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CharacterizationofproductionofPaclitaxelandrelatedTaxanesinTaxusCuspidataDensiformissuspensionculturesbyLC,LC/MS,andLC/MS/MSCHAPTERTHEREPLANTTISSUECULTUREⅠ.PotentialofPlantcellCultureforTaxaneProductionSeveralalternativesourcesofpaclitaxelhavebeenidentifiedandarecurrentlythesubjectsofconsiderableinvestigationworldwide.Theseincludethetotalsynthesisandbiosynthesisofpaclitaxel,theagriculturesupplyoftaxoidsfromneedlesofTaxusspecies,hemisynthesis(theattachmentofasidechaintobiogeneticprecursorsofpaclitaxelsuchasbaccatinⅢor10-deacetylbaccatinⅢ),fungusproduction,andtheproductionoftaxoidsbycellandtissueculture.Thisreciewwillconcentrateonlyonthelatterpossibility.Planttissuecultureisoneapproachunderinvestigationtoprovidelargeamountsandastablesupplyofthiscompoundexhibitingantineoplasticactivity.Aprocesstoproducepaclitaxelorpaclitaxel-likecompoundsincellculturehasalreadybeenparented.Thedevelopmentoffastgrowingcelllinescapableofproducingpaclitaxelwouldnotonlysolvethelimitationsinpaclitaxelsuppliespresentlyneededforclinicaluse,butwouldalsohelpconservethelargenumberoftreesthatneedtobeharvestedinordertoisolateit.Currently,scientistsandresearchershavebeensuccessfulininitiatingfastplantgrowthbutwithlimitedpaclitaxelproductionorviceversa.Therefore,itistheobjectiveofresearcherstofindamethodthatwillpromotefastplantgrowthandalsoproducealargeamountofpaclitaxelatthesametime.Ⅱ.FactorsInfluencingGrowthPaclitaxelContentA.ChoiceofMediaforGrowthGamborg's(B5)andMurashige&Skoog's(MS)mediaseemtobesuperiorforcallusgrowthcomparedtoWhite's(WP)medium.ThemajordifferencebetweenthesetwomediaisthattheMSmediumcontains40mMnitrateand20mMammonium,comparedto25mMnitrateand2mMammonium.ManyresearchershaveselectedtheB5mediumovertheMSmediumforallsubsequentstudies,althoughtheyachievesimilarresults.Gamborg'sB5mediawasusedthroughoutourexperimentsforinitiationofcallusculturesandsuspensionculturesduetosuccessfulpublishedresults.Itwassupplementedwith2%sucrose,2g/Lcaseinhydrolysate,2.4mg/Lpicloram,and1.8mg/Lα-naphthaleneaceticacid.Agar(8g/L)wasusedforsolidcultures.B.InitiationofCallusCulturesPreviousworkindicatedthatbarkexplantsseemtobethemostusefulforestablishingcallus.TheageofthetreedidnotappeartoaffecttheabilitytoinitiatecalluswhencomparingbothyoungandoldtreematerialsgrownonGamborg'sB5mediumsupplementedwith1-2mg/Lof2,4-dichlorophenoxyaceticacid.Callusculturesinitiatedandmaintainedintotaldarknessweregenerallypale-yellowtolightbrownincolor.Thisresultedinsufficientmassesoffriablecallusnecessaryforsubculturewithin3-4weeks.However,thegrowthratecandeclinesubstantiallyfollowingtheinitialsubcultureandresultinveryslow-growing,brown-coloredclumpsofcallus.Ithasbeenpresumedthatthesebrown-coloredexudatesarephenolicinnatureandcaneventuallyleadtocelldeath.Thiscommonphenomenonistotallyrandomandunpredictable.Oncethisphenomenonhasbeentriggered,thecellscouldnotbesavedbyplacingtheminfreshmedia.However,addingpolyvinylpyrrolidonetotheculturemediacanhelpkeepthecellsaliveandgrowing.Ourexperiencewithcallusinitiationwassimilartothosestudies.Ourstudieshavefoundthatcalluswhichinitiatedearly(usuallywithin2weeks)frequentlydidnotproliferatewhensubculturedandturnedbrownandnecrotic.Incontrast,calliwhichdevelopedfrom4weeksto4monthsafterexplantswerefistplacedoninitiationmediawereabletobecontinuouslysubculturedwhentransferredat1-2monthintervals.Thepresenceofthesurvivalofcallusaftersubsequentsubculturing.Therelationshipbetweenpaclitaxelconcentrationandcallusinitiation,however,hasnotbeenclarified.C.EffectofSugarSucroseisthepreferredcarbonsourceforgrowthinplantcellcultures,althoughthepresenceofmorerapidlymetabolizedsugarsuchasglucosefavorsfastgrowth.Othersugarssuchaslactose,galactose,glucose,andfructosealsosupportcellgrowthtosomeextent.Ontheotherhand,sugaralcoholssuchasmannitolandsorbitalwhicharegenerallyusedtoraisethesugarsaddedplayamajorroleintheproductionofpaclitaxel.Ingeneral,raisingtheinitialsugarlevelsleadtoanincreaseofsecondarymetaboliteproduction.Highinitiallevelsofsugarincreasetheosmoticpotential,althoughtheroleofosmoticpressureonthesynthesisofsecondarymetabolitesisnotcleat.Kimandcolleagueshaveshownthatthehighestlevelofpaclitaxelwasobtainedwithfructosel.Theoptimumconcentrationofeachsugarforpaclitaxelproductionwasfoundtobethesameat6%inallcases.WickremesinheandArtecahaveprovidedadditionalsupportthatfructoseisthemosteffectiveforpaclitaxelproduction.However,othercombinationsofsugarssuchassucrosecombinedwithglucosealsoincreasedpaclitaxelproduction.Thepresenceofextracellularinvertaseactivityandrapidextracellularsucrosehydrolysishasbeenobservedinmanycellcultures.Thesereportssuggestthatcellssecreteorpossessontheirsurfaceexcessamountsofinvertase,whichresultinthehydrolysisofsucroseatamuchfasterrate.Thehydrolysisofsucrosecoupledwiththerapidutilizationoffructoseinthemediumduringthelatterperiodofcellgrowth.Thisperiodofincreasedfructoseavailabilitycoincidedwiththefastergrowthphaseofthecells.D.EffectofPicloramandMethylJasmonatePicloram(4-amino-3.5.6-trichloropicolinicacid)increasesgrowthratewhilemethyljas