Priyadarsini et al.
Acclimatization of plantlets
Catalase
Theȱsomaticȱembryo-derivedȱtolerantȱplantletsȱwereȱ Freshȱ Callusȱ sampleȱ (500mg)ȱ fromȱ eachȱ treatmentȱ
transferredȱ toȱ theȱ soilȱ mixtureȱ atȱ theȱ ratioȱ ofȱ 1:ȱ 2:ȱ wereȱ collectedȱ atȱ 4-weekȱ intervals,ȱ homogenizedȱ
1ȱ(Sand:ȱIron-richȱSoil:ȱCowȱdung)ȱandȱkeptȱinȱtheȱ inȱ 0.1ȱ Mȱ sodiumȱ phosphateȱ bufferȱ (pHȱ 7.0)ȱ andȱ
greenhouseȱwithȱ85%ȱrelativeȱhumidity.ȱTheȱnutrientȱ centrifugedȱatȱ1000ȱxȱgȱforȱ10ȱminȱatȱ4 0 C.ȱOneȱmilliliterȱ
mediumȱwithȱ12ȱmMȱFeȱandȱwithoutȱorganicȱisȱgivenȱ ofȱ supernatantȱ wasȱ addedȱ toȱ theȱ reactionȱ mixtureȱ
toȱtheȱplantsȱinȱeveryȱtwo-dayȱintervals.ȱAboutȱ70%ȱ containingȱ1ȱmlȱ0.1ȱMȱH O ȱandȱ3ȱmlȱ0.1ȱMȱsodiumȱ
2 2
plantletsȱsurvivedȱinȱtheȱgreenȱhouseȱandȱfloweredȱ phosphateȱbufferȱ(pHȱ7.0).ȱTheȱreactionȱwasȱstoppedȱ
afterȱtwoȱmonthsȱofȱtheȱtransfer.
byȱaddingȱ1.0ȱmlȱ2%ȱH 2 SO 4 ȱafterȱ1ȱminȱincubationȱatȱ
20 0 C.ȱTheȱacidifiedȱreactionȱmixtureȱwithȱorȱwithoutȱ
Chlorophyll and protein estimation
theȱsupernatantȱwasȱtitratedȱagainstȱ0.01ȱMȱKMnO 4
Callusȱ samplesȱ (500ȱ mgȱ freshȱ weightȱ basis)ȱ fromȱ
toȱ determineȱ theȱ quantityȱ ofȱ H O utilizedȱ byȱ theȱ
2 2ȱ
eachȱtolerantȱandȱnon-tolerantȱsourceȱareȱcollectedȱ
enzyme.ȱ Theȱ catalaseȱ activityȱ isexpressedȱ asȱ µmolȱ
atȱ4ȱweekȱintervalsȱforȱtheȱestimationȱofȱchlorophyll.ȱ
H O destroyed/min/mg.protein.
2 2ȱ
Theȱ callusȱ wasȱ homogenizedȱ withȱ 80%ȱ acetoneȱ inȱ
theȱ dark.ȱ Theȱ amountȱ ofȱ chlorophyllȱ isȱ estimatedȱ
Statistical analysis
accordingȱ toȱ Vernonȱ (1960).ȱ Pigmentȱ contentȱ wasȱ Theȱdataȱpertainingȱtoȱmeanȱpercentageȱofȱexplantsȱ
expressedȱasȱmg/gmȱfreshȱweightȱofȱtheȱsample.ȱForȱ withȱ calluses/treatment,ȱ regenerationȱ frequency,ȱ
theȱanalysisȱofȱproteinȱcontent,ȱfreshȱweightȱsamplesȱ chlorophyllȱcontent,ȱtotalȱproteinȱcontentȱandȱcatalseȱ
(100ȱ mg)ȱ wereȱ analyzedȱ byȱ conventionalȱ micro-
andȱperoxidaseȱactivityȱofȱmetalȱtolerantȱandȱnon-
Jeldahlȱmethodȱforȱtheȱestimationȱofȱtotalȱnitrogen.ȱ tolerantȱ sourcesȱ wereȱ statisticallyȱ analysedȱ byȱ
Solubleȱ nitrogenȱ wasȱ determinedȱ byȱ thisȱ methodȱ analysisȱ ofȱ variance.ȱ Betweenȱ theȱ treatments,ȱ theȱ
afterȱ precipitatingȱ theȱ proteinȱ inȱ theȱ extractȱ ofȱ theȱ averageȱfiguresȱfollowedȱbyȱtheȱsameȱletterȱwithinȱaȱ
freshȱmaterialȱwithȱtrichloroaceticȱacidȱ(Anonymousȱ columnȱinȱtheȱtablesȱwereȱnotȱsignificantlyȱdifferentȱ
1970).
atȱtheȱPȱ<ȱ0.05ȱlevelȱ(Post-HocȱMultipleȱComparisonȱ
Tests).
Enzyme Extraction and Assay
Results and Discussion
Peroxidase
Freshȱcallusȱsamplesȱ(500ȱmg)ȱfromȱeachȱtreatmentȱ
Embryogenic Callus induction
wereȱcollectedȱatȱtwoȱweeksȱintervals,ȱhomogenizedȱ Theȱpresentȱstudyȱindicatesȱtheȱestablishmentȱofȱtheȱ
withȱ mortarȱ andȱ pestleȱ inȱ coldȱ 0.1ȱ Mȱ phosphateȱ efficientȱ protocolȱ onȱ theȱ plantȱ regenerationȱ systemȱ
bufferȱ (pHȱ 6.1)ȱ containingȱ 30ȱ mgȱ ofȱ insolubleȱ throughȱ somaticȱ embryogenesisȱ ofȱ riceȱ genotypesȱ
polyvinylpyrrolidoneȱandȱ15ȱmgȱsodiumȱascorbate.ȱ toleranceȱtoȱiron.ȱBothȱcytokininsȱandȱauxinsȱwereȱ
Theȱhomogenateȱwasȱfilteredȱthroughȱfourȱlayersȱofȱ usedȱforȱdevelopmentȱofȱembryogenicȱcallus.ȱAmongȱ
Micaclothȱandȱcentrifugedȱatȱ12,000ȱxȱgȱforȱ10ȱminȱatȱ theȱtwoȱcytokininsȱandȱauxinsȱtested,ȱkinetinȱaloneȱ
4 0 ȱC.ȱTheȱsupernatantȱwasȱusedȱforȱtheȱperoxidaseȱ orȱ kinetinȱ inȱ combinationȱ withȱ 2,4-Dȱ showedȱ theȱ
assay.ȱTheȱassayȱmixtureȱcontainedȱ0.1Mȱphosphateȱ positiveȱeffectȱonȱcallusȱproliferationȱfromȱimmatureȱ
bufferȱ(pHȱ6.1),ȱ4mMȱguaiacol,ȱ3mMȱH O ȱandȱ0.4ȱmlȱ zygoticȱ embryos.ȱ Theȱ effectȱ ofȱ growthȱ regulatorsȱ
2 2
ofȱcrudeȱenzymeȱextract.ȱTheȱtotalȱreactionȱvolumeȱ onȱ embryogenicȱ calliȱ developmentȱ isȱ presentedȱ inȱ
wasȱ1.2ȱml.ȱTheȱrateȱofȱchangeȱinȱabsorbanceȱatȱ420ȱ Table.ȱ1.ȱInitially,ȱsmallȱyellowishȱwhiteȱfriableȱcalliȱ
nmȱ wasȱ measuredȱ usingȱ aȱ UVȱ spectrophotometer.ȱ developedȱ fromȱ immatureȱ zygoticȱ embryosȱ withinȱ
Theȱlevelsȱofȱenzymeȱactivitiesȱareȱexpressedȱasȱµmolȱ fourȱweeksȱofȱcultureȱonȱMSȱmediumȱsupplementedȱ
H O destroyed/min/mg.protein.
2 2ȱ
withȱ0.5–1.5ȱmg/lȱKnȱandȱ2.0–4.0ȱmg/lȱ2,4-Dȱ(Figureȱ1ȱ
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