Table Of ContentSecondary Metabolite Production
in Transformed Cultures
Stevioside Glycosides Production from Stevia rebaudiana
Hairy Root Cultures
Madhumita Kumari and Sheela Chandra
Contents
1 Introduction................................................................................... 3
2 DistributionandLocalizationofDiterpeneGlycosidesinS.rebaudiana................... 5
3 BiosyntheticPathwayofSteviolGlycosides................................................. 6
3.1 CellularComponentsInvolvedinBiosynthesis....................................... 7
3.2 TransportofSteviolGlycosidestoVacuole........................................... 8
4 BiotechnologicalApproachestoImproveSteviosidesGlycosidesProduction
usingInVitroCultures........................................................................ 8
5 BiotechnologicalApproachesInvolvingGeneticTransformationStudiesin
S.rebaudiana................................................................................. 9
5.1 EstablishmentofHairyRootCulturesThroughInfectionwithAgrobacterium
rhizogenesonAsepticExplantsofS.rebaudiana..................................... 9
5.2 SteviolGlycosidesProductioninHairyRootCulturesofS.rebaudiana............. 10
5.3 ExtractionandAnalysisofSteviosideGlycosidesThroughHPLC................... 10
5.4 StatisticalAnalysis..................................................................... 11
6 HairyRootInductionandSteviosideGlycosideProduction................................ 11
7 EffectofDifferentConditionsonHairyRootsgrowthandSecondaryMetabolites
Production.................................................................................... 12
7.1 EffectofMurashigeandSkoog(MS)andGamborg’sB5MediaonBiomass
andSecondaryMetaboliteContentofS.rebaudianaHairyRoots................... 12
7.2 EffectofLightandDarkConditions................................................... 14
7.3 EffectofTemperatureonBiomassandSecondaryMetaboliteContentof
HairyRoot.............................................................................. 15
8 Conclusions................................................................................... 16
References........................................................................................ 16
M.Kumari(cid:129)S.Chandra(*)
DepartmentofBio-Engineering,BirlaInstituteofTechnology,Mesra,Ranchi,Jharkhand,India
e-mail:[email protected]
#SpringerInternationalPublishingAG2016 1
S.Jha(ed.),TransgenesisandSecondaryMetabolism,ReferenceSeriesin
Phytochemistry,DOI10.1007/978-3-319-27490-4_1-1
2 M.KumariandS.Chandra
Abstract
The study elucidates the production of hairy roots through Agrobacterium
rhizogenes mediated transformation of leaf and stem explants of S. rebaudiana
forsecondarymetaboliteproduction.Hairyrootculturewasestablishedsuccess-
fully using leaf and stem explants of S. rebaudiana. Hairy roots grown in
MurashigeandSkoog(MS)mediaattemperature25(cid:1)Ckeptin16hphotoperiod
provedbestforsteviosideglycosidesproduction.AmongdifferentstrengthofMS
and B5 media, full strength MS media produced stevioside
0.412 (cid:3) 0.008 mg mL(cid:4)1 and Reb A 0.100 (cid:3) 0.008 mg mL(cid:4)1, whereas hairy
roots on B5 media produced stevioside 0.383 (cid:3) 0.002 mg mL(cid:4)1 and Reb A
0.098 (cid:3) 0.005 mg mL(cid:4)1. Growth of hairy root was found to be maximal at
25 (cid:3) 2(cid:1)Candwiththeincreaseoftemperature,growthofhairyrootsdecreases.
Secondary metabolite production was not much affected by increase in temper-
ature up to 31 (cid:1)C with approximately similar stevioside content detectable at
25(cid:1)C(0.425 (cid:3) 0.08mgmL(cid:4)1),28(cid:1)C(0.415 (cid:3) 0.08mgmL(cid:4)1),andat31(cid:1)C
(0.386 (cid:3) 0.02 mg mL(cid:4)1). At 35 (cid:1)C, stevioside content decreases rapidly
(0.090 (cid:3) 0.02mgmL(cid:4)1)andRebAwascompletelynotdetected.Resultswere
validated through ultra high performance liquid chromatography-electrospray
ionizationmassspectrometry(LC-ESI/MS)studies.
Keywords
Bioproduction (cid:129) Stevia rebaudiana (cid:129) In vitro cultures (cid:129) Steviosides glycosides (cid:129)
Biosyntheticpathway
Abbreviations
(cid:1)C DegreeCelsius
% Percent
2,4-D 2,4-Dichlorophenoxyaceticacid
Ads Adeninesulphate
B5 GamborgB5medium
BAP 6-Benzyladenine
HPLC Highperformanceliquidchromatography
h Hour
IAA Indole-3-aceticacid
IBA Indole-3-butyricacid
Kn Kinetin
min Minutes
MS MurashigeandSkoog’smedium
PGRs Plantgrowthregulators
rpm Rotationperminute
SD Standarddeviation
μL Microlitre
SecondaryMetaboliteProductioninTransformedCultures 3
1 Introduction
Stevia rebaudiana (Bertoni) is a small perennial herb, belonging to the asteraceae
family.ItisnativetocertainregionsofParaguayandBrazilinSouthAmerica.The
plant is also known as a honey leaf and candy leaf [1]. S.rebaudiana contains
diterpene glycosides viz. stevioside and rebaudioside A, which are responsible for
itssweettastewithzerocalories[2]andareestimatedtobe250–300timessweeter
than sucrose. These glycosides possess a number of therapeutics properties in
addition to their sweetness value. They regulate the blood glucose level by stimu-
lating insulin secretion so that they can be used as an alternative sweetener by
hyperglycemic patients [3, 4]. Steviol glycosides can also be used as an antihyper-
tensive [5], antitumor [6], vasodilator [7], antimicrobial, [8] and neuroprotective
drugs [9]. They are heat- and pH-stable with a good shelf life and can be added in
cooking,baking,orinbeverages.Inanumberofcountries,Steviawasapprovedas
dietarysupplements.Itmightbeasourceofanumberofpharmaceuticaldrugs.Since
Steviaishighlyversatileasanadditive,ithasgainedagreatboostinpopularityinthe
past few years and is progressively becoming the focal point of attention amongst
foodandbeverageproducers.Thus,varioustherapeuticsandsweeteningproperties
arethemostimportantattributesofStevia,whichmakesitacommerciallyimportant
plant.
Forcommercialcultivation,homogenousrangeofimprovedplantsisrequiredand
plantsgerminatedfromseedsshowadegreeofvariability.Alsoinfieldconditions,a
widerangeofvariationoccursduetoexternalenvironmentalconditionssuchasplant
pathogen,temperature,drought,andwaterlogging,whichleadstovariationincom-
position and sweetening levels [10]. Seeds of Stevia have very poor germination
potential [11–13]. In today’s world, production of stress tolerant varieties, enhanced
plant biomass, and production of medicinally important secondary metabolites are
considerableissues.Earlier,vegetativepropagationwasgenerallyusedforcultivation
ofStevia.Althoughthistechniqueislimited,numbersofexplantswereobtainedfrom
a single plant and that may raise possibilities of pathogen accumulation in tissues.
Theseinvitrotissueculturetechniquesmightproveanalternativetooltoconventional
methods for comparatively rapid multiplication of elite medicinal plantlets, which
givesdisease-free,resistantplantwithhighbiomassandsecondarymetabolites.
In plant cell cultures, secondary metabolite augmentation has limiting culture
parameters and therefore requirement of knowledge and introduction of new tech-
niques are necessary. Many plant secondary metabolites get accumulated in hairy
roots.Therefore,intherecentpasthairyrootculturehasreceivedalotofattentionin
researchfield.Theneedoftakingtheresearchformlaboratorytoindustryhasledto
the development of this new technology. Genetic engineering of hairy roots has
playedakeyroleandhasprovidednewdirectiontohairyrootresearch.Hairyroot
cultures are preferred over other methods of transformation as they have fast,
hormone-independent growth, are highly branched, lack geotropism, shows lateral
branching,aregeneticstabilityandduetotheeaseofelicitortreatment[14].
4 M.KumariandS.Chandra
Table1 Secondarymetabolitesproductioninhairyrootculturesofdifferentplants
Plantspecies Secondarymetabolite References
Atropabelladonna Scopolamine [16]
Artemisiaannua Artemisinin [17]
Camptothecaacuminata Camptothecin [18]
Daturainnoxia Hysocyamineandscopolamine [19]
D.quercifolia Scopolamineandhysocyamine [20]
D.candida Scopolamineandhysocyamine [21]
Duboisialeichhardtii Scopolamine [22]
Droseraburmanii Plumbagin [23]
Echinaceapurpurea Cichoricacid [24]
Ginkgobiloba Ginkgolides [25]
Glycyrrhizaglabra Glycyrhizin [26]
Hyoscyamusniger Hysocyamineandscopolamine [19]
Hyoscyamusniger Scopolamine [27]
Papaversomniferum Morphine,codeine [28]
Panaxginseng Ginsenosides [29]
Psoraleacorylifolia Isoflavones [30]
Przewalskiatangutica Tropanealkaloids [31]
PodophyllumhexandrumRoyle Podophyllotoxin [32]
Plumbagorosea Plumbagin [33]
Rauvolfiamicrantha Ajmalicine,Ajmaline [34]
Rubiatinctoria Anthraquinone [35]
Rubiacordifolia Anthraquinones [36]
Solanumkhasianum Solasodine [37]
Steviarebaudiana Chlorogenicacid [38]
Tylophoraindica Tylophorine [39]
Withaniasomnifera WithanolideA [40]
In vitro culture of plant cells is now a mature technology with successful
applications in crop improvement. The major limitation to wide industrial applica-
tionofplantcellcultureisthemaintenanceofstablecelllines[15].Hairyrootscan
synthesize more than a single metabolite and so prove economical for commercial
production.Anumberofsecondarymetaboliteshavebeenreportedtobeproduced
fromhairyrootcultures(Table1).
Overthelastdecade,transformedhairyrootshavebeendevelopedinnumberof
importantmedicinalplants[41].Differenttypesofexplantslikehypocotyls,cotyle-
dons, petioles, and young leaves are most frequently used for Agrobacterium-
mediated transformation [42, 43]. The extent of secondary metabolite release in
hairy root cultures variesbetween different plant species. As aconsequence,much
efforthasbeenputintotheuseofinvitroculturesasoneattractivebiotechnological
strategyforproducingthisnaturalcompoundofcommercialinterest.
SecondaryMetaboliteProductioninTransformedCultures 5
Fig.1 Stevioside HO
HO
OH
O HO
HO
O OH
O
HO O
HO O O
HO O
HO OH
Fig.2 RebaudiosideA OH
HO
HO OH
O
H O
HO
O H
O
HO H
O OH
O HO OH
OH
H3C CH2
H
HO O O H
CH
3
HO O
OH
OH
2 Distribution and Localization of Diterpene Glycosides
in S. rebaudiana
The sweet diterpene glycosides of Stevia have been the subject of a number of
reviews.TheleavesofS.rebaudianacontainatleasteightditerpeneglycosides,viz.,
steviosideandrebaudiosides.In1931,isolationofsteviosidewasdonebyBrideland
Lavieille [2]. In1952,the chemical structure of stevioside(Fig. 1) was established
and described as an aglycon, steviol with glycoside of three glucose molecules.
Duringthe1970s,othercompoundswereisolated,includingrebaudiosideA(Fig.2),
withsweetnesspotencyevenhigherthanstevioside.
6 M.KumariandS.Chandra
OPP
OPP
CPS KS
Kaurene
Geranylgeranyl diphosphate (-)-copalyl diphosphate
KO
OH
O-glc
UGT85C2
KAH
COOH
COOH
COOH
Steviolmonoside Steviol
(-)-Kaurnoicacid
UGT Kaurenoic acid
O-glc-glc 7-oxidase
COOH COOH
Steviolbioside COOH
GA12
UGT74G1
glc
O-glc-glc O-glc-glc
Gibberllins
UGT76G1
COO-glc COO-glc
Stevioside Rebaudioside A
Fig. 3 Biosynthetic Pathway of Steviol Glycosides (Redrawn from Brandle and Telmer [44]).
(Abbreviations:copalyldiphosphatesynthase(CPS),kaurenesynthase(KS),kaureneoxidase(KO),
kaurenoicacid13-hydroxylase(KAH))
3 Biosynthetic Pathway of Steviol Glycosides
Steviol glycosides biosynthesis pathway shares some common steps with GA
(Gibberellicacid)biosynthesis.Steviolglycosidebiosynthesisoccursinleavesand
transported to different parts. In vivo labeling with [1-13C] glucose and NMR
SecondaryMetaboliteProductioninTransformedCultures 7
Fig.4 Cellularcomponentsinvolvedinbiosynthesisofsteviolglycosides
spectroscopy showed that main precursor steviol is synthesized via the plastid
localizedmethylerythritol4-phosphate(MEP)pathway(Fig.3)[44].
Aglyconesteviol is glycosylated by various glucosyltransferases present in the
cytoplasm. Steviol has two hydroxyl groups, one present at C-19 of C-4 carboxyl
and other at C-13. Glycosylation starts at C-13 by UGT85C2 which produces
steviolmonoside.Shibataetal.[45]used13-O-and19-O-methylsteviolassubstrates
for crude Stevia leaf enzyme extracts to determine which active group is
glucosylated first. They found that only 19-O-steviol could serve as a substrate
and concluded that synthesis of SGs starts with the glucosylation of the
13-hydroxyl of steviol. Steviolmonoside is then glycosylated to produce
steviolbioside. UGT (uridine diphosphate-dependent glycosyltransferase) of this
step is not yet identified. Finally, stevioside is produced by UGT74G1 by
glucosylation at C-19 position (Fig. 3). Rebaudioside A is synthesized by
glucosylationofsteviosideatC-13byUGT76G1[45].
3.1 Cellular Components Involved in Biosynthesis
The precursorof diterpenoids,kaurene issynthesizedin thechloroplast byterpene
cyclases (Fig. 4). Like all diterpenes, steviol is synthesized from GGDP (geranyl
8 M.KumariandS.Chandra
geranyl diphosphate), first by protonation-initiated cyclization to copalyl diphos-
phate (CDP) by CDP synthase (CPS). Next, kaurene is produced from CDP by an
ionization-dependantcyclizationcatalysedbyKS(kaurenesynthase).
Kaurene is then converted to steviol by the activity of enzymes present at the
membraneofendoplasmicreticulum.Itisoxidizedinathree-stepreactiontokaurenoic
acid, by kaurene oxidase (KO), a P450 mono-oxygenase that also functions in GA
biosynthesis. Steviol biosynthesis diverges from GA biosynthesis with the hydroxyl-
ationofkaurenoicacidbyKAH(ent-kaurenoicacid13-hydroxylase).Thisisthefirst
committedstepandtheenzymeisofsignificantinterestforuseinbiotechnology.Steviol
synthesizes various steviol glycosides in the cytosol that ultimately accumulate in the
vacuole.Theaglyconesteviolhastwohydroxylgroups,oneattachedtotheC-19ofthe
C-4carboxylandtheotherattachedtotheC-13,bothofwhichcanbeglycosylated.
3.2 Transport of Steviol Glycosides to Vacuole
Thefinalphaseofglycosideaccumulationisthetranslocationofglycosylatedsteviol
out of the cytosol and accumulation in the vacuole. In Stevia, steviol glycosides are
knowntooccurinthevacuole,butthemechanismbywhichtheyaretraffickedintothe
vacuoleisnotyetunderstood[46].Vesicle-mediatedtraffickingofmetabolitesseems
likeapossiblescenarioforbiosyntheticpathwayswhicharephysicallyassociatedwith
theER(endoplasmicreticulum)ororganizedinER-associatedmetabolons[47].
RecentworkhasrevealedanequallyimportantrolefortheATPBindingCassette
(ABC) superfamily of transporters. ABC transporters are directly energized by the
hydrolysis of ATP [48] and have been shown to transport a diverse array of
compounds across the vacuolar membrane in plants including glutathione-
conjugatedagrichemicalsanthocyanins[49]andflavoneglucuronides.Theenerget-
ics of accumulation of steviosides in Stevia need to be investigated to confirm a
directcarrier-mediatedmechanismandtoidentifytheclassoftransporterinvolvedin
theuptakeofsteviolglycosides.
4 Biotechnological Approaches to Improve Steviosides
Glycosides Production using In Vitro Cultures
Several efforts have been dedicated to the use of plant in vitro cultures as a
biotechnologicalstrategy toproducesecondarymetabolitesofcommercialinterest.
The advantages for industrial production of these compounds include uniform
product quality, independence from climate and seasonal changes, supply stability,
and a closer relationship between supply and demand. Several studies have been
doneinSteviainvitroculturesforglycosidesproduction.
Recent studies of Kumari and Chandra [50] revealed micropropagation in
S.rebaudianafromleafandnodalexplantsandproductionofhigh-valuesecondary
metabolites. A combination of PGRs (plant growth regulators) proved better than
single for both callusing and direct-shoot multiplication from leaf explants.
SecondaryMetaboliteProductioninTransformedCultures 9
Treatment of Kn and IAA (1.5 mg L(cid:4)1) each showed best callusing response
(85.5 (cid:3) 0.33 %). For shoot proliferation from callus, Kn (2.5 mg L(cid:4)1) with IAA
(1.5 mg L(cid:4)1) showed maximum number of shoots (5.3 (cid:3) 0.3) proliferating from
callus with longest set of 9.03 (cid:3) 0.14 cm. Direct organogenesis from leaf explant
andKn(1.5mgL(cid:4)1)withBAP(2.5mgL(cid:4)1)gavemaximumnumber(8.6 (cid:3) 0.33)
ofshootsfromleafexplantwithlongestshootlength(5 (cid:3) 0.11)cm.HPLCstudies
showedthatboththesecondarymetabolites(stevioside,0.451 (cid:3) 0.001mgg(cid:4)1,and
Reb A, 0.131 (cid:3) 0.005 mg g(cid:4)1) are higher in in vitro shoots developed through
organogenesisfromcalluscultures[50].
5 Biotechnological Approaches Involving Genetic
Transformation Studies in S. rebaudiana
5.1 Establishment of Hairy Root Cultures Through Infection
with Agrobacterium rhizogenes on Aseptic Explants
of S. rebaudiana
S.rebaudianaisanimportantmedicinalplantandonlyfewreportsareavailableon
hairy rootcultureofthis plant.No studyhasbeendoneonimprovement ofsteviol
glycoside production through hairy root culture. The following experiments were
performed to establish a transformation system so that in future transgenic
S.rebaudianacouldbedevelopedforvariouseconomicandmedicinalpurposes.
Agrobacterium rhizogenes strain ATCC 15834 was used for the study. The
bacterial cultures were grown in 50 ml of yeast extract broth (YEB) supplemented
with 50 mg L(cid:4)1 rifampicin. Plant material of S. rebaudiana was collected from
IndigenousMedicinalPlantGardenofBirlaInstituteofTechnology,Mesracampus,
andthenstemandshoottipswereselectedforsurfacesterilizationinlaminarairflow.
Acultureofbacteriawasinoculatedin50mLYEBmediaandincubatedinarotary
shakerfor24hat28(cid:1)Cat100rpminthedark.Afterattainingoptimumgrowth(O.D
0.9), bacterial suspension was then centrifuged at 4000 rpm for 10 min and pellet
wascollected.Theresultingpelletfrom50mlsuspensionwassuspendedin250ml
MSliquidmediaandwasacclimatizedfor4hat28(cid:1)Cindark.Thiswasfurtherused
for co-culture. Surface sterilized explants were wounded and co-cultured in MS
liquidmediacontainingbacterialculturefor30min.Infectedexplantswerewashed
with autoclaved distilled water for 1–2 times and excess water was removed by
blotting paper. All the explants were then inoculated on hormone-free MS agar
mediumandculturedinthedarkat25 (cid:3) 2(cid:1)C.After2daysofco-cultivationofplant
tissuesandbacterialcells,theexplantsweretransferredontohormone-freeMSagar
mediumwith500mgL(cid:4)1cefotaxime,abacteriostaticagent,andculturedfor7days.
Oneweeklater,theexplantswereretransferredontohormone-freeMSagarmedium
with250mgL(cid:4)1cefotaxime.Thisprocedurewasrepeatedfor2or3timeswithan
intervalof1week,andgraduallytheconcentrationofcefotaximewasreduced.The
numberofresponsiveexplantsandnumberofhairyrootsperexplantswererecorded
after15,30,and45days.
10 M.KumariandS.Chandra
Table2 PrimersdesignedforamplificationofrolCgene
S.No. Primername Primersequence
1 RolF 5’-TGTGACAAGCAGCGATGAGC-3’
2 RolR 5’-GATTGCAAACTTGCACTCGC-3’
The hairy roots so obtained from the above transformation procedure were
subjected to molecular biology studies to confirm integration of Ri plasmid into
thehostgenome.DNAisolationfromhairyrootswasdoneusingDNAisolationkit
(QIAGENDNeasyplantmini-kit)followingthemanufacturerinstructions.
Polymerase chain reaction (PCR) identification of the rooting locus gene rol C
wasperformedusingDNAfromthehairyrootsastemplateandthenon-transformed
rootsascontrol,respectively.Primers(rolCFandrolCR,detailsshowninTable2)
and100ngofgenomicDNAisolatedfromhairyrootswereusedforamplification.
Amplifications were performed using a GeneAmp PCR System 9700 Thermo-
cycler (Applied Biosystems, Foster City, CA, USA) that was programmed for an
initialdenaturationstepof3minat94(cid:1)Cand35cycles(eachconsistingof1minat
94(cid:1)C,1minat53.5(cid:1)C,and1minat72(cid:1)C),followedbyafinalextensionat72(cid:1)C
for 6 min. Amplified products were resolved on 1.4 % agarose gel by
electrophoresis.
5.2 Steviol Glycosides Production in Hairy Root Cultures
of S. rebaudiana
Purifiedhairy roots werecutby sterilized scalpel under laminar airflow andinocu-
lated on hormone-free liquid MS medium. After 3 weeks, regenerated hairy roots
were transferred to different media according to test conditions. Some physical
parameters like effect of culture media, temperature, and light on steviol glycoside
productionwasalsoobserved.
5.3 Extraction and Analysis of Stevioside Glycosides
Through HPLC
Hairyrootsfromdifferentsetsofflaskswerecollectedanddriedtoaconstantweight
at40(cid:1)Candgroundedtomakefinepowder.Forextractionofsteviosideglycosides,
driedpowderwasdippedinmethanol/waterinthe4:1.Themixturewasleftatroom
temperature for overnight. Extract was then filtered next day using Whatmann
number 1 filter paper and dried. These sample extract were redissolved in distilled
waterandfilteredthrougha0.2μmMilliporefilterforanalysisbyHPLC.Stevioside
glycosides were separated using a Waters HPLC system (WatersCorporation, Mil-
ford,MA)indC18column(WatersAtlantis,4.6mm (cid:5) 150mm;4μmparticlesize).
20 μL of extracts were injected in a HPLC system. The solvents optimized for
isocratic elution consisted of acetonitrile and MilliQ water with 0.1 % ortho-
phosphoricacidintheratioof20:30 withflowrateof0.5mLmin(cid:4)1.Thedetector
wassetat210nm.Thecompoundsfromsampleswereidentifiedbycomparingthe
