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Seed propagation of Nelumbo nucifera is limited by strong physical dormancy due to its hard, impermeable seed coat. This study evaluated the efficacy of four scarification treatments-mechanical nicking, sand abrasion, hot water immersion and concentrated sulphuric acid (98%, 1 h)-compared with an untreated control, on germination, biochemical activity and early seedling growth under ambient conditions of Ananthapuramu, India (latitude 14°41′ N, longitude 77°36′ E) during June 2025. Scarification significantly enhanced imbibition, seed coat permeability, α-amylase activity, germination percentage and seedling vigour (p < 0.05). Among treatments, acid scarification was most effective, achieving 78.4% germination, the shortest mean germination time (4.6 days), highest enzymatic activity (5.76 μmol maltose min⁻¹ g⁻¹ FW) and superior seedling growth (plumule: 9.4 cm; radicle: 12.6 cm). Mechanical nicking and sand abrasion moderately improved germination, whereas hot water immersion showed limited effectiveness. Root-to-shoot ratios remained relatively stable across treatments, indicating balanced biomass allocation. These results demonstrate that optimised acid scarification efficiently overcomes physical dormancy, accelerates metabolic activation and enhances seedling establishment in N. nucifera , providing a reliable, cost-effective method for large-scale propagation and wetland restoration programs.
Keywords: Dormancy; Germination; Nelumbo nucifera; Seed scarification
Thesacredlotus(NelumbonuciferaGaertn.),belongingtothefamilyNelumbonaceae, isanecologically significantaquaticperennialwidelydistributedacross tropicalandsubtropicalAsia,includingIndia,China, Japan,KoreaandAustralia.Itisoneofthemostculturallyreveredplantsinhumancivilisation,holdingdeep symbolicimportanceinHinduism,BuddhismandancientEgyptianculture,wherelotusmotifswereprominentlyemployedinthedecorationoftemples,palaces, tombsandceremonialarchitecture(1,2). InIndia,N. nucifera holds the distinction of being the national floweranditsrepresentationinreligiousiconography, artandliteraturehaspersistedformillennia.Thelotus flowerisregardedasasymbolofpurity,prosperityand spiritualenlightenmentanditsaestheticappealcontinuestomakeitapreferredornamentalplantinwater gardens,pondsandlandscapehorticultureworldwide (3).Beyonditsculturalandornamentalvalue,thelotus isaplantofremarkableeconomicandpharmaceuticalimportance.Allpartsoftheplant-flowers,leaves, seeds,seedpods,rhizomes,stamensandembryosareutilisedacrossfood,medicineandcosmeticindustries.Lotusseeds(Nelumbinissemen)arenutritionally rich,containingproteins,carbohydrates,dietaryfibre, alkaloids,flavonoids,polyphenolsandproanthocyanidinsandhavebeenextensivelyreviewedasanemergingtherapeuticfoodingredientwithantioxidant,antiobesityandanti-inflammatoryproperties.Theseeds havealsodemonstratedtheabilitytoprotectagainst oxidativestress-inducedDNAdamageinhumanlymphocytes,reinforcingtheirnutraceuticalsignificance. Lotusseedstarchexhibitsuniquestructuralandphysicochemicalpropertiesofrelevancetofoodprocessing andthechemicalcompositionofseedsvariessignificantlywithmaturityandharvestingtime,influencing bothnutritionalqualityandpropagationpotential.The metabolomicsoflotusseedshasemergedasanactive areaofresearch,withstudiesdocumentingawidearrayofbioactivesecondarymetabolitesofpharmaceuticalinterest(4,5).TheflowersofN.nuciferaareequally valuedinthecosmeticsandpharmaceuticalsectors. Alkaloid-richflowerbudextractshavebeenshownto possessmelanogenesisinhibitoryandhyaluronidase inhibitory activities, supporting their application in skin-whiteningandanti-ageingformulations.Thefloralscentoflotus,composedofcomplexvolatilecompounds,isalsoexploitedintheperfumeryindustry.Reviewsontheuseoflotusinherbalcosmeticsconfirm itsgrowingacceptanceasamulti-functionalbotanicalingredient(3,6,7).Fromanecologicalstandpoint, thelotuscontributesmeaningfullytowetlandecosystemhealthbystabilisingsediments,reducingturbidity Thethermogeniccapacityofitsflowers,whereinfloral temperatureisactivelyregulatedaboveambientlevelsduringanthesis,isauniquephysiologicaltraitthat facilitatespollinatorattractionandsuccessfulfertilisation.Athoroughunderstandingoflotusfloralbiology, includingflowering, pollinationandseedformation processes,isthereforeessentialforoptimisingseed productionandpropagationstrategies(8,9,10). The lotusisincreasinglybeingrecognisedasanemerging horticulturalmodelplant,withadvancesinmolecular biology,metabolomicsandtissueculture-includingefficientcallusinductionsystems-expandingthescope ofgeneticresearchandimprovementinthisspecies (2,4,5).Innaturalandcultivatedsystems,lotusisprimarilypropagatedthroughvegetativemeans,particularlyrhizomedivision,whichensuresuniformityand rapidestablishment. However, dependenceonvegetativepropagationimposespracticalconstraintson large-scaleproduction,includinglimitedavailability ofhealthyrhizomeplantingmaterial,highlabourinputsandriskofpathogentransmissionthroughclonal propagules.Seed-basedpropagation,bycontrast,offersseveraldistinctadvantages-itenablesthegenerationofgeneticvariability,facilitateseaseofstorageand long-distancetransportandisparticularlysuitedto massmultiplicationprogrammesforwetlandrestorationandbiodiversityconservation. Notably,ancient sacredlotusseedsrecoveredfromsedimentdeposits inChina,estimatedtobeseveralcenturiesold,have beensuccessfullygerminatedundercontrolledlaboratoryconditions,demonstratingtheextraordinaryseed longevitycharacteristicofthisspeciesanditsinherent potentialforlong-termexsituconservation(11,12).Despitethesepropagationadvantages,seedgermination inN.nuciferaisseverelyconstrainedbythepresence ofstrongphysicaldormancy. Theseedcoatoflotus isextremelyhard,thickandimpermeable,composed oftightlyinterlockedsclerenchymatouscellsthatrestrictwaterabsorption,impedegaseousexchangeand therebydelayorcompletelyinhibitgerminationundernaturalconditions.Withoutdeliberatedormancybreakingintervention,germinationratesinuntreated lotusseedsremaincharacteristicallylow,erraticand unpredictable,limitingthereliabilityofdirectsowing forcommercialorrestorationpurposes(1,3).Various pre-sowingscarificationtreatmentshavebeenevaluatedtoovercomethisphysicaldormancybymechanicallyorchemicallydisruptingtheseedcoatandenablingwateruptakeandembryoactivation. Among chemicalapproaches,sulphuricacidscarificationhas beenstudiedinwhitelotusandfoundtoproduceuniform testa erosion, facilitating rapid imbibition and metabolicactivation.Comparativestudiesonchemical versusmechanicalscarificationin N.nucifera haveconfirmedthesuperiorityofacidtreatmentforimproving germinationspeedanduniformity.Mechanicalscarificationthroughnicking,filingorsandabrasionhasalso beenexplored,withresultsvaryingdependingonthe precisionandextentofabrasionapplied[?, ?]. Hotwaterimmersion,anotherphysicalapproach,has demonstrated inconsistent outcomes, as excessive thermalexposurecandamagetheembryoratherthan promote germination. The efficacy of scarification has further been linked to measurable changes in seedmembranepermeability,electrolyteleakageand the activation of α-amylase, a key enzyme responsible for starch hydrolysis and energy mobilisation duringgermination.Studiesonhard-seededaquatic macrophytesandtropicaltreespecies—including Acacia nilotica, Prosopis cineraria, Pongamia pinnata, Luffacylindrica and Albizialebbeck—haveconsistently demonstrated that appropriate scarification treatments,whetherchemical,mechanicalorthermal,significantlyenhancegerminationpercentage,germinationspeedandsubsequentseedlingvigour.
Investigationsonseedcoatscarification,primingand coldstratificationintropicalwaterlilyhybridshavesimilarlyunderlinedthespecies-specificandconditionspecificnatureofdormancy-breakingresponses,reinforcingtheneedforlocallyvalidatedprotocols[?, ?]. Thelongevityofscarifiedseedsunderdifferentstorage conditionsisalsoanimportantpracticalconsideration, particularly for conservation seed banks and largescalenurseryoperations.Recentworkonenhancing lotusseedgerminationthroughmechanicalandchemicaltreatmentshasreaffirmedtherelevanceofstandardisedscarificationprotocolsforreliableseedling production. Drone-basedfieldmonitoringtechnologieshavealsobeenappliedtocountlotusflowersand seedpodsatthecanopylevel,reflectingtheexpanding precisionhorticultureinterfacewithlotuscropmanagement(8,17,18).Althoughconsiderableresearchhas beenconductedonlotusbiologyandseeddormancy globally,optimisedandregionallyvalidatedscarificationprotocolsforN.nuciferaunderIndianagroclimatic conditions,particularlyinsemi-aridenvironments,remaininsufficientlydocumented.Giventhattemperature,humidity,soilpropertiesandwaterqualitysignificantlyinfluencetheoutcomeofpre-sowingtreatments, standardisationofdormancy-breakingmethodsunder localconditionsisessentialforthesuccessfuldeploymentofseed-basedlotuspropagationatacommercial andecologicalscale(19,20). Thepresentstudywas thereforeundertakentocomparativelyevaluatethe efficacyoffourscarificationtechniques-mechanical nicking,sandabrasion,hotwaterimmersionandsulphuricacidtreatment-againstanuntreatedcontrol, withrespecttotheirinfluenceonseedgerminationand seedlingperformanceofNelumbonuciferaunderthe conditionsofAnanthapuramu,AndhraPradesh.
TheexperimentwasconductedintheFloricultureand LandscapeArchitectureLaboratoryatSriKrishnadevarayaCollegeofHorticulturalSciences,Ananthapuramu ( ◦ ′ N, ◦ ′ E; elevation m above 14 41 77 36 ≈ 460 meansealevel)duringJune2025. Freshlymatured lotus( Gaertn.)seedswerecollected Nelumbonucifera fromawell-maintainedcultivatedpondwithinthecollegecampus. Theseedswerehand-harvestedon10 June 2025, thoroughly cleaned to remove adhering pericarptissuesandair-driedundershadedconditions fortwodaysbeforetheinitiationoftreatments.Five
≈ 28 ± 2 anyscarification;T (MechanicalNicking):seedswere firmly held in vise grips and a small nick was made carefully at the micropyle end using a metal file untilthewhitishinnertissuewasexposed,followedby gentlerinsingintapwater;T (SandAbrasion):seeds wererolledslowlyanduniformlyovercoarsesandpaper until a lighter inner surface appeared, ensuring thattheseedcoatwasweakenedwithoutinjuringthe embryo,thenrinsedthoroughly;T (HotWaterImmersion):seedswereimmersedinhotwatermaintained at ◦Cfor2min,afterwhichtheywereimmediately transferred to room-temperature water and soaked for 24 h to cool and hydrate; and T (Acid Scarification): seeds were soaked in concentrated sulphuric acid(H SO ,98%)for1hourwithgentleandcontin- 2 4 uousstirringtoetchtheseedcoatuniformly,thenrepeatedlywashedunderrunningtapwaterfor10min toremoveacidresiduesandsoakedinambientwater foranadditional1hourbeforefurtherincubation. Aftertreatment,allseedswereplacedin500mltransparent glass beakers containing 300 ml of dechlorinatedtapwatermaintainedatapproximately ◦Cunderambientlaboratoryconditions.Thebeakerswere looselycoveredwithperforatedlidstominimizecontaminationandevaporation.Waterwasreplaceddaily tomaintainoxygenationandpreventmicrobialgrowth. Germination was monitored daily for 15 days and a seedwasconsideredgerminatedwhentheradicleprotrudedatleast2mmfromtheseedcoat.
MeanGerminationTime(MGT)wascalculatedusing theformula:
(cid:80) MGT =
where n representsthenumberofseedsgerminated i onday .
ti Germination percentage and MGT were computed for each replicate to assess treatment effects. The dataweresubjectedtoone-wayanalysisofvariance (ANOVA)andtreatmentmeanswerecomparedusing Tukey’sHonestSignificantDifference(HSD)testata significancelevelof p < 0.05. Allstatisticalanalyses wereperformedusingSPSSsoftware.
Scarificationtreatmentssignificantlyinfluencedgermination,biochemicalactivityandseedlinggrowthof Nelumbonucifera (p < 0.05)(Tables1and2).Theuntreatedcontrol(T )exhibitedlowimbibition(18.6%), poorseedcoatpermeability(0.21),reduced α-amylase activity(2.36 molmaltosemin−1 g−1 FW),delayed µ germination(10.4days)andlowgerminationpercentage(22.4%),confirmingstrongphysicaldormancyimposedbythehardandimpermeableseedcoat.Similar dormancy-relatedconstraintsinlotusandotherhardseededaquaticspecieshavebeenreportedbyearlier workers(21,22).
Allscarificationtreatmentssignificantlyenhancedwateruptake,metabolicactivationandgerminationcomparedtothecontrol.Amongthem,sulphuricacidscarification(T )wasthemosteffective,recordingthehighestimbibitionrate(68.9%),seedcoatpermeabilityindex (0.79), germination percentage (78.4%) and the shortesttimeto50%germination(4.9days).Thesuperiorresponseofacid-treatedseedsisattributedtouniformerosionoftheseedcoat,whichfacilitatedrapid wateruptakeandembryoactivation.SimilareffectivenessofacidscarificationinovercomingphysicaldormancyhasbeendocumentedinIndianstudiesonhardseededspeciesaswellasinlotus(23,24,25).
Mechanicalnicking(T )andsandabrasion(T )resulted 2 3 inmoderateimprovementingermination,whilehot water immersion (T ) showed limited effectiveness. Thesefindingsareconsistentwithearlierreportswho observedvariablesuccessofphysicalandthermalscarificationduetounevenweakeningoftheseedcoator potentialthermalinjury(26,27).
Electricalconductivityofseedleachatesincreasedsignificantlyinscarifiedtreatments, particularlyunder sand abrasion and hot water immersion, indicating enhancedmembranepermeabilityfollowingtestadisruption.Suchincreasesinconductivityarecommonly associatedwithrapidhydrationanddormancyrelease (ni × ti) inhard-seededspecies(28,29). Correspondingly, - (cid:80) α ni amylaseactivityincreasedsignificantlyunderscarification,withthehighestvaluerecordedinacid-treated seeds (5.76 mol maltose min−1 g−1 FW), followed µ bysandabrasion(4.32 molmaltosemin−1 g−1 FW), µ whileuntreatedseedsshowedthelowestactivity.Elevated α-amylaseactivityreflectsenhancedstarchmobilisationandenergysupplynecessaryforrapidgerminationandearlyseedlinggrowth,inagreementwith earlierresearchers(5,6,30).
Seedlinggrowthandvigourparameterscloselyparalleledgerminationresponses.Acid-scarifiedseeds(T ) producedthehighestseedlingvigourindex(1724.5), germinationindex(7.86)andmaximumplumule(9.4 cm)andradiclelengths(12.6cm),indicatingrapidand uniformseedlingestablishment.Comparableimprovementsinseedlinggrowthfollowingscarificationhave been reported by earlier workers. Root-to-shoot ratiovariedonlyslightlyamongtreatments(1.23–1.44), withthecontrolshowingamarginallyhigherratiodue to restricted shoot elongation, while scarified treatments—particularly acid scarification—maintained balanced biomass allocation, indicative of healthy seedlingdevelopment(31,32).
Overall,sulphuricacidscarification(98%,1h)proved tobethemostefficientmethodforbreakingphysical dormancyin N.nucifera,resultinginenhancedimbibition, enzymaticactivation, rapidgerminationand superiorseedlingvigourunderAnanthapuramuconditions.Mechanicalnickingandsandabrasionweremoderatelyeffective,whilehotwaterimmersionshowed inconsistentresults.ThefindingscorroborateearlierIndianandinternationalstudiesandhighlightthepotentialofoptimisedacidscarificationasareliable,rapid andscalablemethodforlarge-scalelotuspropagation, wetland restoration and conservation programmes (33,34,35).
Table 1. Effectofscarificationtreatmentsonseedcoat andgerminationparameters
T1 18.6±1.4a 0.21±0.02a 92.4±4.6a 2.36±0.12a 10.4±0.5a 22.4±2.1a T2 42.8±2.1b 0.48±0.03b 138.6±6.2b 3.84±0.18b 7.6±0.4b 45.2±3.0b T3 51.4±2.4c 0.56±0.04c 152.3±5.8c 4.32±0.20c 6.8±0.3c 53.6±2.8c T4 36.2±1.9b 0.41±0.03b 165.4±7.1c 3.42±0.16b 8.3±0.5b 39.2±2.5b d d b d d d T5 68.9±2.8 0.79±0.05 128.7±5.4 5.76±0.22 4.9±0.2 78.4±3.4 CD(P=0.05) 3.01 0.066 10.84 0.38 0.55 4.52 SEd 1.42 0.031 5.12 0.18 0.26 2.14 Treatmentsdetails:T1:Control,T2:MechanicalNicking,T3: SandAbrasion,T4:HotWaterImmersionandT5:Acid Scarification Effectofscarificationtreatmentsonseedcoat
andgerminationparameters
T1 174.1± 15.2a 2.14±0.18a 9.8±0.4a 3.2±0.3a 4.6±0.4a 1.44±0.08a T2 592.6± 28.4b 4.36±0.26b 7.2±0.3b 5.8±0.4b 7.3±0.5b 1.26±0.06b T3 827.5± 35.6c 5.18±0.31c 6.4±0.3c 6.9±0.5c 8.5±0.6c 1.23±0.05b T4 472.3± 22.7b 3.82±0.21b 7.8±0.5b 5.2±0.4b 6.8±0.5b 1.31±0.07b T5 1724.5± 68.3d 7.86±0.42d 4.6±0.2d 9.4±0.6d 12.6±0.7d 1.34±0.06b CD(P=0.05) 73.2 0.51 0.60 0.78 0.88 0.09 SEd 34.6 0.24 0.28 0.37 0.42 0.04
Treatmentsdetails:T1:Control,T2:MechanicalNicking,T3: SandAbrasion,T4:HotWaterImmersionandT5:Acid Scarification.
Seedcoatscarificationsignificantlyimprovesgerminationperformance,enzymaticactivationandinitial seedlinggrowthin Nelumbonucifera.Amongthemethodsevaluated,concentratedsulphuricacid(98%,1hr) was the most effective in overcoming physical dormancy,resultinginthehighestimbibitionrate,seed coat permeability, α-amylase activity, germination percentageandseedlingvigour. Mechanicalnicking andsandabrasionprovidedmoderateimprovements, whereashotwaterimmersionshowedlimitedefficacy. Theenhancedenzymaticactivityandbalancedrootto-shootgrowthobservedunderacidscarificationindicateefficientmobilisationofstoredreservesandacceleratedmetabolicactivation.Thesefindingsestablishsulphuricacidscarificationasareliable,rapidand scalablestrategyforlarge-scalepropagation,wetland restorationandconservationof N.nucifera undersemiaridIndianconditions,withpotentialforoptimisation in terms of exposure duration and concentration to enhancesafetyandsustainability.