Priyadarsini et al.

100ȱmillionȱhectaresȱofȱtheseȱlandsȱ(BeckerȱandȱAsch,ȱ andȱSkoogȱ(1962)ȱbasalȱmediumȱsupplementedȱwithȱ

2005;ȱ Aschȱ et al. ȱ 2005).ȱ Inȱ India,ȱ totalȱ 11.7ȱ millionȱ variousȱconcentrationsȱofȱBAȱorȱKnȱ(0,ȱ0.25,ȱ0.5,ȱ1.0,ȱ

hectareȱofȱlandȱisȱaffectedȱbyȱironȱtoxicity.ȱIronȱtoxicityȱ 1.5,ȱ2.0ȱmg/l),ȱNAAȱorȱ2,ȱ4-Dȱ(0,ȱ0.5,ȱ1.0,ȱ1.5,ȱ2.0,ȱ3.0ȱ

interfereȱaȱrangeȱofȱnutrientȱdisordersȱandȱdeficienciesȱ mg/l)ȱ aloneȱ orȱ inȱ combinationȱ forȱ callusȱ inductionȱ

particularlyȱ potassium,ȱ phosphorus,ȱ calcium,ȱ andȱ plantȱ regeneration.ȱ Theȱ pHȱ ofȱ theȱ mediaȱ wasȱ

magnesiumȱ andȱ zincȱ inȱ plantȱ metabolismȱ (Ottowȱ adjustedȱtoȱ5.7ȱusingȱ0.1NȱNaOHȱorȱ0.1NȱHClȱpriorȱ

et al .ȱ 1983;ȱ Pathiranaȱ et al .ȱ 2002;ȱ Royȱ andȱ Mandal,ȱ toȱtheȱadditionȱofȱ0.8%ȱ(w/v)ȱagarȱ(Qualigen,ȱIndia).ȱ

2005;ȱMehrabanȱ et al .ȱ2008).ȱ In vitro ȱcultureȱtechniqueȱ Routinely,ȱ15ȱmlȱofȱmoltenȱmediumȱwasȱdispensedȱ

isȱanȱalternativeȱmethodȱforȱinductionȱofȱironȱtolerantȱ intoȱ 25ȱ xȱ 150ȱ mmȱ glassȱ testȱ tubesȱ (Borosil,ȱ India),ȱ

plantsȱforȱgeneticȱimprovementȱprogram.ȱDuringȱlastȱ cappedȱ withȱ non-absorbentȱ cottonȱ plugsȱ wrappedȱ

twoȱ decades,ȱ plantȱ regenerationȱ viaȱ organogenesisȱ withȱ oneȱ layerȱ ofȱ cheesecloth.ȱ Theȱ culturesȱ wereȱ

asȱ wellȱ asȱ somaticȱ embryogenesisȱ usingȱ variousȱ sterilizedȱatȱ121ȱ 0 Cȱandȱ104ȱkPaȱforȱ15ȱmin.

explantsȱasȱwellȱasȱfromȱcellsȱandȱprotoplastsȱofȱriceȱ

areȱwellȱdocumentedȱ(Yamadaȱ et al .ȱ1986;ȱAbdullahȱ Development of Fe- tolerant Callus Culture

et al .ȱ1986;ȱChenȱ et al .ȱ1985;ȱRoyȱandȱMandal,ȱ2005).

Friableȱ embryogenicȱ callusesȱ (500ȱ ±ȱ 20ȱ mg)ȱ wasȱ

Theȱabilityȱofȱtheȱtargetedȱplantȱcellsȱtoȱregenerateȱ

transferredȱ toȱ MSȱ mediumȱ supplementedȱ withȱ

intoȱplantletȱandȱsubsequentlyȱdevelopȱintoȱaȱmatureȱ

differentȱconcentrationsȱofȱkinetinȱandȱ2,4-Dȱsinglyȱ

plantȱisȱpre-requitedȱforȱgeneticȱimprovement.ȱThisȱ

orȱinȱcombinationsȱwithȱvariousȱconcentrationȱofȱFeȱ

canȱ happenȱ onlyȱ ifȱ theȱ callusesȱ areȱ embryogenicȱ

(1.5ȱmMȱnormalȱFeȱcontentȱinȱMSȱmediumȱ(Control),ȱ

i.e.ȱtheȱcallusesȱareȱcapableȱofȱformingȱembryos.ȱInȱ

4ȱ,ȱ8,ȱ10,ȱ12ȱandȱ14ȱmM)ȱforȱselectionȱofȱironȱtolerantȱ

general,ȱIndicaȱriceȱhasȱaȱlowȱregenerationȱpotentialȱ

calli.ȱStockȱsolutionȱinȱtheȱformȱofȱFeSO .4H Oȱwereȱ

4

2

asȱcomparedȱtoȱJaponicaȱriceȱ(Ruebȱ et al .ȱ1994;ȱGairiȱ

filterȱsterilizedȱbyȱ0.45ȱµmȱMilliporeȱmembraneȱfilter.ȱ

andȱ Rashid,ȱ 2004).ȱ Theȱ developmentȱ ofȱ efficientȱ

Theȱconcentrationȱofȱtheȱtestȱmetalȱwasȱselectedȱ(i.e.ȱ

protocolȱonȱsomaticȱembryogenesisȱisȱaȱprerequisiteȱ

LD ,ȱ sub-lethalȱ dosage:ȱ twoȱ concentrationsȱ belowȱ

50

forȱ cropȱ improvementȱ researchȱ andȱ alsoȱ valuableȱ

LD ;ȱ theȱ concentrationsȱ aboveȱ LD )ȱ onȱ theȱ basisȱ

50

50

toolsȱ inȱ understandingȱ theȱ basicȱ plantȱ biology.ȱ Soȱ

ofȱ preliminaryȱ experiments).ȱ Theȱ culturesȱ wereȱ

far,ȱthereȱareȱscantyȱreportsȱonȱ in vitro ȱselectionȱandȱ

incubatedȱunderȱ16hȱphotoperiodȱwithȱlightȱintensityȱ

regenerationȱofȱtheȱheavyȱmetalȱtolerantȱplant.ȱInȱtheȱ

ofȱ55µmolȱm -2 s -1 providedȱbyȱcool,ȱwhiteȱfluorescentȱ

presentȱinvestigation,ȱweȱreportȱonȱtheȱselectionȱofȱ

lampsȱ(Phillips,ȱIndia)ȱatȱ25ȱ±ȱ2 0 C.ȱSubculturingȱwasȱ

ironȱtolerantȱcallusȱlineȱasȱwellȱasȱplantȱregenerationȱ

madeȱ everyȱ fourȱ weekȱ intervals.ȱ Theȱ mediaȱ wereȱ

ofȱIndicaȱriceȱvarietiesȱ‘Khandagiri’ȱandȱ‘Jagannath’ȱ.

solidifiedȱwithȱ0.8%ȱ(w/w)ȱagar-agar.ȱMorphologicalȱ

observationsȱandȱcallusȱgrowthȱmeasurementsȱwereȱ

Materials and Methods

determinedȱatȱ4-weekȱintervals.ȱPre-weighedȱcultureȱ

Induction of embryogenic callus

vialsȱ containingȱ 15ȱ mlȱ ofȱ cultureȱ mediumȱ wereȱ

inoculatedȱ withȱ similarȱ quantitiesȱ ofȱ callus,ȱ andȱ

Semi-matureȱseedsȱofȱ Oryza sativa vars.ȱKhandagiriȱ theȱinoculatedȱvialsȱwereȱre-weighedȱtoȱobtainȱtheȱ

andȱJagannathȱwereȱcollectedȱfrom germplasmȱcentreȱ initialȱfreshȱweightȱofȱtheȱcallusȱinoculum.ȱTheȱfinalȱ

ofȱOrissaȱUniversityȱofȱAgricultureȱandȱTechnology,ȱ weightȱminusȱinitialȱweightȱofȱcallusesȱinȱdifferentȱ

Bhubaneswar,ȱOdisha. Theȱseedsȱwereȱwashedȱwithȱ treatmentsȱwasȱusedȱtoȱcalculateȱtheȱpercentageȱofȱ

2% (w/v)ȱ detergentȱ solutionȱ (Teepol)ȱ forȱ 30ȱ min,ȱ callusȱ growthȱ againstȱ control.ȱ Theȱ culturesȱ wereȱ

further ringedȱ withȱ 70%ȱ (v/v)ȱ ethanolȱ forȱ 5ȱ min,ȱ incubatedȱatȱ25±2 0 Cȱinȱcool,ȱwhiteȱfluorescentȱlightȱ

surfaceȱsterilizedȱwithȱ0.2%ȱ(w/v)ȱaqueousȱsolutionȱ (55µmolȱ m -2 s -1 )ȱ underȱ aȱ 16hȱ photoperiodȱ forȱ fourȱ

ofȱmercuricȱchlorideȱforȱ30ȱmin,ȱfollowedȱbyȱthreeȱ weeks.ȱTheȱexperimentȱhadȱ15ȱculturesȱperȱtreatmentȱ

rinsesȱ inȱ sterileȱ distilledȱ water.ȱ Immatureȱ zygoticȱ andȱrepeatedȱthreeȱtimes.

embryosȱ wereȱ asepticallyȱ culturedȱ onȱ Murashigeȱ

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