plant in vitro culture
TRANSCRIPT
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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
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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.