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tAvailableonlineaScholarsResearchLibraryArchivesofAppliedScienceResearch,2015,7(1):22-27(http://scholarsresearchlibrary.
com/archive.
html)ISSN0975-508XCODEN(USA)AASRC922ScholarsResearchLibraryPlantregenerationofA.
lakoochafromencapsulatednodalexplantsShivKumarVerma*,DhirajKumarChoudhary,Ashish,AnnandKumarandMotiLalSchoolofBiochemicalEngineering,IIT(BHU),VaranasiABSTRACTOneofthealternativemethodsadoptedinrecentyearsistousebiotechnologicalapproachesforimprovingthetreespecies.
AproficientprotocolforencapsulationofnodalsegmentsofArtocarpuslakoochaRoxbhasbeendevelopedforplantregenerationthroughnon-embryogenicsyntheticseeds.
Concentrationsofsodiumalginateandcalciumchloridegreatlyaffectedmorphologyandtextureencapsulatinggel.
3%sodiumalginatewith100mMCaCl2hasbeenfoundtobebestpossibleconcentrationfortheproductionofidenticalsyntheticseeds.
25daysoldinvivoplantletofA.
lakoochawasusedforobtainingnodalsegments,usedinallexperiments.
FiveexperimentswereperformedusingMSmedium(agarsolidifiedandliquid)supplementedwith1,3and5mg/lBAP.
Concentrationof1mg/lBAPwasobservedtobemoreeffectiveforachievinghighestregenerationfrequencyincomparisontootherconcentrationofBAP(3and5mg/l).
InthirdexperimentonlydifferentstrengthofMSmediumwasused,inlasttwoexperimentsnodalsegmentspriorencapsulationandencapsulatednodalsegmentsweregivenpulsetreatmentwith(IBA)for48hoursandconcentrationof3mg/lBAPwasobservedtobemosteffectiveforsyntheticseedregeneration.
Datawererecordedafter4weeksofculture.
Keywords:Pulsetreatment,MSmedium,BAP,IBA,Acclimatization,syntheticseeds.
INTRODUCTIONThereisworldwidesusceptibilityofplantgeneticdiversityduetounmatchedperturbations,habitatlossanddestructionrates.
Variousspeciesaredescribedasrareorendangered,andasaresultintegratedprogramsarerequiredtodefendandconservebiodiversityofcurrentlyavailablespecies[1].
ArtocarpuslakoochaRoxb,aspeciesoffamilyMoraceae,isanimportanttropicalmedicinaltreespeciesnativetoIndiaandusedforvariousimportantpurposeslike;fruit,furniture,timber,andfeed.
Ripfruitsoflakoochaaregenerallyeatenfresh.
Eachfruitgenerallycontains20–30seedsthatarefleshywiththinseedcoat.
Theediblefruitpulpisbelievedtocontainessentialingredientswhichplaykeyroleinfunctioningiftheliver.
lkoochaseedsandmilkylatexarepurgativeinnature.
Seedscontainartocarpins(ALAIandALAII),theisolectinswhichexhibithighhaemagglutinationactivity(Wongkham1995).
However,theagglutinin(ALA)fromArtocarpuslakoochaisnotorganspecificintheplant.
Moreover,thehaemagglutinationactivityofALAwasdemonstratedinvariousorgansoftheplantexceptfruitflesh[2].
Traditionallytheplantispropagatedthroughseedbutseedpropagationisnotrapidandseasondependent.
Extensiveimprovementhasbeenmadeinthepropagationofthisplantthroughplantcell,tissueandorganculture[3-4].
However,inordertocompletethedemandofpharmaceuticalindustries,continuoussupplyofplantthroughoutyear,thereisneedtodevelopamethodofconservationandtransportofhealthyplant.
Inthiscontext,forrapidpropagation,encapsulationtechnologymaybeanalternativemethodofconservationandgermplasmexchangeofthismedicinallyimportantplant[3].
Inrecentyears,syntheticseedtechnologyusingencapsulationofinvitro-derivednon-embryogenicpropaguleshasbecomeanimportantassettomicropropagation(Naiketal.
2006).
InShivKumarVermaetalArch.
Appl.
Sci.
Res.
,2015,7(1):22-2723ScholarsResearchLibraryaddition,syntheticseedtechnologycouldbeusefulingermplasmconservationofelite,endangeredandcommerciallyimportantplantsbyusingappropriatestoragetechniqueaswellasexchangeofaxenicplantmaterialbetweenlaboratoriesandpharmaceuticalindustries[5]thatcanbeusedifstockplantsorproliferationculturesbecomeinfestedwithbacteria,fungi,orarthropods.
Successfulcasesofsyntheticseedproductionandplantletregenerationhavebeenreportedforawiderangeofplantsincludingcereals,vegetables,fruits,ornamentals,medicinalplantsandforesttree,[7].
Someotherpotentialadvantagesofsyntheticseedtechnologyareeaseinhandlingduetosmallsizeofcapsules,reductionincosts,geneticuniformityofplantsanddirectdeliverytothefield[8]Anartificialseedpreparedbycoatingasomaticembryousingapolymermatrixisatrueseedanalog.
Animmobilizedsomaticembryocangerminateundersuitablegrowthconditionsandbecomeacompleteplant.
Syntheticseedtechnologyusingencapsulationofvegetativepropagulesofwoodyplantspecieshasbecomeapotentiallycosteffectiveclonalpropagationsystem.
Successfulplantregenerationfromsyntheticseedshasbeenreportedinseveralplantspecies.
However,inmostofthecases,theembryogenicpropagulessuchassomaticembryoswereusedforsyntheticseedsproduction.
Thereareonlyfewreportsonencapsulationofvegetativepropagules[9-14].
Encapsulationofvegetativepropagulescouldbeusedformassclonalpropagationatareasonablecost[14].
Therehasbeennoreportonutilizingsomaticembryostoproducesyntheticseeds.
Thedevelopmentofsyntheticseedsfromsomaticembryoscouldofferapracticalmeansformasspropagationofbanana.
Inthisstudy,encapsulationofsomaticembryosdevelopingfromembryogenicsuspensionculturesinanimportantIndiancultivarofbanana,Rasthali(AABwithtwoAAandoneBgenome),andconversionoftheencapsulatedembryosintoplantsarereported(Ganpatietal.
2001).
Amongseveralnon-embryogenicpropagules,shoottipexplantsaremoreresponsivethanotherexplantsbecauseofgreatermitoticactivityinthemeristem[15].
Althoughtherearemanyreportsonencapsulationofshoottipsobtainedfrominvivoraisedplants[7,16].
Inmanyoftheworks,somaticembryoshavealreadybeenusedintheencapsulationprocess.
However,encapsulationofsomaticembryoswererestrictedmostlytoplantsinwhichsomaticembryogenesishasbeendocumented.
But,incurrenttime,useofnon-embryogenicvegetativepropaguleslikeapicalshootbuds,axillarybuds,nodalsegments,etc.
,fortheencapsulationhavealsobeenextendedasasuitablealternativetosomaticembryos[6,9,11,14,17,18,20,21].
ThepresentstudywasaimedtoinvestigateencapsulationofsomaticembryosofArtocarpuslakoochinsodiumalginatebeadsandconversionofencapsulatedsomaticembryosintoplantlets.
EffectofdifferentconcentrationofBAP,wasalsostudiedtoexplorethemorphogeneticresponsesofencapsulatedshoottips.
Validationofthepossibilitytostoretheencapsulatedshoottipsfortimeperiodsufficientforexchangesanddistributionofgermplasmwasdiscussed.
Inthepresentinvestigation,wedescribertheencapsulationofshoottipsforthedevelopmentofsyntheticseedinA.
lakoocha.
MATERIALSANDMETHODS2.
1.
PreparationofexplantsandcultureconditionsSeedsofripenfruitsofA.
lakoochawereobtainedfromagriculturefieldofChandraShekharAjadAgricultureUniversity,Kanpur,India.
SeedsweregrownonagarsolidifiedMSmedium.
25daysoldplantletswereusedformakingnodalsegments.
Nodalsegments(0.
5cm)ofA.
lakoochawereprepared.
Initiallytheexplantswerewashedinrunningtapwaterfor20–30mintominimizemicrobialconcentrationonthesurfaceofnodalsegments.
Afterthatsurfacesterilizedinlaminarairhoodwith70%ethanolfor30-40sfollowedby0.
05%mercuricchloride(Hi-Media,India),for3–4min,andrinsed4–5timeswithsteriledoubledistilledwater[imp].
Forshootmultiplication,nodalsegmentswereculturedonMS,medium[22]supplementedwithsucrose(30g/l).
Themediaweresolidifiedwith0.
8%(w/v)agar(Hi-Media,India).
ThemediawereadjustedtopH5.
7using1NNaOHor0.
1NHClbeforeautoclavingat1210Cfor15min.
Culturesweremaintainedat25±20Cwitha16hphotoperiodataphotonfluxdensityof50–70mmolm-2s-1fromcoolwhitefluorescenttubes(Philips,India).
Nodalsegmentsexcisedfrominvitroproliferatedplantletswereusedasexplantsforthesyntheticseedproduction.
2.
2.
EncapsulationofnodalsegmentsandpreparationofsodiumalginatebeadsForencapsulationofnodalsegments,sodiumalginate(Himedia,india)waspreparedintherangeof2.
0,3.
0,4.
0,or5.
0%(w/v),whereascalciumchloride(CaCl2.
2H2O)solutionwaspreparedin100,mM(w/v)indouble-distilledwaterorliquidMS(MurashigeandSkoog,1962)mediumwithoutanyplantgrowthregulator.
Boththegelmatrixandcomplexingagentwereautoclavedat121°Cfor15min.
Encapsulationwasaccomplishedbymixingthenodalexplantintothesodiumalginatesolutionanddroppingtheseexplantsintothecalciumchloridesolution.
Thebeadscontainingthesomaticembryoswereheldfor20–30mininthecalciumchloridesolutionandafterhardeningoftheShivKumarVermaetalArch.
Appl.
Sci.
Res.
,2015,7(1):22-2724ScholarsResearchLibrarybeads,encapsulatedsomaticembryoswerewashedwithsterilizeddistilledwatertwotimestotakeawaytracesofcalciumchloride.
(A)(B)(C)(D)(E)Figure1-SyntheticseedpreparedindifferentsodiumalginateandcalciumchloridecompositionPlantletregenerationinagarsolidifiedMSmedium(D)andMSmediumsupplementedwith1mg/lBAP(E)3.
ExperimentsDifferentfactorsaffectingconversionwereevaluatedandthefollowingexperimentswereperformed.
Tomaintaintheculture,samelightandtemperatureconditionswereusedinthegrowthroomasdescribedpreviously.
Dataforconversionfrequencyandshootlengthwasrecordedafter3weeksofculture.
3.
1EffectofBAPinagarsolidifiedMSfullstrengthMediumSyntheticseedswerepreparedusinginsodiumalginate(3%w/v)and100mMCaCl2,seedswereinoculatedinsolidifiedMSmediumsupplementedwithBAPattheconcentrationof1,3and5mgl-1.
AfterthreeweekspercentseedgerminationwasrecordedinallthreesupplementofBAP.
3.
2EffectofBAPinliquidMSfullstrengthMediumInthisexperimentsamecompositionofsyntheticseedandsameconcentrationofBAPwasused.
LiquidMSmediumwasused.
AfterthreeweeksinoculationpercentseedgerminationwasrecordedinallthreesupplementofBAP.
3.
3EffectofCompositionofEncapsulatingGelSodiumalginatebeadscomplexedwithnodalexplantswereinoculatedinMSfullstrength,1/2MSand1/4MSstrength.
Percentseedgerminationwasrecordedafterthreeweeksofinoculation.
ShivKumarVermaetalArch.
Appl.
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,2015,7(1):22-2725ScholarsResearchLibrary3.
4EffectofPulsetreatmentofIBA(IndoleButyricAcid)BeforeEncapsulationofnodalExplantNodalexplantswerepulsetreatedfor48hours,withIBA(3mgl-1),beforepreparationofsyntheticseeds.
Aftersyntheticseedpreparation,seedswereinoculatedinfullstrengthliquidMSmediumsupplementedwith,3and5mgl_1BAP.
3.
5EffectofPulsetreatmentofIBA(IndoleButyricAcid)AfterEncapsulationofnodalExplantInthisexperimentafterencapsulation,nodalexplantsweresubjectedto48hourspulsetreatmentwithsamegrowthregulatorandsameconcentration.
SeedswereinoculatedinfullstrengthliquidMSmediumsupplementedwith,3and5mgl-1BAP.
3.
6AcclimatizationofplantsinsoilArtificiallygrownplantletsregeneratedfromencapsulatednodalsegmentsweretransferredtoplasticpotscontaining3:1(w/w)mixtureofsterilesandandsoilmoistenedwithliquidMSmedium.
Plantletswerecoveredwithpolyethylenebagstomaintainhighhumidityandirrigatedwithtapwater.
Potswithplantletswerekeptunderlaboratoriesconditionat250Cinartificiallight(irradianceof60mmolm-2s-1)providedbycoolwhitefluorescenttubesfor4Weeksandthenthepotsweretransferredtofieldlevels.
RESULTSANDDISCUSSIONAtpresentdaysnewrouteinsyntheticseedtechnologyhasbeenreportedwiththeuseofnonembryogenicplantpropagulesviz;rootstemleaves(StandardiandPiccioni,1998).
Themostimportantbenefitofusingvegetativepropagulesforthepreparationofsyntheticseedswouldbeinthosecaseswheresomaticembryogenesisisnotwellestablishedorsomaticembryosdonotgerminateintocompleteplantlets(Raietal.
,2009).
Insuchcases,syntheticseedscanbeproducedfromshoottipsforcost-effectivemassclonalpropagation,potentiallong-termgermplasmstorage,anddeliveryoftissue-culturedplants.
Inthepresentexploration,nodalsegmentsexcisedfrominvivoproliferatedsamallplantletswereusedasanexplantforthedevelopmentofsyntheticseedinA.
lakoocha.
inmanyexperimentalstudiesSimilarobservationswerealsomadeinDalbergiasissooRoxb(Chandetal.
2004),Ceropegiabulbosavar.
bulbosa(Dhiretal.
2013),VitexnegundoL.
(Ahmadetal.
2010).
EvaluationofconcentrationofsodiumalginateandcalciumchloridewhichareeffectiveforthegellingpropertiesofthematrixandeventuallyfortheexcellenceofcalciumalginatebeadsisanimportantaspectforthesuccessfulinvitropropagationofplantsthroughencapsulationmethodsConcentrationofsodiumalginateandcalciumchloridegreatlyaffectedtheencapsulationofnodalsegmentsdifferedqualitativelywithrespecttotexture,shape,andtransparency.
Table1.
EffectofBAPinagarsolidifiedMSfullstrengthMediumConcentrationofBAP(mgl-1)+MS(S)PercentresponseofsyntheticSeedGerminationDays1+MS(S)91213+MS(S)81215+MS(S)7321Inourstudy3.
0%sodiumalginateand100mMCaCl2.
2H2Owasfoundmostsuitableforformationofidealcalciumalginatebeads.
ConcentrationofBAPalongwithMS(MurashigandSkoog1962.
)mediumgreatlyaffectedthepercentconversionfrequencyofencapsulatednodalsegmentsofA.
lakoocha,highestconversionfrequency(91%)wasobservedonsolidMSmediumsupplementedwith1mg/lBAP(6-BenzyleAminoPurine)followedby3and5mg/lBAPwithpercentsyntheticseedgerminationof89%and72%respectively(Table1).
Table2.
EffectofBAPinliquidMSfullstrengthMediumConcentrationofBAP(mgl-1)+MS(L)PercentresponseofsyntheticSeedGerminationDays1+MS(L)96213+MS(L)84215+MS(L)7821WehaveobservedinourexperimentthatwhensameexperimentwasperformedwithliquidMSmediumwithsameconcentrationofBAP,percentconversionfrequencywasobservedhigherthantheearlierexperiment.
Itwas96%onliquidMSmediumsupplementedwith1mg/lBAP.
LiquidMSmediumsupplementedwith3and5mg/lBAPshowedconversionfrequencyof84%and78%respectively(Table2).
thisdifferenceofconversionfrequencyintwoconditionsofmediummaybeduetofactthatinliquidMSmediumnutrientcomponentsaremoreeaslyandrapidlyShivKumarVermaetalArch.
Appl.
Sci.
Res.
,2015,7(1):22-2726ScholarsResearchLibraryabsorbedthroughthesurfaceofsodiumalginatebeadswhileinsolidMSmediumnutrientcomponentsareslowlyabsorbedwhichisresponsibleforslowregenerationofnodalsegments.
Table3.
EffectofCompositionofEncapsulatingGelStrengthofMS(L)PercentresponseofsyntheticSeeGerminationDaysMS(FS)8721MS(1/2S)5421MS(1/4S)4821WhenonlydifferentstrengthofliquidMSmedium(MSFS,MS1/2SandMS1/4S)wasuseditwasobservedthatsyntheticseedregenerationfrequencyoffullstrengthliquidMSwashighest(87)amongthreedifferentstrengthofliquidMSmedium.
Possiblereasonofthisdifferenceofregenerationfrequencymaybeduetoinsufficientconcentrationofallnutrientcomponents,becauseinsideaplantcellforabiochemicalreactionparticularconcentrationofnutrientcomponent(metalionsetc.
)isneeded.
DuetothisfactinMS1/2SandMS1/4S)regenerationfrequencywasobservedtobe54%and48%respectively(Table3).
Table4.
EffectofPulsetreatmentofIBA(IndoleButyricAcid)BeforeEncapsulationofnodalExplantsConcentrationofBAP(mgl-1)+MS(L)PercentresponseofsyntheticSeeGerminationDays1+MS(L)81213+MS(L)87215+MS(L)7821When,beforeencapsulation,nodalsegmentswerepulsetreatedwithIBA(IndoleButyricAcid)for48hrsandthenencapsulatedinsodiumalginatebeadsandinoculatedinmediumcompositionasdescribedintable2syntheticseedregenerationfrequencyobservedwasdifferent.
InthisexperimentwasobservedthatliquidMSmediumsupplementedwith3mg/lBAPshowedhighestregenerationfrequencyof87%incomparisontoMSmediumsupplementedwith1and5mg/lBAPwhichshowedregenerationfrequency81%and78%respectively(Table4).
Inourlastexperimentafterencapsulation,beadswerepulsetreatedwithIBAforsametimeperiodasinexperimentTable5.
EffectofPulsetreatmentofIBA(IndoleButyricAcid)AfterEncapsulationofnodalExplantsConcentrationofBAP(mgl-1)+MS(L)PercentresponseofsyntheticSeedGerminationDays1+MS(L)81213+MS(L)87215+MS(L)6821Samepatternofresultswereobtainedwithdifferenceinregenerationfrequencyvalue.
Thesevalueswere81%,87%and68%onMSmediumsupplementedwith1,3and5mg/lBAPrespectively(Table5).
PossiblereasonofeffectofPulsetreatmenttoaffectregenerationfrequencyisnotknown.
Allexperimentswereperformedintriplicatetominimizeerrorduringdatacalculation.
CONCLUSIONTheartificialseedtechnologyprovidesanoptionalmethodofmicropropagationforawidearrayofmedicinalplants,especiallydesirableelitegenotypes.
However,successfulplantretrievalfromencapsulatedvegetativemicropropagulesfollowingshort-termstorageismostlydependsonplantspecies,matrixcompositionandperiodofstorage.
Treesgenerallyhavealonggenerationtimeandaremostlyheterozygous.
Duetothesereasons,geneticdevelopmentinthesespecieshasbeenamostimportanthindrance.
Largescaleclonalpropagationofsuperiorclonesalongwithacceleratedtreeimprovementprogramsisnecessaryforsuccessfulandrapidexplorationoftreeplant.
Itispossibletouseeasy-to-obtainsomaticfragmentsoftheplantsbynodalsegmentcuttingsbyapplyingartificialseedtechnologyinA.
lakoocha.
Oneoftheadvantagesofsyntheticseedtechnologyisthattheriskofsomaclonalvariationwouldalsobereducedbecausemutationsareknowntooccurmorefrequentlywhenthede-andre-differentiationprocessesoccurduringinitialgrowth.
However,there-growthabilityoftheunipolarpropagulesafterencapsulationhinderedbyfactorssuchasprecociousgrowthandlackofataproot.
Encapsulatednodalsegmentsorapical/auxiliaryshootbudsareconsideredtobemoreeffectivecomeintoviewifwemaketherelativeeaseandextensibilityofthetechnique,aswellasthepossibilityofundamagedanduninfectedmovingsmall-sizedmicroShivKumarVermaetalArch.
Appl.
Sci.
Res.
,2015,7(1):22-2727ScholarsResearchLibrarypropagatedplantmaterialbetweenlaboratoriesandcountries,whilereducingphytosanitaryandquarantineproblems(Bapat,1993.
,Mathuretal.
,1989;HasanandTakagi,1995;Maruyamaetal.
,1997b.
,PiccioniandStandardi,1995).
Theresultspresentedinthispapersupportthefeasibilityofthiscost-effectivemethod,foralargescalepropagationofthisecologicallyandmedicinallypotentplantspecies.
Oneofthedrawbackofsyntheticseedtechnologyisthatonstorageofseeds,percentregenerationfrequencyisgreatlyreducedTherefore,forlarge-scaleapplicationofthissyntheticseedtechnology,furtherexperimentsarenecessary;toaccomplishahigherpercentageofconversionmakethemviableevenafterlongtimestorageofencapsulatednodalsegmentsofA.
lakoocha.
Further,encapsulatedsomaticembryoscouldofferanimportantsystemforthetransportationandexchangeofgermplasminasafeandeconomicalmanner.
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