Plant Material

2.1.1.Establishmentandmaintenanceofstockplants

ThewhiteclovercultivarGrasslandHuiawasusedasthegeneticbackgroundindifferentsets

ofexperiments.Toestablishstockplants,seeds(collectedfromAgResearchGrasslands,

PalmerstonNorth)wereplantedinsoil(PremierSeedRaisingMix,Daltons,Matamata,New

Zealand)andtheemergedseedlingsmaintainedinaglasshouse.Afteronemonth,andto

maintainasinglegeneticline,aplantarisingfromasingleseedwasselectedrandomlyand

maintainedasastockplant,eventuallyin5ͲLͲcapacitypots.Vegetativepropagationfromthis

stockplantwascarriedoutatleasttwiceayear,andtheoldstockplantsdiscarded.To

propagatestockplants,theapicalpartofthestolonwasexcisedjustproximaltonode4,all

leavesexceptthefirstemergedleafwereremovedandthecuttingsplantedintosoil.Plants

weresupplementedwithnutrientsonceamonthwiththeadditionofThriveRSolubleAll

PurposePlantFood(YatesNewZealand,Auckland,NewZealand).

2.1.2Vegetativepropagationofplantmaterialforexperimentaluse

Toprovideplantmaterialfordifferentsetsofexperiments,stolonswithfournodeswere

excisedfromthestockplantsasdescribed(2.1.1).Thecuttingswerethenplacedinpots

containingvermiculiteandwateredregularlywithhalfstrength(0.5x)Hoagland’ssolution

(Appendix8)androotedforatleastoneweek.Thehealthyandmorphologicallysimilarrooted

stolonswerethenusedfordifferentsetsofexperiment.

2.1.3Experimentalplantsandtreatments

Fordifferentsetsofphysiologicalexperiments,generally,theplantsweregrownina

controlledtemperatureroomorConthermBiosynSeries6000PlantGrowthChambers(Model

620RHS,ConthermScientificLtd,Wellington,NewZealand).Thegrowthconditionswere

typicallymaintainedat22qcduringthedayand140cduringthenight,withaconstantrelative

humidity(RH)of65%andwithalightintensityof180μEovera14hphotoperiod.

2.1.3.1Spodopteraliturafeedingexperiments

Thegeneralistherbivoretobaccocutworm(Spodoptera.litura)wasusedinthestudyto

monitortheexpressionpatternofTrͲKPIsunderherbivoryattack.Forthefeedingexperiments,

thestolonsweregrownasdescribed(2.1.3).The24hinsectfeedingexperimentwas

conductedatPlantandFoodResearch,Mt.Albert,Sandringham,Auckland.Forthis,thelarvae

wereraisedonlimabeanartificialdietwherethefollowingingredientswereused:

Ingredients Weight(g)

Limabeanpowder 60g

Agar 32g

Brewer'syeast 40g(rollerdried,unsalted)

Wheatgerm 48g

Theaboveingredientswerestirredin800mLoftapwater,microwavedonhighpowerfor7Ͳ8

minoruntilwellboiled,withstirringat1to2minintervals.Thefollowingingredientswere

thoroughlymixedinto200mLofwaterandaddedtothemicrowavedcomponentsoncethey

hadcooledto650C:

Ingredients Measurement

Flaxseedoil 0.8mL

Wheatgermoil 1.6mL

Vanderzantvitaminmixture 8g

Ascorbicacid 3.2g Methylparaben(Nipagin) 1.6g Sorbicacid 0.8g Penicillin 280mg Streptomycin 280mg Prochloraz(Octave50W) 16mg

2.1.3.2Nematodefeedingexperiment

ThenematodeinfestationexperimentwasperformedatAgResearchGrasslandswherestolon

cuttingsofcv.Huiaandresistant(17R)andsusceptible(23S)linesforcystnematodewere

rootedinsoilin300x450mmtrays.Thecuttingsweregrowninthegreenhouseat20Ͳ250Con

aplantheatingmatfor20days.Wellestablishedstolonswereselectedandplantedinto60

mmdiameterplasticcupscontainingsandandweresuppliedwithnutrientsolution(0.5x

Hoagland’ssolution)onaregularbasis.Theeggsofwhiteclovercyst(Heteroderatrifolii)and

rootͲknot(Meloidogynetrifoliophila)nematodesweresuppliedbyAgResearchGrasslands.To

infesttheplants,aftertwodaysofestablishmentinsandaholewasmadeinthemiddleof

eachcupandinoculumwereaddedundertheplantatarateof4000eggs/3mLofwaterfor

M.trifoliophilaand3000eggs/3mLofwaterforH.trifolii.Theinfectedplantswereallowedto

growforeightdaysandtheplanttissues(leafandroot)wereharvestedafter4and8daysof

inoculation.Harvestedrootswereexcisedandwashedinrunningtapwatertoremovesand

particles,blotdriedandsnapfrozeninliquidnitrogenandstoredatͲ80qCuntilrequired.

Fordetectionofnematode,someoftheharvestedrootsafter8daysofinoculationwere

stainedusingthefollowingprotocol:

•Infectedrootsthatwerewashedunderrunningtapwaterwereplacedinabeakercontaining

1.5%(v/v)NaOCl(45mLwater+25mLJanola)andleftfor5minwithagitation.

•Afterthat,therootswererinsedinrunningwaterfor30secandallowedtostandintap

waterfor15minandblotdried.

•Therootswerethenincubatedinboilingstain[0.05%(w/v)anilinebluein33%(v/v)glycerol,

33%(v/v)lacticacid]for1min,cooledtoroomtemperature,rinsedinrunningwaterandthen

blotdried.

•Tocountnematodesunderamicroscope,therootswereplacedinacidifiedglycerol[afew

dropsoflacticacidaddedto50%(v/v)glycerol].

2.1.3.3Waterdeficitexperiment

Awaterdeficittreatmentwasimposedbythecompletewithholdingofwatertotheplants.To

dothis,fournodedstolons(excisedasdescribedin2.1.2)wererootedina1:1vermiculiteand

perlitemixturein1.2ͲLͲcapacitypotsandwateredregularlywith0.5xHoagland’ssolution.

treatmentswereapplied:(i)nonpreͲstressed(NPS)(withoutapreviousperiodofwater

withholding)and(ii)preͲstressed(PS)(involvingaoneweekperiodofwaterwithholding,a

weekofrehydrationandthenthewaterwithholdingperiod(outlinedinFigure3.24).ForqRTͲ

PCR,sampleswerecollecteddailyuntilthemoisturecontentdroppedtoapproximately32%

forboththePSandNPStreatments.Beforetheexperimentstarted,excesswaterwasdrained

awayfromthemediainthepots,andthemoisturecontentofvermiculiteandperlitemixture

wasdeterminedusingthegravimetricmethodofRobinson(1974)andwasexpressedasa

percentageusingthefollowingformula:

Ⱥ=(WwͲDw)/Wwx100

Where,

Ww:isthewetweightofvermiculite:perlitemixtureandwater Dw:isthedryweightofvermiculite:perlitemixture

Attheappropriateharvestingintervals,themoisturecontentwasmeasuredusingthesame

method.

2.1.3.4Phosphoruslimitingexperiment

Forthephosphoruslimitingexperiment,stolonswereexcisedandrootedinvermiculite

supplementedwith0.5xHoagland’smedium(asdescribedin2.1.2).After5days,thestolons

weretransferredto0.5xHoagland’smediain0.6ͲLͲcapacityPVCpipes(Figure3.31).After14

days,thestolonswereeithertreatedwithHoagland’smediumcontaining5μMP(Pideficiency

treatment)or0.5mMP(Pisufficienttreatment)overthetimeͲcourseoftheexperiment.