinesitislessclear whetheradecreaseinshootbiomassand/oraparticularsensitivity ofreproductiontosalinityisthemaincauseforthedecreaseinseed yield.Thisagreeswellwithrecentfindingsthatinearly-flowering chickpealines,salinitytoleranceisexplainedbothbythe mainte-nanceofshootgrowthandbythemaintenanceofarelativelylarge numberofseeds,whereasinlate-floweringlinesitisonlythelatter trait,relatedtothesuccessofreproductivebiology,thatisthebasis ofsalttolerance(Vadezetal.,2011).

4.2. Constitutivetraitsthatexplaingenotypicdifferencesin salinitytolerance

4.2.1. Highernumberofflowers

Probablythemostimportantfindingofthisworkwasthat toler-antlinesproducedamuchlargernumberofflowersthansensitive linesundernon-salinecontrolconditions.Thiswasconfirmedin allthree experiments,althoughwithvariationinthedegreesof differenceacrossexperiments.Alsoimportantwastheobservation thattheflowernumberdidnotdecreaseunderthesalttreatment appliedatthetimeofflowering,neitheroutdoors(Exp.1),norin theglasshouse(Exp.3),whichsuggestthatsalinityhadnoeffecton theproductionofflowersinmostlines.Theflowernumbereven increasedunderSFtreatmentinthetolerantlineICC3512(Table5). Theflowernumberinthesalttreatmentappliedatsowingdidnot decreaseintheoutdoorexperiment(Exp.1),whereasitdecreased inthegreenhouseexperiment(Exp.3)comparedtothenon-saline control,wherethedecreaseintheflowernumbercouldhavebeen aconsequenceofthelimitedgrowthintheSStreatment.The tag-gingprocedurethen allowedflowerstobefollowedthroughto maturity.Theflowerabortionpercentagewassimilarintolerant andsensitivelinesacrossalltreatments.Sohere,flowerproduction wasanimportantfactorforseedyieldundersalinity,whichdiffers fromLeportetal.(2006)wherepodabortionwasthekeyfactorfor seedyielddecreaseunderdrought.Thelargernumberofflowers wasverycloselyrelatedtothenumberofseedsundernon-saline conditions(R2=0.70)andundersalineconditions(R2=0.55). 4.2.2. Tertiarybranches

Anotherstrikingresultwasthefactthatplantarchitectureof tol-erantlinesdifferedfromthatofsensitivelines.Sensitivelineshad alargernumberofprimarybranches,buthadmuchfewertertiary branchesthanthetolerantlines.Therewasinfactahighly signifi-cantrelationshipbetweenthenumberofflowersandthenumber oftertiarybranches(R2=0.34),although thenumber offlowers showedaplateauoncethenumberoftertiarybrancheswasabove 20.Thisisthefirsttimethatsalinitytoleranceinacrophasbeen reportedlyrelatedtoplantarchitecture.Asfloweringnodesoccurin leafaxils,ahighernumberoftertiarybrancheswouldexpectedly relatetoalargernumberofflowers.TolerantICC9942,ICC1431, ICC7819hadindeedlargenumberoftertiarybranches.

4.3. Stressresponsivetraitsthatexplaingenotypicdifferencesin salinitytolerance

4.3.1. Numberofseedsperplant

Tolerantlineshadonaveragealargernumberofseedsthanthe sensitivelinesundercontrolconditionsand,asdiscussedabove, thiswasrelatedtothelargernumberofflowersoftolerantlines

undernon-salinecontrolconditions(R2=0.70).However,the dif-ferencesinseednumberbetweentolerantandsensitivelineswere increasedunderbothsalinetreatmentsinExp.1.Thiswasinpart relatedtotheevenlargernumberofflowersinthetolerantthan inthesensitivelinesundersaltstress(R2=0.55).Therefore,these resultssuggestthatinadditiontoconstitutivedifferenceof sensi-tivelinesproducinglessflowers,therearealsodifferencesinthe numberofseedsthattolerantandsensitivelinesmanagetoretain undersaltstress.

4.3.2. Unrelatedparameters(flowerandpodabortion)

Surprisingly,the largernumber of seedsundersalt stressin tolerantlinesunderbothsalinetreatmentswasnotrelatedto dif-ferencesinflowerandpodabortion.Thenumberofemptypodswas alsonotsignificantlydifferentbetweenthetolerantandthe sensi-tivelines.Seednumberperpodundersaltstressdidnotappearto becloselyrelatedtotoleranceorsensitivity,althoughtherewasa tendencytohavelargenumberofseedsperpodinatleastsomeof thetolerantlinesundersaltstress.Thepossibilitythattheflowering orthepoddingdurationmaybedifferentiallyaffectedintolerant andsensitivelineswasalsoconsidered.However,whilethelength offloweringwasdecreasedinthesaltstressappliedatsowing,but notbythesaltstressappliedatflowering,andthelengthofpodding wasreducedinbothsalinetreatments,noneofthese developmen-talperiodsshowedlargeenoughdifferencesbetweentolerantand sensitivelines.However,theseednumberperplantwasclearly higherinthetolerantthaninthesensitivelines,especiallyunder SSandSFconditions.Ourinterpretationisthatthenumberofempty pods,thepercentageofpodabortion,thenumberofseedsperpod andsmalldifferencesinfloweringandpoddingtimesare compo-nentsoftheseednumberperplantandmaynothave,individually, anysignificanteffect,buthaveanadditiveeffect,resultingin sig-nificantdifferencesinthenumberofseedbetweentolerantand sensitivelines.

4.3.3. Canopytranspirationandrelationtoseedfill

Differences in the transpiration response to salt application were found in three pairs of tolerant/sensitive lines in which therelativetranspiration ofthetolerantlinewasabove thatof thesensitiveline(Fig.2).Therewere usuallylarge experimen-talerrorsassociatedwithsuchmeasurements.Nevertheless,the resultsindicatedthattolerantlineswerebetterabletomaintain highassimilaterates(proxiedherebycanopytranspiration).An interestingfactwasalsothehigherrootmassofsensitivelines underthenon-salinetreatment,butthelackofsignificant differ-encesintherootmassoftolerantandsensitivelinesunderSSand SFtreatment.Sincesaltstressinducesanosmoticeffectonplants (MunnsandTester,2008),wemayinterpretthatwaterabsorption couldhavebeenroot-limitedundersaltstressinthesaline treat-mentinsomesensitivelines.Inagreementwiththatinterpretation wasthefactthatthethreesensitivelineshavinglowerNTRthan theirrespectivetolerantcounterparts wereICC6263,ICCV2,and ICC15802,andallhadsignificantrootdrymassreductionunderSF treatment.Thesedatawouldalsoagreewiththeprevious hypoth-esisthatsalinestressreduceswaterabsorptionbecauseoflimited rootgrowth(Whishetal.,2007).

5. Conclusion

Thisworkledtotwomajorfindings:(i)thatconstitutivetraits largelyaffectthedegreeoftoleranceofchickpea germplasmto saltstress(i.e.grainmassinsalineconditions),althoughthistrait discriminationisincreasedundersalineconditions;(ii)that repro-ductionis indeedaffectedmore bysalinity insensitivethan in tolerantlines.Theconstitutivetraitswerethecapacityoftolerant

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linestoproducealargernumberoftertiarybranchesandalarger numberofflowers,regardlessoftreatment.Thestress-responsive traitswererelatedtothecapacityoftolerantmaterialsto main-tainalargernumberofseedsundersaltstressthansensitivelines, which werelatetoa higher reproductivesuccess,likely during thefertilization/developmentoftheembryos.Thepresentfindings identifyprioritiesforanumberofmappingtargetsforthesetraits withthreeimmediatepossibilities:(i)numberoftertiarybranches; (ii)numberofflowers;(iii)numberofseedsperplantundersaline conditions.

Acknowledgements

The work was supported by Australian Research Council LP0776586grantandCOGGO(CouncilofGrainGrower Organisa-tionsLtd),andfromsupportfromtheIndianCouncilofAgriculture Research(ICAR)fromtheGovernmentofIndia.Authorsare thank-fulfortheexperttechnicalassistanceofMr.NJangaiahatICRISAT.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.eja.2012.03.008.

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