plant tissue culture techniques in education

plant tissue culture

1. Plant Tissue Culture
Dr. MadhaviPatel, Associate Professor
ParulInstitute of Pharmacy,
ParulUniversity
4/10/2021

2. Content
Introduction
Concept of Totipotencyand Explant
History
Culture (Nutrient) Media
Types of Culture
Applications and Future prospects
Micropropagation

3. Plant tissue culture (PTC) ?

4. Introduction
Planttissueculture(PTC)
Invitrocultivationofplantcellortissueunderasepticandcontrolled
environmentalcondition,inliquidoronsemisolidwelldefinednutrient
mediumforproductionofprimaryorsecondarymetaboliteorregenerate
plant.

5. Totipotency
❖Theabilityofacelltodifferentiateanddevelopintoawholeplantwhen
giventhecorrectconditions.
❖Thisisbecauseeverycellhasthegeneticpotentialoftheparentplant.

6. Explant
❖Livingtissuetransferred
fromaplanttoanartificial
mediumforculture.
❖Itcanbeanyportionofthe
shoot,leaves,roots,flower
orcellsfromaplant.
❖Sterilizationiscarriedout
forutilization

7. HistoryofPTC
❖AnAustrianbotanist Prof. Gottlieb
Haberlandt(1854–1945):Throughhisinitial
attempttocultureisolatedplantcellsinvitro
providedthefirstexperimentalapproachtothe
fieldofPlantTissueCulturein1902.
❖HereportedtheCultureofleafmesophylltissue
andhaircells.Unfortunatelythecellsthat
Haberlandtcultureddidnotcometosuccess.
Thepossiblereasonforthisfailuremaybelack
ofknowledgeaboutplantgrowthregulators
neededforcelldivisionandembryoinduction.
❖CelltheorybySchwannandSchliden:Cellulartotipotencyforplant
regenerationifprovidedwithproperexternalconditions.

8. Historyof PTC
1902Haberlandt Culturingofsinglecellsisolatedform
planttissuesinsimplenutrientmedia
1904Haning Embryoculture
1934White Establishmentofactivelygrowing
tomatoroots
1937White DiscoveryoftheimportanceofVitamin
Bforthegrowth
1937WentandThimannRoleofAuxininplattissueculture
1939White,Guatheret EstablishmentofCallusculture
1954Muir,Haberlandt EstablishmentofCellSuspension
culture

9. 1960Bergmann Singlecellcloning
1960Cocking Isolationofprotoplasts
1962Murashigaeand
Skoog
Mediumforthegrowthofcallus
1964Morel Micropropagation
1977Street Cultivationofsinglecell
1978Melchersand
Holders
ProductionofSomaticembryo
1983Chilton TransformedTobaccoplants
1994Calgene,USA Productionofnoveltransformedplants
Historyof PTC

10. Themilestonesfortheproductionof2
0
metabolitesfromPTC
Year Milestones
1934 Planttissuecultureobtained(Gautheret)
1939 DiscoveryofAuxin(Gautheret)
1942 Secondarymetabolite(Diosgenin)producedincallus
culture(Gautheret)
1954 Cellsuspensionculture(Muir)
1955 Productionofsecondarymetabolitesincellcultures
(MothesandKala;RoutienandNickell)
1959 Largescalecultivationofplantcells(Tuleckeand
Nickell)
1967 YieldsofPhytopharmaceuticalsequalstointactplant
(KaulandStaba)
1977 Cultivationoftobaccocellsin20,000literbioreactor
(Noguchietal.,)

11. Year Milestones
1979 ImmobilizationofplantcellsbySodiumalginate
(Brodeliusetal.,)
1981 Useofhollowfiberreactorforsecondarymetabolite
Production(Shuler)
1983 Firstplanttissuecultureprocesscommercialized
(Curtin)
1995 Taxolproducedfromplantcellcultureoncommercial
scale
Themilestonesfortheproductionof2
0
metabolitesfromPTC

12. PTC Technique
•Modernplanttissuecultureisperformedunderaseptic
conditionsunderfilteredair.Livingplantmaterialsfromthe
environmentarenaturallycontaminatedontheirsurfaceswith
microorganisms,sosurfacesterilizationofstartingmaterials
(explants)inchemicalsolutions(usuallyalcoholorbleach)is
required.
•Explantsarethenusuallyplacedonthesurfaceofasolidculture
medium,butaresometimesplaceddirectlyintoaliquidmedium
(Suspensionculture).
•Asculturesgrow,piecesaretypicallyslicedoffandtransferredto
newmedia(subcultured)toallowforgrowthortoalterthe
morphologyoftheculture.

13. PTC Technique

14. PTC laboratory
BasicRequirements
▪Equipmentandapparatus
▪Washing andstoragefacilities
▪Mediapreparationroom
▪Sterilizationroom
▪Asepticchamberforculture
▪Cultureroomsorincubatorfullyequippedwithtemperature
light and humiditycontrolleddevice.
▪Observation andrecordingareawithcomputer.

15. GeneralProcedureInvolvedinPTC
Sterilizationofglasswareandtools
Mediapreparationandsterilization
Preparationandsterilizationof explant
Aseptictransfer
Productionofcallusfromexplant
and its proliferation
Subculturingofcallus
Suspensionculture

16. CultureMedia

17. CultureMedia
•Culturemediumisdefinedasaliquidorgelatinoussubstancecontaining
nutrientsinwhichtissuesarecultivatedforscientificpurposes
Culture Media
1. Inorganicessentialelements,ormineralions
a) Macro-nutrients
b) Micro-nutrients
2. Organic supplement
(Growthregulators, Carbohydrates, Vitamins, Amino acids,
Antibiotics)
3. Miscellaneousmediacomponents

18. CultureMedia
1.Inorganicessentialelements,ormineralions
Macro-Nutrients
Nitrogen→Componentofproteins,nucleicacids&somecoenzymes
Potassium→Regulatesosmoticpotential,principalinorganiccation
Calcium→Cellwallsynthesis,membranefunction,cellsignalling
Magnesium→Enzymecofactor,componentofchlorophyll
Phosphorus→Component ofnucleicacids,energytransfer,
componentofintermediatesinrespirationandphotosynthesis
Sulphur→Componentofsomeaminoacids(methionine,cysteine)
andsomecofactors

19. CultureMedia
1.Inorganicessentialelements,ormineralions
Micro-Nutrients
Chlorine→Requiredforphotosynthesis
Manganese→Enzymecofactor
Cobalt→Componentofsomevitamins
Copper→Enzymecofactor,electron-transferreactions
Zinc→Enzymecofactor,chlorophyllbiosynthesis
Molybdenum→Enzymecofactor,componentofnitratereductase
Iron→Electrontransferasacomponentofcytochromes

20. CultureMedia
2. OrganicSupplements
Plant Growth Regulators
Plant Growth regulators Main effects in culture
Auxins: IAA, IBA,
NAA,
2, 4-D
Adventitious root formation (at high concentration)
Induction of somatic embryos (particularly 2, 4-D)
Cell Division
Differentiation of vascular tissue
Callus formation of outgrowth of auxillarybuds
Inhibition of shoot formation
Cytokinins: Stimulation of shoot initiation/bud formation
Inhibition of root formation
Cell division
Callus formation and growth
Promotion of rejuvenation of mature shoots
Stimulation of outgrowth of auxillarybuds
Inhibition of leaf/shoot senescence
Promotion of some stages of root development
Stimulation of the dark-germination of light-development
seeds.

21. CultureMedia
2. OrganicSupplements: Plant Growth Regulators
Plant Growth regulators Main effects in culture
Gibberellins Promotion of internode elongation
Loss of dormancy in seeds, somatic embryos, apical buds
and bulbs
Inhibition of adventitious root formation
Synthesis of inhibitors which promote tuber, corm and
bulb formation
Abscisic acid Maturation of somatic embryos.
Promotion of desiccation tolerance of the somatic embryos
Promotion of the accumulation of storage protein during
embryogenesis.
Bulb and tuber formation.
Promotion of the development of dormancy and
senescence.
Inhibition of elongation.
Ethylene Controlling fruit riping
Promotion of adventitious regeneration depending upon
the species and phase of culture.
Breaking of seed and bud dormancy in some species.
Induction of adventitious roots and root hairs.

22. CultureMedia
2. OrganicSupplements:
❑Carbohydrates: Sucrose, D-Mannitol, Sorbitol
❑Vitamins: B complex, Folic acid, Ascorbic acid, Tocopherols
❑Amino acids: Alanine, Arginine, Asparagine, Cysteine,
Glutamine, Lysine, Tyrosine
❑Antibiotics: Ampicillin, Carbenicillin, Streptomycine(6-
400mg/ltr)

23. CultureMedia
3. Miscellaneousmediacomponents
❑Gelling/solidifying agent: Agar, Gelatin, Alginate
❑Activated charcoal: Added to rooting media to absorb root
inhibiting agents
❑Natural Complexes: Yeast extract, banana and tomato like
fruit juices, malt extract
❑Antioxidants: Citric acid, Ascorbic acid, Pyrogallol,

24. CultureMedia
Additionalmediarequirements
❑Light: Intensity and Period vary depending on plant
❑Temperature: 25
0
C–27
0
C
❑Correct pH: 5.6-6
❑Humidity: 70-75%
❑Oxygen: By agitation or aeration

25. CultureMedia
E.g. Composition of Murashigeand Skoog Medium

26. TypesofCulture
Culturesaregenerallyinitiatedfromsterilepiecesofawholeplant
i.e.explant
Culture
types
Callus
cultures
Cell-
suspensio
n cultures
Microspore
culture
Embryo
culture
Hairy
root
culture
Shoottip
and
Meristem
culture
Root
cultures
Protoplasts
cultures

27. CallusCulture
TypesofCulture
❑Callus is an amorphous aggregate of loosely arranged parenchyma
cells, whichproliferatefrom mother cells.
❑Cultivationof callususually on solidnutrientmediumunder
asepticcondition.
❑The unique feature of callus is its ability to develop normal root
and shoot, ultimately forming a plant.

28. CallusCulture
TypesofCulture
❑It is a good source for the establishmentofsuspensionculture.
Callusisformedthroughthreestagesofdevelopment.
•Induction, Celldivision, Celldifferentiation.
❑Forthemaintenanceofgrowthincultureitbecomesnecessaryto
subculturethecallusintoafreshmedium.
Callustissuemayappearofthedifferentcolours
•White:Ifgrownindarkand duetoabsentofchlorophyll.
•Green:Ifgrowninlight.
•Yellow:More carotenoid pigment development
•Purple:duetotheaccumulationof cyaninsinvacuole.
•Brown:Excretionofphenolicsubstance& formationof quinones.

29. SuspensionCulture
TypesofCulture
•Suspensionculturecontainsauniform
suspensionofseparatecellsinliquidmedium.
•Forthepreparationofsuspensionculture,
callusfragmentsistransferredtoliquid
medium(withoutagar),whichisagitated
continuoustokeepthecellsseparate.
•Agitationcanbeachievedbyrotaryshaker
systemattachedwithintheBODincubatorata
rateof50-150rpm.
•Aftersufficientnumberofcellsareproduced,
subculturingcanbedoneinfreshliquid
medium.
•Thegrowthofsuspensioncultureishigherthancallusculture,and
thereforeitrequiresrapidsubculture(7-21days)ascomparedto
callusculture(4-8weeks).

30. ProtoplastCulture
TypesofCulture
•Protoplastisacellwithoutcellwallwhicharecultivatedinliquid
aswellsolidmedium.
•Inplants,thecellsareinvariablysurroundedbythickcellwall
madeofcelluloseandpectin.
•Duringtheprocessoftissuecultureusingitforcropimprovement,
cellwallactsasabarriertoinducemutationsorsometimesto
developthecallus.
•Insuchsituations,cellwallisdissolvedbytheuseofcellulaseand
pectinaseandanakedprotoplastcanbefreed.
•Suchprotoplastitselfcanbedevelopedintoindividualplantsor
theycouldbeusedtofusewithnakedprotoplastofothervarietiesor
speciestoproducewhatarecalledassomaticembryo.

31. ProtoplastCulture
TypesofCulture

32. TypesofCulture
ProtoplastCulture
Products obtained from protoplast culture

33. Root culture
TypesofCulture
•Rootculturescanbeestablishedinvitrofromexplantsoftheroottip
ofeitherprimaryorlateralrootsandcanbeculturedonfairlysimple
media.
•Thegrowthofrootsinvitroispotentiallyunlimited,asrootsare
indeterminateorgans.

34. Shoot tip and meristem culture
TypesofCulture
•Thetipsofshoots(whichcontaintheshootapicalmeristem)canbe
culturedinvitro,producingclumpsofshootsfromeitheraxillaryor
adventitiousbuds.
•Thismethodcanbeusedforclonalpropagation.
•Shootmeristemculturesarepotentialalternativestothemore
commonlyusedmethodsforcerealregenerationastheyareless
genotype-dependentandmoreefficient.

35. Micropropagation
•Micropropagationisthepracticeofrapidlymultiplyingstockplant
materialtoproducealargenumberofprogenyplants,usingmodern
planttissueculturemethods.
•Microproopagationisafielddealingwiththeabilitytoregenerate
directlyfromexplants.
•Micropropagationisusedtomultiplynovelplants,suchasthosethat
havebeengeneticallymodifiedorbredthroughconventionalplant
breedingmethods.
•Itisalsousedtoprovideasufficientnumberofplantletsforplanting
fromastockplantwhichdoesnotproduceseeds,ordoesnotrespond
welltovegetativereproduction.
•Thispropagationisnowcommonlyacceptedandadoptedwidely
withlotifsignificanceincommercialhorticulture.
Technique

36. Micropropagationsteps

37. WhyPlantTissueCulture?
❖Toproduceofexactcopiesofplantsthatproducegoodflowers,fruitsor
desirabletraits.
❖Naturalcompoundproductionundercontrolledasepticcondition
independentofsoilandclimaticcondition.
❖Tostudybiogenesisof secondarymetabolites:It is
possible tofeed labeledprecursortocellcultureand
predictmetabolicpathwayofdesiredcompound.
❖NaturalproductsfromPTCcaneasilypurified-absenceofsignificant
amountofpigmentsandotherundesirableimpurities.
❖ByPTC itispossibletoproducethosechemical
compoundwhichareimpossibletosynthesis.

38. WhyPlantTissueCulture?
❖Toquicklyproducematureplants.
❖Theproductionofmultiplesofplantsintheabsenceofseeds.
❖Theregenerationofwholeplantsfromcellsthathavebeen
genetically modified.
❖To produceplantswithreducedchancesof transmittingdiseases
and pathogens.
❖Toproduceplantswhichhaveverylowchancesofgermination.

39. ApplicationsofPTC
❑ProductionofPhytopharmaceuticals
❑Betterqualityofplant
❑Micropropagation
❑ProductionofImmobilizedPlantcells.

40. FutureProspects
1.Pharmaceuticalfoodstuffs;oralvaccinationwithtransgenic
plants
2.Plantbiotechnologyandgenemanipulation
3.Useofplantrootsforenvironmentalremediationandchemical
manufacturing
4.Signalpathwaysinplantdiseaseresistance
5.Towardssustainablecropsviainternationalcooperation
6.Achievementsandopportunitiesinplantbiotechnologyand
micropropagation.Theuseofliquidimmersionsystemand
largescalemicropropagationformanymorespecies
7.Genetransfertechnologyforgeneticengineeringofplants.
8.Somaticembryogenesisandsyntheticseedtechnology

41. Any questions?