PlantTissueculture,
Micropropaga3on
ofPitahaya,Dragonfruit.
PlantTissueCulture
•  Micropropaga3onistheproduc3onof
wholeplantsfromsmallsec3onsofplant
suchasastem3p,node,meristem,
embryo,orevenaseed
•  Plant3ssuecultureisbasicallythesame
thing,exceptthatitimpliestheuseof
callus3ssuegeneratedfromplantcells
culturedin-vitro.
•  Micropropaga3onandplant3ssuecultureareused
toproducelargenumbersofplantsfromsmallpieces
ofthestockplantinrela3velyshortperiodsof3me.
•  Whydoesmicropropaga3on
work?
•  Plantcellshavetheabilityto
reproducethewholeplant
fromsinglecells.Thisiscalled
to3potency.
•  To3potencyistheabilityofa
singlecelltoexpressthefull
genomeinthecellstowhich
itgivesrisebycelldivision.
PlantTissueCulture
Whydowedoit?
•  Toregenerateplantsfromsinglecellsor
plant3ssues
•  Tocul3vateundersterilecondi3ons(=in
vitro)
•  Toproducelargequan33esofiden3cal
plants
•  Tocul3vatewithouttheimpactof
environmentalcondi3ons
PlantTissueCulture
•  Tosavespeciesfromex3nc3on
•  Toisolatediseasefromplants
•  Toproduceplantswithenhancedstressor
pestresistance
•  Tocreatenewplantvarie3es
•  Tomakemoney
Plant Tissue Culture
•  TheBasicsofMicropropaga3on
•  TheFourstages
•  Stageone–Explantestablishmentorini3a3on
•  Stagetwo–Mul3plica3on
•  Stagethree–Roo3ng
•  Stagefour–Acclima3za3onorhardeningoff
Whatpartoftheplantcanbeusedfor
micropropaga3on?
• Virtuallyanypartofaplantcanbeused.
• Meristems,shootandroot3ps,leaf3ssue,
anthers,embryos,flowers,virtuallyallparts
ofaplantcanbeused.
Surface Disinfesta3on
•  Typically!0%BleachSolu3onfor20minutes.
•  SurfactantoWen3mesu3lized.
•  Cantreatwithan3oxidantsolu3on.
•  Othermaterialscanbeused.
•  ABalancingactnottodamaging3ssuewhileatthe
same3megeYnga“clean”culture.
•  Endophy3corganisms?Dr.Mar3nStoner.
Plant Tissue Culture
•  Meristema3c3ssue-whichareundifferen3atedcellsfrom
shootandroot3psthathavenotbeenprogrammedfor
theirul3matedevelopment
•  Parenchymacells–themostcommontypeofplantcell,
whichcanregenerateanddifferen3atetoini3atethe
growthofnewandvaried3ssueandorgans
•  Adven33ousgrowthisthedevelopmentofnewshoots,
buds,roots,orleavesfromatypicalorunusualloca3ons
Plant Tissue Culture
•  Alimita3onisthateachplantispropagated
differentlyandnoteveryplantwillrespondthe
sameway.
•  Eachgenus,speciesandvarietymayrequirea
different3ssuewhichwillobtainthebestresults.
•  Differentplantsmayrequireverydifferentmedia
aswellasenvironmentalcondi3ons.
•  Someplantsarebe\ersuitedfora3ssueculture
systemthanothers.
Plant Tissue Culture
•  TissueCulture/Micropropaga3onhastwogeneral
formsofplantletproduc3on.
• Callusproduc3on
• MicrocuYngs
Plant Tissue Culture
•  Callusproduc3onisthe
dedifferen3a3onofaplant
cellintocallus.
•  Callusisthenexpandedinto
alargermassof
undifferen3atedcells.
•  Callusisthenac3vated,by
selec3veuseofplant
hormonestodifferen3ate
toproduce,shoots,roots
andul3mately,plantlets
Micro
cuttings
Somehow, this culture is
Growing underneath the
Surface of the medium!
What a survivor.
This one is by far the
Largest shoot so far.
Major Cons3tuents
•  SaltMixtures
•  OrganicSubstances
•  NaturalComplexes
•  GrowthRegulators
•  InertSuppor3veMaterials
Macro-nutrient salts
•  NH4NO3 Ammoniumnitrate
•  KNO3
Potassiumnitrate
•  CaCl2-2H2O
Calciumchloride(Anhydrous)
•  MgSO4-7H2OMagnesiumsulfide(EpsomSalts)
•  KH2PO4 •  ToomuchNH4+maycausevitrifica3on,butisneededfor
embryogenesisands3mulatesadven33ousshootforma3on
Micro-nutrient salts
•  FeNaEDTAor(Na2EDTAandFeSo4)
•  H3BO3
BoricAcid
•  MnSO4-4H2O Manganesesulfate
•  ZnSO4-7H2O Zincsulfate
•  KI Potassiumiodide
•  Na2MoO4-2H2OSodiummolybdate
•  CuSO4-5H2O Cupricsulfate
•  CoCl2-H2O
Cobaltchloride
2. Organic Compounds
• CarbonSources
• 1.Sucrose(1.5to12%,3%Ireferencepoint)
• 2.Glucose(Some3mesusedwithmonocots)
• 3.Fructose
• WhiteVitamins
• -Thiamine 1.0mg/l
• -Nico3nicAcidandPyroxidine 0.5mg/l
• -Glycine 2.0mg/l
• VitaminC(an3oxidant)100.0mg/l
Organic compounds cont.
•  AminoAcidsandAmides
•  AminoacidscanbeusedasthesolesourceofNitrogenbutnormally
tooexpensive
•  2aminoacidsmostcommonlyused
•  L-tyrosine,enhancesadven33ousshootform.
•  L.Glutamine,mayenhanceadven33ousembryogenesis
Salt Mixtures
•  M.S.(MurashigiandSkoog)
•  Gamborg
•  NitschandNitsch(SimilartoM.S.)
•  White
•  Knudson
Salt Mixtures
•  M.S.(MurashigiandSkoog)
•  Gamborg
•  NitschandNitsch(SimilartoM.S.)
•  White
•  Knudson
Natural Complexes, Can Tissue Culture be
done Organically?
•  -Coconutendosperm
•  -Fishemulsion
•  -Proteinhydrolysates
•  -Tomatojuice
•  -Yeastextracts
•  -Potatoagar
Plant Growth Regulators/Hormones
•  Auxins
•  IAA
•  IBA
•  NAA
•  2,4-D
•  Cytokinins
•  BAPorBA
•  Kine3n
•  TDZ
•  Zea3n
Growth regulators
•  -auxin
•  -cytokinin
•  -gibberellin
•  -abscisicacid
•  -ethylene
Gelling Agents
•  -Agar-extractfromMarineredagar
•  -Phytagar
•  -Taiyo
•  -Difco-Bacto
•  -TCAgar
•  -Agarose
•  -Hydrogels
•  -Gela3n
•  Gelrite
Equipment, Sterile Condi3ons
•  Autoclave
•  LaminarFlowHood
Growing Area