Rev. bras. farmacogn. vol.27 número5

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Yeast

extract

elicitation

increases

vinblastine

and

vincristine

yield

in

protoplast

derived

tissues

and

plantlets

in

Catharanthus

roseus

Mehpara

Maqsood,

Mujib

Abdul

∗

JamiaHamdard,HamdardNagar,NewDelhi,India

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l

e

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f

o

Articlehistory:

Received17January2017 Accepted23May2017 Availableonline29July2017

Keywords: Embryogeniccallus Elicitor

Protoplastisolation Vinblastine Vincristine Yeastextract

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Catharanthusroseus(L.)G.Don,Apocynaceae,isanimmenselyimportantmedicinalplant,producesa varietyofanticancerouscompounds.Theyieldoftwomostinvestigatedalkaloidsvinblastineand vin-cristineisunfortunatelyverylow.Avastarrayoftechnologiesincludingelicitationhaverecentlybeen usedtoenrichCatharanthusalkaloidinculture.Yeastextractisabioticelicitor,thepolysaccharideand thepeptidemoietyhavebeenrecognizedasasignallingelementinenrichingsecondarymetabolites.In thisstudy,theyeastextractelicitationonvinblastineandvincristinewasstudiedinvariousprotoplast derivedtissuesandplantlets.Fourdifferentyeastextracttreatments(T1=0.5g/l,T2=1.0g/l,T3=1.5g/l andT4=2.0g/l)werepreparedandused.Thealkaloidwasquantifiedandacomparativeaccountofyield werepresentedbytheuseofHighperformancethinlayerchromatography.Theyeastextractamendment inmediumimprovedvinblastineandvincristineyieldincultivatingtissues,maximumbeingin germinat-ingembryosandininvitroraisedleaf.ThehighestyieldwasinT3(1.5mg/l)inwhich22.74%vinblastine and48.49%vincristineenrichmentwasnotedingerminatingembryos;theenhancementwashowever, treatment-specific.Antioxidantenzymessuchassuperoxidedismutase,catalase,ascorbateperoxidase andglutathionereductaseactivitieswereinvestigatedasadditionofyeastextractcausedcellularstress andhadenrichedlevelofalkaloids.

Introduction

Catharanthusroseus(L.)G.Don(Madagascarperiwinkle)belongs tothefamilyApocynaceae.Itisanimportantmedicinalplantand it contains a wide variety of anti-cancerousalkaloids (Van Der Heijdenetal.,2004;Mujibetal.,2012).Amongthose,vinblastine (VB)andvincristine(VC)arethemostimportantdrugs(Moreno, 1995)andthesetwoalkaloidsareusedasatherapeuticagentto treatagainstanumberofcancers(Mukherjeeetal.,2001).Thelow yieldofalkaloidsinplanthasencouragedresearcherstoexplore otheralternativeproduction methods byusinginvitro cultures (Verpoorteetal.,2002;Nazetal.,2015),metabolicengineering (DeLucaandStPierre,2000),semi-synthesis(Kutneyetal.,1991) or even total chemical synthesis (Kuboyama et al.,2004).In C. roseus,varioussemi-syntheticproceduresweredevelopedbyusing chemicalsandenzymaticcouplingofcommerciallyavailable catha-ranthineandvindoline(Goodbodyetal.,1988;Morenoetal.,1995). In recent years, various culturalpractices such asoptimization ofculturemedia,selection ofhighalkaloid producingcelllines,

∗ _{Corresponding}_author.

E-mail:amujib@jamiahamdard.ac.in(M.Abdul).

precursor feeding,bioconversionand metabolicengineeringare successfullyutilizedforenrichingalkaloidsincultivatedcells(Van DerHeijdenetal.,2004;ZarateandVerpoorte,2007;Mujibetal., 2012;TeixeiradaSilvaandDobranszki,2016).Ithasearlierbeen notedthatanexogenousamendmentofspecificgroupsof com-pounds tomedium drasticallyincreasedthe yieldof secondary metabolites(Ibrahimetal.,2009;Murthyetal.,2014).These exter-naltriggersarebioticandabioticinnature;inthebiotictypesthe microbial extractssuchas Pythium,Botrytis, Phytophthora, Pseu-domonas and Aspergillus are observed to be very effective in a varietyofcellculturesincludingC.roseus(Valluri,2009;Mustafa etal.,2009;Diptietal.,2016).

Yeastextract(YE)isanimportantelicitorandisfoundtoberich invitaminB-complex.Italsocontainsessentialcomponentslike chitin, N-acetyl-glucosamineoligomers, ␤-glucan, glycopeptides andergosterol(Boller,1995);thesecompoundselicitplantdefense responsesbytriggeringmetabolitesynthesis(Putalunetal.,2007; Caietal.,2012).Inotherculturalexperiments,complexbiological preparationshave alsobeenusedaselicitorswhere the molec-ularstructure of active compound isnot fully known(Radman etal.,2003;Zubeketal.,2012).TheadditionofYEhassuccessfully beenusedincultureandoverproductionofimportant phytocom-poundwasobservedinseveralstudiedplantgenera(Prakashand

http://dx.doi.org/10.1016/j.bjp.2017.05.008

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Srivastava, 2008;Zhao et al., 2010;Cai et al.,2012).The addi-tionof external compounds causesstress in cultivating tissues andimprovesalkaloidbiosynthesis(Elmaghrabietal.,2013;Samar etal.,2015;Diptietal.,2016).Inducedstresshasbeenmeasured bystudyingantioxidantenzymesactivityandotherstressmarkers inculturedtissuesthatoverproduceimportantphyto-compounds (Feher,2015).InthispresentarticleofC.roseus,wetherefore exam-inedtheroleofYEonVBandVCyieldoncultivatedtissues/plants developedfromprotoplast.

Materialandmethods

Plantmaterial

Catharanthusroseus(L.)G.Don,Apocynaceae,seedswere col-lectedfromherbalgarden,HamdardUniversity,NewDelhi, and wasidentifiedby taxonomist(Dr.M.P. Sharma),Department of Botany;thespecimenwasdepositedinDepartmentalarchivefor futurereference.Theseedsweresurface-sterilizedwith0.1%(w/v) HgCl2for2min,rinsedfour-timeswithsterilizeddouble-distilled waterandwereplacedinaconicalflaskcontaining50mlofMS medium(MurashigeandSkoog,1962)withoutorganiccompounds andplant growthregulators(PGR).The seedsweregerminated andtheseedlingsweregrownfor3weeks.Variousexplants(e.g., hypocotyls,leavesandnodalsegments)wereexcisedandcultured onsameMS,amendedwithvariousPGRforcallus/embryogenic induction.Thedetailedprocedurewasdescribedpreviously(Junaid etal.,2006)and wasfollowed.ThemediumpHwaskeptat5.7 beforesterilizationat121◦_C_temperature._The_cultures_were_kept inanincubationroomwith25±2◦_C_and_16-h_photo_period_regime providedbycoolwhitefluorescenttubes(100␮molm−2_s−1_).

Protoplastisolationandculture

Protoplastswereisolatedfrom4-day-oldembryogenic suspen-sion.Suspended cells (0.5gfresh weight) were incubatedwith 10mlof enzymesolutioncontaining2% cellulase,1% pectinase, 0.02%(w/v)macerozyme(SigmaAldrich)and0.5%(w/v)driselase (SigmaAldrich)alongwithCaCl2·2H2Oandsorbitol.Themixture waslaterincubated onarotary shaker(50rpm)for 12hunder constantlightconditions at25◦_C _for_liberating _protoplasts. Iso-latedprotoplastswereculturedinliquidmediumatadensityof 5×105_{protoplasts/ml.}_Protoplasts_were_poured_into_a_conical_flask

containing25ml MS liquidmedium amended withPGR, which

facilitatedprotoplastdivision.

Micro-colony,callus,embryoformationfromprotoplasts

Suspended protoplasts were cultured on medium

supple-mented with appropriate PGR concentrations, i.e. 0.25mg/l l_{-naphthaleneacetic} _acid _(NAA), _0.50_mg/l _NAA, _0.25_mg/l

2,4-dichlorophenoxyaceticacid(2,4-D),0.50mg/l2,4-D,and combina-tionof0.25mg/lNAA+0.25mg/l2,4-D,0.50mg/lNAA+0.50mg/l 2,4-Dfordevelopingmicrocolonies.Theinducedcolonies trans-formedintocallus,whichlaterconvertedintoembryogeniccallus. The embryogenic calli were placed on MS, added with 1mg/l NAA+1.5mg/l 6-benzylaminopurine (BA) for differentiation of embryos. Later, protoplasts-derived embryos were cultured in optimizedgibberellicacid(GA3)amendedmedium(1.5mg/l)for embryomaturationandsubsequentgermination/plantrecovery. Theculturedtissuesweresubculturedregularlyatanintervalof4 weeks,unlessmentionedotherwise.

Elicitortreatment

VariousinvitrogrowntissuesweresubjecttoYEtreatments. Four different concentrations of YE (Merck, India) were pre-pared and were designated as: T1=0.5g/l, T2=1g/l, T3=1.5g/l andT4=2g/l.The abovedesignateddoses ofYEwereamended toMSfollowing Darshanieetal.method(1991).AfterYE addi-tion,thepHwasmaintainedat5.7andthemediumwassterilized at15lb(121◦_C)._The_yield_of_alkaloid_was_measured_at_different tissues/embryos,andplantletsoriginatedfromprotoplasts.

QuantificationofVBandVCusinghighperformancethinlayer chromatography

Extraction

TheVBandVCcontentsweremeasuredindifferenttissuesusing standardVBandVC,procuredfromSigma-Aldrich(St.Louis,MO, USA).Select tissues(25 weeksold)were culturedin optimized mediaand wereharvested whenthetissues wereattheirbest growth.Tissuesample(onegram[g],dryweight)wasrefluxedin 30mlofmethanolforabout5–6handtherecoveredsupernatant washeatedat60◦_C_until_the_volume_reduced_to_1–2_ml.

Preparationofstocksolutionandcalibrationcurve

OnemgofVBandVCwasdissolvedin1mlofmethanoltomake astocksolutionofconcentration1mgml−1_._From_stock_solution, differentdilutionsweremade(minimumfive)toobtain200,400, 600,800and1000␮gperbandofstandardandanalyzed indepen-dentlybyHPTLC.Standardcurvewasplottedbetweenpeakarea (y-axis)andconcentration(x-axis).

HPTLCandquantificationindifferentsamples

Pre-coatedThinLayerChromatography(TLC)Aluminiumsheets (20×10cm)withSilicagel(60F254,Merck)wereusedas station-aryphase.Themobilephasewasfreshlyprepared,consistingof toluene,carbinol,acetoneandammoniaintheratioof40:20:80:2. Sampleswereappliedbythehelpof100␮lmicrosyringe(CAMAG) usingLinomat3(CAMAG)applicator.Aftertheapplicationof sam-ples,thesilicaplateswereairdriedfor10–15minandplacedina developingchamber(TwinthroughChamberCAMAG20×10cm) containingmobile phase. Solventsystemwas allowedtomove upwardsupto85mm.Theplatewasremovedfromthechamber and air dried again for 10–20min. Silica gel plates were

doc-umented by using the Reprostar (CAMAG) in the presence of

UV-light.StationaryphasealongwiththeVBandVCwasscanned byusingtheScanner3(CAMAG).VBwasscannedat280nmand theVCwasscannedat300nm.Alkaloidcontentwasmeasuredas ␮gg−1_dry_weight_basis._The_peak_of_VB_and_VC_was_fixed_and_the detectionofthesamealkaloidsinthesampleswascomparedwith thepeaksofstandard.

Antioxidantenzymaticactivity

Freshplantmaterialwashomogenizedin2mlof0.1M com-monextractionbuffer(pH7.5,0.1MK-phosphate,0.5mMEDTA, 1.0mMascorbicacid)andcentrifugedat8608.6×gfor20minat 4◦_C._Supernatant_(enzyme_extract)_was_taken_for_enzyme_analysis andtheactivityofenzymeswasexpressedinEnzymeUnit(EU) mg−1_protein_min−1_.

Catalase

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0.1mlH2O2.Thereactionwasrunfor3min.Theenzyme activ-itywasexpressedinEUmg−1_protein_min−1_._One_unit_of_enzyme determinestheamountnecessarytodecompose1␮molofH2O2 permin.CATactivitywascalculatedbyusingtheco-efficientof absorbance0.036mM−1_cm−1_.

Superoxidedismutase

ThemethodofDhindsaetal.(1981)wasfollowedwithslight modificationtoestimatesuperoxidedismutase (SOD)activities. Embryos(0.1g) atdifferentdevelopmentalstageswere homog-enized in 2ml of extraction mixture containing 0.5M sodium phosphatebuffer(pH7.3),3mMEDTA,1.0%(w/v) polyvinylpyrolli-done(PVP),1.0%(v/v)TritonX-100,andcentrifugedat8608.6×gat 4◦_C._The_SOD_activity_in_the_supernatant_was_assayed_by_its_ability toinhibitthephoto-chemicalreduction.Theassaymixture consist-ingof1.5mlreactionbuffer,0.2mlmethionine,0.1mlofenzyme extractwithequalvolumeof1MNaCO3and2.25mMNitroBlue Tetrazolium(NBT)solution,3mMEDTA,riboflavin,1mlof Milli-poreH2Owastakenintest tubesandincubatedunderlightfor 10min at25◦_C. _A _50% _reduction _in _colour _is _one_unit _and _the enzymeactivitywasexpressedinEUmg−1_protein_min−1_.

Ascorbateperoxidase

ThemethoddevelopedbyNakanoandAsada(1981)wasused todeterminetheactivityofascorbateperoxidase(APX).The reac-tionmixturecontained1mlof0.1Msodiumbuffer(pH7.2),0.1ml of EDTAand 0.1ml of tissue extract. Ascorbatewasadded and thereactionwasrunfor3minat25◦_C._Ascorbate_oxidation_was monitoredspectrophotometrically at 290nm by a reduction of absorbanceandusingtheabsorptioncoefficientof2.8mM−1_cm−1_. TheactivityofAPXwasexpressedinEUmg−1_protein_min−1_._One unitof enzymeistheamountnecessarytodecompose1␮mof ascorbatepermin.

Glutathionereductase

Theglutathionereductase(GR)activitywasdeterminedbythe methodofFoyerand Halliwell(1976). Theenzymeextractwas rapidlyassayed for theGR activity bytaking 0.1ml of enzyme extractand1mlofreactionbuffer(Tris0.1M/pH7.0,to100ml ofTris0.2mMNADPHand0.5mMGSSGwasadded).Thereaction wasrunfor3minat25◦_C._The_GR_activity_was_calculated_by_using theco-efficientofabsorbance6.22mM−1_cm−1_._The_GR_activity_was determinedthroughglutathione-dependentoxidationofNADPHat 340nm.TheactivityofGRwasexpressedinEUmg−1_protein_min−1_. Oneunitofenzymedeterminestheamountnecessaryto decom-pose1␮molofNADPHpermin.

Statisticalanalysis

ThedataontheeffectsofYEonprotoplastcallus,embryogenesis, alkaloidyieldandonenzymeactivitywereanalyzedbyone-way analysisofvariance(ANOVA)followedbyleastsignificant differ-ence(LSD)test.Valuesareexpressedasmeans±standarderrors ofthreereplicatesoftwoexperiments.Thepresentedmeanvalues wereseparatedusingLSDatp≤0.05level.

Results

Protoplastisolation,callusformation,embryoformationand plantletregeneration

Thehypocotylinducedembryogeniccalluswasusedfor pro-toplast isolation study; this embryogenic callus henceforth is calledas normal embryogeniccallus(NEC). Protoplastisolation wascarriedfromNECsuspensionbyusingvariouswall degrad-ingenzymes.Significantnumbersofprotoplasts(datanotshown

here)wereobtainedontreatmentwithcellulase(2%),pectinase (1%),driselase(0.5%),macerozyme(0.02%)andsorbitol(0.6mM) (Fig.1a).Repeateddivisionsofprotoplastsformedmicro-colonies; andthemaximumnumbersofmicrocolonies/calliwereinduced

on 0.50mg/l NAA+0.50mg/l 2,4-D added medium within 12

weeks ofincubation. Protoplastoriginatedmicro-calli gradually transformedintoembryogeniccalli, calledasprotoplastderived embryogeniccalli(PDEC).The micro-callihad slowgrowth ini-tially, butwithtime thetissues showednormal fastgrowthas thosecultivatedcellsovercamestressgradually,causedbyenzyme mixturesandosmoticumtreatment.Themicro-calliofPDEC ori-ginstartedtoinducesomaticembryoswithin8weeksofculture (inductionstage).InMSembryoproliferationmedium,a hetero-geneous mixture of embryos was formed (proliferation stage). Globular andheart-stageembryosweremorein numbers com-paredtotorpedoandcotyledonaryembryos,whichdifferentiated ingoodnumberslater.Theembryomaturationwasveryhighon 1.5mg/l GA3addedmediumand withtimethematuration per-centageimproved.Theembryosstartedtoregenerateplantsinthe samematurationmedium.Theentireprocessfrom‘protoplastto plantlet’recoverywasachievedthroughsomaticembryogenesis followingembryoinduction,proliferation,maturationandembryo germinationmethods(Fig.2aandd).Thewholeprocesstookabout 40weekstoobtainaplantfromprotoplast.

VBandVCcontent(gg−1dryweight)

The yield of VB and VC was measured in different tissues. ThemobilephaseofstandardsproducedasharpandcompactVB (Rf=0.28)andVC(Rf=0.46)peak(Fig.1aandc,respectively). Scan-ningof VBand VC wasmadeat 280and 300nm,respectively. TheregressionanalysisshowedagoodlinearrelationshipwithVB r=0.999(r2₌_0.998)_and _VC_r₌_0.994₍_r2₌_0.989)_with_respect_to peakareaandconcentrationrangeof200–1000␮gperspot.The regression equationofVBwasY=943.7X+180.0andof VCwas Y=813.5X+1684.

From Tables 1 and 2, it is clear that VB was maximum (9.32␮gg−1_dry_weight)_in_germinating_embryos_and_the_yield_was improved(11.44␮gg−1_dry_weight)_with_YE_elicitation,_especially inT3treatments.ComparedtoVB,theyieldofVCwaslowand ingerminatingembryos,theyieldwashigh.WithYEelicitation, VCyieldwasfurtherimproved,maximumbeinginT3(3.46␮gg−1 dry weight). We alsoquantified VBand VCyield in protoplast regeneratedleaves.Invitroleaftissuehadmaximumamountof VBcomparedtoothertissues/embryos.T3(1.5mg/lYE)harvested leaveshadhighestyieldofVB(15.47␮gg−1 _dry_weight)_and_VC (4.14␮gg−1_dry_weight),_followed_by_T4_treatment₍_Fig.₁_b_and_d). TheVBandVClevelincreased22.74%and48.49%,respectively,with YEelicitationingerminatingembryosovernon-treatedcontrol tis-sue.

CAT,APX,SODandGRactivities

The addition of elicitors often cause stress in

cultivat-ing tissue we therefore analyzed and compared antioxidant

enzyme activities (CAT, APX, SOD and GR) as an indicator of stress. Protoplast derived embryogenic tissue showed higher

antioxidant enzyme activities compared to normal

embryo-genic tissue as these tissues derived from digestion with wall

degrading enzymes, which caused moderate to severe stress

to cells/tissues. With YE elicitation, the anti-oxidant enzyme activities were further up suggesting extra stress on tissues. Comparedtoembryoinductionandproliferationstages,the

alka-loid yield was high on maturing- and germinating embryos,

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Track 1 , ID: ∨inblastine Track 13 , ID: ∨incristine

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Fig.1.(a)Standardpeakofvinblastine,(b)peakshowingthemaximumyieldofvinblastineinYEtreatedleavesatT3,(c)standardcurveofvincristineand(d)peakshowing themaximumyieldofvincristineinYEtreatedleavesatT3(x-axispresentspeakareaandy-axispresentsRf).

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Table1

Vinblastine(VB)content(␮gg−1_dry_weight)_in_protoplast_derived_{embryos/tissues}_in_Catharanthus_roseus._Tissues_were_added_with_different_treatments_of_YE.

Sample Vinblastine(␮gg−1_dry_weight)

Induction Proliferation Maturation Germination Leaves(invitro)

C 1.82±0.015d _4.44_±_0.022e _7.45_±_0.025e _9.32_±_0.028e _13.10_±_0.007d

T1 1.93±0.018c _4.81_±_0.043d _7.65_±_0.025d _9.64_±_0.029d _13.25_±_0.031c

T2 2.04±0.022b _5.25_±_0.015c _7.87_±_0.033c _10.34_±_0.015c _13.24_±_0.094c

T3 2.13±0.019a _6.03_±_0.015a _8.06_±_0.036a _11.44_±_0.014a _15.47_±_0.018a

T4 1.94±0.023c _5.86_±_0.044b _7.97_±_0.014b _11.07_±_0.019b _14.21_±_0.007b

LSDat5% 0.048 0.426 0.071 0.051 0.108

Valuesareexpressedasmeans±standarderrorsofthreereplicatesoftwoexperiments.Dataweretakenafter6thofculture.Differentsuperscriptletters(a–f)indicate

significanceatp≤0.05levelaccordingtoLSD.T1=0.5mg/l;T2=1.0mg/l;T3=1.5mg/l;andT4=2.0mg/l.C,control,i.e.protoplastderivedembryogenictissue.

Table2

Vincristine(VC)content(␮gg−1_dry_weight)_in_protoplast_derived_{embryos/tissues}_in_Catharanthus_roseus_._Tissues_were_added_with_different_treatments_of_YE.

Sample Vincristine(␮gg−1_dry_weight)

Induction Proliferation Maturation Germination Leaves(invitro)

C 0.79±0.058de _1.79_±_0.011d _2.21_±_0.047e _2.33_±_0.014d _3.65_±_0.022e

T1 0.95±0.018c _2.08_±_0.022c _2.54_±_0.018d _2.94_±_0.025c _3.82_±_0.011d

T2 1.09±0.027b _2.46_±_0.029b _2.99_±_0.018c _3.27_±_0.011b _3.96_±_0.022c

T3 1.18±0.011a _2.68_±_0.018a _3.35_±_0.016a _3.46_±_0.016a _4.14_±_0.015a

T4 0.83±0.025d _2.33_±_0.011b _3.13_±_0.015b _3.35_±_0.015b _4.07_±_0.011b

LSDat5% 0.068 0.048 0.058 0.281 0.040

Valuesareexpressedasmeans±standarderrorsofthreereplicatesoftwoexperiments.Dataweretakenafter6thofculture.Differentsuperscriptletters(a–f)indicate

significanceatp≤0.05levelaccordingtoLSD.T1=0.5mg/l;T2=1.0mg/l;T3=1.5mg/l;andT4=2.0mg/l.C,control,i.e.protoplastderivedembryogenictissue.

Table3

Enzymeactivities(EUmg−1_protein_min−1₎_at_maturation_stages_of_embryo;_MS_medium_was_added_with_0.5_mg/l_GA

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Sample CAT APX SOD GR

NEC 1.02±0.025d _0.55_±_0.018d _2.87_±_0.018d _0.21_±_0.014d

C 1.46±0.022c _0.74_±_0.011c _4.27_±_0.033c _0.31_±_0.011c

T3 1.77±0.015b _1.01_±_0.018b _6.42_±_0.048b _0.67_±_0.011b

T4 1.85±0.018a _1.14_±_0.022a _7.05_±_0.016a _0.73_±_0.019a

LSDat5% 0.041 0.033 0.080 0.031

Valuesareexpressedasmeans±standarderrorsofthreereplicatesoftwoexperiments.Dataweretakenafter6thofculture.Differentsuperscriptlettersindicatesignificance

atp≤0.05levelaccordingtoLSD.T3=1.5mg/landT4=2.0mg/l.C,control,i.e.protoplastderivedembryogenictissue;NEC,normalembryogenictissue.

Table4

Enzymeactivities(EUmg−1_protein_min−1₎_at_germination_stages_of_embryos;_MS_medium_was_added_with_0.5_mg/l_BA₊_0.5_mg/l_NAA.

NEC 1.67±0.018c _0.65_±_0.015d _3.20_±_0.011d _0.29_±_0.018d

C 1.99±0.022a _0.86_±_0.021c _5.05_±_0.018c _0.39_±_0.011c

T3 1.89±0.018b _1.18_±_0.011b _7.24_±_0.029b _0.68_±_0.029b

T4 2.03±0.014a _1.26_±_0.022a _7.47_±_0.021a _0.85_±_0.014a

LSDat5% 0.052 0.045 0.057 0.035

Table5

Enzymeactivities(EUmg−1_protein_min−1₎_of_in_vitro_harvested_leaf_tissues,_cultivated_on_MS₊_0.50_mg/BA₊_0.50_mg/l_NAA.

NEC 1.05±0.029d _1.04_±_0.036d _2.10_±_0.029d _0.19_±_0.014d

C 3.27±0.018c _1.57_±_0.018c _3.15_±_0.018c _0.29_±_0.002c

T3 6.02±0.032b _1.96_±_0.015b _6.06_±_0.022b _0.52_±_0.001b

T4 7.52±0.835a _2.09_±_0.011a _7.06_±_0.011a _0.62_±_0.002a

LSDat5% 0.063 0.057 0.039 0.039

in maturing- and germinating embryos, respectively, at high YE (T4) treatment (Tables 3 and 4). The APX activity was alsosimilarly morein maturating-(1.14EUmg−1_protein_min−1₎ andgerminatingembryos(1.26EUmg−1_protein_min−1_)._The_SOD

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CAT,APX,SODandGRactivities,whichyieldedhighestlevelofVB andVC(Table5).

Discussion

The present study was carried out to investigate the effect ofYEonVBandVCinprotoplast derivedtissues/plantletsinC. roseus.WenotedenhancedyieldofVBandVCinresponsetoYE elicitation.ThereareseveralsimilarreportsonYE-induced syn-thesisoftriterpenoidsandothermetabolitesincultivatedplant tissuessuchasinScutellariabaicalensis(Yoonetal.,2000),Panax ginseng(Luetal.,2001),Centellaasiatica(Kimetal.,2007), Angel-icagigas(Rheeetal.,2010)andPuerariacandollei(Korsangruang etal.,2010).Itisknownthattheplants’responsetopathogenis basedonitsabilitytorecognizesignalmoleculesofforeignagent andthesemoleculesorelicitorsareprimarilyproteinsand/orcell wall-derived oligosaccharides (Neurnberger, 1999; Mahalingam andFedoroff,2003).Elicitorsarecapableofevokingavarietyof plantdefenseresponsesincludingthesynthesisofreactive oxy-genspecies (ROS),thehypersensitiveresponse,thesynthesis of phytoalexin,antimicrobialsecondarycompoundsandother man-ifestationsof defensivenature(Montesanoetal.,2003).Several valuablebioactivecompoundslikeazadirachtin,artemisininand tanshinonesaccumulationwerereportedtobestimulatedbyYE elicitor(Putalunetal.,2007;PrakashandSrivastava,2008;Zhao etal.,2010).Themechanismofelicitationwas,however,variedin differentplantgroupsandinmostcases‘elicitor-receptor’ com-plexwasformedandavastarrayofbiochemicalresponseswere manifested(Radmanetal.,2003;Caietal.,2012).

TheactivitiesofantioxidantenzymessuchasAPX,SODandCAT areoftenusedasmarkerinevaluatingphysiological,biochemical alterationsof plants inresponse tostresses (bioticand abiotic) andinstudieswithplants’systemic acquiredresistancesystem (Tanabeetal.,2008);andtheseenzymeactivityincreaseatthe onsetand increasinglevelsof stress(Renet al.,2002).Inplant systems,themajorROSscavengingmechanismsincludethe activ-ityofSOD,APXandCAT(Mittler,2002).InEuphorbiapekinensis, increasedCATactivityandsynthesisofH2O2werenotedpost elici-tortreatmentincultivatedtissues(Gaoetal.,2011).Theprotoplast derivedtissueshadhighenzymaticactivityandtheactivitywas evenmoreonYEaddedconditions;thisincreasemaybedueto overproductionofROS.InOryzasativacultivar,increased antiox-idativeenzymeactivityscavengedcellularROSsuccessfully and preventedmembraneinjurybyimprovingoxidativestress toler-ance(Hassanein,2000).FoyerandNoctor(2005)notedthatthe enzymeglutathionereductase(GR)–akeyascorbate-glutathione cycleenzyme,helpedmaintainabalanceofGSH/GSSGratioand protected cells from oxidative damage. Current proteomic and molecularanalysessuggestthatSaltOverlySensitive1(SOS1)gene, genesencodingtranscriptionfactors,stressproteins,enzymes cat-alyzingsecondarycompoundsgenesarenotedtobeupregulated bystressorstressinducers(Pazminoetal.,2012;Maetal.,2014). Protoplast derived embryogenic tissue showed an enriched level ofalkaloid, the yield ofVB and VCwaseven more in YE amended conditions. The action of YE was earlier reported to beassociatedwithactivationofterpenoidindolealkaloids(TIA) biosyntheticgenesSTRandTDC encodingstrictosidinesynthase andtryptophandecarboxylaseinbiosynthesisofalkaloids(Pauw etal.,2004).Asemi-syntheticmethodofchemicalorenzymatic couplingofmonomericindolealkaloidsvindolineand catharan-thinein formingvinblastine wassuccessfullymade inprevious studies(Goodbodyetal.,1988;Kutney,1990)inwhichperoxidase catalyzedcouplingreactionstepofsynthesis(Smithetal.,2003). Inthispresentstudy,weobservedincreasedenzymeactivityon applicationofYEthatmighttriggerenhancedlevelofVBandVC

synthesisinculture.Abrahametal.(2011)studiedYEeffectin Cur-cumamanggaandhigherantioxidantenzymeactivitywasnoted tobedue toenrichedlevel ofphenolicaccumulationintissues. Thecorrelationbetweenaccumulatedphenoliclevelswithradical scavengingwasearliernotedindifferentberryspeciesandthus, theantioxidantenzymeactivitiesandthesynthesisofsecondary metaboliteareconsideredtobeinvariablyrelated(Amakuraetal., 2000).In Catharanthus,there is enrichmentof alkaloids,sothe studyisimportantasitistheonlysourceofexpensive,important chemotherapeuticdrugs withanti-cancerousactivity,whichare presentinaverytrace(0.0005%DW)amount(SchmellerandWink, 1998).Thesynthesisofrosmaricacid(RA)andphenoliccompounds inSalviamiltiorrhizahairyrootswassimilarlystimulatedbyYEand Ag+_,_an_abiotic_elicitor₍_Qiong_et_al.,₂₀₀₆_)._The_biotic_and_abiotic elicitorinduced(YEandMJ)RAaccumulationwasobservedinother investigatedplantssuchasOrthosiphonaristatus(Mizukametal., 1992)andColeusblumei(Szaboetal.,1999).InL.erythrorhizon,the additionofYEinsuspensionrapidlyandtransientlyenhancedRA accumulation(Ogataetal.,2004).Sanchez-Sampedroetal.(2005) reportedabout three-foldproduction of silymarin followingYE treatmentinSilybummarianum.TheamendmentofYEat0.5g/l wasfoundtobeanoptimumtreatmentforelicitationinGymnema sylvestreinwhich over5.25foldsof gymnemicacidproduction wasnotedwithin20daysofculture(Veerashreeetal.,2012).Over 113.3%increaseintotalphenoliccontentovercontrolwasreported in YE treated cells in Astragalus chrysochlorus (Ozgur and Sule, 2009).Inourstudy,enrichedVBandVCalkaloidyieldwasnoticed onYEtreatmentsand1.5g/lwasobservedtobetheoptimumdose. YEcontainsseveralcomponentslikechitin,N-acetylglucosamine oligomers,␤-glucan,glycopeptidesandergosterol(Boller,1995) butthespecificfractiontriggersthesynthesisofalkaloidsisstill notelucidatedclearly. Thepeptideandpolysaccharidemoieties presentinYEhavebeensuggestedtobethemoleculesof stimulat-ingnature(Menkeetal.,1999;Zhaoetal.,2011).Theprotoplasts and theprotoplast derived tissues showedstress as this tissue underwentadversecellularconditions,causedbywalldegrading enzymes,PGRsandinflux/effluxofrequiredions.Theadditionof YEinmediummayraisestresslevelfurther(aswasevidencedfrom increasinglevelsofstressmarkerenzymes)thatmayhelpin syn-thesizingenhancedlevelofVBandVCinculturedtissues.Thisis thefirsteverstudyofsystematicmonitoringofVBandVCin proto-plastregeneratedtissuesinCatharanthusunderYEinfluence.This studymaycomplementtoothereffortsaimingtoenrichVBandVC yieldinculture.

Conclusion

ThisisthefirstreportontheeffectofYEonVBandVCyieldin protoplastregeneratedplantsinC.roseus.Theobservationsuggests that YE efficiently enhanced alkaloid yield however, the accu-mulationwastreatment-specific.Theidentificationofsignalling componentofYEinbiosynthesiscouldbeaveryeffectiveapproach forlargescale augmentationofalkaloidyieldofpharmaceutical importance.

Authorship

Thefirstauthorconceivedtheideaandperformedmostofthe experimentsandthesecondauthoreditedthemanuscript.

Conflictsofinterest

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Acknowledgements

We are grateful to University Grant Commission, India, for receivingfinancialassistance.WearealsothankfultoDepartment ofBotany,Central InstrumentationFacility,HamdardUniversity (JamiaHamdard)forprovidingotherfacilities.

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