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Plant Tissue Culture

1. PLANTTISSUECULTURE
Dr.RohiniC.Kolhe
Assist.prof.
Departmentofpharmacognosy

2. PLANTTISSUECULTURE
•Planttissuecultureisacollectionof
techniquesusedtomaintainorgrow
plantcells,tissuesororgansunder
sterileconditionsonanutrientculture
mediumofknowncomposition.
•Planttissuecultureiswidelyusedto
produceclonesofaplantinamethod
knownasmicropropagation.

3. HISTORICALDEVELOPMENTOF
PLANTTISSUECULTURE

5. AdvantagesofPlantTissue
Culture
•Theproductionofexactcopiesofplants
thatproduceparticularlygoodflowers,
fruits.
•Toquicklyproducematureplants.
•Theproductionofmultiplesofplantsin
theabsenceofseeds.
•Theregenerationofwholeplantsfrom
plantcellsthathavebeengenetically
modified

6. •Theproductionofplantsinsterilecontainers
thatallowsthemtobemovedwithgreatly
reducedchancesoftransmittingdiseases,pests,
andpathogens.
•Theproductionofplantsfromseedsthat
otherwisehaveverylowchancesof
germinatingandgrowing,i.e.:orchidsand
.
•Toclearparticularplantsofviralandother
infectionsandtoquicklymultiplytheseplants
as'cleanedstock'forhorticultureand
agriculture.

7. EXPLANT
•Anypartofaplanttakenoutand
grownintesttubeundersterile
conditionsinspecialnutrientmedia
iscalledexplant.

8. METHODSOFPLANTTISSUE
CULTURE
Planttissuecultureincludestwomajor
methods:
•(A)Typeofinvitrogrowth-callusand
suspensioncultures.
•(B)Typeofexplant—singlecellculture,shoot
androotcultures,somaticembryoculture,
meristemculture,anthercultureandhaploid
production,protoplastcultureandsomatic
hybridisation,embryoculture,ovuleculture,
ovaryculture,etc.

9. Environmental
Conditions
•Therearethreeimportantaspectsin
vitroculture
1)Nutrientmedium
2)Asepticcondition
3)Aerationofthetissue

10. NutrientMedium
•Mediumdependsuponthetypeofplant
tissueorcellusedforculture
•Generallynutrientconsistof
inorganicsalts(bothmicro&macroelements)
acarbonsource(usuallysucrose)
Vitamins(eg.nicotinicacid,thiamine,pyridoxine
Aminoacids(eg.arginine)
Growthregulators(eg.auxins)
•AnoptimumpH(5.7)isalsovaryimportant

11. Aseptic
Condition
•Nutrientmediumcontainssugarwhich
increasesgrowthofmicrobes
•Thesemicrobescompetewithgrowing
tissueandfinallykillit.
•Itisimportanttomaintainaseptic
condition.
•Sterilizationisveryimportanttostopthe
growthofmicrobes.

12. Aerationofthe
Tissue
•Properaerationoftheculturedtissueis
alsoanimportantaspectofculture
technique.
•Itisachievedbyoccasionallystirringthe
mediumbystirringorbyautomaticshaker.

13. TypesofPlant
Tissue
Culture

16. CallusCulture
•InCallus culture,celldivisionin
explantforms acallus.
•Callusisirregularunorganizedand
undifferentiatedmassofactivelydividingcells.
•Darkness&solidmediumgelledbyagar
stimulatescallusformation.
•ThemediumcontainstheauxinsandBAP
(Benzylaminopurines).Botharegrowth
regulators
(Hormones).
•Thisstimulatescelldivisioninexplant.
•Callusisobtainedwithin2-3weeks.

17. CalluscultureandSub
culture

18. Maintenanc
e
•Aftersufficienttimeofcallusgrowthon
thesamemediumfollowingchangeswill
occur:
❑Depletionofnutrientinthemedium
❑Graduallossofwater
❑Accumulationofmetabolictoxins
•Henceformaintenanceofgrowthincallus
itisnecessarytosubculturethecallus.
•Subcultureshouldberepeatedafter4-5
weeks

19. CellSuspensionCulture
•Asstatedearlier,cellsderivedfroma
singlecellthroughmitosisconstitutea
cloneandtheprocessofobtainingclones
iscalledcloning(asexualprogenyofa
singleindividualmakeupaclone).

32. Roottipculture
•Tipsofthelateralrootsaresterilized,excised
andtransferredtofreshmedium.
•Thelateralrootscontinuetogrowand
provideseveralroots.
•After7daysthatareusedtoinitiatestockor
experiment.
•Thustherootmaterialderivedfromasingle
radical.
•Suchgeneticallyuniformrootculturesare
referredtoasacloneofisolatedroots.

33. Leaves
culture
•Leaves(800µm)maybedetachedfrom
shoots,surfacesterilizedandplacein
healthyconditionforlongperiod.
•Growthrateintheculturedependsontheir
stagesofmaturityatexcision.
•Youngleaveshavemoregrowthpotential
thenthenearlymatureones.

34. Shoottip
culture
•Theexcisedshoottips(100-1000µm
long)ofmanyplantspeciescanbe
culturedonrelativelysimplenutrient
media.
•Thismediamustcontainsgrowth
hormonesandwilloftenformrootsand
developintowholeplants.

35. Completeflower
culture
•Flowers(2daysafterpollination)are
excised,sterilizedbyimmersionin5%
calciumhypochloride,washedwith
sterilizedwater.
•Transferthistoculturetubescontaining
anagarmedium.
•Fruits,whichdeveloparesmallerthan
theirnaturalcounterpart,sizecanbe
increasesbysupplementingthemedium
withappropriatecombinationofgrowth
hormones.

36. AntherCulture
•Youngflowerbudsareremovedfromthe
plant&surfacesterilized.
•Theanthersarethenexcisedand
transferredtoanappropriatenutrient
medium.
•Theplantletareformedafter4-5weeksof
inoculation.
•Manyplantletsareproducedfromthe
singleanther.

37. Pollens
culture
•Pollengrainsareremovedfromtheanther.
•Anthersareplacedina5mlliquidmedium
inpetridish.
•Petridishescontainingthepollengrainsin
theculturemediaaresealedwithparafilm
&incubatedat28ºCindarkfor14days.
•3-4weeksmayberequiredtoobtain
haploidplantlets.

41. Applicationoftissue
culture
•Tissuecultureisusedtoconservetherare
speciesintheforest.
•Aplantbreedermayusetissuecultureto
screencellsratherthanplants.
•Large-scalegrowthofplantcellsinliquid
cultureasasourceofsecondaryproducts,
likerecombinantproteinsusedas
biopharmaceuticals.

42. •Tocrossdistantlyrelatedspeciesby
protoplastfusion.
•Helpincropimprovement.
•Creationofadditionalgeneticvariation.
•Selectionofplantsresistanttotoxins,
virusesetc.
•Suitableforbreedingoftreespecies.
•Productionofmanyplantsthatareclone
toeachother.
•Productionofdiseasefreeplants.

43. •Tissueculturehaveaadvantageoftime
sever,
Withthehelpofrootcultureplantsareready
togrowwhichsavetime.

44. POLYPLOID
•Polyploidyreferstowhenanorganismhas
morethantwocompletesetsofchromosomes.
•Thisconditionisfrequentlyfoundinplants.
Polyploidscanbedividedintotypesbasedonthe
numberofchromosomestheycarry
1.Triploids(threesets)
2.Tetraploids(foursets)
3.Pentaploids(fivesets)
4.Hexaploids(sixsets)
5.Octoploids(eightsets)
6.Decaploids(tensets)
7.Dodecaploids(twelvesets)

46. ImportanceofPolyploids
1)Identifygeneticoriginofcrops.
2)Generatenewplantgenotypesand
species.
3)Enableintrogressionofgenesfrom
relatedspecies.
4)Selffertilization.
5)Heterosis(crossbredplantspecieshavingsuperior
qualitiesthanthoseofeitherofitsparents).
6)Completetheno.ofchromosomes
havingunevensetsofchromosomes.

47. 7)Aplantmaybemadesterilebydevelopinga
triploid-fertiletetraploidsanddiploidsare
hybridizedtogettriploids.
7)Polyploidycanbeusedtoobtainseedlessfruits,
suchastheseedlesswatermelon.Anotherexample
ofsuccessfulpolyploidyisthebanana,whichisa
triploid,seedlessandsterile.
SeedlessWatermelon

48. 7)Aswithhybrids,polyploidyhavebeen
observedtobebiggerandmorerobustthan
theirdiploidparents.Thismayincludebigger
leaves,flowersandfruits,aswellasan
increaseinheight.Anexampleisthe
strawberryplant,diploidvarietiesof
strawberryhavesmallerfruitwithlessyields,
whereasthetetraploidvarietieshavebigger
andbetteryields.Thepolyploidscanalsobe
morediseaseandpestresistant.

50. 8)Itexhibitsusefuleffectsonmedicinalplantslike
digitalis,mentha,plantswithtropanealkaloidsetc.

51. Mutation
•Mutationistheinheritablechangeinthe
charactersoforganisms.
•Mutationcanoccurspontaneouslyinnature
ortheymaybeinducedexperimentally.
•Mutationcanbebeneficialorharmfultoan
individual.

54. ImportanceofMutation
•Evolutionofnewandbetterfoodplants
havinggooddesirablecharacterssuchas
higheryieldandgreaterresistance.
•Productionofnewspecies.
•Agriculturalrevolution.
•Mutationbreedingoftenprovidesolutionfor
somespecificplantbreedingproblems.
•Canproduceimprovedvarietyofplantsthan
thetraditionalvariety.
•Lesstimeconsuming.

55. •Improvementingeneticcharacterssothat
thenewspeciesgivedesirableeffects.
•Toincreasecropyield.

57. Hybridization
•Hybridizationistheprocessofcrossingtwogenetically
differentindividualstoresultinathirdindividualwitha
differentone.
•Planthybridizationistheprocessofcrossbreedingbetween
geneticallydissimilarparentstoproduceahybrid.It
frequentlyresultsinpolyploidoffspring.

58. TYPESOFHYBRIDIZATION
Basedonthetaxonomicrelationshipoftheparents
involvedinhybridization,itisoftwotypes.
1.Intervarietal/IntraspecificHybridization-Whenparents
areofthesamespeciesthatmaybetwostrains,
varietiesorracesofthesamespecies.Thisismost
commonlyusedincropimprovement.Thistypeof
hybridizationmaybesimpleorcrossdependson
numberofparentsinvolved.
2.DistantHybridization-Herecrossesaremadebetween
differentspeciesofthesamegenusorofdifferent
genera.Interspecificcrossinvolvedcrossingbetween
twodifferentspeciesofthesamegenus,whereas,
intergenericcrossmeansthecrossbetweentwo
differentgenera.Generallythismethodisusedto
transferoneorfewsimplyinheritedcharacterslike
diseaseresistancetoacropspecies.

62. APPLICATIONOF
PLANTTISSUECULTURE
INPHARMACOGNOSY

69. EDIBLEVACCINES

73. Theconceptofediblevaccineswas
developedbyArntzeninthe1990s.