MitosisinOnionRootTips

Takenfrom:http://www.huntington.org/Education/FIBR/download/2004/04PlantCell.pdf
Objectives
Youwillfix,stain,andmakeslidesofonionroottips.Theseslideswillbestudiedforthe
presenceofcellsinthefourstagesofmitosis.
Atthecompletionofthislaboratoryyoushouldbeableto
Staintissuefortheidentificationofcellsinthevariousstagesofmitosis
Identifythestagesofmitosisinplantcells

Introduction
Allnewcellscomefrompreviouslyexistingcells.Newcellsareformedbytheprocessofcell
divisionwhichinvolvesbothreplicationofthecellsDNAanddivisionofthecytoplasm
(cytokinesis)toformtwogeneticallyidenticaldaughtercells.Therearetwotypesofnuclear
division:mitosisandmeiosis.Mitosistypicallyresultsinnewsomatic(body)cellsorisaform
ofasexualreproductioninunicellularorganisms.Formationofanadultorganismfroma
fertilizedegg,asexualreproduction,regeneration,andmaintenanceorrepairofbodypartsis
accomplishedthroughmitoticcelldivision.Meiosis,ontheotherhand,resultsintheformation
ofeithergametes(inanimals)orspores(inplants).Thesecellshavehalfthechromosome
numberoftheparentcell.
Wheredoesonefindcellsintheprocessofmitosis?Plantsandanimalsdifferinthisrespect.In
higherplantstheprocessofformingnewcellsisrestrictedtospecialgrowthregionscalled
meristems.Theseregionsusuallyoccuratthetipsofstemsorroots.Inanimals,celldivision
occursalmostanywhereasnewcellsareformedorasnewcellsreplaceoldones.Inbothplants
andanimals,though,tissuesrarelydivideoncetheorganismismature.
Tostudythestagesofmitosis,youneedtolookfortissueswheretherearemanycellsinthe
processofmitosis.Inplants,thisrestrictsyoursearchtogrowingtips,suchastheonionroottip.
Thephasesofplantmitosisare:
Thenondividingcellisinastagecalledinterphase.Thenucleusmayhaveoneormoredark
stainednucleoliandisfilledwithafinenetworkofthreads,thechromatin.Interphaseisessential
tocelldivisionbecausethegeneticmaterial(DNA)isduplicated(replicated)duringthisstage.
Thefirstsignofadivisionisprophase,inwhichathickeningofthechromatinthreadsoccurs.
Thickeningcontinuesuntilitisevidentthatthechromatinhascondensedintochromosomes.
Withsomewhathighermagnificationyoumaybeabletoseethateachchromosomeiscomposed
oftwochromatids.Asprophasecontinues,thechromatidscontinuetoshortenandthicken.In

lateprophasethenuclearenvelopeandnucleoliarenolongervisible,andthechromosomesare
freeinthecytoplasm.Justbeforethistimethefirstsignofaspindleappearsinthecytoplasm;
thespindleapparatusismadeupofmicrotubules,anditisthoughtthatthesemicrotubulesmay
pullthechromosomestowardthepolesofthecellwherethetwodaughternucleiwilleventually
form.
Atmetaphase,thechromosomeshavemovedtothecenterofthespindle.Oneparticular
portionofeachchromosome,thecentromere,isattachedtothespindle.Thecentromeresofall
thechromosomeslieataboutthesamelevelofthespindle,onanimaginaryplanecalledthe
metaphaseplate(equator).Atmetaphaseyoushouldbeabletoobservethetwochromatidsof
somechromosomes.
Atthebeginningofanaphase,thecentromereregionsofeachpairofchromatidsseparateand
aremovedbythespindlefiberstowardoppositepolesofthespindle,draggingtherestofthe
chromatidbehindthem.Oncethetwochromatidsseparate,eachiscalledachromosome.The
daughterchromosomescontinuepolewardmovementuntiltheyformtwocompactclumps,
oneateachspindlepole.
Telophase,thelaststageofdivision,ismarkedbyapronouncedcondensationofthe
chromosomes,followedbytheformationofanewnuclearenvelopearoundeachgroupof
chromosomes.Thechromosomesgraduallyuncoiltoformthefinechromatinnetworkof
interphase,andthenucleoliandnuclearenvelopereappear.Thecelldevelopsintotwonewcells.
Inplants,anewcellwallislaiddownbetweenthedaughtercells.Inanimalcells,theoldcell
willpinchoffinthemiddletoformtwonewdaughtercells.Thisdivisionofthecytoplasm,in
contrasttonucleardivision(mitosis),iscalledcytokinesis.

Preparinganonionroottipsquash
Itispossibleforyoutomakeyourownstainedpreparationsofonionroottipsandobserve
mitoticfigures.Onionbulbshavebeenrootedinwater.Growthofnewrootsisduetothe
productionandelongationofnewcells.Mitoticdivisionsareusuallyconfinedtothecellsnear
thetipoftheroot.Followtheprocedureoutlinedbelowtomakeyourownroottippreparation.

Procedure
1.Obtainanonionbulbthathasbeenrootedinwater.Cut2or3rootsoffnearthebaseofthe
bulb.
2.Then,cutoffthebottom1or2mmoftheroottipandplaceinapetridish.Withapasteur
pipette,addasmallpuddleof1MHClandlettheroottipstandfor4min.
3.RemovetheroottipfromtheHClwithforcepsandplaceonaslide.
4.Covertheroottipwith2dropsof1%toluidineblueandletsitfor2minutes.
5.Carefullyblotaroundtheroottoremoveexcessstain.Rinsewithwaterandblotuntilthe
waterisclear.Addonedropofwaterandapplyacoverslip.
6.Placetheslide,coverslipdownonapapertowel.Usingapencileraser,carefullyapply
pressuretothecoverslipareainordertosquashandspreadtheroottiptissue.[Thistakesalittle
practice,soifyourfirstsquashdoesnotyieldgoodresults,tryanotheroneortwo.]
7.Mounttheslideonyourmicroscope.
8.Usethelowpowerobjectiveonyourmicroscopetolookforthinlayersofcellsandthenuse
the40Xpowerobjectivetoobservemitoticstagesinindividualcells.
9.Identifychromosomesatthevariousstagesofmitosis.
10.Compareyourslidetothepreparedslidesthatcontainstainedsectionsofroottips.
11.Makesketchesofthemitoticstagesobservedonthenextpage.

Usethissideofpagetodrawsketchesof
whatyouseeinthemicroscope.

Usethissideofpagetonotestagesand
descriptions.