Rev. bras. farmacogn. vol.27 número1

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

Original

Article

Morpho-anatomical

study

of

Stevia

rebaudiana

roots

grown

in

vitro

and

in

vivo

Rafael

V.

Reis

_,

_Talita

_P.C.

_Chierrito

_,

_Thaila

_F.O.

_Silva

_,

_Adriana

_L.M.

_Albiero

_,

_Luiz

_A.

_Souza

_,

José

E.

Gonc¸

alves

_,

_Arildo

_J.B.

_Oliveira

_,

_Regina

_A.C.

_Gonc¸

_alves

a_{ProgramadePós-graduac¸ãoemCiênciasFarmacêuticas,UniversidadeEstadualdeMaringá,Maringá,PR,Brazil} b_{DepartamentodeFarmácia,UniversidadeEstadualdeMaringá,CampusUniversitário,Maringá,PR,Brazil} c_{DepartamentodeBiologia,UniversidadeEstadualdeMaringá,CampusUniversitário,Maringá,PR,Brazil} d_{ProgramadeMestradoemPromoc¸ãodaSaúde,CentroUniversitáriodeMaringá,Maringá,PR,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received16May2016 Accepted14August2016 Availableonline7October2016

Keywords: Steviarebaudiana Adventitiousrootcultures Morpho-anatomical Chemicalprofile

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b

s

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Steviarebaudiana(Bertoni)Bertoni,Asteraceae,isusedasafoodadditivebecauseitsleavesareasource ofsteviolglycosides.Thereareexamplesoftissueculturebasedonmicropropagationandphytochemical productionofS.rebaudianaleavesbuttherearefewstudiesonadventitiousrootcultureofS.rebaudiana. Morethan90%oftheplantsusedinindustryareharvestedindiscriminately.Inordertoovercomethis situation,thedevelopmentofmethodologiesthatemploybiotechnology,suchasrootculture,provides suitablealternativesforthesustainableuseofplants.Theaimofthisstudywastocompare morpho-anatomicaltransversesectionsofS.rebaudianarootsgrowninvitroandinvivo.Theinvitrosystemused tomaintainrootculturesconsistedofagyratoryshakerunderdarkandlightconditionsandarollerbottle system.TransversesectionsofS.rebaudianarootsgrowninvitrowerestructurallyandmorphologically differentwhencomparedtothecontrolplant;rootsartificiallymaintainedinculturemediacanhave theirdevelopmentaffectedbythedegreeofmediaaeration,sugarconcentration,andlight.GC–MSand TLCconfirmedthatS.rebaudianarootsgrowninvitrohavetheabilitytoproducemetabolites,whichcan besimilartothoseproducedbywildplants.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Steviarebaudiana(Bertoni)Bertoni,Asteraceae(Asterales),has longbeenknowntotheIndians,whocallitka’ahe’ê(“sweetherb”). Steviaisanerect,taprootandperennialherbaceousspeciesthat reachesupto90cminheight,withsmallleavesthatmeasurefrom 3to5cminlengthand1to1.5cminwidth,beingsimpleandsessile onoppositeandalternatevertices.Theflowersarewhiteandthe petalsaregroupedinterminaloraxillaryracemes(Magalhãesetal., 2000).

The herb is native of the Amambay region, in northeastern Paraguay,andisalsofoundinArgentinaandBrazil(Tavariniand Angelini,2013).Steviaisknownasthesweetherbof Paraguay, sweetleaf,candyleaf,andhoneyleaf(Soejarto,2002;Brandleand Telmer,2007;Madanetal.,2010).

Theplanthasbeenusedcommerciallysincethe1970s,when theJapanesedevelopedprocessestoextractandrefinestevioside

∗ Correspondingauthor.

E-mail:[email protected](R.A.Gonc¸alves).

fromtheleaves(Dacomeetal.,2005).ThemajorproducersofStevia areChinaandSouthAsia.Currently,thereisnolarge-scaleStevia farming,howevertheSteviamarkethasbeengrowingsincethe steviolglycosidesextractedfromtheleaveswereapprovedasa foodadditive(sweetener),in2011(TavariniandAngelini,2013).

Therearemorethan150Steviaspecies,howeveronlyS. rebau-dianahassignificantsweeteningproperties,althoughotherspecies containchemicalsofinterest(Soejartoetal.,1982).Therearefew chemicalstudiesavailablewithregardstotheroots.Inthemain phytochemical studies,the following substances were isolated: longipinenediesters,whichwereisolatedfromS.lucida( Guerra-Ramírezetal.,1998), S.serrata(Sánchez-Arreolaetal.,1995),S. porphyrea(Sánchez-Arreolaetal.,1999),andS.vicida(Románetal., 1995),andglycosidestevisalioside,whichwasisolatedfromS. sali-cifolia(Mataetal.,1992).

Thedevelopment of methodologiesthat employ biotechnol-ogy,suchasplanttissueculture,micropropagation,rootculture, andtransformedrootculturerepresent analternativeapproach tothesearchforsecondarymetaboliteswithspecificproperties, andtheyallowgeneticstabilitytobemaintained(Thiyagarajan andVenkatachalam,2012).Thus,withthedevelopmentofrapid

http://dx.doi.org/10.1016/j.bjp.2016.08.007

rootgrowthinvitrosystems,itwouldbepossibletoobtainenough materialfortheproductionofextractswithouttheneedforlarge agriculturalareasandthedestructionofnature,forthecommercial productionofcompoundsofinterest.

Considering the promising results from previous studies on S.rebaudiana roots,reportedbyourresearchgroup(Reisetal., 2011;Oliveira etal.,2011; Lopeset al.,2015), theexistenceof fewmorpho-anatomicalstudiesregardingS.rebaudianaroots,and the search for new ways of obtaining primary and secondary metabolites,S.rebaudianarootsinvitrorepresenta biotechnolo-gicalalternativeforobtainingthesemetabolites.Thus,themainaim ofthisstudywastocompareS.rebaudianaroot(invitroandinvivo) morpho-anatomicaltransversesectionsandpreliminarychemical analysisbyGC–MSandTLC.Theresultsofourstudyprovidecrucial informationforboththeoptimizationandtechnological develop-mentofadventitiousrootsofS.rebaudianafortheproductionof secondaryandmainlyprimarymetabolitesinbioreactors.

Materialsandmethods

Plantmaterial

SteviarebaudianaBertoni(Bertoni),Asteraceae,rootsandshoots werecollectedattheMedicinalPlantsTeachingGarden,attheState UniversityofMaringa(HDPM-UEM).Avoucherspecimen (14301-HUEM)wasdeposited attheHerbarium oftheStateUniversity ofMaringa,Maringa,Brazil,andwasidentifiedbyJimiNakajima (UniversidadeFederaldeUberlandia).

Seedlingsgrowninvitro:S.rebaudianashootsweretakenfrom plants fromHDPM-UEM. Shoots weresubjected to disinfection (Patrãoetal.,2007)andtransferredtoMSmedia(Murashigeand Skoog,1962)andsupplementedwith0.8%agar(w/v)and30gl−1

sucrose.Shootswerecultivatedunderlightconditions,witha pho-toperiodof14h(45␮molm−2s−1)at25±1◦_{C,obtainingseedlings.}

In vitro adventitious roots: Seedlings were grown and their roots(withabout90and300mgfreshweight)wereremovedand transferredtoliquidMSmediasupplementedwith2.0mgl−1

␣

-naphthaleneaceticacid(NAA)and30gl−1_{sucrose.Seedlingswere}

alsocultivatedunderdarkandlightconditions,at25±1◦_C,ina

gyratoryshaker(90rpm)andinarollerbottlesystem,underdark conditions(Reisetal.,2011).

Preparationoftheextracts

Crudeextractsfromroots(control):Rootscollectedatthe HDPM-UEMweredried inacirculatingair dryingovenat45◦_{Cfor 15}

days andpowdered. The powder(200g) was extractedusing a Soxhletextractorwith800mlofhexanefor4h.Theprocedurewas repeatedthreetimesandthehexaneextractswereconcentrated underreducedpressureat45–50◦_{Conarotaryevaporator,yielding}

4.13gofcontrolcrudehexaneextract(CHEC).

Crude extract from roots grown in vitro in agyratory shaker, underlightanddarkconditions:Rootsofplantsgrowninthe gyra-toryshakerwerelyophilizedafter42daysandsubjected(5g)to Soxhletextractionusinghexane, as describedabove. 255mg of gyratoryshakercrudehexaneextract(CHES)wereobtainedfrom rootsgrowninagyratoryshaker,underlightconditions.Thesame procedurewasperformedwithrootsgrowninagyratoryshakerin thedark,yielding230mgofgyratoryshakercrudehexaneextract inthedark(CHESD).

Crudeextractofinvitrorootsgrownintherollerbottlesystem: After42daysofgrowth,rootswerelyophilizedand5gwere sub-jectedtothesameSoxhletextractionprocess,yielding235mgof rollerbottlesystemcrudehexaneextract(CHEB).

TLCanalysis

Thin-layer chromatography (TLC) was used to compare the chromatographicprofilesofrootextractsgrownindifferentculture typesandinthecontrol(invivo).Silicagel60GF254(Merck®_)was

usedforastationaryphase.Hexaneextractswereanalyzedusing hexane:ethyl acetate 95:5(v/v) as eluent. Visualizationof sub-stancespotsonTLCplateswasperformedusingUVlight(:254and 366nm)andalsowith4%vanillinsulfuricacid,followedby heat-ingat150◦_{Cfor2–4min(GibbonsandGray,1998).Extractswere}

appliedonto TLCplates in knownconcentrations:10mg CHEC, CHES,CHESDandCHEBdissolvedinhexane(1ml).

Morpho-anatomicalanalysis

Roots were fixed in Bouin solution (saturated picric acid:formaldehyde:glacial acetic acid 7:2:1, v/v/v) (Kraus and Arduin,1997)andtheirfragmentswerekeptinthefixativefor5 days.Afterthisperiod,theywerewashedwith70%ethanoland putin60%ethanolfor1hfordehydration.Afterwards,theywere keptin70%ethanolforpreservation.

Therootswereembeddedinacrylichystoresin,inaccordance withGerrits(1991)andthemanufacturer’sguidelines.The embed-dedmaterialwassectionedintotransversesectionswitharotary microtome,andthesectionswerestainedwith0.1%ofblue tolui-dine(O’Brienetal.,1964).SlidesweremountedinPermount®_.

Illustrationswereobtainedviaphotomicrographimagecapture usingaCanonPowerShotA95(ZoomBrowserEX4.6)digital cam-era,andillustrationscaleswereobtainedusingamicrometerscale. Thesameopticalconditionswereusedforeachcase.

Gaschromatography–massspectrometry(GC–MS)

GC–MSanalyseswereperformedusingagaschromatograph (Thermo Electron Focus) equipped with a TR5MSSQC fused

CHEC CHES CHESD CHEB

2.1

ep

ep

ep

pe

mx

mx pe en

fp px

es

en

ex ep

ep ex

ex

p p

p

p

fp _pe

en

mx mx

px

px

2.3

2.5

2.6

2.11

2.4

2.9

2.12

2.10

2.2

2.7

2.8

Fig.2.Steviarebaudiana.(A)(2.1)GeneralaspectoftherootspecimengrownatHDPM-UEM(control).(2.2)Generalaspectoftherootcultivatedintherollerbottlesystem (bar–1cm).(2.3and2.5)DetailofthecortexandthecentralcylinderofthecrosssectionoftherootgrownatHDPM-UEM.(2.4and2.6)Detailofthecrosssectionofthe rootcultivatedintherollerbottlesystem(bar–50␮m)(en,endodermis;ep,epidermis;ex,exodermis;fp,primaryphloem;mx,metaxylem;p,parenchyma;pe,pericycle;

px,protoxylem).(B)(2.7)Generalaspectoftherootgrowninarotarygyratoryshakerinthedark.(2.8)Generalaspectoftherootgrowninagyratoryshakerinthepresence oflight(bar–1cm).(2.9and2.11)Detailofcortexandcentralcylinderofthecrosssectionoftherootgrowninagyratoryshakerinthedark.(2.10and2.12)Detailofthe cortexandcentralcylinderofthecrosssectionoftherootgrowninagyratoryshakerinthepresenceoflight(bar–50␮m)(en,endodermis;ep,epidermis;es,Casparian

strips;ex,exodermis;fp,primaryphloem;mx,metaxylem;p,parenchyma;pe,pericycle;px,protoxylem).

silicacapillary column(20m×0.25mm,0.25␮mfilmthickness)

andinterfacedwithaThermoDSQ-IImassspectrometer.Theoven temperaturewasprogrammedwithaninitialtemperatureof60◦_C,

heldfor4minand60–220◦_Cat4◦_Cmin−1_{,held for28min;an}

injectortemperatureof250◦_{C;atransferlinetemperatureof250}◦_C

andanionsourcetemperatureof250◦_{C;heliumasacarriergas;a}

linearfluxadjustedto1mlmin−1_{;a1:30splitratio;70eV}

ioniza-tionenergyandfullscanmode.

Resultsanddiscussion

PreliminaryTLCanalysis

Preliminarycharacterizationofthemajorcompoundspresent intheextractswasperformedusingtheTLCofhexaneextracts obtainedfromtheS.rebaudianarootsgrownatHDPM-UEM(CHEC) (control)andtherootsgrowninvitro(CHES,CHESD,CHEB).TLC qualitativeanalysisshowedthatrootsgrowninvitrohavethe abil-itytoproducesecondarymetabolitesandcanbesimilartothose producedinparentplants(Fig.1).

Morpho-anatomicalstudy

The rootsystem of S. rebaudiana (in vivo)grown at HDPM-UEM(control)isbranched(Fig.2.1),andtherootsintheprimary structurearecylindrical,asobservedinthetransversesection.The epidermisisuniseriatewithunicellularhairs,aparenchymatous cortexwithanexodermisandendodermis(Fig.2.3),acentral cylin-derwithaonelayerpericycle,axylemwithtwoprotoxylempoles (diarchroot),andtwophloematicstrands(Fig.2.5).

RT:10.00 - 65.00

10 15 20 25 30 35 40 45 50 55 60 65

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

A

B

C

85 90 95 100

Relative abundance

55.75

49.11

51.44

27.51 61.08

55.33 61.76

51.77

41.91 56.27 62.17

16.90 _60.80

62.48 15.97 17.68 24.66 27.87 31.43 52.03

10.1512.42 20.23 33.7836.23 41.47 45.2548.00

10 15 20 25 30 35 40 45 50 55 60 65

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Relative abundance

49.11

44.97 _56.27

30.60

42.21 31.13

27.61

26.55 27.87

61.06 26.41

26.22 39.90

25.61

36.89 24.69 34.17

61.74 38.33

42.74 57.88 31.74

55.72 15.16

36.24

20.85_{23.00 48.98}

12.95 _16.42

54.22 48.82 53.30

20.14 63.49

10.56

10 15 20 25 30 35 40 45 50 55 60 65

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Relative abundance

44.96

36.21

55.73

56.27 42.19

33.66

37.32 _61.0961.75 41.99

49.84

37.46 57.68

11.24 14.5915.0817.7721.03 _26.1926.5227.4830.59 33.77 38.32 42.7345.2549.11 53.3054.22 62.02

Therootshadalsobranchedinthegyratoryshakerunderdark conditions (Fig. 2.7), in the primary structure, and the epider-miswasglabrous,comprisingalayerofregularisodiametriccells (Fig.2.9).Thecortexwasparenchymatous,withanexodermisand endodermiswithCasparianstrips(Fig.2.11).Thecentralcylinder showedauniseriatepericycleandavascularsystemconsistingof fourprotoxylempolesandaroottetrarch,whichisdifferentfrom thecontrolplant.

Therootsystemwasequallybranchedunderlightconditions (Fig.2.8),andtherootsshowedalowertransversesection diam-eter,theexodermisandepidermiswereslightlydifferent,andthe corticalparenchymahadlargeintercellularspaces(Fig.2.10).The centralcylindershowedapoorlydevelopedvascularsystem, char-acterizedbytriarchroot(Fig.2.12).

Theinvitroplantsaresubjectedtoanenvironmentwithhigh rel-ativehumidityandlowlightintensity.AccordingtoWetzsteinand Sommer(1982),theseconditionscouldpossiblyleadtostructural changes,whichcanbeobservedwhencomparingthetransverse sectionsoftherootsgrowninagyratoryshakerunderdarkand lightconditions.Inthepresenceoflight,therootistriarchandthe corticalparenchymashowedlargeintercellularspaces,whileinthe absenceoflight,therootsaretetrarchandtherearenointercellular spacesinthecorticalparenchyma(Fig.2.9and2.10).

AccordingtoMayeretal.(2008),similarresultswerefoundfor CymbidiumplantsHort.(Orchidaceae)invitro,whichalsoshoweda lessdevelopedcortexwithintercellularspaces,unlikeplantsgrown intheirnaturalenvironment.ThevascularbundlesofS. rebaudi-anagrowninvitroareless developedthanthose ofthecontrol plant,aswasalsonotedforthevascularsystemsofRolliniamucosa leaves(Albarelloetal.,2001).StreetandMcgregor(1952)reported thatrootsartificiallymaintainedinculturemediacouldhavetheir developmentaffectedbythedegreeofmediaaeration,sugar con-centration,light,andotherfactors.

Themodificationofdiarchtotriarchrootconditionsnoticedin thegyratoryshakerwasreportedbyTorrey(1955)forpearoots growninculturemedia.RootsofS.rebaudianagrowninagyratory shakerwentfromdiarch(control)totriarchrootinthepresenceof light,andfromdiarch(control)totetrarchrootintheabsenceof light.

Gaschromatography–massspectrometry(GC–MS)

The GC–MS analyzes of hexane extracts (Fig. 3) show the chromatographicprofileofHDPM(Fig.3a)and theinvitroroot culturesofS.rebaudiana(Fig.3bandc).Itispossibletoobserve thattheinvitrorootculturehasagreatervarietyofcompounds thantheHDPMand thatthehexaneextractsfromthegyratory shaker in the dark (Fig. 3b) have a higher diversity of com-poundswhencomparedtothechromatogramofrootsgrownin therollerbottlesystem.GC-MS analysisofthehexane extracts ofHDPM(Fig.3a)andtheinvitrorootculturesofS.rebaudiana (Fig.3bandc)showsacomplexmixtureofcompoundswithtypical fragmentationoflongipinenederivatives(Sánchez-Arreolaetal., 1999;Cerda-García-Rojasetal.,2006),thedataforwhichisnot shown.SimilarresultswereobtainedbyReis(2009),whoisolated andestablishedalongipinenederivativefromS.rebaudianaroots grownat HDPM-UEM(CHEC) using spectroscopicand chemical methods.

Highermetabolite production variability may be the conse-quenceofthecultureconditionsemployed(Murthyetal.,2016) and/ormorphologicalandanatomicalalterations,whichweremore significantinthegyratoryshaker(inthedark)thanintheroller bottlesystem(Reisetal.,2011).Furtherphytochemicalstudiesare necessaryforthecompleteidentificationoftheseunknown com-pounds.

Conclusion

AftercomparingS.rebaudianarootcrosssections,itwaspossible toreportthatrootsgrowninvitroarestructurallyand morpholog-icallydifferentwhencomparedtocontrolplants.Factorssuchas light,degreeofaeration,nutrientconcentrationandothers,which areinaccordancewithseveralreportsintheliterature,makeS. rebaudianainvitrorootsdifferent.Chromatographicprofilingby TLCandGC–MSanalysisshowedthatrootsofplantsgrowninvitro havetheabilitytoproducesecondarymetabolites,whichcanbe similartothoseproduced byplantsinnature(invivo),but ina sustainableway.

Authors’contributions

TFOSandRVR(MScstudents)carriedoutthelaboratorywork (preparingherbariasamples,chemicalextraction,obtaining chro-matograms, and plant anatomy studies). TPCC contributed to carryingouttherootculturelaboratoryworkandanalysis.ALMA andLAScontributedinplantanatomystudies.JEGcontributedto thechromatographicanalysis.AJBOandRACGdesignedthestudy, supervisedthelaboratorywork,andwrotethemanuscript.Allthe authorshavereadthefinalmanuscriptandapprovedthe submis-sion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsaregratefultotheCNPqandCAPES,Brazilian fund-ingagencies.

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