plant in vitro culture

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PLANT BIOTECHNOLOGY

http://biotechnology4u.com/plant_biotechnology.html

PLANTINVITROCULTURE

INTRODUCTION

Animportantaspectofallbiotechnologyprocessesisthecultureofeithertheplant

cellsoranimalcellsormicroorganisms.Thecellsinculturecanbeusedfor

recombinantDNAtechnology,geneticmanipulations,amongothers.

Plantcellcultureisbasedontheuniquepropertyofthecelltotipotency.CELL

TOTIPOTENCYistheabilityoftheplantcelltoregenerateintowholeplant.This

propertyof

the

plant

cells

has

been

exploited

to

regenerate

plant

cells

under

the

laboratoryconditionsusingartificialnutrientmediums.Withtheadvancesmadein

geneticengineering,itbecamepossibletointroduceforeigngenesintocellandtissue

culturesystems.ThisledtothedevelopmentofGENETICALLYMODIFIED(GM)OR

TRANSGENICCROPSwhichhadimprovedtraitsandcharacteristics.

Historyofcellculture

Intheearly19thcentury,SchleidenandSchwannproposedtheconceptofthe'cell

theory'.In1902,GottliebHaberlandt,thegermanbotanistandregardedasthefather

ofplant

tissue

culture,

first

attempted

to

cultivate

the

mechanically

isolated

plant

leaf

cellsonasimplenutrientmedium.Hedidnotsucceedinachievingthegrowthand

differentiationoftheculturedcells;however,hepredictedtheconceptofgrowth

hormones,theuseofembryosacfluids,thecultivationofartificialembryosfrom

somaticcells,etc.

Duringtheperiod1902 1930,attemptsweremadetoculturetheisolatedplant

organssuchasrootsandshootapices(organculture).Hanning(1904)isolated

embryosofsomecrucifersandsuccessfullygrewonmineralsaltsandsugarsolutions.

Simon(1908)successfullyregeneratedabulkycallus,buds,androotsfromapoplar

treeonthesurfaceofmediumcontainingIAAwhichinducedcelldivision.Gautheret,

Whiteand

Nobecourt

(1934

1940)

largely

contributed

to

the

developments

made

in

planttissueculture.White(1939)culturedtobaccotumortissuefromthehybrid

Nicotianaglauca,andN.Langsdorffii.

Theperiodof1940 1970ssawthedevelopmentofsuitablenutrientmediatoculture

planttissues,embryos,anthers,pollen,cellsandprotoplasts,andtheregenerationof

completeplants(invitromorphogenesis)fromculturedtissuesandcells.In1941,van

Overbekandcoworkersusedcoconutmilk(embryosacfluid)forembryo

developmentandcallusformationinDatura.StewardandReinert(1959)first

discoveredsomaticembryoproductioninvitro.MaheswariandGuha(1964)

developedtheantherculturefortheproductionofhaploidplants.SkoogandMiller

(1957)advanced

the

hypothesis

of

organogenesis

in

cultured

callus

by

varying

the

ratio

ofauxinandcytokinininthegrowthmedium.Muir(1953)developedasuccessful

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techniqueforthecultureofsingleisolatedcellswhichiscommonlyknownaspaper

raftnursetechnique(placingasinglecellonfilterpaperkeptonanactivelygrowing

nursetissue).In1952,thePfizerInc.,NewYork(U.S.A)gottheUSpatentandstarted

producingindustriallythesecondarymetabolitesofplants.Thefirstcommercial

productionofanaturalproductshikoninbycellsuspensionculturewasobtained.

In1980s

using

Genetic

engineering,

for

the

first

time,

it

was

possible

to

introduce

foreigngenesintocellandtissueculturesystemstodevelopplantswithimproved

characteristics(transgeniccrops)whichmaycontributetothepathtowardsthe

secondgreenrevolution.

PLANTCELLANDTISSUECULTURETECHNIQUES

Thewholeplantscanberegeneratedvirtuallyfromanyplantpart(referredtoas

explants)orcells.

Planttissue

culture

techniques

involve

the

following

steps:

a)Preparationandselectionofsuitablenutrientmedia

b)Selectionofexplantssuchasshoottip.

c)Surfacesterilizationoftheexplantsbydisinfectantse.g.sodiumorcalcium

hypochloritesolution0.3 0.6%)followedbywashingtheexplantswithsterile

distilledwater.

d)InoculationorTransferoftheexplantsontothesuitablenutrientmedium(sterilized

byautoclaving)inculturevesselsundersterileconditions(usinglaminarflowhood).

e)Incubationorgrowingtheculturesinthegrowthchamberorplanttissueculture

roomatoptimumphysicalconditionsoflight(16hoursofphotoperiod),diurnal

illumination,temperature

(25+/

20C

and

relative

humidity

(50%

60%).

f)Regenerationofplantsfromculturedplanttissues.

g)Hardening:itisthegradualexposureofplantletsforacclimatizationto

environmentalconditions

h)Transferofplantstothefieldconditionsfollowingtheacclimatization/hardeningof

theregeneratedplants.

NUTRIENTMEDIA

Theintactplantscanmaketheirownfoodbuttheinvitrocultureofplantpartsorcells

requiresavariety

of

nutrients

and

suitable

physical

conditions

for

their

growth.

The

compositionofplanttissueculturemediumdependsuponthetypeofplanttissuesor

cellsthatareusedforculture.Nosinglemediumcanbeusedforalltypesofplantsand

organs,sothecompositionoftheculturemediumforeachplantmaterialhastobe

workedout.

Atypicalnutrientmediumconsistsofthefollowingcomponents:

a) Inorganicnutrients(bothmicro andmacroelements C,H,O,N,P,S,Ca,K,Mg,

Fe,Mn,Cu,Zn,B,Mb),thesixelementsnamelynitrogen,phosphorus,potassium,

calcium,magnesium

and

sulfur

are

the

essential

macronutrients

for

tissue

culture.

Theidealconcentrationofnitrogen,andpotassiumisaround25mmoll1whilefor

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calcium,phosphorus,sulfurandmagnesium,itisintherangeof13mmoll1.

Amongthemicronutrients,ironrequirementisverycritical.Chelatedformaofiron

andcopperarecommonlyusedinculturemedia.

b) Acarbonsourceandenergysource(usuallysucrose)) Plantcellsandtissuesinthe

culturemediumareheterotrophicandthereforedependontheexternalcarbonfor

energy.Among

the

various

energy

sources,

sucrose

is

the

most

preferred.

During

thesterilizationofthemedium,sucrosegetshydrolysedtoglucoseandfructose

andtheplantcellsutilizefirsttheglucoseandthenthefructose.Theother

carbohydratessuchaslactose,maltose,galactoseetchavebeenusedinculture

mediabutwithlimitedsuccess.

c) Organicsupplementsvitamins(e.g.nicotinicacid,thiamine,pyridoxineandmyo

inositol),aminoacids(e.g.arginine)Theplantcellsincultureareabletosynthesize

vitaminsjustlikenaturalplants,butinsuboptimalquantitieswhichdoesnot

supportpropergrowthofcellsinculture.Thereforethemediumissupplemented

withvitamins

to

achieve

good

growth

of

cells.

Similarly

amino

acids

are

added

to

thecellculturestostimulatethecellgrowthandestabilishthecelllines.Organic

acidsespeciallytheintermediatesofkrebscyclee.g.citrate,malate,succinate,

pyruvatealsoenhancesthegrowthofplantcells.Sometimesantibiotics(e.g.

streptomycin,kanamycin)arealsoaddedtothemediumtopreventthegrowthof

themicroorganisms.

d) Growthregulators(e.g.auxins,cytokininsandgibberellinsPlanthormonesplayan

importantroleingrowthanddifferentiationofculturedcellsandtissues.The

growthhormonesincludedinculturemediainvolve:auxins,cytokinins,and

gibberellins.The

auxins

facilitate

the

cell

division

and

root

differentiation.

The

cytokininsinducecelldivisionanddifferentiationandthegibberellinsismainlyused

toinduceplantletformationfromadventiveembryosformedinculture.

Auxinsinducecelldivision,cellelongation,andformationofcallusincultures.2,4

dichlorophenoxyaceticacidisoneofthemostcommonlyaddedauxinsinplantcell

cultures.Cytokinins,promotesRNAsynthesisandstimulateproteinandenzyme

activitiesintissues.Kinetinandbenzylaminopurinearethemostfrequentlyused

cytokininsinplantcellcultures.Theratioofauxinsandcytokininsplayanimportant

roleinthemorphogenesisofculturesystems.Whentheratioofauxinstocytokinins

ishigh,embryogenesis,callusinitiation,androotinitiationoccur.Foraxillaryand

shootproliferation,

the

ratio

of

auxins

to

cytokinins

is

kept

low.

Among

the

gibberellins,gibberellinA3(GA3)isthemostcommonlyusedfortissueculture.GA3

enhancescallusgrowthandinducesdwarfplantletstoelongate.

e) Solidifyingagentslikeagar.Generallyagellingagentagar(apolysaccharide

obtainedfromredalgae,Gelidiumamansil)isaddedtotheliquidmediumforits

solidification..Theagarobtainedfromseaweedsprovidessolidsurfaceforthe

growthofcellsbecauseintheliquidmedium,thetissuewillbesubmergedanddie

duetolackofoxygen.Cellsaregrowninsuspensionmediumwithoutagarbutsuch

culturesareaeratedregularlyeitherbybubblingsterileairorbygentleagitation.

Some

other

less

frequently

used

solidifying

agents

are

biogel

(polyacrlyamide

pellets),phytagel,gelrite,andpurifiedagarose.

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f) Othercompoundslikecaseinhydrolysate,coconutmilk,maltextract,yeast

extract,tomatojuice,etc.maybeaddedforspecificpurposes.

g) pH AnoptimumpH(usually5.7)isalsoveryimportant.AtpHhigherthan7.0and

lowerthan4.5,theplantcellsstopgrowingincultures.

Themost

extensively

used

nutrient

medium

is

MS

medium

(developed

by

MurashigeandSkoogin1962).

MAJORTYPESOFMEDIA

Whitesmedium isoneoftheearliestplanttissueculturemedia

MSmedium formulatedbyMurashigeandSkoog(MS)ismostwidelyusedfor

manytypesofculturesystems

B5medium developedbyGamborgforcellsuspensionandcallusculturesand

atpresentitsmodifiedformusedforprotoplastculture

N6

medium

formulated

by

Chu

and

used

for

cereal

anther

culture

NitschsmediumdevelopedbyNitschandNitschandusedforantherculture

PREPARATIONOFMEDIA

Themethodologyformediapreparationinvolvespreparationofstocksolutions(inthe

rangeof10xto100xconcentrations)ofhighlypurifiedchemicalsanddemineralized

water.Thestocksolutionsarestoredinglassorplasticcontainersandfrozentill

furtherrequirement.Nowdays,planttissueculturemediaarecommerciallyprepared,

andareavailableinthemarketasdrypowders.Theculturemediaisusuallysterilized

inanautoclaveat1210Cand15psifor20minutes.Hormonesandotherheatsensitive

organic

compounds

is

filter

sterilized

and

added

to

the

autoclaved

medium.

MAINTENANCEOFASEPTICENVIRONMENT

Itisveryimportanttomaintainasepticenvironmentduringtheinvitrocultureofplant

cellsandtissues.Followingaresomeofthemethodsadoptedforsterilization:

(a)SterilizationofGlassware Theglasswarecanbesterilizedinahotairovenat160

1800Cfor24hours.

(b)Sterilizationofinstruments Themetallicinstrumentsareincineratedbydipping

them

in

75%

ethanol

followed

by

flaming

and

cooling.

(c)Sterilizationofnutrientmedia Theculturemediaaretransferredintoglass

container,pluggedwithcottonorsealedwithplasticclosuresandsterilizedby

autoclavingat15psifor30min.Theautoclavingdenaturesthevitamins,plant

extracts,aminoacidsandhormonesthereforethesolutionofthesecompoundsare

sterilizedbyusingMilliporefilterpaperwithporesizeof0.2micrometerdiameter.

(d)Sterilizationofplantmaterials Thesurfaceoftheplantmaterialismadesterileby

usingdisinfectantse.g.sodiumhypochlorite,hydrogenperoxide,mercuricchloride,or

ethanol.Thetransferofsterileplantmaterialontothenutrientmediumisdoneunder

thecabinetoflaminarairflow.

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(e)SterilizationofCultureroomandtransferarea thefloorandwallsoftheculture

roomshouldbewashedwithdetergentfollowedby2%sodiumhypochloriteor95%

ethanol.ThesterilizationcanalsobedonebyexposuretoUVlight.Thecabinetof

laminarairflowissterilizedbyexposingtoUVlightfor30min.and95%ethanol15

minutesbefore

starting

the

work.

TYPESOFCULTURE

OrganCulture

Itdealswiththecultureoftheisolatedorgans(roots)underlaboratoryconditions(in

vitro).Differentnamesaregivendependingupontheorganusedfortheculture.For

instancethecultureofroots,endosperm,ovary,andovulearecalledasrootculture,

endospermculture,ovarycultureetc.ItwasSkoog(1944),whoforthefirsttime

suggested

that

the

organogenesis

could

be

chemically

controlled.

Skoog

and

Miller

(1957)alsodemonstratedthatahighratioofauxin:cytokininstimulatedtheformation

ofrootintobaccocallus,butalowratioofthesameinducedshootinformation.

Explantculture

Thecultureofplantparts(explants)isknownasexplantculture.Theexplantscanbe

anypartoftheplante.g.thepieceofstem,leaf,hypocotyl,etc.Theexplantcultures

aregenerallyusedtoinducecallusorplantregeneration.

Callusculture

Callusreferstoanunorganizedmassofcellsgenerallyparenchymatousinnature.

Theuniquefeatureofcallusisthattheabnormalgrowthhasbiologicalpotentialto

developnormalroot,shoots,andembryoids,ultimatelyformingaplant.Naturally,the

callusisformedduetotheinfectionofmicroorganismsfromwoundsdueto

stimulationbyendogenousgrowthhormones,theauxinsandcytokinins.However,it

hasbeenpossibletoartificiallydevelopcallusbyusingtissueculturetechniques.

Auxinsareaddedtoculturemediumforcallusinductionbutthenatureandquantityof

auxinadded,

depends

on

the

nature

and

source

of

explant

and

its

genotype

besides

otherfactors.Callusculturescanbemaintainedforprolongedperiodsbyrepeated

subculturing.Callusculturesareusedfora)plantregeneration,b)preparationof

singlecellsuspensionsandprotoplasts,and,c)genetictransformationstudies.

FACTORSAFFECTINGCALLUSCULTURE

thesourceandthegenotypeoftheexplant

compositionofthemedium(mostcommonlyusedMSmedium)

temperature(22280Csuitableforcallusformation)

growthregulatorse.g.auxins,cytokininsaloneorcombinationofthese.

Age

of

the

plant

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Locationoftheexplant

Physiologyandgrowthconditionoftheplant

Cellsuspension

cultures

Cellsuspensionispreparedbytransferringafragmentofcallustotheliquidmedium

andagitatingthemasepticallytomakethecellsfree.

Singlecellscanbeisolatedfromeithercallusoranyotherpartoftheplantand

culturedinliquidmediumusingbothmechanicalandenzymaticmethods.Mechanical

methodsinvolvegrindingofthetissuetoafinesuspensioninabufferedmedium

followedbyfiltrationandcentrifugationtogetridofcelldebris.Theenzymaticmethod

usestheenzymes(pectinaseormacerozyme)todissolvethemiddlelamellabetween

thecells.Aftertheisolationofthecells,theyareculturedbybatchculturesor

continuouscultures.

As

the

medium

is

liquid

in

nature,

the

pieces

of

callus

remain

submergedwhichcreatesanaerobicconditions.Toovercomethisproblem,the

suspensionculturesareagitatedbyarotaryshakerwhichdispersesthecellsand

exposethemtoair.

Theadvantagesofcellsuspensionculturesoverthecallusculture:

a)Thesuspensioncanbepipetted.

b)Theyarelessheterogeneousandcelldifferentiationislesspronounced.

c)Theycanbeculturedinvolumesupto1,500liters.

d)Theycanbesubjectedtomorestringentenvironmentalcontrols.

e)Themanipulationsfortheproductionofnaturalproductsbyfeedingprecursors,is

possible.

Batchculturesareinitiatedassinglecellsin100 250mlflasksandarepropagatedby

transferringregularlysmallaliquotsofsuspensiontoafreshmedium.Continuous

culturesaremaintainedinasteadystateforlongperiodbydrainingouttheused

mediumandaddingfreshmedium.

Thecellsuspensionculturescanbeusedfora)inductionofsomaticembryos/shoots,

b)invitromutagenesisandmutantselection,c)genetictransformation,d)production

ofsecondarymetabolites.

Masscellculture

Plantcellsareculturedinspeciallydesigned'plantbioreactors'whichessentiallydo

nothaveastirrerasplantcellsareshearsensitive.Inplaceofstirrer,gasisgently

bubbledwhichprovidesstirringaswellasmeetthedemandofahigheroxygensupply.

Protoplastculture

Protoplastsareplantcellswithoutcellwallandcanbeisolatedbyusingenzymeslike

cellulases,pectinases)fromleaf,seedling,calli,pollengrains,embryosacsetc.The

protoplastsregenerate

cell

wall,

undergo

cell

division,

and

form

callus.

The

callus

can

alsobesubcultured.Someoftheexamplesofplantspeciesthathavebeen

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regeneratedfromprotoplastsare Cucumissativus,Capsicumannum,Ipomoea

batata,Glycinemax,Chrysanthemumsp.Theseculturesareusedfora)various

biochemicalandmetabolicstudies,b)fusionoftwosomaticcellstocreatesomatic

hybrids,c)fusionofenucleatedandnucleatedprotoplaststocreateCybrids

(cytoplasmichybrids),d)geneticmanipulation,ande)drugsensitivitytests.

Bergmannscellplatingtechnique(cultureofsinglecells)

Inthistechnique,freecellsaresuspendedinaliquidmedium.Equalvolumesofliquid

andagarmediaaremixedandrapidlyspreadinpetridish,whichmakesthecells

evenlydistributedinathinlayeraftersolidification.AftersealingthePetridisheswith

parafilm,theyareexaminedundertheinvertedmicroscopetomarkthesinglecells.

Platesareincubatedindarkat250Candcellcoloniesdevelopingfrommarkedsingle

cells,areusedtoobtainsinglecellcultures.

EMBRYOCULTURE

Besides,roots,shoots,andpollen,embryoscanalsobeculturedtoproducehaploid

plants.Theembryocultureisveryusefulinconditionswhereembryofailstodevelop

duetodegenerationofembryonictissues.Ithasbeenusedasaroutinetechniquein

orchidpropagation,inbreedingofspeciesshowingdormancy.

EMBRYORESCUE

Ithasbeenobservedthatsometimes,inspiteofsuccessfulpollinationandfertilization,

theembryosdonotdevelop.Theincompatibilitybetweentheembryoandthe

endospermorsomeinherentdeficiencyalsoresultsintheunderdevelopmentof

embryo.These

immature

embryos

can

be

dissected

out

from

the

seeds

and

can

be

grownartificiallyonculturemedium.Theseembryosdifferentiateintoshoot,rootand

plantletsundercultureconditions.Thistechniqueofgrowingimmatureembryois

termedasembryorescue.Thistechniqueisveryusefulinhybridization,breaking

dormancyofcertainseeds,andtoachievecompletegrowthofembryointoaplant.

ANTHERANDPOLLENCULTURE(PRODUCTIONOFHAPLOIDPLANTS)

Haploidplantspossessasinglesetofchromosomes(gametophyticnoofchromosomes

i.e.n)inthesporophyteincontrasttodiploidswhichcontaintwosetsofchromosomes

(2n).TheexistenceofhaploidplantswasreportedbyBergner(1921)inDatura

stramonium.Tulecke(1951)culturedthepollengrainsofGinkobiloba(gymnosperm)

andsucceededininducingthedevelopmentofhaploidcallus.GuhaandMaheswari

(1964)reportedthedirectdevelopmentofhaploidembryosandplantletsfrom

microsporesofDaturainoxiabytheculturesofexcisedanthers.In1967,Bourginand

HitschobtainedthefirstfullhaploidplantsfromNicotianatabacum.

Haploidplantsareveryusefulin:

a)Directscreeningofrecessivemutationbecauseindiploidorpolyploidscreeningof

recessivemutationisimpossible.

b)Development

of

homozygous

lines

in

ashort

span

of

time.

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c)Thegenerationofexclusivemaleplantsbytheprocessofandrogenesisusing

techniquestodoublethechromosomenumbers.

d)Productionofdiseaseresistantplantsbyintroducingdiseaseresistantgenese.g.

BarleyaccessionQ21681resistanttostemrust,leafrust,andpowderymildew.

e)Cytogeneticresearchwhichincludesproductionofaneuploids,determinationof

originand

basic

number

of

chromosome

numbers,

induction

of

genetic

variability.

Atpresent,morethan247plantspeciesandhybridsbelongingto38generaand34

familiesofdicotsandmonocotshavebeenregeneratedusingantherculturetechnique

e.g.rice,wheat,maize,coconut,rubbertreesetc.TheInstituteofCropBreedingand

Cultivation(China)hasdevelopedthehighyieldingandblastresistantvarietiesofrice

zhonghuaNo.8andzhonghuaNo.9throughtransferofdesiredaliengene.

ANDROGENESIS

Inandrogenesis,themalegametophyte(themicrosporeorimmaturepollen)produces

haploidplantsbystoppingthedevelopmentofpollencellintoagameteandforcingit

todevelop

into

ahaploid

plant.

INVITROANDROGENESIS

Invitroandrogenesisistheformationofsporophytefromthemalegametophyteon

artificialmediumandismostcommonlyfoundinfamilySolanaceaeandPoaceae

(Graminae).

DIRECTANDROGENESIS

In

the

pollen

derived

embryogenesis,

also

called

direct

androgenesis,

the

pollen

directlyactsasazygoteandpassesthroughvariousembryogenicstagessimilarto

zygoticembryogenesis.Directandrogenesisisverycommoninmanyplantsofthe

familySolanaceaeandBrassicaceae.

INDIRECTANDROGENESIS

Theprocesswherethepollengrainsinsteadofnormalembryogenesis,divide

erraticallytodevelopcallusiscalledindirectandrogenesise.g.inbarley,wheat,Coffee

etc.

THETECHNIQUE

OF

ANTHER

CULTURE

Theantherswiththeirfilamentsareremovedfromtheflowerbudsaftersurface

sterilization.Underasepticconditions,theanthersareexcisedandcrushedin1%

acetocarminetotestthestageofpollendevelopment.Theanthersincorrectstageof

developmentareseparatedandinoculatedonanutrientmedium.Theanthercultures

aremaintainedat280Candalternatingphotoperiodsoflight(1218hrs)anddarkness

(612hrs).Theanthersproliferateandproducecalluswhichformsanembryoandthe

embryosubsequentlydevelopsintoahaploidplant.

THETECHNIQUE

OF

POLLEN

CULTURE

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Thepollensareextractedbypressingandsqueezingtheantherswithaglassrod

againstthesidesofthebeaker.Theanthertissuedebrisisremovedbyfilteringthe

pollensuspensionandlargeandhealthypollenarewashedandcollected.Thesepollen

areculturedonasolidorliquidmediumandthecallusortheembryoformedis

transferredtoasuitablemediumtoproduceahaploidplant.

Thefactorsthataffectandrogenesisarethegenotypeandthephysiologicalstateof

thedonorplants,compositionoftheculturemedium,themethodadoptedinthe

pretreatmentofantherstodevelopintohaploidplantsandthestageofthe

microsporeorpollenselectedforculture.

Thehaploidareidentifiedbylookingattheirmorphologicalfeaturesorbyusing

geneticmarkerse.g.a1markerforbrowncoloredaleuronetodetecthaploidsinmaize

plant.

LIMITATIONSIN

HAPLOID

PRODUCTION

a)Duetothelowfrequencyofhaploidproductiontheselectionisverydifficult.

b)Haploidswithdeleterioustraitsfrequentlydevelopincultures.

c)Itissometimesdifficulttoisolatehaploidsfromtheculturesincethepolyploids

outgrowhaploids.

d)Thedoublingofhaploids(diploidization)doesnotalwaysleadtotheformationof

homozygousplant.

e)Theembryosderivedfromhaploidsoftengetaborted.

REGENERATION

PATHWAYS

OF

PLANTS

Theplantscanberegeneratedbya)organogenesisandb)somaticembryogenesis.

Organogenesisreferstotheformationoforgansfromtheculturedexplants.Theshoot

budsstructuresareformedbymanipulatingtheratioofcytokinintoauxininthe

cultures.

InSomaticembryogenesis,thetotipotentcellsmayundergoembryogenicpathwayto

formsomaticembryoswhicharegrowntoregenerateintocompleteplants.Itwas

demonstratedforthefirsttimeincarrots(Daucuscarota),wherebipolarembryos

developedfrom

single

cells.

The

somatic

embryogenesis

is

influenced

by

plant

extracts,

growthregulators,andbythephysiologicalstateofcalli.

APPLICATIONSOFCELLANDTISSUECULTURE

Micropropagation/ClonalPropagation

Clonalpropagationreferstotheprocessofasexualreproductionbymultiplicationof

geneticallyidenticalcopiesofindividualplants.Thevegetativepropagationofplantsis

labor

intensive,

low

in

productivity

and

seasonal.

The

tissue

culture

methods

of

plant

propagation,knownasmicropropagation,utilizethecultureofapicalshoots,axillary

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budsandmeristemsonsuitablenutrientmedium.Theregenerationofplantletsin

culturedtissuewasdescribedbyMurashigein1974.Fossard(1987)gaveadetailed

accountofstagesofmicropropagation.

Themicropropagationisrapidandhasbeenadoptedforcommercializationof

importantplantssuchasbanana,apple,pears,strawberry,cardamom,many

ornamentals(e.g.

Orchids)

and

other

plants.

The

micro

propagation

techniques

are

preferredovertheconventionalasexualpropagationmethodsbecauseofthe

followingreasons:(a)Inthemicropropagationmethod,onlyasmallamountoftissue

isrequiredtoregeneratemillionsofclonalplantsinayear.,(b)micropropagationis

alsousedasamethodtodevelopresistanceinmanyspecies.,(c)invitrostockcanbe

quicklyproliferatedasitisseasonindependent,(d)longtermstorageofvaluable

germplasmpossible.

Thestepsinmicropropagationmethodare:a)Initiationofculture fromanexplant

likeshoottiponasuitablenutrientmedium,b)multipleshootsformationfromthe

culturedexplant,

c)

rooting

of

in

vitro

developed

shoots

and,

d)

transplantation

transplantationtothefieldfollowingacclimatization.

Thefactorsthataffectmicropropagationare:(a)genotypeandthephysiological

statusoftheplante.g.plantswithvigorousgerminationaremoresuitableformicro

propagation,(b)theculturemediumandthecultureenvironmentlikelight,

temperatureetc.Forexampleanilluminationof16hoursadayand8hoursnightis

satisfactoryforshootproliferationandatemperatureof250Cisoptimalforthe

growth.

Thebenefitsofmicropropagationthismethodare:

a)rapidmultiplicationofsuperiorclonescanbecarriedoutthroughouttheyear,

irrespectiveofseasonalvariations.

b)multiplicationofdiseasefreeplantse.g.virusfreeplantsofsweetpotato(Ipomea

batatus),cassava(Manihotesculenta)

c)multiplicationofsexuallyderivedsterilehybrids

d)Itisacosteffectiveprocessasitrequiresminimumgrowingspace.

Somaclonalvariation

The

genetic

variations

found

in

the

in

vitro

cultured

cells

are

collectively

referred

to

as

somaclonalvariationandtheplantsderivedfromsuchcellsarecalledassomaclones.

Ithasbeenobservedthatthelongtermcallusandcellsuspensioncultureandplants

regeneratedfromsuchculturesareoftenassociatedwithchromosomalvariations.Itis

thispropertyofculturedcellsthatfindspotentialapplicationinthecropimprovement

andintheproductionofmutantsandvariants(e.g.diseaseresistanceinpotato).

LarkinandScowcroft(1981)workingatthedivisionofPlantIndustry,C.S.I.R.O.,

Australiagavetheterm'somaclones'forplantvariantsobtainedfromtissueculturesof

somatictissues.Similarly,ifthetissuefromwhichthevariantshavebeenobtainedis

havinggametophyticoriginsuchaspollenoreggcell,itisknownas'gametoclonal'

variation.Theyexplainedthatitmaybedueto:(a)reflectionofheterogeneitybetween

thecellsandexplanttissue,(b)asimplerepresentationofspontaneousmutationrate,

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and(c)activationbycultureenvironmentoftranspositionofgeneticmaterials.

Shepardetal.(1980)alsocontributedbyscreeningabout100somaclonesproduced

fromleafprotoplastsofRussetBurbank.Theyfoundthattherewasasignificant

amountofstablevariationincompactnessofgrowthhabit,maturity,date,tuber

uniformity,tuberskincolourandphotoperiodicrequirements.

SomaclonalVariationshasbeenusedinplantbreedingprogrammeswherethegenetic

variationswithdesiredorimprovedcharactersareintroducedintotheplantsandnew

varietiesarecreatedthatcanexhibitdiseaseresistance,improvedqualityandyieldin

plantslikecereals,legumes,oilseedstubercropsetc.Somaclonalvariationis

applicableforseed

APPLICATIONSOFSOMACLONALVARIATIONS

a)Methodologyofintroducingsomaclonalvariationsissimplerandeasieras

comparedto

recombinant

DNA

technology.

b) Developmentandproductionofplantswithdiseaseresistancee.g.rice,wheat,

apple,tomatoetc.

c) Developbiochemicalmutantswithabioticstressresistancee.g.aluminium

toleranceincarrot,salttoleranceintobaccoandmaize.

d) Developmentofsomaclonalvariantswithherbicideresistancee.g.tobacco

resistanttosulfonylurea

e) Developmentofseedswithimprovedqualitye.g.anewvarietyofLathyrus

sativaseeds(LathyrusBioL212)withlowcontentofneurotoxin.

f) Bio13AsomaclonalvariantofCitronellajava(with37%moreoiland39%more

citronellon),amedicinal

plant

has

been

released

as

Bio

13

for

commercial

cultivationbyCentralInstituteforMedicinalandAromaticPlants(CIMAP),Lucknow,

India.

g) Supertomatoes HeinzCo.andDNAplantTechnologyLaboratories(USA)developed

supertomatoeswithhighsolidcomponentbyscreeningsomacloneswhichhelpedin

reducingtheshippingandprocessingcosts.

Productionofvirusfreeplants

Theviraldiseasesinplantstransfereasilyandlowerthequalityandyieldoftheplants.

It

is

very

difficult

to

treat

and

cure

the

virus

infected

plants

therefore

te

plant

breeders

arealwaysinterestedindevelopingandgrowingvirusfreeplants.

Insomecropslikeornamentalplants,ithasbecomepossibletoproducevirusfree

plantsthroughtissuecultureatthecommerciallevel.Thisisdonebyregenerating

plantsfromculturedtissuesderivedfroma)virusfreeplants,b)meristemswhichare

generallyfreeofinfection Intheeliminationofthevirus,thesizeofthemeristem

usedinculturesplayaverycriticalrolebecausemostofthevirusesexistby

establishingagradientinplanttissues.Theregenerationofvirusfreeplantsthrough

culturesisinverselyproportionaltothesizeofthemeristemused.,c)meristems

treatedwithheatshock(34360C)toinactivatethevirus,d)callus,whichisusually

virusfreelikemeristems.e)chemicaltreatmentofthemedia attemptshavebeen

madetoeradicatethevirusesfrominfectedplantsbytreatingtheculturemedium

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withchemicalse.g.additionofcytokininssuppressedthemultiplicationofcertain

viruses.

Amongtheculturetechniques,meristemtipcultureisthemostreliablemethodfor

virusandotherpathogenelimination.

Viruseshave

been

eliminated

from

anumber

of

economically

important

plant

species

whichhasresultedinasignificantincreaseintheyieldandproductione.g.potatovirus

Xfrompotato,mosaicvirusfromcassavaetc.Thesevirusfreeplantsarenotdisease

resistantsothereisaneedtomaintainstockplantstomultiplyvirusfreeplants

wheneverrequired.

Productionofsyntheticseeds

Insyntheticseeds,thesomaticembryosareencapsulatedinasuitablematrix(e.g.

sodiumalginate),alongwithsubstanceslikemycorrhizae,insecticides,fungicidesand

herbicides.These

artificial

seeds

can

be

utilized

for

the

rapid

and

mass

propagation

of

desiredplantspeciesaswellashybridvarieties.Themajorbenefitsofsyntheticseeds

are:

a)Theycanbestoreduptoayearwithoutlossofviability

b)Easytohandleandusefulasunitsofdelivery

c)Canbedirectlysowninthesoillikenaturalseedsanddonotneedacclimatizationin

greenhouse.

Mutantselection

Animportant

use

of

cell

cultures

is

in

mutant

selection

in

relation

to

crop

improvement.Thefrequencyofmutationscanbeincreasedseveralfoldthrough

mutagenictreatmentsandmillionsofcellscanbescreened.Alargenumberofreports

areavailablewheremutantshavebeenselectedatcellularlevel.Thecellsareoften

selecteddirectlybyaddingthetoxicsubstanceagainstwhichresistanceissoughtin

themutantcells.Usingthismethod,celllinesresistanttoaminoacidanalogues,

antibiotics,herbicides,fungaltoxinsetchaveactuallybeenisolated.

Productionofsecondarymetabolites

The

most

important

chemicals

produced

using

cell

culture

are

secondary

metabolites,

whicharedefinedasthosecellconstituentswhicharenotessentialforsurvival.

Thesesecondarymetabolitesincludealkaloids,glycosides(steroidsandphenolics),

terpenoids,latex,tanninsetc.Ithasbeenobservedthatasthecellsundergo

morphologicaldifferentiationandmaturationduringplantgrowth,someofthecells

specializetoproducesecondarymetabolites.Theinvitroproductionofsecondary

metabolitesismuchhigherfromdifferentiatedtissueswhencomparedtonon

differentiatedtissues.

Thecellculturescontributeinseveralwaystotheproductionofnaturalproducts.

Theseare:(a)anewrouteofsynthesistoestablishproductse.g.codeine,quinine,

pyrethroids,(b)arouteofsynthesistoanovelproductfromplantsdifficulttogrowor

establishe.g.thebainfromPapaverbracteatum,(c)asourceofnovelchemicalsintheir

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ownrighte.g.rutacultinfromcultureofRuta,(d)asbiotransformationsystemseither

ontheirownoraspartofalargerchemicalprocesse.g.digoxinsynthesis.

Theadvantagesofinvitroproductionofsecondarymetabolites

a)The

cell

cultures

and

cell

growth

are

easily

controlled

in

order

to

facilitate

improved

productformation.

b)Therecoveryoftheproductiseasy.

c)Asthecellculturesystemsareindependentofenvironmentalfactors,seasonal

variations,pestandmicrobialdiseases,geographicallocationconstraints,itiseasy

toincreasetheproductionoftherequiredmetabolite.

d)Mutantcelllinescanbedevelopedfortheproductionofnovelandcommercially

usefulcompounds.

e)Compoundsareproducedundercontrolledconditionsasperthemarketdemands.

f)Theproductiontimeislessandcosteffectiveduetominimallaborinvolved.

APPLICATIONSOFSECONDARYMETABOLITES

Manyofthesesecondaryproductsespeciallyvariousalkaloidsareofimmenseusein

medicine.Theyieldofthesechemicalsincellcultureisthoughgenerallylowerthanin

wholeplants,itissubstantiallyincreasedbymanipulatingphysiologicaland

biochemicalconditions.

ShikonineisadyeproducedbythecellsLithospermumerythrorhizononacommercial

scale.Besidesthisthereareanumberofsecondarymetaboliteproductsthatarebeing

widelyusedforvariouspurposes.Vincristineisusedasanticanceragent,digoxin

controlscardiovascular

disorders,

pyrithrins

is

an

insecticide

etc.

The

production

of

specialtychemicalsbyplantshasbecomeamultibillionindustry.

Pleaserefertothetableforsomesecondarymetabolitesandtheiruses.

TABLESHOWINGPLANTSPECIESANDSECONDARYMETABOLITESOBTAINEDFROM

THEMUSINGTISSUECULTURETECHNIQUES

Product Plantsource Uses

Artemisin Artemisiaspp. Antimalarial

Azadirachtin

Azadirachta

indica

Insecticidal

Berberine Coptisjaponica Antibacterial,antiinflammatory

Capsaicin Capsicumannum CuresRheumaticpain

Codeine Papaverspp. Analgesic

Camptothecin Campatothecaaccuminata Anticancer

Cephalotaxine Cephalotaxusharringtonia Antitumour

Digoxin Digitalislanata Cardiactonic

Pyrethrin Chrysanthemumcinerariaefolium Insecticide(forgrainstorage)

Morphine Papaversomniferum Analgesic,sedative

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Quinine Cinchonaofficinalis Antimalarial

Taxol Taxusspp. Anticarcinogenic

Vincristine Cathranthusroseus Anticarcinogenic

Scopolamine

Datura

stramonium

Antihypertensive

ProductionofSomatichybridsandcybrids

TheSomaticcellhybridization/parasexualhybridizationorProtoplastfusionoffersan

alternativemethodforobtainingdistanthybridswithdesirabletraitssignificantly

betweenspeciesorgenera,whichcannotbemadetocrossbyconventionalmethodof

sexualhybridization.

SOMATICHYBRIDIZATION

Somatichybridization

broadly

involves

in

vitro

fusion

of

isolated

protoplasts

to

form

a

hybridcellanditssubsequentdevelopmenttoformahybridplant.Theprocess

involves:a)fusionofprotoplasts,(b)Selectionofhybridcells,(c)identificationof

hybridplants.

Duringthelasttwodecades,avarietyoftreatmentshavebeenusedtobringaboutthe

fusionofplantprotoplasts.Protoplastfusioncanbeachievedbyspontaneous,

mechanical,orinducedfusionmethods..Thesetreatmentsincludetheuseoffusogens

likeNaNO3,highpHwithhighCa2++ionconcentration,useofpolyethyleneglycol

(PEG),andelectrofusion.Theseinducingagentsusedinprotoplastfusionarecalled

fusogen.

PEGtreatmentisthemostwidelyusedmethodforprotoplastfusionasithascertain

advantagesoverothers.Theseare:(a)itresultsinareproduciblehighfrequencyof

heterokaryonformation,(b)ThePEGfusionisnonspecificandthereforecanbeused

forawiderangeofplants,(c)Ithaslowtoxicitytothecelland(d)Theformationof

binucleateheterokaryonsislow.

MECHANISMOFFUSION

Thefusionofprotoplaststakesplaceinthreephases agglutination,plasmamembrane

fusionand

formation

of

heterokaryons.

When

the

two

protoplasts

come

in

close

contactwitheachother,theyadheretoeachother.Thisagglutinationcanbeinduced

byPEG,highpHandhighCa2+.Theprotoplastmembranesgetfusedatlocalizedsites

atthepointofadhesion.Thisleadstotheformationofcytoplasmicbridgesbetween

protoplasts.HighpHandhighCa2+ionsneutralizethesurfacechargesonthe

protoplastswhichallowclosercontactandmembranefusionbetweenagglutinated

protoplasts.Thefusedprotoplastsbecomeroundasaresultofcytoplasmicbridges

whichleadstotheformationofsphericalhomokaryonorheterokaryon.

SELECTIONOFHYBRIDCELLS

Themethodsusedfortheselectionofhybridcellsarebiochemical,

visualandcytometricmethodsusingfluorescentdyes.Thebiochemicalmethodsfor

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selectionofhybridcellsarebasedontheuseofbiochemicalcompoundsinthe

medium.Thedrugsensitivitymethodisusefulfortheselectionhybridsoftwoplants

species,ifoneofthemissensitivetoadrug.Anothermethod,auxotrophicmutant

selectionmethodinvolvestheauxotrophswhicharemutantsthatcannotgrowona

minimalmedium.Thereforespecificcompoundsareaddedinthemedium.The

selectionof

auxotropic

mutants

is

possible

only

if

the

hybrid

cells

can

grow

on

a

minimalmedium.Thevisualmethodinvolvestheidentificationofheterokaryons

underthelightmicroscope.Insomeofthesomatichybridizations,thechloroplast

deficientprotoplastofoneplantspeciesisfusedwiththegreenprotoplastofanother

plantspecies.Theheterokaryonsobtainedarebiggerandgreenincolourwhilethe

parentalprotoplastsareeithersmallorcolourless.Thecytometricmethodusesflow

cytometryandflourescentactivatedcellsortingtechniquesfortheanalysisofplant

protoplasts.

APPLICATIONSOFSOMATICHYBRIDIZATION

a)CreationofhybridswithdiseaseresistanceManydiseaseresistancegenes(e.g.

tobaccomosaicvirus,potatovirusX,clubrotdisease)couldbesuccessfully

transferredfromonespeciestoanother.E.gresistancehasbeenintroducedin

tomatoagainstdiseasessuchasTMV,spottedwiltvirusandinsectpests.

b)Environmentaltolerance usingsomatichybridizationthegenesconferring

toleranceforcold,frostandsaltwereintroducedine.g.intomato.

c)Cytoplasmicmalesterility usingcybridizationmethod,itwaspossibletotransfer

cytoplasmicmalesterility.

d)Qualitycharacters somatichybridswithselectivecharacteristicshavebeen

developede.g.

the

production

of

high

nicotine

content.

CHROMOSOMENUMBERINSOMATICHYBRIDS

Thechromosomenumberinthesomatichybridsisgenerallymorethanthetotal

numberofbothoftheparentalprotoplasts.Ifthechromosomenumberinthehybrid

isthesumofthechromosomesofthetwoparentalprotoplasts,thehybridissaidto

besymmetrichybrid.Asymmetrichybridshaveabnormalorwidevariationsinthe

chromosomenumberthantheexacttotaloftwospecies.

In1972,Carlsonandhisassociatesproducedthefirstinterspecificsomatichybrid

between

Nicotiana

glauca

and

N.

langsdorffii.

In

1978,

Melchers

and

his

co

workers

developedthefirstintergeneticsomatichybridsbetweenSolanumtuberosum

(potato)andLycopersiconesculentum(tomato).ThehybridsareknownasPomatoes

orTopatoes.

LIMITATIONSOFSOMATICHYBRIDIZATION

a)Somatichybridizationdoesnotalwaysproduceplantsthatgivefertileandvisible

seeds.

b)Thereisgeneticinstabilityassociatedwithprotoplastculture.

c)Therearelimitationsintheselectionmethodsofhybrids,asmanyofthemarenot

efficient.

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d)Somatichybridizationbetweentwodiploidsresultsintheformationofan

amphidiploidwhichisnotfavourablethereforehaploidprotoplastsare

recommendedinsomatichybridization.

e)Itisnotcertainthataspecificcharacterwillgetexpressedinsomatichybridization.

f)Regeneratedplantsobtainedfromsomatichybridizationareoftenvariabledueto

somaclonalvariations,

chromosomal

elimination,

organelle

segregation

etc.

g)Protoplastfusionbetweendifferentspecies/genusiseasy,buttheproductionof

viablesomatichybridsisnotalwayspossible.

CYBRIDS

Thecytoplasmichybridswherethenucleusisderivedfromonlyoneparentandthe

cytoplasmisderivedfromboththeparentsarereferredtoascybrids.Theprocessof

formationofcybridsiscalledcybridization.Duringtheprocessofcybridizationand

heterokaryonformation,thenucleiarestimulatedtosegregatesothatoneprotoplast

contributestothecytoplasmwhiletheothercontributesnucleusalone.Theirradiation

withgammaraysandXraysanduseofmetabolicinhibitorsmakestheprotoplasts

inactiveand

non

dividing.

Some

of

the

genetic

traits

in

certain

plants

are

cytoplasmicallycontrolled.Thisincludescertaintypesofmalesterility,resistanceto

certainantibioticsandherbicides.Thereforecybridsareimportantforthetransferof

cytoplasmicmalesterility(CMS),antibioticandherbicideresistanceinagriculturally

usefulplants.CybridsofBrassicaraphanusthatcontainnucleusofB.napus,

chloroplastsofatrazincresistantB.capestrisandmalesterilityfromRaphanussativa

havebeendeveloped.

INVITROPLANTGERMPLASMCONSERVATION

Germplasmreferstothesumtotalofallthegenespresentinacropanditsrelated

species.Theconservationofgermplasminvolvesthepreservationofthegenetic

diversityofaparticularplantorgeneticstockforitsuseatanytimeinfuture.Itis

importanttoconservetheendangeredplantsorelsesomeofthevaluablegenetic

traitspresentintheexistingandprimitiveplantswillbelost.Aglobalorganization

InternationalBoardofPlantGeneticResources(IBPGR)hasbeenestablishedfor

germplasmconservationandprovidesnecessarysupportforcollection,conservation

andutilizationofplantgeneticresourcesthroughouttheworld.Thegermplasmis

preservedbythefollowingtwoways:

a)Insituconservation Thegermplasmisconservedinnaturalenvironmentby

establishingbiospherereservessuchasnationalparks,sanctuaries.Thisisusedin

thepreservationoflandplantsinanearnaturalhabitatalongwithseveralwild

types.

(b)Exsituconservation Thismethodisusedforthepreservationofgermplasm

obtainedfromcultivatedandwildplantmaterials.Thegeneticmaterialintheform

ofseedsorinvitroculturesarepreservedandstoredasgenebanksforlongterm

use.

Invivogenebankshavebeenmadetopreservethegeneticresourcesby

conventionalmethodse.g.seeds,vegetativepropagules,etc.Invitrogenebanks

16


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17

havebeenmadetopreservethegeneticresourcesbynon conventionalmethods

suchascellandtissueculturemethods.Thiswillensuretheavailabilityofvaluable

germplasmtobreedertodevelopnewandimprovedvarieties.

Themethodsinvolvedintheinvitroconservationofgermplasmare:

(a)Cryopreservation Incryopreservation(Greekkrayosfrost),thecellsare

preservedinthefrozenstate.Thegermplasmisstoredataverylowtemperature

usingsolidcarbondioxide(at790C),usinglowtemperaturedeepfreezers(at

800C),usingvapournitrogen(at 1500C)andliquidnitrogen(at1960C).Thecells

stayincompletelyinactivestateandthuscanbeconservedforlongperiods.Any

tissuefromaplantcanbeusedforcryopreservatione.g.meristems,embryos,

endosperms,ovules,seeds,culturedplantcells,protoplasts,calluses.Certain

compoundslike DMSO(dimethylsulfoxide),glycerol,ethylene,propylene,sucrose,

mannose,glucose,praline,acetamideetcareaddedduringthecryopreservation.

Theseare

called

cryoprotectants

and

prevent

the

damage

caused

to

cells

(by

freezingorthawing)byreducingthefreezingpointandsupercoolingpointof

water.

(b)ColdStorage Coldstorageisaslowgrowthgermplasmconservationmethod

andconservesthegermplasmatalowandnonfreezingtemperature(190C).The

growthoftheplantmaterialissloweddownincoldstorageincontrasttocomplete

stoppageincryopreservationandthuspreventscryogenicinjuries.Longtermcold

storageissimple,costeffectiveandyieldsgermplasmwithgoodsurvivalrate.Virus

freestrawberryplantscouldbepreservedat100Cforabout6years.Severalgrape

plantshave

been

stored

for

over

15

years

by

using

acold

storage

at

temperature

around90Candtransferringtheminthefreshmediumeveryyear.

(c)Lowpressureandlowoxygenstorage Inlow pressurestorage,theatmospheric

pressuresurroundingtheplantmaterialisreducedandinthelowoxygenstorage,

theoxygenconcentrationisreduced.Theloweredpartialpressurereducesthein

vitrogrowthofplants.Inthelowoxygenstorage,theoxygenconcentrationis

reducedandthepartialpressureofoxygenbelow50mmHgreducesplanttissue

growth.DuetothereducedavailabilityofO2,andreducedproductionofCO2,the

photosyntheticactivityisreducedwhichinhibitstheplanttissuegrowthand

dimension.

This

method

has

also

helped

in

increasing

the

shelf

life

of

many

fruits,

vegetablesandflowers.

Thegermplasmconservationthroughtheconventionalmethodshasseveral

limitationssuchasshortlivedseeds,seeddormancy,seedbornediseases,andhigh

inputsofcostandlabor.Thetechniquesofcryopreservation(freezingcellsandtissues

at1960c)andusingcoldstorageshelpustoovercometheseproblems.

plant in vitro culture

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