学习植物组织培养技术Learn plant tissue culture techniques.docx

学习植物组织培养技术Learn plant tissue culture techniques.docx

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学习植物组织培养技术Learnplanttissueculturetechniques

学习植物组织培养技术（Learnplanttissueculturetechniques）

I.teachingaims

1.understandthebasicprinciples,objectives,requirementsandmethodsofplanttissueculturetechniques.

2.mastertheoperationskillsofplanttissueculture.

Two,referencematerials

Mediumpreparation,acommonmediuminplanttissueculture,istheMSmedium.ThepreparationoftheMSmediumcomprisesthefollowingsteps.

PreparationandpreservationofMSculturemediumcontainingnearly30kindsofnutritionalcomponentsofbaseliquor,inordertoavoideverymediumpreparationshouldbecarriedoutontheweighingdozensofingredients,canbeculturedinthemediumofvariouscomponents,accordingtotheoriginalamountof20timesor200timesrespectivelyweighing,withconcentratedliquid,thisconcentratedsolutioniscalledculturebaseliquor.Inthisway,eachuse,takethetotalamountof1/20（50mL）or1/200（5mL）,dilutewithwater,intotheculturemedium.Thequantitiesofvarioussubstancesrequiredforthepreparationofthemotherliquidoftheculturemediumarelistedforuseinpreparation.

Alargenumberofelements（motherliquorI）mg/L

NH4NO333000

KNO338000

CaCl22H2O8800

MgSO47H2O7400

KH2PO43400

Traceelements（MotherLiquorII）

KI166

H3BO31240

MnSO44H2O4460

ZnSO47H2O1720

Na2MoO42H2O50

CuSO45H2O5

CoCl26H2O5

Ironsalt（motherliquorIII）

FeSO47H2O5560

Na2-EDTA2H2O7460

Organicingredients（motherliquorIV）

IVA

Inositol20000

IVB

Niacin100

Pyridoxinehydrochloride（vitaminB6）100

Thiaminehydrochloride（vitaminB1）100

Glycine400

Theamountofallthenutrientsmentionedabovewas200timesofconcentratedliquidexceptformotherliquor,whichwas20timesconcentratedliquid.

Eachofthesemotherliquidsshallbeindividuallyseparatedinto1Lstocksolution.Thepreparationmethodofmotherliquor,liquorandliquorare:

IIIVweighingseveralcomponentseachinthemotherliquorafterrespectivelywithdistilledwatertodissolveasmallamount,andthentheyareimmiscible,thefinalvolumeto1L.

Thepreparationmethodis:

LiquorIIIwillbecalledFeSO47H2OandNa2-EDTA2H2Orespectivelyin450mLdistilledwater,heatingandstirringtodissolvethem,thenthetwosolutionsaremixed,andwillbeadjustedtopH5.5,thefinalvolumeto1L,storedinbrownglassbottle.

Afterallthemotherliquorhasbeenprepared,theyarestoredinglassbottles,labeledwithmother'snumbers,preparationtimes,datesandsoon.Theyarekeptintherefrigerator.

MSmediumadding24-,twochlorophenoxyaceticacid（2,4-D）,NAA,6-BA（NAA）6-（6BA）andotherplantgrowthregulators,andrespectivelywithmotherliquor（0.1mg/mL）.Thepreparationmethodis:

the3substanceswerecollectedevery10mg,2,4-DandNAA（1mL）withasmallamountofethanolpredissolving,6BA（1mL）withasmallamountofsubstanceisdissolvedinNaOHsolutionfor0.1mol/L,thedissolutionprocessrequiresconstantvolumeheatinginawaterbath,atlastto100mL,theconcentrationof0.1mg/mLsolution.

Preparationofliquidorusingapipettetoremovetherequiredamountfromavarietyofmotherliquorrespectively:

motherliquorwas50mL,II,IIIandIVliquorAandIVB5mL.Take24-D,5mL,1NAAmL,andallkindsofliquortogetherintoabeaker.

Shouldpayattentiontopreparationofmedium:

intheuseofadvancepreparationofmotherliquor,shouldtakeallkindsofliquorintheamountbefore,gentlyshakethebottlecontainingmotherliquor,iffoundabottleofprecipitation,suspendedormicrobialcontaminationshouldbeimmediatelyeliminated,themotherliquor,repreparation;usingapipetteortheamountoftrainingbaseliquorbefore,mustbetakenbythevolumeofthecylinderorpipetteliquorGuanRunxi2times;theamountofliquor,theliquorwillbeallthebestaccordingtotheamountoftheorderwrittenonpaper,Volume1,crossed1,inordertoavoidmistakes.

Meltagarwithcoarsebalancewereweighed,steamedsugaragar9g30g1000mLintotheenamelcup,thenadd750mLofdistilledwater,withelectricheating,heatingandstirringwithaglassroduntiltranslucentliquid.Thenaddthepreparedmixturetotheboilingagar,andfinallyaddthedistilledwaterto1000mL,stirringevenly.

Itisimportanttonotethattheoperatormustnotleavetheagarwhenitisheatedandthemediumisprepared,otherwiseboilingagaroverflows,

Itneedstobereweighedandprepared.Inaddition,ifthereisnoenamelcupsavailableinsteadofbeaker.Butweshouldpayattentiontotheoutersurfaceofthebottomofthebeakercannottouchwater,otherwisewhenheatingthesolutionbeakereasilycracked,overflow,scald.

PHwiththedropperabsorbamountofsubstanceconcentrationofNaOHsolutionwas1mol/L,eachmediumdropsintothemelt,whiledropswhilestirring,andatanytimewiththepHtestprecision（5.4~7）measuringthepHofthemedium,untilthepHofthemediumwas5.8sofar（thepHofthemediummustbestrictlycontrolin5.8）.

Mediumshouldbepackedseparatelywhenheated.Whenloading,themediumispouredintothebeakerandthemediuminthebeakerispouredintoaconicalflask（50mLor100mL）.Becarefulnottoleavetheculturemediumtothemouthofthebottleandthewallofthebottle.Theamountofthemediuminaconicalflaskisabout1/5to1/4ofthecapacityofaconicalflask.Each1000mLmediumcanbedividedinto25~30bottles.

Afterthemediumispacked,thebottlemouthshouldbesealedintime.2piecesofparchmentpaper（eachaboutthesizeof9cm*9cm）sandwichedbetween1thinlayersofkraftpapercoverthebottle,andbinding.Finally,labeltheouterconeoftheconicalflask.

Autoclavingofhigh-pressuresterilizationmediuminvolvesthefollowingsteps.

First,puttheflask.Aconicalflaskcontainingaculturemediumisplacedinasmallmetalbasketandplacedinaautoclave.Ifthereisnosmallmetalbasket,itcanbeseparatedbyaglassplatebetweenthetwoconicalbottles.

Secondplaceotheritemsthatneedtobesterilized.Otheritemsalsoneedsterilizationinhighpressuresteamsterilizationpot,suchasconicalflask,filledwithdistilledwaterwithascrewtopglassbottle,beaker,jar（theaboveitemsaretobewrappedinkraftpaper,kraftpapercoatedwithsealing）Petridishes,scissors,tweezers,scalpel,filterpaperandpenciletc..

Third,sterilization.Tobesterilizeditemsstackedaftercover.At98kPaand121.3DEGC,20minwassterilized.

Aftersterilization,removetheconicalflaskandallowthemediumtocoolandsolidifynaturally.Betterplace1dbeforeusing.

FumigationsterilizationinoculationboxwillbePotassiumPermanganateinaglassorceramiccontainer,andthenpourtheformaldehyde.Twotheamountisusually6~8percubicmeterofspacewiththemassfractionofmLis16%ofFormaldehydeSolution（knownasFaureMarin40%）,4~5gPotassiumPermanganate.Thefumigationmethodisirritatingtotherespiratorytractandeyes,sotheoperatorshouldleaveimmediatelyafterthemixturehasbeenmixed.

Whentheexplantsarerequiredtousecarrotrootsasexplants,thecarrotsshouldbewashedandpeeledbeforebeingsterilized,andbeleftinthemiddleandcutintosmallpieces.Whenthechrysanthemumisusedasexplants,itsyoungleavesandtenderstemsarerecommended.Theexplantsmustpassthroughthesurfacedisinfectantbeforeuse,inadditiontotheuseofsurfacedisinfectantsodiumhypochlorite,canalsobeusedwithmassfractionof9%~10%calciumhypochloritesolution5~30min,orwiththevolumefractionof10%~12%hydrogenperoxidetreatment5~15minsterilization.

Vaccinationrequirementsandprecautions

Theexplantswerepreparedundersterileconditionsandthesterilizedexperimentalmaterialswerecutintosmallpiecestoprepareexplants.Forexample,preparationmethodofcarrotrootis:

withascalpelshortofcarrotrootaroundtheleftlayertoformpartoftheintermediatezone,cutinto1cm*1cmslices.Asanotherexample,preparationmethodis:

CutChrysanthemumpetiole,leafveinsfromthecut,thencutinto3~4mm2needstocutsmallpieces;tendersteminternodesbetweentwosections,about1cm.Theexcisedexplantsshouldbeputintosterileculturedishes.

Attentionshouldbepaidtotheinoculation.First,eachinoculumshouldbedisinfected1timesinanalcoholicsolutionof75%alcohol,andthenburnedontheflameofanalcohollamp,thencooledandtheninoculated.Notethatthealcoholstainedtweezerswaituntilthealcoholevaporatesandthenburnonthealcohollightflame.

Second,explantsshouldbeleveledwhenplacedinculturemedium.Thenumberofexplantsplacedineachbottleshouldbedeterminedaccordingtothesizeofthecone.Generallyplaced3~4carrots,6~8stemsorleavesofchrysanthemum.Notethatexplantsarenotplacedtoosmalltotakefulladvantageofthenutrientsintheculturemedium.

Third,thealcoholusedforvaccination,theflameshouldnotbetransferredtoohigh,vaccinationshouldbeclosetothealcohollampflameoperation,vaccinationspeed.

Fourth,vaccinationtopreventfireinthebox.

CultureofcalliandPlantlets

Calluscultureusuallytakes2weeksfrominoculationofexplantstotheemergenceofcallus.Youcanseetheexplantgrowgraduallynodularcallusofwhiteoryellowishwhitefor2weeks.Whencallusisfirstcultivated,thedooroftheincubatorshouldbeclosedwithoutlight,becausethecallusgrowsfasterwithoutlight.2weekslater,

Sincethenutrientcontentintheculturemediumisnearlyexhausted,itisnecessarytoreplacethemediumforsubculture.

Thesubculturemediumforcallussubculturewasthesameasthemediumusedforcallusculture.U