1/9/2014

BT3026 HERBAL BIOTECHNOLOGY

Module1
(11hours)
Introductionandfutureprospectsofherbalbiotechnology,Useofbiotechnologicaltoolsin
medicinalplantscience,Biotechnologicalapproachesfortheproductionofplantbased
chemotherapeuticsandsecondarymetabolites,Combinatorialbiosynthesisasanewtoolin
thegenerationofnovelnaturalproducts,Bioprocesstechniquesinherbalmedicines,
Module 2
Module2
(11 hours)
(11hours)
Bioanalyticalmethodsandmetabolomics.Endophytes:plantassociatedmicrobesasanew
sourceofbioactivecompounds,Bioprospecting:thesearchforbioactiveleadstructures
fromherbs,selectionofhighyieldingcelllines,GeneticmodificationsandTransgenic
herbalplants,Productionoftherapeuticantibodiesinplants.
Module3
(10hours)
Breedingofmedicinalplants,Improvementofaplantpopulationbyselection,
Improvementofselectionresponsebyspecifictechniques,Characterizationofmedicinal
plants, Biochemistry of flavor compounds and essential oils, Biotechnological applications
plants,Biochemistryofflavorcompoundsandessentialoils,Biotechnologicalapplications
offlavorcompounds.
Module4
(9hours)
Intellectualpropertyprotectionofplantbiotechnology,Utilitypatents,TradeRelated
AspectsofIntellectualPropertyRights(TRIPS),Plantvarietyprotection,Ethicsofpatenting
plantbiotechnology.

TEXTBOOKS
1. D.J.ChadwickandJ.Marsh,Ethnobotany andtheSearchforNewDrugs,JohnWiley
&Sons,Chichester,1994.
2. L.ZhangandA.L.Demain,NaturalProducts DrugDiscoveryandTherapeutic
Medicine,HumanaPress,Totowa,2005.
3. C.BaconandJ.F.White,MicrobialEndophytes.MarcelDekker,Inc.,NewYork,2000.
4. P.M.Dewick,MedicinalNaturalProducts.ABiosyntheticApproach,Wiley,NewYork,
2002.
5. OliverKayser andWim J.Quax,MedicinalPlantBiotechnologyFromBasicResearchto
IndustrialApplicationsVolume1,WILEYVCHVerlag GmbH&Co.2007

REFERENCEBOOKS
1. K.G.Ramawat,BiotechnologyofMedicinalPlants:Vitalizer andTherapeutic,
IllustratedEdn.,SciencePub.,2004.
2. M.Spinella,ThePsychopharmacologyofHerbalMedicine,1st Edn.,TheMITpress,
2001.
3. K.OksmanCaldentey andW.H.Barz,PlantBiotechnologyandTransgenicPlants,1st
Edn.,CRCpress,2002.

1/9/2014

Herbs
Aherbisaplantthatisvaluedforflavor,scent,orotherqualitiesthatareessential
formedicinalandspiritualpurposes.
Thereisagreatincreaseintheuseofmedicinalplantsacrosstheworld.
AccordingtotheWorldHealthorganization(WHO),approximately80%ofthe
g
g
(
), pp
y
worldspopulationcurrentlyusesherbalmedicinesdirectlyasteas,decoctsor
extractswitheasilyaccessibleliquidssuchaswater,milk,oralcohol.
Althoughmodernsyntheticdrugsaremostlyusedindevelopedcountries,the
useofherbaldrugsinthewesternworldiswellaccepted,andacontinuouslyhigh
demandforplantmaterialandextractednaturalproductscanbeobserved.
Thetop10rankedplantsthathavereceivedgreatestinterestintheUSAand
Europeoverthepast30years,andaccountforover50%oftheoverthecounter
(OTC)
(OTC)marketareasfollows.
k t
f ll

The top 10 ranked plants in terms of market potential

S. No.

Species

Uses

1
2
3

Hypericum perforatum
Echinacea purpurea
Ginkgo biloba

4
5
6

Sabal serrulata
T
Tanacetum
t
parthenium
th i
Allium sativum (Garlic)

Anxiety, depression, insomnia

Immune stimulation
Dementia,
Alzheimers
disease,
tinnitus
Benign prostatic hyperplasia
Mi i prophylaxis
Migraine
h l i
Lipidlowering,
antithrombotic,
fibrinolytic, antihypertensive, anti
atherosclerotic

7
8

Zingiber officinalis (Ginger)

Panax ginseng

Antiemetic
Tonic,
performance
enhancer,
adaptogen, mood enhancer

9
10

Valeriana officinalis
Ephedra distachya,
distachya
Ephedra sinica

Sedative, hypnotic, anxiolytic

Asthma, rhinitis,
Asthma
rhinitis common cold,
cold weight
loss; enhancer of athletic performance

Tanacetum parthenium

Hypericum

Echinacea
purpurea

Ginkgo biloba

Sabal

Panax ginseng

Valeriana officinalis

Ephedra sinica

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TopRankedMedicinalplantsintheIndianMarket
S. No.

Species

Uses

Ashwagandha

W. somnifera

Satavari

Neem

Amla

Tulasi

Brahmi

Bringaraj

Rootused as an antiinflammatory, anticancer, rheumatism; and as a

sedative and hypnotic in anxiety. Leafantiinflammatory,
hepatoprotective, antibacterial. Fruits and seedsdiuretic.
Asparagus racemosus Female Tonic/Womens Healthcare, disorders of female genitourinary
tract; an intestinal disinfectant and astringent in diarrhoea; as a
nervine
i tonic,
t i
Azadirachta indica
Leaf, barkantimicrobial, antifungal, anthelmintic, insecticidal,
antiviral, antipyretic, antimalarial, mosquito larvicidal, anti
inflammatory, antifertility, spermicidal, hypoglycaemic; used in
inflammation of gums, gingivitis, periodonitis, protect Healthy Skin
Emblica officinalis
Rejuvenative,
Fruitantianaemic,
anabolic,
antiemetic,
antihaemorrhagic, antidiarrhoeal, diuretic, antidiabetic, carminative,
antioxidant.
Ocimum sanctum
Leafcarminative, stomachic, antispasmodic, antiasthmatic,
antirheumatic, expectorant, stimulant, hepatoprotective,
Bacopa monnieri
Mental Clarity, diuretic, sedative, potent nervine tonic, antianxiety
agent (improves mental functions, used in insanity,
epilepsy), antispasmodic
Eclipta alba
Liver Tonic, used in jaundice

Arjuna

Terminalia arjuna

Barkused as a cardioprotective and cardiotonic in angina

and poor coronary circulation; as a diuretic in cirrhosis of liver and for
symptomatic relief in hypertension; externally in skin diseases,
herpesand leukoderma.

Majorplantdrugsforwhichnosyntheticoneiscurrentlyavailable
S. No.
1
2
3
4

Drug
Vinblastine
Ajmalacine
Reserpine
Quinine

Plant
Catharanthus roseus
Catharanthus roseus
Rauvolfia serpentina
Cinchona sp.

5
6
7
8
9

Pilocarpine
Cocaine
Morphine
Codeine
Atropine

10

Taxol

11

Berberine

Pilocarpus jaborandi
y
y
coca
Erythroxylum
Papaver somniferum
Papaver somniferum
Atropa belladonna,
Hyoscyamus niger
Taxus baccata
T. brevifolia
Berberis aristata

12

Plumbagin

Plumbago indica

13
14
15

E ti
Emetine
Glycyrrhizin
Digitoxin,
Digoxin
Podophyllin
Camptothecine

C h li ipecacuanha
Cephaelis
i
h
Glycyrrhizia glabra
Digitalis purpurea

Breast and ovary cancer,

antitumour
For leishmaniasis,
Antimicrobial, antihelminthic
Antibacterial, antifungal,
anticancer
A
Amoebiasis
bi i
Antiulcer
Cardio tonic

Podophyllum emodi
Camptotheca acuminata

Anticancer
Anticancer

16
17

Uses
Anticancer
Anticancer, hypotensive
Tranquilizer
Antimalarial,
amoebic dysentery
Antiglucoma
Topical
p
anaesthetic
Painkiller
Anticough
Spasmolytic, cold

1/9/2014

Itshouldbenotedthattheseplantsareonlypartlyofinterestfor
biotechnologyandgeneticmodification,andtodayapproachesofmedicinal
plantbiotechnologyfocusmoreondistinctnaturalproductsandbiosynthetic
pathways.
pathways
Themainreasonforthisisthatgeneticallymodifiedplantsasasourcefor
extractpreparationsandformanufacturedpharmaceuticalsavailableinthe
pharmacyarenotacceptedbypatientsandconsumers.
Becauseofagainingpopularityforphytotherapy,andthewishfulthought
thatproductsobtainedfromNaturearesafe,patientsdonotconsider
geneticallymodifiedplantsasapartofthisphilosophy.
Hence,theapproachesofmedicinalplantbiotechnologycurrentlyfocus
moreondistinctnaturalproductsandbiosyntheticpathways.

Today,only10%ofallmedicinalplantspeciesusedarecultivated,withby
farthelargermajoritybeingobtainedfromwildcollections.
Harvestingfromthewildmay,however,becomesproblematic,duetoloss
ofgeneticdiversityandhabitatdestruction.
Examples,Podophyllum
Examples Podophyllum andTaxus
and Taxus species,Pipermethysticum
species Piper methysticum
Thus,thedomesticcultivationofmedicinalplantsisawellacceptedwayin
whichtoproduceplantmaterial.
Moreover,suchanapproachalsohelpstoovercomeotherproblems
inherentinherbalextracts,suchasthestandardizationofextracts,
variabilityoftheplantmaterial,minimizationoftoxicconstituentsand
contaminations,increasingthecontentofthedesiredconstituents,and
breedingaccordingtointernationallyacceptedGoodAgriculturalPractice
(GAP)guidelines
Hypericum perforatum andGingkobiloba aretwoexamplesoftopselling
plantswhichhavebeencultivatedformanyyears

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ApplicationsofBiotechnologyintheherbalindustry
Whetherthereisaneedforbiotechnologyandgenetechnologyformedicinalplants?
Fromitsnarrowdefinition,biotechnologydoesnotfocusonmedicinalplants,and
thereforeitshouldbeacceptedinabroadersense.
Withregardtomedicinalplants,biotechnologycouldbedescribedasamethodfor
enhancingtheformationandaccumulationofdesirablenaturalproducts,withpossible
productmodificationinmedicinalplants.
Micropropagation,cellandhairyrootcultureaswellasgenetechnologyareall
importanttechniquesforplantpropagation,butthesearemostlyusedtoimprovethe
productionandyieldofdesirednaturalproducts.
Twowelldescribedexamplesofthisareartemisinin andpaclitaxel,bothofwhichare
availableinplants,albeitinonlysmallquantities.
Inanattempttoovercometheseproblemsforbothdrugs,intensiveresearchisbeing
carriedoutworldwide,includingcombinatorialbiosynthesis,orimprovedbioprocessing in
bioreactorsforbothartemisinin andpaclitaxel (Taxol)
Th
Theapplicationofbiotechnologicaltechniquestomedicinalplantshasreceived
li i
f bi
h l i l h i
di i l l
h
i d
considerableinterest,especiallywhenthefinalproductisdefined,purified,andnatural.
Themanipulationofmedicinalplantsiswellknownandacceptedbothbyscientistsand
consumers,ifthepathwaysandproductyieldcanbeoptimizedtocreateprecursorsfor
semisyntheses (e.g.,baccatinIIItopaclitaxel),foodcomponents(e.g.,vitamins),pesticide
residence(e.g.,Atropa belladonna),andcellularstorageconditions,withenhanced
resistanceagainstfungalattackandabiotic stress.

Future Prospects
Thecommercialviabilityofbiotechnologyandgenetechnologyinmedicinalplant
researchisstronglyinfluencedbythecommonperceptionofboth,theplantand
biotechnology.
Geneticallymodifiedmedicinalplantslosetheirnaturalstatus,andareconsidered
erroneously bythepublicasunsafeanddangerous.
Thecropindustrylearneditslessonfollowingtherejectionofgeneticallymodified
cropsacrosstheworldthatwereintroducedintothefoodchain.
Clearly,companiesproducingherbalcompoundswouldfacethesameproblemsin
obtainingpermissiontoconductfarmscaletrails,todocumentthesafetyofthefinal
product,andtoovercometheinprincipleresistanceoftheconsumerasastrongand
perhapsimmovablebarrier.
Withinanopenscientificenvironment,thediscoveryanddevelopmentofbotanical
therapeuticsandmedicinalplantbiotechnologymustbeaccepted,asitsexpansionis
very unlikely to cease
veryunlikelytocease.
Itisdifficulttopredictthefutureformedicinalplants,butitislikelythatherbaldrugs,
isolatednaturalproductsandrecombinantlow andhighmolecularweightproductswill
holdatleastthesamesignificance.
Plants,asarenewablesourcewithlowenergyconsumptionthatcanoffercomplex
biochemicalsyntheses,willbeevenmorecompatibleinthefuture.

1/9/2014

Useofbiotechnologicaltoolsinmedicinalplantscience
BiotechnologicalApproachesfortheProductionofsomePromisingPlantBased
Chemotherapeutics:
Today,naturalproductsfromplantsprovidebettertemplatesforthedesignofpotential
chemotherapeuticagentsthansyntheticdrugs.
Paclitaxel (Taxol),podophyllotoxin andcamptothecin aresomeleadmoleculeswhichare
widelyusedinthetreatmentofcancer.

Tomeeteverincreasingdemands,biotechnologicalmethodsofferanexcellentalternative,
i
i d
d bi
h l i l
h d ff
ll
l
i
buttheeconomyofsuchaproductionisthemajorhurdletobeovercome.
Thesuccessfulindustrialapplicationofplantcellcultivationfortheproductionofthese
therapeuticcompoundswilltriggerfurtherresearchonotherpromisingplantbased
chemotherapeutics.
Thecompletepathwayforthebiosynthesisofpodophyllotoxin hasalreadybeenestablished,
butalternativepathwaysofdifferentmetabolicfatesoflignans,theprecisesequenceofthe
laterstepsintaxol biosynthesis,andtheiridoid sectionofcamptothecin biosynthesishaveyet
to be realized
toberealized.
Understandingofbiochemistry,enzymology,physiology,bioreactordesignandthe
applicationofproteomicsandgenomicsareotherareasonwhichtofocus.
Severalpointsinagivenmetabolicpathwaycanbecontrolledsimultaneously,eitherby
overexpressing and/orsuppressingseveralenzymes,orthroughtheuseoftranscriptional
regulatorstocontrolendogenousgenes.
Thus,multipointmetabolicengineeringoffersnewperspectivesforimprovementsinthe
productionofplantbasedchemotherapeutics

Inspiteofthespectacularadvancesmadebymedicalscienceduringthepastcentury,
thetreatmentofcancerremainsanenigma.
Inrecentyears,effortshavebeenmadetosynthesizepotentialanticancerdrugs;
consequently,hundredsofchemicalvariantsofknownclassesofanticancertherapeutic
agentshavebeensynthesized.
Ithasbeenrecognizedthatasuccessfulanticancerdrugshouldbeone,whichkills
cancercellswithoutcausingexcessivedamagetothenormalcells.
Thiscriterionisdifficult,orperhaps,impossible
Whilevastamountsofsyntheticchemistryhaveprovidedrelativelysmallimprovements
overtheprototypedrugs,thesynthesisofmodifiedformsofknowndrugscontinuesas
animportantaspectofresearch.
Thereexistsaneedfornewprototypesandnewtemplatesforuseinthedesignof
potentialchemotherapeuticagents.
Naturalproductsarecapableofprovidingsuchtemplates.
Pl
Plantsarethemostexclusivesourceofdrugsforthemajorityoftheworldspopulation,
h
l i
fd
f h
j i
f h
ld
l i
andplantproductsconstituteabout25%ofprescribedmedicines
Theimpactofnaturalproductsuponanticancerdrugdiscoveryanddesigncanbe
gaugedbythefactthatapproximately60%ofalldrugs,nowinclinicaltrialsforthe
treatmentofcancer,areeithernaturalproducts,compoundsderivedfromnatural
products,orcontainpharmacophores derivedfromnaturalproducts

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Theanticancerplantsandtheiractiveconstituents
Plant
Camptothecaacuminata

Activeconstituent(s)
Camptothecin(Pyrraloquinolillealkaloid)

Catharanthus roseus

Vinblastine,Vincristine(Bisindolealkaloid)

Podophyllum hexandrum

Podophyllotoxin,Peltatin (Lignan)

Colchicumautumnale
Taxus baccata
Taxus brevifolia
Maytenus buchananii

Colchicine (alkaloid)
10Deacetylbaccatin,Docetaxel
Paclitaxel
Maytansine (Ansa macrolide)

Concentrationsofantitumorcompoundspresentinhigherplants:

No.

Antitumorcompound

Concn.
[drywt.%]

1
2
3
4
5
6

Camptothecin
Ellipticine
Maytansine
Podophyllotoxin
Vinblastine,Vincristine
Taxol

5.0103
3.2105
2.0105
6.4101
5.0103
5.0101

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Duringthepastdecade,renewedinterestininvestigatingplantbasedproducts
hasledtotheadventofseveralimportantanticancersubstances.
Mostimportantarepaclitaxel (taxol)fromTaxus brevifolia L.,podophyllotoxin from
Podophyllum peltatum L.,andcamptothecin fromCamptotheca acuminata Decne.
Interestingly,thesesubstancesembracesomeofthemostexcitingnew
chemotherapeuticagentscurrentlyavailableforuseinclinicalsettings.
Theanaloguesofpodophyllotoxin,taxol andcamptothecin

Podophyllotoxin

Docetaxel
Taxol

camptothecin

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Somechemotherapeuticproductsfromnaturalsources,andtheirmodeofaction.
Source
Podophyllum
spp.

Taxus baccata

Naturalcompounds
oritsderivatives
Podophyllotoxin
(Natural)
Etoposide and
teniposode
(Semisynthetic
derivatives)

Modeofaction

Mitoticspindlepoison bindsthe
microtubuleand causesmitoticarrestin
metaphase.
Induceapremitotic blockageinthecell
cycle,attwospecificplaces,eitherinlate
SphaseorinearlyGphase,bybindingto
andstabilizingthecleavablecomplexof
DNAtopoisomerase II.
10Deacetylbaccatin, Promotestubulin assemblyandinhibition
Docetaxel
ofmicrotubuledepolymerization;alsoacts
asamitoticspindlepoisonandinduced
mitoticblockinproliferativecells

Taxus brevifolia

Paclitaxel
(Natural)
(Natural)

Promotesassemblyofmicrotubules,
stabilizes them against depolymerization;
stabilizesthemagainstdepolymerization;
andinhibitscellreplication;causescell
apoptosis

Camptotheca
acuminata

10hydroxy
camptothecin,
(Natural)
Irinotecan,SN38
(Semisynthetic
derivatives)

Inhibitsactionoftopoisomerase I,
preventsreligation ofDNAstrand,results
incelldeath

Source

Naturalcompounds
oritsderivatives
Vinblastine and
vincristine

Modeofaction

Catharanthus
roseus

Mitoticspindlepoison bindsthe
microtubuleand causesmitotic
arrestinmetaphase.

Cancer
Inhibited
Lung
Lung,testicular,
leukemias

Breast,ovarian,
nonsmall cell
lung,headand
neck,colorectal
melanoma
Advancedbreast,
ovarian
ovarian,
adenocarcinoma,
andothersolid
tumors
Liver,colorectal,
headandneck
cancer,
leukemia

Cancer
Inhibited
Vinblastine ismainly
usedinthe
treatmentofHodgkins
disease,acancer
affectingthelymph
nodes,spleenand
liver.

Vincristine ismainly
usedinthe
treatmentof
childhood
leukaemia.

Catharanthus roseus

Taxusbrevifolia

1/9/2014

Someofthemajorlimitationstomeetthedemandofpodophyllotoxin arethe
inaccessibleregionfromwheretheplantisobtained,itsendangeredstatusduetoover
exploitationofnaturalresources,alongjuvenilephase,poorfruitsettingability,along
periodofgerminationoftheseeds,thecontinuingdemandforthedrugandvery
complicatedandratherdifficultchemicalsynthesisbecauseofthepresenceoffourchiral
centers,arigidtranslactone andanaxiallylocated1arylsubstituent.
Themostdifficultproblemsencounteredinpaclitaxel productionaresupplylimitation
arisingfromitsverylowconcentrationinthebarkoftheT.brevifolia (0.01%dryweight),
andtheveryslowgrowthoftheyewtree.
Inordertoisolate1kgoftaxol,10000kgofdriedbarkfrom3000T.brevifolia trees
mustbeextracted,whilstalmost2goftaxol isneededtotreatonepatient.
Ontheotherhand,certainsecondarymetabolitessuchascamptothecin are
accumulatedonlyafteracertainageormaturityoftheplant.
Hence it is difficult to increase the area under plantation
Hence,itisdifficulttoincreasetheareaunderplantation.
Toovercomeallthesehurdles,theindustryrequiresalternativemethodsofsupplyof
uniformmaterialthroughouttheyear.Plantcellculturetechnologyisundoubtedlyone
oftheappropriateapproachestosolvetheabovedescribedproblems.

ProductionbyPlantCellCultures
Itisnotaneasytasktoproducethesecompoundseconomicallybyextractionfrom
intactplantsandmeettheeverincreasingdemand.
Thismaybeduetoverylowconcentrationsoftheseactivecompoundsinplants,the
slowgrowthrateofplants,complexaccumulationpatterns,andhighsusceptibilityto
geographicalandenvironmentalconditions.
Otherpossiblereasonsarethenonavailabilityofuniformandunadulteratedquality
plantmaterialinquantitiessufficientforindustrialproductionanduneconomical
chemicalsynthesis,particularlyforlargecomplexmolecules.
Therefore,biotechnologicalmethodsofferanexcellentalternativeforproductionof
suchcompounds.
Antitumorcompoundsfromcellculturesversusintactplant.
Plant

Antitumorcompound

Camptothecaacuminata Camptothecin

Plant.
[
[%DW]
]
5.0103

Culturedcells
[[%DW]]
2.5104

Podophyllumspp.

Podophyllotoxin

6.4101

7.1101

Taxusspp.

Taxol

5.0101

153mgL1

10

1/9/2014

BiotechnologicaltoolsandtechniquesforMedicinalplants
Today,thepotentialofplantcellstoproducesecondarymetabolitesindedifferentiated
culturesisusedextensivelytoproduceplantderiveddrugs.
Becauseplantsecondarycompoundsareoftenproducedonlyinsmallquantitiesina
particulartypeofcellsofrareplantspecies,itisnotalwaysfeasibletoisolatesecondary
compoundsfromintactplants.
Plantcellculturecanbeanalternativewaytoproducethesecompoundscontinuously
underartificiallycontrolledconditions.
Plantgrowthregulators
Thegrowthanddevelopmentofplantsisregulatedbyanumberofchemicalsubstances
whichtogetherexertacomplexinteractiontomeettheneedsoftheplant.
Fivegroupsofplanthormonesarewellestablished;theyareauxins,gibberellins,
cytokinins abscisic acidanditsderivativesandetheylene.
cytokinins,abscisic
acid and its derivatives and etheylene

Callus Culture
GrowthofExcisedPlantCallusinanArtificialNutrient,isknownascallusculture.
Exceptforthosecaseswhereaxenic materialisused,therespectiveexplantsare
surfacesterilizedandplacedonagarsolidifiednutrientmedia.
Thewoundcallusformedatsitesoftissueinjurycaneasilyberemovedfromthe
initialexplant,anditsfurtherdevelopmentcanbecontrolledbyexogenous
phytohormones orsyntheticgrowthregulators.
or synthetic growth regulators
Duringprolongedcultureinvitrocellsmayachievehormoneautonomy,andoftenbe
distinguishedaseithercytokinin orauxin autotrophic,orcompletelyhormone
autotrophic.
Thisphenomenoniscalledhormonehabituation,andhasbeenreportedincultures
ofseveralspecies.
Hormonehabituationisreversibleinsomecases,andhenceepigeneticratherthan
geneticchangesmayberesponsible.
Plant cell culture media are composed of many different compounds, including
Plantcellculturemediaarecomposedofmanydifferentcompounds,including
inorganicmacronutrients(saltsofN,P,S,K,Na,Ca,Mg)andmicronutrients(saltsofe.g.,
I,Ni,Fe,Cu,B,Mn,Co),aswellasvitaminsandacarbohydratesource(usually25%
sucrose).ThemostcommonmediaforplantcellculturearethosedevisedbyMurashige
andSkoog,Gamborg etal.,SchenkandHildebrandt,andWhite.

11

1/9/2014

Initiationofcallusculturesandinductionoforganogenesis.
Morphogenesiscanbetriggeredbychangingthephytohormone regime.

Plantcellculturemediasupportthegrowthofmicrobialcells,andthereforeany
contaminationmustbeavoidedinordertoprovideanasepticenvironment.
Thestandardsterilizationtechniquesthatareusedinmicrobiologycanbeapplied.
Usually,plantcellsandtissuesarehandledunderlaminarflowinsuitablecabinets.
Culturemediaareautoclaved(121C,15min),whilethermolabile compounds
(e.g.,phytohormones)canbefiltersterilizedandthenaddedtotheautoclaved
media.
media.
Varioustypesofvesselsareusedforcelllinepreservationandsubcultivation,
forexampledisposable,sterilePetridishes,tubesorErlenmeyerflasks.
Glassflasksandtubesmaybesealedwithfoamorcottonbungs,whilemetalcaps,
aluminumfoilorsterileclingwrap areusedtocoverthevesselstoavoidthembecoming
contaminated.

12

1/9/2014

Cell Suspension Cultures:

Suspension cultures are established by transferring the callus tissue into
liquid medium of the same composition as used for callus tissues and
agitating the culture on a rotatory or reciprocal shaker
shaker.
Suspension cultures generally comprise more homogeneous and less
differentiated cell populations and grow more rapidly than their parent callus
cultures.
Furthermore, cell suspensions are suitable for continuous and/or chemostat
culture and easy to feed various chemical factors during the culture.
These properties make suspension cultures the material of choice for
biochemical and molecular biological investigation of plant secondary
metabolism.
Scaling up from flask to bioreactor for the production of phytochemicals is
always performed using suspension cultures.

ImmobilizedCultures
Overthelasttwodecades,immobilizedculturesystemshaveattractedmuchattention
forefficientproductionofplantsecondarymetabolites.
Culturedcellsfromhighdensitysuspensionculturesaretrappedinaninertmatrix
suchascalciumalginategelbeads,stainlesssteel,andfoamparticles.
The immobilized entities are cultured in shaken flasks or aerated bioreactors
Theimmobilizedentitiesareculturedinshakenflasksoraeratedbioreactors.
Alternatively,thecellencapsulatedbeadscanbepackedintoacolumn,whichis
percolatedwithnutrientmedium.
Themajoradvantageoftheimmobilizedculturesystemisthatcellgrowthand
secondarymetaboliteproductioncanbeseparatedbytheprecisemanipulationofthe
chemicalenvironment,allowingcontinuousorsemicontinuous operation.
However,establishingtheimmobilizedculturesystemforlargescaleproductionof
phytochemicals isexpensive.
Inaddition,forefficientoperationoftheimmobilizedsystem,permeationofthe
productfromthecellstothemediumisnecessary,whichhasnotyetbeenfullyachieved.

13

1/9/2014

OrganCultures:
Inspiteofprolongedandconcentratedefforts,manyvaluablephytochemicals
suchasmorphinan alkaloidsofPapaver somniferum,tropane alkaloidsofvarious
solanaceous species,anddimeric indole alkaloidsofCatharanthus roseus cannotbe
producedbycallusandcellsuspensioncultures.
Becausemostofthesecompoundsstarttoaccumulatewhentheproperorgansare
regeneratedfromtheculturedcells,productionofthesecompoundsinculturedcells
requiresdecouplingofbiochemicaldifferentiationfrommorphologicaldifferentiation,
whichhassofarbeenunsuccessful.
Thissituationmakesorganculturesafavoredoption.
Onemajordisadvantageoforganculturesisreducedproductivityinbioreactorsbecause
thephysicalstructureofshootsorrootsresultsinvariousdifficultiesincludinghandling
problemsatinoculationandshearoftheorgansduringculture.

ShootCultures
Multipleshootsregeneratedeitherfromcallusculturesordirectlyfromexplants
includingapicalbudsareculturedinsolidorliquidmedium.
Shootcultureshavebeenconsideredappropriatewhenthetargetsecondary
metabolitesareproducedinaerialpartsofplants.
Monoterpenoid essentialoilflavors,whicharenotproducedindedifferentiatedcallusor
cellsuspensionculturesbecauseoflackofoilsecretory tissues,havebeenreportedto
accumulate in shoot cultures
accumulateinshootcultures
Productionofasesquiterpene lactone artemisinin thatexhibitspotentantimalarial
activityinshootculturesofArtemisiaannua hasalsobeenactivelyinvestigated.
Adimeric indole alkaloidanhydrovinblastine thatisadirectprecursorofantileukemic
indole alkaloids,vinblastine andvincristine,accumulatedinshootculturesof
Catharanthus roseus atalevelsimilartothatintheleavesofintactplants.
Vindoline andcatharanthine,precursorsofthedimeric indole alkaloids,werealso
producedinmultipleshootculturesofC.roseus.
Itisinterestingtonotethatprovidingtheculturedshootswithenvironments
similartothoseoftheintactplantssometimesresultsinenhancementoftheparticular
secondarymetabolism.
Forexample,mentholproductioninMentha arvensis shootculturesincreasedwithlight
illumination.
Dimeric indole alkaloidproductioninC.roseus shootcultureswasalsostimulatedby
nearultravioletlightirradiation.Rootingwasalsoreportedtoenhanceartemisinin
formationinculturedshootsofA.annua

14

1/9/2014

RootCulture
Therearetwotypesofrootculture,untransformedrootcultureandhairyrootculture,
whichareobtainedbytransformationwithAgrobacterium rhizogenes.
Hairyrootsgenerallyshowmorevigorousgrowththanuntransformedroots.
However,untransformedrootssometimesshowvigorousgrowthtoanextentsimilarto
thatoftransformedrootswhenculturedinauxincontainingmedium.
Production of hyoscyamine andscopolamine,pharmacologicallyactivetropane
Productionofhyoscyamine
and scopolamine pharmacologically active tropane
alkaloids,wasmoreactiveinnormalrootculturesofDuboisia myoporoides thaninthe
hairyroots.
ScalingupoftransformedAtropa belladonnarootcultureswasattainedwithoutany
reductionintropane alkaloidproductivitybycombiningcuttingtreatmentofseedroots
withuseofastirredbioreactorwithastainlesssteelnet.
Itshouldalsobepointedoutthattheresearchontropane alkaloidproductioninroot
culturesofsolanaceous plantsstartinginthemid1980shasfruitedasmolecular
g
g
g
g
p
alkaloidbiosynthesis
y
biologicalcharacterizationandgeneticengineeringoftropane

Organogenesis
Socalledadventitiousrootsorshootsareformedwhenorgandevelopmentisinducedin
nonmeristematic areasofagivenplanttissue.
Inthisprocess,specializedcells(e.g.,epidermiscells)mayturnmeristematic,usually
passingthroughashortperiodofcallusformation.
Thoseplantswhichcanbepropagatedvegetatively areespeciallysusceptibleto
adventitiousorganformationinvitro.
Plantregenerationthroughmultipleadventitiousshootdifferentiationhasbeen
establishedinmanyspecies.
Mostoften,leafexplantsareusedandshootformationistheninducedbyasuitable
phytohormone treatment.
Explantsofyoungerplantsusuallyrespondbetterthanolderones,andherbaceousplants
asarulerespondbetterthanwoodyplants.
Adventitiousrootorshootformationcanalsobeinducedinotherplantorgansorin
callus.
Depending on the starting material growth regulators such as ben yladenine 4
Dependingonthestartingmaterial,growthregulatorssuchasbenzyladenine,4
dichlorophenoxyaceticacid,indole3butyricacid,naphthaleneaceticacid,2
isopentenyladenineorthidiazuron areaddedtothebasalgrowthmediatoinduceshoot
formation.
Shootsarethenpropagatedandrootedasdescribedforshootcultures
Changesoflightconditions,photoperiod,temperature,and/orreductionofthenutrients
intheculturemediummayhelptoachievehighrootingefficiency.

15

1/9/2014

SomaticEmbryogenesis
Whenrootsandshootsdevelopsimultaneouslyandfromacommonorigin,a
differentiationprocessisseenthatleadstoshootandrootformationinacoordinated
manner.
Thisprocessresemblesthedevelopmentofzygoticembryos,andistermedsomatic
embryogenesis.
The structures formed from callus or suspension cells are therefore called somatic
Thestructuresformedfromcallusorsuspensioncellsarethereforecalledsomatic
embryosor,moreprecisely,somaticembryoids (embryolikestructures).Globular
somaticembryoids,whichcanbemaintainedincultureoverlongperiodsoftime,can
differentiateintoheartshapedandlatertorpedoshapedembryoids
Theregenerationofproperlydevelopedsomaticembryoids tointactplantsisquite
simple.
Forcarrotwhichservesasamodelsystemforstudyingsomaticembryogenesis it
wascalculatedthatabout50000somaticembryoids perlitermediumareformed
y
eachday.
Hence,somaticembryogenesisisregardedasasystemofchoiceformass
propagation,butforthispurposetheprocessmustbesynchronized.
Itmustalsobeconsideredthatembryogenic cellculturesmaylosethisquality
duringprolongedcultivation.
Theadvantagesofsomaticembryoids includehighmultiplicationratesandthe
potentialforscaleupinbioreactors.

Somaticembryoids canbeencapsulatedinalginateassingleembryobeadsto
producesocalledartificialseeds.
Thequalityofartificialseedsdependsonthesupplyofgrowthregulatorsand
nutrients.
Artificialseedscanbedesiccatedinthisway,therebyfacilitatingyearround
production,storage,anddistribution

Morphogenesisfromroot,stemorleafexplantsviaadventitiousorganformation
andsomaticembryogenesisfromembryogenic callusviasomaticembryos.

16

1/9/2014

In-vitro culturing techniques of medicinal plants

Basic Methods and Techniques
SeedGerminationInVitro:
Seedgerminationunderaxenic (germfree)conditionsisthesimplestinvitroculturing
techniqueused
Surfacesterilizedseedsofalmostanyplantspeciescanbegerminatedonagarsolidified
mediaonwhichtheydevelopintosmallsterileplantlets.
These in turn can be used to establish plant tissue cultures such as callus or organ cultures
Theseinturncanbeusedtoestablishplanttissuecultures,suchascallusororgancultures.
Sinceaggressivechemicals(e.g.,hypochloriteorhydrogenperoxide)aregenerallyusedfor
decontamination,itmaybeofadvantagetotreattheratherrobustseedsinsteadofthemuch
softerexplants,suchasleaforstempieces.
Themethodhasabroadapplicationinthecommercialpropagationoforchids
InNature,orchidswillnotgerminateunlessinfectedbyafungus,whichfeedstheyoung
plantsuntiltheyaretallenoughtoproducetheirownfood.
Oncetheseedhasgerminatedtheembryodevelopsintoasphericalprotocorm thatwill
continue to grow for weeks, months or years, until large enough to differentiate into an orchid
continuetogrowforweeks,monthsoryears,untillargeenoughtodifferentiateintoanorchid
plant.
Whengerminatedundersterileconditions(i.e.,without thefungus)onsuitablemedia,the
nutrientsprovidedwillsubstituteforthefungusandallowtheseedstogerminateandgrow
withoutit.
Sterileseedsareusuallyisolatedfromsurfacesterilizedimmaturecapsules;thisprocedure
hastheadvantagethattheseedsthemselveshavenottobesterilized.

EmbryoCulture
Embryoscanbeisolatedfromseedsandcultivatedunderaxenic conditionsinvitro.
Grainfruitscontainrelativelylargeembryoswhichcanbeisolatedquiteeasily,butif
theembryosaretootinytobepreparedsafely,thecompleteovulecanbeisolated
instead.
Thecompositionofculturemediaonwhichisolatedembryoswilldevelopisa
problematicissue,andrequiresmuchexperimentaleffort;however,iftherespective
d
demandsaremet,thenregenerationtointactplantsiseasilyachieved.
d
t th
ti t i t t l t i
il
hi d
Byapplyingtheembryoculturetechnique,itispossibletoshortengenerationtimes
bybreakingseeddormancy.
Moreover,embryospresentinseedsobtainedfromsexualcrossingsthatarenotable
togerminatecanberescuedandstimulatedtodevelop.
Embryoscanevenbepreparedfromoneseedandthentransplantedintothe
endospermofanurseseedwhich,togetherwiththenutrientsaddedtotheculture
medium,supportsfurtherdevelopmentoftheembryo.

17

1/9/2014

HaploidTechnology
Amongthedifferentusesofhaploids,theiruseincropimprovementisconsideredtobe
themostsignificantandstandsoutasthemostimportantreasonforemphasisonhaploid
research.
Thetechniqueallowsforasignificanttimereductionintheachievementofhomozygous
breedinglinesincropimprovement.
Someoftheimportantcrops,wherehaploidplantshavebeenproducedareAlalfa,
Cucumber,Rye,Asparagus,Barley,Sugarcane,Broccoli,Sunflower,Citrus,Pepper,Sweet
Potato,Potato,Tomato
Haploidsaresporophytes withthechromosomenumberofgametophytes,andhaploid
cellsareformedduringmeiosis.
Haploidplantcellsarepresentintheembryosack(megagametophyte)orthepollen
(microgametophyte),andconsequentlyhaploidscanbeobtainedviagynogenesis from
cellsoftheembryosack,orbyandrogenesis frompollen.
Inthisway,monohaploid anddihaploid plantscanbeobtainedfromdiploidand
tetraploid plants,respectively.
plants respectively
Colchicine canbeusedtoinducechromosomedoublingbyinhibitingchromosome
segregationduringmeiosis,andinthiswayhomozygouscellscanbeobtained.
Thesemayformaregenerable callusthatcanbestimulatedtodevelopintohomozygous
(dihaploid)plants,whilehaploidcalluscanbeproducedviagynogenesis or
androgenesis,thelattermethodbeingthepreferredoneinpractice.
Inandrogenesis,twobasictechniquescanbedistinguished,namelyanthercultureand
microsporeculture.

ProtoplastTechnology
Plantcellspossessathickandquiterigidcellwallwhichiscomposedofcelluloseand
otherpolysaccharides,suchaspectin.
I h
Inhypertonicsolutions,theplasmamembranesofcellscontractfromtheirwalls,with
t i
l ti
th l
b
f ll
t tf
th i
ll
ith
removalofthewallmaterialreleasinglargepopulationsofspherical,osmotically fragile
structures,termedprotoplasts.
Themainobjectivesinusingprotoplastisolationandculturetechniquesareto:
regenerateanintactplantfromasinglecellforprovingtheconceptoftotipotency;
fuseprotoplastsofdifferentoriginwithaviewtoproducingahybridcellwhich
subsequentlyregeneratesintoahybridplantthatcannotbeobtainedbysexualcrossing
(somatichybridization);and
obtain cells accessible for genetic manipulation
obtaincellsaccessibleforgeneticmanipulation.

18

1/9/2014

Duringrecentyearstheisolationofprotoplastshasbecomeroutine,ifchoppedplant
tissueistreatedwithpectinase andcellulase,itwillreleaseprotoplasts.
Aslightlyhypertonicmediumduringtheisolationandcultivationprocessis
mandatory,asotherwisetheprotoplastswillburst.
Whenprovidedwiththecorrectchemicalandphysicalstimuli,eachprotoplastis
capable(intheory)ofregeneratinganewwallandundergoingrepeatedmitotic
divisiontoproduceacallusfromwhichfertileplantsmayberegenerated.
Isolatedprotoplastsbegincellwallregenerationalmostimmediatelyaftertheir
isolation.
Toachievethistheyrequireosmoticprotectionuntiltheirnewcellwallscan
withstandthenormalcellturgor,andthisisgenerallyprovidedbytheadditionofnon
metabolizable sugaralcohols(e.g.,mannitol orsorbitol).

Isolation of Protoplast
(Separartion of protoplasts from plant tissue))

1. Mechanical Method

2. Enzymatic Method

MechanicalMethod
Cells Plasmolysis

Plant Tissue

Microscope Observation of cells

Cutting cell wall with knife

R l
Release
off protoplasm
l

Collection of protoplasm

19

1/9/2014

MechanicalMethod
Usedforvacuolatedcellslikeonionbulbscale,
radishandbeetroottissues
di h d b t
t ti
Lowyieldofprotoplast
Laboriousandtediousprocess
Lowprotoplastviability

EnzymaticMethod
Leaf sterilization, removal of
epidermis

Plasmolysed
cells

Plasmolysed
cells
Pectinase +cellulase

Protoplasm released

Pectinase

Protoplasm
released

Release of
isolated cells
cellulase

Isolated
Protoplasm

20

1/9/2014

EnzymaticMethod

Used for variety of tissues and organs including leaves, petioles, fruits, roots,
coleoptiles hypocotyls,
coleoptiles,
hypocotyls stem,
stem shoot apices,
apices embryo microspores
Mesophyll tissue most suitable source
High yield of protoplast
Easy to perform
More protoplast viability

SomaticHybridization
Somatichybridsobtainedthroughthefusionofplantprotoplastshavewidenedthegenetic
variabilityofplants.
Severalgroupshavereportedthegenerationofhybridplantsthroughprotoplastfusion,and
someimprovementshavebeendescribedinfusiontechnologysincetheearlydayswhen
plantprotoplastswerefusedusingthepolyethyleneglycol(PEG)method.
ThischemicallyinducedfusionwasachievedathighpH(10.5)inthepresenceofcalcium
(10 mM) and PEG (1050%)
(10mM)andPEG(10
50%).
Intheelectrofusion method,Protoplastsareheldinanappropriateelectricfield,andmade
tofusetogether
Electrofusion isatwostepprocedure.
Inthefirststep,anonuniform,alternatingelectricfieldisappliedwhichcausesthe
protoplaststolineupperpendiculartotheelectrodes.
Theprotoplastschainlengthisinfluencedbythedistancebetweentheelectrodesandthe
celldensity,butideallytheprotoplastsagglutinatepairwise.
Inthesecondstep,fusionisinducedbyasinglehighvoltageDCpulse.
Itisthoughtthatthechargedifferencebetweentheoutsideandinsideoftheplasma
membranefinallycrushesthemembrane.
Thiscausestheelectricpotentialtobreakdown,whereuponthemembranestructurecan
bereformed,includingthemixingofmembranesofagglutinatedprotoplasts;theresultiscell
fusion.
Somatichybridizationwasaimedeitheratmodifyingthecompositionoftheoilinthe
Mentha spp.oratcombiningessentialoilqualitywithdiseaseresistance

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1/9/2014

Somatichybridizationtechnique
1. isolation of protoplast

2. Fusion of the protoplasts of desired species/varieties

3. Identification and Selection of somatic hybrid cells

4. Culture of the hybrid cells

5. Regeneration of hybrid plants

Advantagesofsomatichybridization
Production of novel interspecific and intergenic hybrid
Production of fertile diploids and polypoids from sexually sterile haploids, triploids
and aneuploids
Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance
and many other quality characters
Production of heterozygous lines in the single species which cannot be propagated
by vegetative means
Examples
Somatic hybridization techniques have been tried with some success in the case of
the potato Solanum tuberosum. This cultivated species with low insect resistance
has been crossed with the wild species Solanum chacoense that is insectresistant.
Other examples include hybrids of Nicotiana tabacum with Petunia hybrid and
Atropa belladonna with Datura inoxia.
Scientists also developed the "Pomato" a cross between a potato and a tomato
by intergeneric somatic hybridization.

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1/9/2014

Bioreactor Cultures
Irrespectiveofsuspendedcells,immobilizedcells,orwholeorgans,itisnecessaryto
establishefficientlargescalebioreactorsystemsforcommercialproductionof
secondarymetabolites.
Productionofcertaincompoundslikeshikonin,taxol,ginsengbiomass,andberberine
havejustreachedsemicommercialproductionlevelsusingbioreactortechnology
Themostcommonlyusedbioreactortypeslikestirredreactor,rotatingdrumreactor,
airliftreactor,bubblecolumn,packedbedreactor,tricklebedreactorareusedforthe
productionofbiomassandsecondarymetabolites
Withregardtoscaleup,themaintenanceofconstantenvironmentalconditionsat
thevariousscalesofoperationissimpleintermsofthesolublecomponents
(nutrients),butmoredifficultwithrespecttothephysicalenvironment(shear,mixing,
gastransfer).
Themajorityoflargescalesystemsworkwithplantcellsuspensionculturesthatcan
be grown in bioreactors in a manner similar to microbial fermentation
begrowninbioreactorsinamannersimilartomicrobialfermentation
Somedifferencesexistbetweenmicrobialandplantcellsconcerningcellsizeand
shape,growthbehavior,andproductformation,allofwhichinfluencecultivation
processtechnology.

Bioprocessing

Studiesonbioreactorproductionofusefulandvaluablemetabolitesinplantcelland
tissueculturesaswellasmasspropagationproceduresonalargescalehavebeencarried
outsincetheendofthe1950s.
Sincemasscultivationofplantcellshasbeenproposedasanalternativewayfor
supplyingimportantphytochemicals,itwasnecessarytodevelopsystemsthatwouldallow
plantcellstobecultivatedonalargescale
Oneadvantageofbioreactorsisthattheyarescalable thatis,itispossibletoreproduce
onalargescalethoseconditionswhichwerefoundconducivetooptimalproductionona
smallerscale.

23

1/9/2014

Comparison of Plant Cell Suspension and Microbial Culture

Thefollowingfourgeneralfactorshavetobeconsideredforcultivationofplantcell
suspensionculturesinbioreactors
1. Homogeneousandlowshearmixingforefficientnutrienttransportwithout
sedimentationand/orclumpingaswellaslossofcellviability
2. Optimalaerationwithlowshearstress
3. Guaranteeofthelongtermsterilityoftheprocessasapracticalconsequenceof
slow growth rates
slowgrowthrates
4. Introductionoflightforheterotrophic,photomixotrophic,andphotoautotrophic
culturesforincreasingthebiosyntheticcapacities
Formoredifferentiatedplantcellandtissueculturessuchasorgancultures(root
cultures,hairyrootcultures,shootandembryogenic cultures)itbecomesnecessary
torealizeincreasedcelltocellcontactaswellasalowershearstressinthe
cultivationsystem.
Thisisduetothemorphology,thegrowthbehavior,andthelowersheartolerance
p
gy,
g
,
oftheseorganculturetypes.

24

1/9/2014

INSTRUMENTATION OF BIOREACTORS FOR PLANT

CELL AND TISSUE CULTURES
Processmonitoringandcontrolisanessentialelementforcontrolofbioreactorprocesses
usingplantcellsasbiocatalysts.
Themajorphysicalprocessparametersinfluencingsuccessfulcultivationofplantcelland
tissueculturearetemperature,viscosity,gasflowrates,andfoaming.
Furthermore,forapplicationofmechanicallyagitatedreactors,agitatorshaftpower
(estimated by wattmeter measures or torsion dynamometers) and impeller speed are also
(estimatedbywattmetermeasuresortorsiondynamometers)andimpellerspeedarealso
importantparameters.
Temperatureisoneimportantparametermeasuredandcontrolledinallplantcell
cultivationprocesses.
BecauseplantcellculturesoftenshownonNewtoniancharacteristics,online
measurementofviscositycanbequitedifficultandofflinemethodsareoftenpreferred.
Acommonmethodformeasuringthegasflowrates(airfeed,exhaustgas)istouseair
flowmeters suchasrotameters,andliquidflowratescanbemonitoredwithelectromagnetic
flowmeters orcapacitanceprobes.
p
p
Foaming,resultingfromextracellularpolysaccharidesoftenexcretedinthemedium,isan
ordinaryprobleminmanycultivationprocesses.
Thefoamcanbedetectedbyacapacitanceorconductivityprobe.
Thefoamformationandfoamingarecontrollablebymechanicalfoamdestructiondevices
(foambreaker)suchassingleorrotatingplates,ultrasonicirradiation,oradditionofsilicon
andpolypropylenebasedantifoamagents.

Bioreactorsforplantcellculturecanbeclassifiedintoeightmainreactortypes
Basedontheworkingprinciple.

StirredReactor
Themostwidelyusedreactor
Theordinarystirredreactorisequippedwithbaffles,airsparger and
radial flow impellers axial flow impellers and impellers distributing the
radialflowimpellers,axialflowimpellers,andimpellersdistributingthe
powerinputoveralargefractionofthetotalreactorvolume.
Itstypicalfermenter geometry(fermenter height/diameterratio)
mustbe2:1or3:1.
Mixingandmassdispersionareachievedbymechanicalagitation.
Differentflowpatternsandshearratesinsidethevesselwillbe
producedbydifferentimpellershapes,sizes,andspacing
Itisalsopossibletodesignandoperatestirredreactorsina
multistagemode.
Temperature,pH,amountofdissolvedoxygen,andnutrient
concentrationcanbecontrolledbetterwithinthisreactorthanany
otherreactor.
Amajordrawbackofthestirredtankreactoristheshearingstress
generatedbyitsstirringdevicetowhichplantcellsmaybesensitive.

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1/9/2014

BubbleColumn
Thebubblecolumnisanotheralternativetothestirredreactorthathasno
mechanicalagitationandisstructurallyverysimple.
Itconsistsofverticalarrangedcylindricalcolumns.
Theintroductionofgastakesplaceatthebottomofthecolumnand
causesaturbulentstreamtoenableanoptimumgasexchange.
Itisbuiltinnumerousformsofconstruction.
The mixing is done by the gas sparging anditrequireslessenergythan
Themixingisdonebythegassparging
and it requires less energy than
mechanicalstirring.
Theliquidcanbeinparallelfloworcountercurrent.
Bubblecolumnreactorsarecharacterizedbyahighliquidcontentanda
moderatephaseboundarysurface.
Thebubblecolumnisparticularlyusefulinreactionswherethegasliquid
reactionisslowinrelationtotheabsorptionrate
Advantagesofthisreactortypearelowcapitalcostandminimized
problemsofsterilitybasedonlackofmovingpartsinsidethereactor.
Bubblecolumnscanbedividedintofivemaintypesofreactorsonthe
basisoftheirstructure:simplecolumnreactors,multistageperforatedplate
columnreactorsaswellasmultistagecolumnreactorswithstaticmixersfor
repeatedgasdispersion,andcolumnreactorswithnozzleaerationand
towerreactors.

AirliftReactor
Thisisthesecondwidelyusedtypeofbioreactors
usedinplantcellcultivation
Asinbubblecolumns,massandenergyinputin
airliftreactorsisaccomplishedpneumaticallywithout
mechanicalagitationandassociatedwithsignificantly
lowershearlevelsthaninstirredreactors.
Inairliftreactor,asitsnameimplies,compressed
airisusedforaerationandmixingofthecontentsof
thereactorvessel.
Itsoperationisbasedonthedraughttube
principle.
Airsparged intothebaseofthereactorlowersthe
densityofthemediumwhichrisesupthedrafttube
pullingfreshmediuminatthebaseand,therefore,a
flowisachieved.
A
Amoreuniformflowpatternisachievedintheair
if
fl
tt
i
hi d i th i
Internalloop
Externalloop
Draughttube
liftreactorcomparedwiththebubblecolumn
reactor,wherearandomflowpatternexists.
Airliftreactorsgenerallyprovidebettermixingconditionsthanthebubblecolumns
Here,agitationiscoupledtoaeration,andconsequentlyitmaybenecessarytousehigher
gasflowratestoprovidemixingthanwouldberequiredforoxygentransfer.
Thedisadvantagesofairliftreactorsarethedevelopmentofdeadzonesinsidethe
reactorandinsufficientmixingathighcelldensities.

26

1/9/2014

RotatingDrumReactor
Therotatingdrumreactorconsistsofahorizontally
rotatingdrumonrollersconnectedtoamotor.
Therotatingmotionofthedrumfacilitatesgood
mixingandaerationwithoutimposingahighshear
stressontheculturedcells.
Bafflesintheinnerwallofthedrumhelptoincrease
oxygensupply.
h
Thistypeofreactorhasthecapacitytopromote
f
h h
highoxygentransfertocellsathighdensity.
IthasbeenusedtogrowculturesofC.roseus andL.erythrorhizon upto1000Linvolume.
Systemswithonereactorchamberworkwithalongaxisdesignedasahollowshaftforair
andgasexchange.
Transportprocessesinsidethereactorcanbevariedbythedrumrotationrate.
Inacomparativestudyoftheperformanceofrotatingdrumandstirredtankreactorsfor
thecultivationofVinca rosea theformerwasfoundtobesuperioronthebasisofincreased
oxygentransferathighcelldensities
f
h h ll d
Therotatingdrumreactorfacilitatesbettergrowthandimpartslesshydrodynamicstress.
Inthestirredtankreactorgrowthratewaslowatlowagitationspeedbecauseof
insufficientoxygensupply,whileathighagitationspeedthecellsdied.
Hence,forcultivationofcellsathighdensities,therotatingdrumreactorwaspreferred.
Therotatingdrumreactorhasalsobeenshowntobesuperiortoairliftandmodified
stirredtankreactorsforthecultivationofL.erythrorhizon
Themajordisadvantageofthisreactortypeistherestrictioninscaleup

Packed Bed Reactor

Inpackedbedreactors,cellsareimmobilizedonlargeparticles
suchaspolymericbeads.
These particles do not move with the liquid
Theseparticlesdonotmovewiththeliquid.
Thenutrientmediumcanbefedeitheratthetoporbottomof
thetubeandiscirculatedthroughthepackedbed.
Packedbedreactorsaresimpletoconstructandoperatebut
cansufferfromblockagesandfrompooroxygentransfer.
Topreventgaspocketsandflowchannelinginthepackedbed,
airissparged indirectlybyaerationofaseparatelyusedstorage
vesselaswellasarecyclingmediumvesselforsomeapplications.
Withrespecttothehighpackingdensityofbiocatalysts,
thepackedbedreactoristhebioreactorconfigurationwiththe
highestachievableproductivityperunitreactorvolume.

27

1/9/2014

Trickle Bed Reactor

The trickle bed reactor is one variation of the packed bed

configuration.
It entails the downward movement of a liquid and gas over
a packed bed of catalyst particles
particles.
It is considered to be the simplest reactor type for
performing catalytic reactions
One or a number of nozzles integrated in the headspace of
the column spray the nutrient continuously or periodically
onto the top of the packaging.
The aeration takes place directly by introducing air at the
base.
A special version of the trickle bed reactor is the mist
reactor.
The nutrient medium is introduced as a mist phase
produced by an injector or ultrasonic nozzle

Fluidized Bed Reactor

Whenpackedbedsareoperatedinupflow mode,
thebedexpandsathighliquidflowratesandfollows
themotionoftheparticles.
gp
p
y
Thisistheworkingprincipleoftheordinaryfluidized
bedreactor,wheretheparticlesareinaconstant
motion.
Aspecialfluidizedbedreactorconfigurationwith
mixingcharacteristicssimilartothoseofstirred
reactorsisdepictedschematicallyinFigure
Consideringthereactorgeometry,thefluidizedbed
reactorswithplugflowaretallerthanfluidizedbed
reactorswithcompletebackmixing

Fluidizedbedreactor
(withhighenergyinput)

Fluidizedbedreactor
(withplugflow)

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1/9/2014

BIOREACTORS FOR PLANT CELL SUSPENSION

Stirredreactors,airliftreactors,rotatingdrumreactors,andfluidizedbedreactorswere
adaptedwithonlyminormodificationsandintroducedtoplantcellcultivationtechniques
withgoodresultsforawiderangeofsuspensioncultures.
Stirredreactorshavingvolumeswithinarangeof75m3 forplantcellandtissuecultures
arewidelyused.
They are generally equipped with aeration and mixing systems producing no or low
Theyaregenerallyequippedwithaerationandmixingsystemsproducingnoorlow
sheardamage.
Incontrasttomostanimalcellcultures,anumberofplantcellandtissueculturesare
notdamagedbyairbubbles.
Directaerationbygasinjectionusingspargers becomespossible.
However,bubblefreeaeration(membraneaeration,externalaeration,indirectaeration
usingspinfiltersorotherassemblies)canhaveadvantagesforculturestendingtofoaming
andflotation
The primary impeller task is to guarantee mass and temperature homogeneity and gas
Theprimaryimpellertaskistoguaranteemassandtemperaturehomogeneityandgas
dispersioninbioreactorcultivationprocessesbasedonplantcellsuspensioncultures.
Gasdispersionplaysasubordinaterole.
Incontrasttoreactorswithhighaerationratesshowingoptimaloperationnearthe
floodingpointsituatedonthetop,plantcellandtissueculturebioreactorsoperatebelow
thispoint.
Suitableimpellersareradialflowimpellers,axialflowimpellers,andimpellersthat
distributethepowerinputoveralargefractionofthetotalreactorvolume

Reactors for Hairy Root Cultures

Suitablebioreactorperformanceforcultivationofhairyrootsshouldconsiderthe
structuralroots'integrityincludingphysiology,morphology,andtheirrheological
properties.
Thegrowthbehavioroftherootscausesessentialdifficultiesconcerningtheinoculation,
g,
p gp
p
harvesting,andsamplingprocedureinthecourseofthecultivationprocess.
Aspecialandenlargedinoculationassemblyintheformofspecialinoculationvessels,
cellbags,andinoculationtubesbecamenecessary.
Anonuniform biomassdistributioninthereactorandinsufficientmasstransferinthe
denselypackedmassofgrowingrootsresultincellnecrosisandautolysisaswellaslossof
biosyntheticcapability.
Itbecameclearthatstandardreactorsaregenerallynotsuitableforhairyrootcultures.
Usually,stainlesssteelornylonmeshes(basket)andpolyurethanefoamareintroduced
intothechosenstandardreactorforrootstoprotectagainstmechanicaland
hydrodynamicshear.

29

1/9/2014

Amodifiedairliftreactorfortheproductionofartimisinin fromA.annua hairyroot

culture

Thebasketmakesitpossibletoseparatethegrowthspaceoftherootsfromthe
aerationandmixingareaandtosupporttheselfimmobilizationoftheroots.
Laboratoryrotatingdrumreactorswerealsoadaptedforhairyrootcultivations.
Therotatingdrumreactorcouldbemodifiedtoproducesthreebasicmovementslike
rotation,translation,andinversion
Theconsequenceisthattherhythmicmovementandlowshearstressdidnotinhibit
thegrowthandbiosyntheticcapabilityofthecultivatedroots.
Biomassgrowthofabout45foldwasachievedin28days
Tricklebedreactorsofrootculture
Itcouldalsobedemonstratedthatworkingtricklebedreactorsinwhichthenutrient
mediumissprayedordispersedoffermoreidealconditionsforgrowthandproductivity
ofhairyrootsthanordinarysubmergedbioreactors.
Althoughmoistureisrequiredbyroottissue,ifthemediumfilmistoothickitlimits
nutrientandgastransfertothetissue.
Th
Thesolutionisadropletormistapplicationcycle,whichdecreasesthedevelopmentof
l i i d l
i
li i
l
hi h d
h d l
f
thickmediumfilmsontheroots.
Laboratorytricklebedreactorswithasupportmatrixusuallywithworkingvolumesof1
to14LhavebeensuitableformasspropagationsofHyoscyamus muticus ,Nicotiana
tabacum,Betavulgaris,andCarthamus tinctorius hairyroots.

30

1/9/2014

FACTORSAFFECTINGSECONDARYMETABOLITE PRODUCTIONBYPLANTCELLCULTURES
PlantGrowthRegulators
Auxin isessentialandcytokinin ispreferabletoinducecelldedifferentiationandto
maintaincellproliferationinvitro.
Inhasalsobeenwidelyrecognizedthattheconcentrationandbalanceofauxin and
cytokinin
k
affectorganregenerationfromculturedcells.
ff
f
l
d ll
Thesegrowthregulatorsregulatesecondarymetabolismininvitroculturedcellsprobably
throughcontrollingcelldifferentiation.
However,theeffectsofauxin andcytokinin arevariablefromspeciestospeciesandfrom
producttoproduct,andthemechanismbywhichtheplantgrowthregulatorup ordown
regulatestheparticularsecondarymetabolismisnotclearinmostcases.
Gibberellin isusuallynotaddedtoculturemedium,andonlyafewreportsdescribeits
effectonnaturalproductbiosynthesis.
Production of berberine inCoptis
Productionofberberine
in Coptis japonicacellcultureswasincreasedbygibberellin.
japonica cell cultures was increased by gibberellin
Incontrast,gibberellin inhibitedshikonin biosynthesisinLithospermum erythrorhizon cell
cultures

MediumNutrients
Themostcommonlyusedculturemediaarebasedontheestablishedformulationsdefined
byGamborg,Heller,Linsmaier andSkoog,Murashige andSkoog,SchenkandHildebrandt,
andWhite.
However,othermediaexistwithvariationsuponthesebasicthemes.
Forexample,thechoiceofnitrogensourcemayvarydependinguponthespeciesbeing
culturedandmaybeintheformofanammoniumornitratesalt,aminoacids,casein
y
,
,
hydrolysate,orurea.
Themostcommoncarbonsourceusedinplantcellculturemediaissucrose,although
glucoseissometimesusedinitsplace,andmaysupportanequalorevenhighergrowthrate
inculture.
However,ahighrateofgrowthisnotalwaysofparamountimportanceforcultures.
Oftentheaimofinitiatingaplantcellcultureistoobtainsecondaryproductsthatare
generallyaccumulatedduringthestationaryphaseofgrowthorindifferentiatedcultures.
Therefore,manyoftheseinvestigationsseemtoindicateanegativecorrelationbetween
cellproliferationandsecondarymetabolism.
ll
lf
d
d
b l
Itmightbepossiblethatanymanipulationforinhibitingcellgrowthleadstoanincrease
intheproductivityofsecondarymetabolites,leadingtoestablishmentofatwostageculture
systemforproductionofphytochemicals wherethecellsarefirstculturedinthemedium
appropriateformaximumbiomassproductionandthentransferredtothegrowthlimiting
mediumformaximumproductivityofsecondarymetabolitesasestablishedforshikonin
productioninLithospermum erythrorhizon cellcultures

31

1/9/2014

AphosphatefreemediumincreasedthealkaloidproductivityofPeganum harmala cells,it

hasbeenrecognizedthatreducingthephosphateconcentrationresultsingrowthlimitation
andaconcomitantincreaseinthelevelofsecondaryproducts.
Nitrogenisessentialtosupportcellgrowthasasourceofproteinandnucleicacid
synthesis,thusaffectingsecondarymetabolism.
Generally,culturemediumcontainsNsourcesasNH4+andNO3,andboththe
+

concentrationoftotalnitrogenandtheratioofNH
f
l
d h
f
d
l
ll
h d
4 andNO
3 regulatecellgrowthand
secondarymetabolism.
Inmanycases,reducingthetotalnitrogenconcentrationinthemediumleadstolowercell
growthandhigherproductformationastypicallyreportedforanthocyanin productionby
Vitis vinifera cellcultures.
However,cellgrowthandproductionofbetacyanin inPhytolacca americana cellcultures
increasedwithanelevatednitrogensupply.
Ifusedasasolenitrogensource,NH4+isoftentoxictocellgrowth.
Shikonin productioninL.erythrorhizon
production in L erythrorhizon cellsuspensioncultureswascompletelyinhibited
cell suspension cultures was completely inhibited
whenthecellswereculturedinNH4+containingmedium.
ItisimportanttofindanoptimumratioofNH4+andNO3 forattainingmaximum
productionofsecondarymetabolites.

32

1/9/2014

Sucroseisutilizedmostasacarbonsource.
Incontrasttophosphateandnitrogen,anincreaseintheinitialsucroseconcentrationin
culturemediumleadstoanincreaseinsecondarymetaboliteproduction.
Theenhancingeffectofsucrosewasmostimpressivelyshowninthecaseofrosmarinic
acidformationinColeusblumei cellsuspensioncultures,wheretherosmarinic content
increasedsixfold inamediumcontaining5%sucrosecomparedwiththatinthecontrol
medium(2%sucrose),reaching12%ofdryweight.
(
),
g
y
g
Thiseffectwasnotduetothehigherosmoticpressurebecauseadditionofmannitol
tolowsucrosemediumdidnotincreaserosmarinic acidproduction.
Incontrast,thestimulatoryeffectofsucroseonanthocyanin productioninVitis vinifera
cellcultureswasshowntobeduetoosmoticstress.
Thecarbontonitrogenratioisalsoanimportantfactorinsecondarymetabolismasshown
byanthocyanin productioninVitis cellcultures.
Althoughlessinvestigatedcomparedwithmacronutrients,micronutrientsarealso
expectedtoaffectsecondarymetabolism.
Infact,theshikonin contentinL.erythrorhizon cellculturesincreaseddrasticallywithan
increasingCu2+levelinthemedium.

Elicitors
Elicitationistheinductionofsecondarymetaboliteproductionbymoleculesor
treatmentsknownaselicitors.
Elicitationisusedtoinducetheexpressionofgenesoftenassociatedwiththeenzymes
responsibleforsynthesisofsecondarymetabolitesbymimickingthepathogendefenseor
woundresponseinplants.
Theelicitorsproducedbymicroorganismsandplantsarereferredasbioticelicitors,while
physicalandchemicalstressessuchasultraviolet(UV)irradiation,heatorcoldshock,and
heavymetalsalsoinduceawiderangeofdefenseresponsesandaredefinedasabiotic
elicitors.
Abiotic elicitorsarethoughttoinducethereleaseofbioticelicitorsfromplantcellwalls.
Ithasbeenshownthatelicitorsarecapableofnotonlyinducingdenovoformationof
phytoalexins butalsoactivatingbiosyntheticpotentialsofvariousconstitutivemetabolites
inculturedplantcells.
Elicitortreatmentincreasedthebiosynthesisofthebenzophenanthridine alkaloid
sanguinarine
i i 26foldinPapaver
26 f ld i P
somniferum
if
cellcultures
ll l
Inductionofsecondarymetabolismbyelicitorsincellsuspensionculturesofvariousplant
specieswascorrelatedwithearlierrapidandtransientaccumulationofjasmonic acidandits
methylestermethyljasmonate,
Jasmonic acidwasproposedtobeakeysignalcompoundinthecellularprocessof
elicitationleadingtotheaccumulationofvarioussecondarymetabolitesinthecultured
plantcells

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1/9/2014

Productionofvariousphytochemicals includingrosmarinic acid,taxol,shikonin,and

stilbene hasbeenreportedtobeinducedbyjasmonic acidormethyljasmonate.
Severalstudieshavedemonstratedthevalueoffungalelicitorsinpromoting
biosynthesisoftheantitumoragent,paclitaxel,byTaxus sp.cellcultures,andthese
elicitorshavebeenderivedfromavarietyofmicrobialsources.
Fungalelicitorshavealsobeenusedtoelevateginsenoside productionincell
suspensionculturesofginseng.
l
f
Methyljasmonate treatmentledtolargeimprovementsinbothcostandefficiency
overtheuseofundefinedfungalextracts;100pM methyljasmonate increasedthe
paclitaxel levelfrom28to110mgL1 inaT.mediacellline,andfrom0.4to48mgL1 in
T.baccata

PhysicalFactors
Physicalfactorscontrollingsecondarymetaboliteproductionsynthesisinculturedplant
cellsincludelight,temperature,mediumpH,aeration,celldensity,etc.
Theeffectoflightonnaturalproductbiosynthesisisquitevaried.
Lightilluminationusuallyinduceschloroplastdifferentiation,whichsometimesleadsto
elevationofsecondarymetabolism.
A lupine alkaloid lupanine was
wasproducedonlyinthegreencallusofThermopsis
produced only in the green callus of Thermopsis
Alupinealkaloidlupanine
lupinoides culturedunderlightillumination.
Lightilluminationisoftenessentialtoinduceanthocyanin biosynthesis,althoughits
biosynthesisisnotlocalizedinchloroplasts.
Incontrast,biosynthesisofnicotineintobaccocellsandshikonin inLithospermum
erythrorhizon cellculturesisinhibitedbylightillumination
TheoptimalculturetemperatureandmediumpHareusuallybetween20and25Cand
between5.6and6.0,respectively.
Aerationisalsoanimportantfactortoregulatebothcellgrowthandproductyield,
especiallyinbioreactorcultures

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BiologicalFactors
Oneofthemostimportantfactorscontrollingsecondarymetaboliteproduction
iscelltocellvariation.
Withinapopulationofculturedcellsthereisadifferenceinmetabolicbehavior,
especiallyinabilitytosynthesizeparticularmetabolitesevenifthecellpopulationwas
inducedfromthesamepieceofexplant andculturedinthesamephysicalandchemical
environments.
environments
Althoughamolecularbiologicalbasisforsuchcellularvariationinsecondary
metabolismhasnotyetbeenclarified,ithasbeenwellrecognizedthatselective
subcultureofcellaggregateswhosecontentofthesecondarymetaboliteishigherthan
otherswilleventuallyresultintheisolationofsocalledhighproducingcelllinesfora
particularsecondarymetabolite.
Anotherimportantbiologicalfactorisstabilityofthebiosyntheticcapability
ofculturedplantcells.
p
g
roseus that wereestablishedby
y
AlkaloidproducingcelllinesofCatharanthus
repeatedselectionlosttheirbiosyntheticabilityduringsubcultures;theindole alkaloid
contentdecreased70foldover8yearsofsubculture.
AsimilarkindofbiochemicalinstabilitywasalsoreportedfornicotineinNicotiana
rustica callus, cardenolides inDigitalispurpurea callus,andcinnamic acidinCapsicum
frutescens cellsuspension.
Theseresultsindicatethatitisimportanttosubculturethecellsunderselection
pressureorwithoccasionalreselection.

Combinatorialbiosynthesis

IntroductionandScopeofCombinatorialBiosynthesis
Anincreasingnumberofnaturalproductsarebeingbiosynthesizedinlowquantities,with
knownexamplesbeingartemisinin,paclitaxel,podophyllotoxin,andVincaalkaloids.
Theuseofgeneticallymodifiedplantcellcultures,suchashairyrootculturesfor
Solanaceae orforartemisinin,offersarationalapproachtoallowtheoverexpression of
genesencodingbiosyntheticenzymes,andtoovercometheratelimitingstepsofthe
biosynthesis.
Combinatorialbiosynthesisisanewtoolinthegenerationofnovelnaturalproducts,as
wellasfortheproductionofrareandexpensivenaturalproducts
Thebasicconceptofcombinatorialbiosynthesisistocombinemetabolicpathwaysin
differentorganismsatthegeneticlevel.
Themainproblemwithcombinatorialbiosynthesis,however,isthatmostbiosynthetic
pathwaysarestillpoorlyunderstoodatthegeneticlevel,withrelativelyfewgenesinvolved
inregulationandbiosynthesisinplantshavingbeensequencedandfunctionallyelucidated.
Therefore,nocompletebiosyntheticpathwayhasbeencompletelytransferredtoa
heterologous host.
host
Consideringtheimportanceofphytochemicals,itisimportanttounderstandtheir
biosyntheticpathwaysonamolecularlevel.
Thisinformationwillaidinthediscoveryofnewcompoundsandallowfordiversificationof
naturalproductscaffoldsbythegeneticmanipulationofmetabolicpathways.
Itwillalsobeusedtoincreasetheproductionlevelswithinnativehostsand/orincrease
thebiotechnologicalproductionofplantnaturalproductsinplantcellculturesand
recombinantmicrobialhosts.

35

1/9/2014

Scheme of the targets for genetic engineering of secondary metabolic

pathway

Tropane Alkaloid Metabolites, Enzymes and Products

Despitethebiosyntheticpathwayoftropane alkaloidsbeingdepictedalmostuniversally
intextbooksofplantnaturalproducts,onlytwoenzymesspecifictothebiosynthesisof
hyoscyamine havebeenisolatedandcharacterized:PMT(EC2.1.1.53)andthetropine
forming tropinone reductase (EC1.1.1.206).
formingtropinone
(EC 1.1.1.206).
Further,hyoscyamine6hydroxylase(EC1.14.11.11),whichcatalyzestheformationof
scopolaminefromhyoscyamine,hasbeenclonedandcharacterizedindetail.
Althoughmostmetabolicstepsintropane alkaloidformationhavebeenelucidatedusing
radioactiveprecursorsandsubsequentmetaboliteanalysis,thismethodispronetoerrors
ifnotinterpretedwithcaution,sinceassumedprecursormoleculesmaybemetabolized
withoutparticipatingintheplantsendogenousbiosyntheticpathway

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1/9/2014

Itisonly15yearsagothathydroxylated nortropane alkaloids,thecalystegines,were

structurallyelucidatedinextractsfromrootsofCalystegia sepium,Convolvulaceae.
Basedonthenortropane structure,itwassoonhypothesizedthatcalystegines are
formedviathetropane alkaloidpathway,andconsequentlytropane alkaloid
containingSolanaceae wereinvestigatedforcalystegines.
Specific extraction and purification schemes were developed for the hydrophilic
Specificextractionandpurificationschemesweredevelopedforthehydrophilic
alkaloids,togetherwithmodifiedchromatographicprocedures.

Putrescine N-methyltransferase (PMT)

Formationofthetropane bicyclus inSolanaceae beginsbymethylation ofthe
ubiquitousdiamine putrescine.
Thisreactioniscommontoboth,tropane andnicotinebiosynthesis,andtheenzyme
PMTwasinitiallyextractedandmeasuredfromtobaccoplantrootsandcalluscultures.
RootculturesofD.stramonium,ofHyoscyamus albus,andofH.niger containPMT
withcatalyticpropertiessimilartothoseofthetobaccoenzyme.
ThecDNA ofthepmtgenewasclonedfromtobaccoandfromNicotiana sylvestris
Thepmt genewasshowntobeexpressedexclusivelyintherootpericycle ofA.
belladonna andintheendodermis,xylemandoutercortexcellsofNicotiana sylvestris
roots.
Expressionwasmonitoredbyfusionofthe5flankingregionsofthePMTgenestothe
glucuronidase reportergene.
Theresultsofferanexplanationfortheinabilityofdedifferentiatedcellsuspensionto
synthesizetropane alkaloids:specificpericycle cellsarerequiredforpmt expressionin
tropane alkaloidcontainingplants.
alkaloid containing plants
Tobaccopmt,incomparison,isexpressedinseveralcelltypes,amongthemrootcortex
parenchymacells,towhichdedifferentiatedsuspensioncellsmaybesimilar.
Intobacco,pmt isstressresponsiveandinduciblebyMethylJasmonate.
Incontrasttothetobaccopmt promoter,noMethylJasmonate responsiveelement
wasidentifiedinthepromoterregionofA.belladonnapmt

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1/9/2014

AdetaileddeletionanalysisoftheN.tabacum pmtpromotershowedthataslittleas111
bp upstreamofthetranscriptionalstartsiteweresufficienttoconferMethylJasmonate
responsiveness.
DeletionofaconservedGboxelement(GCACGTTG)at103to96bp completely
abolishedMeJAresponsiveness.
Furthermutagenesisstudiesrevealedthat,inadditiontoafunctionalGbox,MeJAS
responsivenessofthePMTpromoteralsorequiredaTArichregionandaGCCmotif
(TGCGCCC)locatedat80to69bp and62to56bp relativetothestartsite,
respectively,indicatingmultipleintersectingsignaltransductionpathwaysanddifferent
transcriptionalregulatoryfactorsinvolvedinMeJasresponseofPMTexpressionintobacco.
Somepmtexpressionwasalsoobservedintobaccoleavesaftermechanicalwounding.
Thisexpressionwashighlylocalizedaroundthewoundsiteandprovedtobetransient,
withlevelsbeingmaximalimmediatelyafterwoundingbutdiminishingafter24h.

Tropinone Reductases
Initiallyitwasthoughtthatduringthecourseofitsbiosynthesis,tropinone wasreduced
stereospecifically totropine (3tropanol)andnottotheisomericpseudotropine (3
tropanol).
MeasurementoftropinonereducingenzymeactivitiesinD.stramonium proteinextracts
confirmedthisview:tropine onlywasfoundasreductionproduct,andpseudotropine was
notformed
Thefirsttropinone reductase purificationfromH.niger,however,yieldedanenzyme
specificforpseudotropine formation.
Inaddition,pseudotropine wasprovednottobeisomerized intotropine inplanttissues.
Consequently,asthatenzymewasnotresponsiblefortropine formation,theexistence
ofanothertropinone reductase formingtropine waspostulated.
Twoseparatetropinone reductases werepurifiedfromH.niger rootcultures,andalso
fromD.stramonium rootcultures
A.belladonnarootculturesalsocontainedtwospecificenzymes;thetropineforming
enzymewastermedTRI(EC1.1.1.206),andthepseudotropineformingenzymeTRII(EC
1 1 1 236)
1.1.1.236).
TRIIactivitywasfoundtobestronginmanySolanaceae tissues;forexample,shortly
aftertheapplicationoftropinone,pseudotropine accumulatedfasterthantropine.
Estersofpseudotropine (e.g.,ofaceticacidortiglic acid)wereidentifiedonlyasminor
alkaloidsinthoseplants,andthemetabolicroleofTRIIandthedestinationof
pseudotropine formationwereenigmatic,untilcalystegines werebroughtintothecontext
oftropane alkaloidbiosynthesis.

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Thestructureofcalystegines containsanequatorialhydroxylgroupinposition3,the
typicalfeatureofpseudotropine.
Accordingly,atypicalTRIIwasisolatedandcharacterizedfrompotatotubersthat
containcalystegines,butnotthetropane estershyoscyamine andscopolamine
ItisnowacceptedthatthepseudotropineformingTRIIisresponsibleforcalystegine
biosynthesis,whileTRIisrequiredfortheformationoftropine,whichisintegratedinto
hyoscyamine andscopolamine.
Thesimilarityinproteinandcatalysisofbothreductases,buttheapparentdifferences
inreactionstereospecificity,wereintriguing.
Differentialtropinone acceptanceandfixationweresuspectedtoberesponsiblefor
theselectiveformationoftropine andpseudotropine.
Reactionvelocity,substrateaffinity,andpHoptimaforTRIandTRIIareconspicuously
different

Hyoscyamine-6-Hydroxylase
Subsequentesterification oftropine wasshowntooccurwithphenyllactic acid,thefirst
esterified alkaloidbeinglittorine,whichalsoaccumulatesinsomeSolanaceae andinroot
culturesoftherespectiveplants.
Rearrangementofthephenyllactic acidmoietyoflittorine toyieldhyoscyamine was
demonstratedbylabeledprecursorsandNMR,butnotelucidatedonanenzymaticlevel.
Hyoscyamine isoxidizedtoformscopolaminebyanoxoglutarate
is oxidized to form scopolamine by an oxoglutaratedependent
dependentdioxygenase,
dioxygenase
thehyoscyamine6 hydroxylase (H6H).
Theenzyme,whichwaspurifiedandcharacterizedfromaH.niger rootculture,performs
atwostepreaction,firsthydroxylating hyoscyamine in6positionandsubsequentlyforming
theepoxygroupofscopolamine
AntibodiesagainstthepurifiedenzymeenabledlocalizationoftheH6Hproteininthe
pericycle ofrootdiametersofH.niger.
ThisfindingsimilartospecificlocalizationofPMTenforcestheconclusionofdifferentiated
roottissuebeingnecessaryfortropane alkaloidbiosynthesis.
Cloningoftheh6hgeneandtransformationofA.belladonnawithh6hcDNA yielded
plantswithadrasticallyincreasedscopolamineproduction

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The shikimate pathway

Theshikimate pathwaybeginswithacouplingofphosphoenolpyruvate (PEP)andD
erythrose 4phosphatetogivethesevencarbon3deoxy Darabinoheptulosonic acid7
phosphate(DAHP)
Thisaldoltypecondensationreactionisknowntobemechanisticallymorecomplexin
theenzymecatalysed version
Elimination of phosphoric acid from DAHP followed by an intramolecular aldol reaction
EliminationofphosphoricacidfromDAHPfollowedbyanintramolecular
reaction
generatesthefirstcarbocyclic intermediate3dehydroquinicacid.However,thisalso
representsanoversimplification.
TheeliminationofphosphoricacidactuallyfollowsanNAD+dependentoxidationofthe
centralhydroxyl,andthisisthenreformedinanNADHdependentreductionreactionon
theintermediatecarbonylcompoundpriortothealdol reactionoccurring.
Allthesechangesoccurinthepresenceofasingleenzyme.
Reductionof3dehydroquinicacidleadstoquinic acid,afairlycommonnaturalproduct
found in the free form, as esters, or in combination with alkaloids such as quinine
foundinthefreeform,asesters,orincombinationwithalkaloidssuchasquinine
Shikimic aciditselfisformedfrom3dehydroquinicacidvia3dehydroshikimicacidby
dehydrationandreductionsteps.
Averyimportantbranchpoint compoundintheshikimate pathwayischorismic acid,
whichhasincorporatedafurthermoleculeofPEPasanenol ethersidechain.PEP
combineswithshikimic acid3phosphateproducedinasimpleATPdependent
phosphorylation reaction.ThiscombineswithPEPviaanadditioneliminationreaction
giving3enolpyruvylshikimicacid3phosphate(EPSP).Thisreactioniscatalysed bythe
enzymeEPSPsynthase.

Catharanthus roseus Alkaloids

Catharanthus roseus plantcellculturesareapotentialcommercialsourceforthe
productionofsomepharmaceuticallyimportantalkaloids.
Catharanthus roseus (L.)G.DonisatropicalplantbelongingtothefamilyApocynaceae
andproducesawiderangeofterpenoid indole alkaloids(TIAs).
More than 130 alkaloids have been isolated from different parts of the plant and many of
Morethan130alkaloidshavebeenisolatedfromdifferentpartsoftheplantandmanyof
themshowbiologicalactivities,suchastheantineoplastic vinblastine andvincristine,the
antihypertensiveajmalicine,andthesedativeSerpentine.
CellsuspensionculturesofC.roseus produceamuchsimpleralkaloidprofile,theyieldis
lessthanthatfromintactplantsandafterprolongedsubculturetheylosetheabilityto
producealkaloidsathighlevelsevenafterextensivescreeningandselectionprocedures.
Therefore,theregulationofthebiosynthesisofTIAsincellculturesofC.roseus isbeing
extensivelystudied
Amajorproblemassociatedwiththeclinicaluseofvinblastine andvincristine isthatonly
verysmallamountsofthesedesirablealkaloidsarepresentintheplant.Althoughthetotal
alkaloidcontentoftheleafcanreach1%ormore,over500kgofcatharanthus isneededto
yield1gofvincristine.Thisyield(0.0002%)isthelowestofanymedicinallyimportant
alkaloidisolatedonacommercialbasis.
Extractionisbothcostlyandtedious,requiringlargequantities

40

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Subcellular Compartmentation of Terpenoid indole alkaloid biosynthesis

in Catharanthus roseus

AS,anthranilate synthase;CR,
NADPH:cathenamine
reductase;DAT,
deacetylvindoiine 17O
17 O
acetyltransferase;ER,
endoplasmicreticulum;G10H,
geraniol 10hydroxylase;GAP,
glyceraldehyde3phosphate;

D4H,desacetoxyvindoline4hydroxylase;MEP;2CmethylDerythritol 4phosphate;NMT, S
adenosylLmethionine methoxy2,16dihydro 16hydroxytabersonineNmethyltransferase;
SGD,strictosidine glucosidase;STR,strictosidine synthase;TDC,tryptophandecarboxylase;
THAS,NADPH:tetrahydroalstonine reductase.
Solidarrowsrepresentasingleenzymaticstepanddashedarrowsrepresentmultipleenzymesteps.

Biosynthesis of terpenoid indole alkaloids in Catharanthus roseus

TheTIApathwayisacomplexmetabolicnetwork.
ThebiosyntheticprecursorsforTIAbiosynthesis
areprovidedbytheshikimate pathwayandthe
terpenoid pathway
Becauseinthetryptophanbranchofthe
shikimate pathwaytryptophandecarboxylase (TDC,
EC4.1.1.28)operatesattheinterfacebetween
primaryandsecondarymetabolism,ithasbeen
demonstratedtobeasiteforregulatorycontrolof
TIAbiosynthesis.
TDCcatalyzestheconversionofLtryptophanto
tryptamine,theindole moietyoftheTIA.
Strictosidine synthase (STR)catalyzesthe
condensationoftryptamine with
secologanin byareactionofthePictetSpengler
type into 3(S) strictosidine the pivotal
typeinto3(S)strictosidine,thepivotal
intermediateoftheTIAs.

STR,strictosidine synthase;TDC,tryptophandecarboxylase;MEP,2CmethylDerythritol4phosphate

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Vinblastine

Vincristine

H3C

H3C

H3C

H3C
CH3

Vinorelbine

AnumberofenzymesoftheTIAbiosyntheticpathwayhavebeencharacterizedand
theencodinggeneshavebeencloned.
Thishasenabledstudiesoftheoverexpression ofthesegenesinvariousplantsand
organisms.
Therearesomevaluableexamplesofwhatmetabolicengineeringcanachievein
secondarymetabolismusingsomeofthesegenes.
Theseedsofcanola(Brassica napus)areusedasanimalfeed,butthepresenceof
indole glucosinolates makesthecroplesspalatable.
AfterthetransformationofB.napus withtheTdc genefrom C.roseus,themature
seedsofthetransgenicplantscontainreducedlevelsofindole glucosinolates,which
increasestheeconomicvalueoftheplant.
AnotherexampleisthefunctionalexpressionoftheStr genefrom Rauwolfia
serpentina inbacteria,yeast,andinsectcells,whichcanbeusedasatoolforproduction
oflargeamountsofSTR.

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Results of overexpressing the enzymes TDC, STR, and TDC and STR in C. roseus
TDC:
Overexpressionofthetdc geneisolatedfromC.roseus inNicotiana tabacum resultedin
theleavesoftransformedplantshaving4to45timesmoreTDCactivitythanthecontrols.
Increaseintryptamine accumulation:>1mg/g.
AnotherresearchgrouphasfoundthattheTDCactivityinthetransformedtobaccowas45
times greater than in the controls and resulted in a > 200 fold increase in the free tryptamine
timesgreaterthaninthecontrolsandresultedina>200foldincreaseinthefreetryptamine
and>30foldinfreetyramine pools.
AnincreaseinTDCproteinlevel,TDCactivityandtryptamine contentbutnosignificant
increaseinTIAproductionwereobserved
STR:Overexpression ofthestr geneisolatedfromC.roseus inNicotiana tabacum had many
foldgreaterSTRactivitythanC.roseus plants,howevertheTIAproductionwasnotincreased
significantly.
TDCandSTR:WhenTDCandSTRareoverexpressed togetherinasinglecellline,despitean
increaseinTDCandSTRactivitiesobservedcomparedwiththenontransformed cultures,TIA
accumulationdidnotincrease.

Identification of Bottlenecks
Asoverexpression ofthegenesTdc andStr didnotresultinamuchincreasedTIAlevel
duringlongtermsubculture,thetotalcapacityofthepathwaywasassessedbyfeeding
theindole andiridoid precursors.
Theuptakeandutilizationratesofthefedprecursorsdifferedamongcelllines
LinesS10,SI,andT22andthewildtypecelllineCRPMwerefedinPMwithvarious
concentrationsandcombinationsoftheindole precursorsLtryptophanandtryptamine
and the iridoid precursorsloganin
andtheiridoid
precursors loganin andsecologanin.
and secologanin
First,theoptimalconditionsforfeedingweredetermined.
Itwasfoundthatadditionofprecursor(s)intheearlycultureandtothePMwasmore
efficientthanadditiontotheGM.
Fromfeedingasingleprecursor,itwaslearnedthatadditionoftheindole preursors
suchasLtryptophanandtryptamine totheculturemediumdidnotincreaseTIA
productionbutadditionoftheiridoids suchasloganin andsecologanin didincrease
alkaloidproduction.
ThisshowsthatthemajorbottleneckforthebiosynthesisofTIAswasintheterpenoid
j
y
p
pathwayforboththetransgeniclinesandthewildtypecelllineCRPM
ConstitutivelyincreasedenzymeactivityintheTIApathway,inthiscaseTDCorSTR,
wasnotsufficienttokeepelevatedTIAlevels.
TDCandSTRdoesnotseemtonotbealimitingfactorintheTIApathway.

43

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FutureProspects
Thefollowingpointsmightbeconsideredinfuturestudies,concerningpossible
physiologicalorfurtherbiochemicalbarriersforTIAaccumulationinC.roseus cell
cultures:
i. Thecellularorsubcellular localizationofenzymes,precursors,andTIAs
ii. FactorsinvolvedintransportoftheprecursorsforTIAs
iii. Effectofcompetitivepathway(s)utilizingTIAprecursors
iv CatabolismofTIAs
iv.
Catabolism of TIAs
v. Factorsthatcontrolthecarbonfluxintothepathway
Asallthefactorsareconnectedwitheachother,furtherstudiesontheregulatorygenes
andtheoverexpression ofsuchgenesseemapromisingapproach.
Thenextstepwillbetointroduceproteinsinvolvedinthebiosynthesisof
Pharmaceuticalssuchasvinblastine,vincristine,ortaxol intheappropriatehostto
producethecompoundsmentionedonanindustrialscalewithplantcellculturesor
g
y
g
p
microorganismsinbioreactorsorbytransgenicplants.
Toaccomplishthisgoal,biosyntheticpathwaysneedtobecompletelyidentifiedon
gene,enzyme,andproductlevels.
Inparticular,identificationoffactorsinvolvedintheregulationofthebiosynthetic
pathwaysseemsaninterestingapproachthatmayleadtoregulatorygenescontrolling
thefluxthroughapathway.
Thiswillmakepossibletheproductionofknownornovelfinechemicalsbyplantcell
cultures.

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