3.培养基中的矿质元素

刘彤石河子大学生命科学学院1NutrientMediaforPlantTissueCultures刘彤石河子大学生命科学学院刘彤石河子大学生命科学学院2•Oneofthefirstdecisionsthatmustbemadewhendevelopingatissueculturesystemiswhatmediumtouse.Nutrientmediaforplanttissueculturearedesignedtoallowplanttissuestobemaintainedinatotallyartificialenvironment.刘彤石河子大学生命科学学院3Functionsofmedium•Providewater•Providemineralnutritionalneeds•Providevitamins•Providegrowthregulators•Accesstoatmosphereforgasexchange•Removalofplantmetabolitewaste4A.MacroelementsconsistofN,K,P,Ca,Mg,andS.•1.Nitrogen(N)-Nitrogenisessentialforplantgrowth.Mostinorganicnitrogenisconvertedtoaminoacidsandthentoproteins.Nitrogenistypicallyaddedtoplantnutrientmediaasthenitrateion(NO3-,oxidized)and/ortheammoniumion(NH4+,reduced),whichareaddedasinorganicsalts.Inorganicnitrogengenerallyrangesfrom25-60mMinnutrientmedia.Indevisingmedia,boththetotalamountofnitrogenaswellastherelativeamountsofNO3-andNH4+areimportant.ThereareusuallylowerlevelsofNH4+thanNO3-inmedium;nitrateisusuallyaddedatconcentrationsbetween25and20mMandammoniumatconcentrationsbetween2and20mM.刘彤石河子大学生命科学学院5•2.Potassium(K)-Potassiumisthemajorioninplantswithapositivecharge,balancingnegativeions.Althoughtheamountofpotassiumrequiredvarieswidelyamongdifferentspecies,inmediapotassiumconcentrationisgenerallycorrelatedwiththatofnitrateandrangesbetween20-30mM.刘彤石河子大学生命科学学院6•3.Phosphorous(P)-Phosphorusisanintegralpartofnucleicacidsandotherstructuralcompounds.Itisaddedtoculturemediumasphosphate(PO4-)insodiumorpotassiumhydrogenphosphatesinconcentrationsrangingfrom1-3mM.•4.Calcium(Ca)-Calciumisaco-factorofmanyenzymesandisparticularlyimportantincellwallsynthesis.Itissuppliedmostlyascalciumchlorideorcalciumnitrate,concentrationsrangingbetween1and3mM.Inplantcultures,calciumdeficiencymayresultinshoottipnecrosis.刘彤石河子大学生命科学学院7•5.Magnesium(Mg)-Magnesiumiscriticalforthefunctioningofenzymes,isanintegralcomponentofthechlorophyllmolecule,andisacationthatbalancesnegativeions.Itisusuallyaddedasmagnesiumsulfateinconcentrationssimilartothatofcalcium.•6.Sulfur(S)-Sulfurisapartofseveralaminoacidsandhasanimportantfunctioninproteinstructure.ItissuppliedastheSO4-ion,generallywithmagnesiumasthecation,inconcentrationsrangingfrom1-3mM.刘彤石河子大学生命科学学院8B.MicronutrientsFe,Mn,Zn,B,CuandMo,Co,andI.•1.Iron(Fe)-Ironisnecessaryforchlorophyllsynthesisandfunctionsinmanyoxidation/reductionreactions.Itisgenerallypresentinmediaatapproximately1M.ThemajorprobleminsupplyingironinvitroisthatitformsinsolublecompoundsinalkalinepH,aproblemthatisparticularlyevidentinliquidculture,whereitmaybeseenasaprecipitate.Theuseofchelatingagents,whichbindmetalions,makesironmorestableandavailabletoplanttissuesoverwiderpHranges.Althoughthereareseveralofthese,thesodiumorpotassiumformofethylenediaminetetraaceticacid(EDTA)ismostoftenusedbecauseitisnotastoxicasotherchelatingagentsanditenablesirontobeavailabletoculturesoverawiderpHrangethanotheragents.Fe-EDTAmaybepurchasedasasaltorpreparedfromferricsulfateandEDTA.刘彤石河子大学生命科学学院9•2.Manganese(Mn)-Manganeseisrequiredforenzymereactions,particularlyinrespiratoryandphotosyntheticprocessesandisusuallyaddedasmanganesesulfateinconcentrationsof5-30M.•3.Zinc(Zn)-Zincisalsorequiredinmanyenzymeactivitiesandisaddedtomediuminconcentrationssimilartothatofmanganese.Themostcommonforminwhichzincisaddedisasthesulfatesalt.•4.Boron(B)-Boronisanessentialelementinvolvedinligninbiosynthesisandmetabolismofphenolicacidsandissuppliedasboricacidinculturemedium(25-100M).Borondeficiencyresultsinthedeathofshoottipmeristems.刘彤石河子大学生命科学学院10•5.Copper(Cu)-Copperiscriticalinmanyenzymereactions,includingthecytochromeoxidasesystem.Itisaddedtoculturemedium(ascupricsulfate)inverylowconcentrations(0.1M),becausehighamountscanbetoxic.•6.Molybdenum(Mo)-Molybdenumfunctionsinthetransformationofnitratetoammonium.Itisaddedassodiummolybdateinlowconcentrations(1M)inculturemedium.•7.Cobalt(Co)-Cobaltisnotconsideredtobeanessentialmineralbyplantphysiologists,butisincludedinmanyofthemostwidelyusedmediaformulations.Cobaltissuppliedinconcentrationssimilartothatofcopper,againbecauseitmaybetoxicathigherconcentrations.•8.Iodine(I)-Iodineisnotconsideredtobeanessentialelement,butitisoftenaddedtoplantculturemedia(5M)becauseithasbeenfoundtoimprovegrowthofrootsandcallusinvitro.刘彤石河子大学生命科学学院11II.OrganicCompounds•Organiccompoundsarealsoaddedtoplantculturemedium.Someofthesecompounds,suchassugars,areabsolutelyneededforgrowth,whileothers,suchasvitamins,undefinedcompounds,andorganicacids,maynotbeessentialbutmayenhancegrowth.刘彤石河子大学生命科学学院12A.Sugars•Mostplanttissueculturesarenothighlyautotrophic,thatis,capableoffixingcarbonthroughphotosynthesis,duetolimitationsincultureofCO2availability,amongotherfactors.Therefore,sugarisaddedtothemediumasanenergysource.Sucroseisthemostcommonsugaradded,althoughglucose,fructose,andsorbitolarealsousedincertaininstances.Sucroseisthesugarformmostcommonlytransportedinplants;itisbrokendownintoglucoseandfructoseduringmetabolism.Itisalsopartiallyhydrolyzedintoglucoseandfructoseduringautoclaving.Theconcentrationofsugarsinnutrientmediagene