14. TETRAZO LIUM TEST FOR SEED VIABILITY AND …

1 ,it ,commonlyknownastheTZtestforseedviabilit yhave,therefore,.beendevelopedtofurnishq uickestimatesofseeds' veryusefulinprocessing,handling, shorttime,testingdormantseedlotS,vigourr atingoftheseedlots,~ (1939-1958),whorecognisedthatalllivingti ssues,whichrespire,arecapableofreducinga colourlesschemical2,3,5triphenyltetrazol iumchlorideorbromide)intoa redcolouredcompoundformazenbyH transferreactionscatalysedbytheenzymedeb ydrogenases.~qN-N-C6H5.+. '+2(;.1/~65\+DEHYDROGENASlsC6 HSG+HCL-N=N-~HS~\N= ~.. ,3,, (COLOURLESS}FO~A!AN( RED)Formazanbeingnon-diffusable, ,thelivingpartsofa ~lutilityofthistestwasrealisedbytheSee9s cientistsandtechnologiststheworldoverand it (1973) "theseedandseedlingstructures, ,abnormalormissingstructuresetc.,inviewa nd, ,dicotsandpolycotsonthebasisorthepresenc eofone, :SeedsofGramineaefamilyarea <; , , :Inthedicotseedswitha lowdegreeofdifferentiationtheembryoisemb eddedinnutritivetissuesandconsistsofa beanseed-a typicaldicotseedwithwelldevelopedembryoi s notverywelldifferentiatedexceptfora (1).)

2 (2)Beakersof100to 250mlcapacityforlargerseeds,(3)Singleedg erazorblade,(4)Needles,(5)Forceps,(6)Mag nifyingdevices,(7)Medicinedropper,(8)Dis pens-ingbottle,(9)Germinatingbl(~tters,f ilterpaperandpapertowel.;;,and(10) a creamorlightyellowcolouredwatersolublepo wdercalled2,3,5, usedforseedsthatarenotbisectedthroughthe embryo,is ,onegramoftetrazoliumsaltis dissolvedin thedistilledortapwaterto make100m!. orlowerwillnotstainevenviableembryosands olutionofpHmorethan8 thepHofthewaterisnotintheneutralrange, ofPotassiumdihydrogenphosphate(KH2PO4)in watertomake1000m!. ( ) ,400mlofsolutionA and( ,dissolve10g darkorambercolouredbottletopreventtherea ction'whichaL<; [orseveralmonthsin a clearingagent,if , consisL<;of:Lacticacid:20parts, :40parL<;andWater:20p~lrts.,' (~ cotyledonsandembryoaxeswhilecuttingseeds , 'whileothersmustbemoistenedslowly~Slowmo isteningis 'Ihiscanbedonebykeepingtheseedontopofori nb~ slightlyhighertemperature(within40 C) :(a)Nomoisteningorpreparationrequired(s~ allseededlegumeswithsoftcoats).)))]

3 (b)Slowmoisteningwithoutanypreparation(l argeseededlegumeswithsoftcoats).(c) 'oftheseedcoat(smallseededgrasses).(d) ,keepingonlyonehalfforthestaining,discar dtheother.(e)Cuttingtheseedslongitudinal lyslightlyoff-centretoavoidcuttingintoth eembryo.(f)Removingtheseedcoat(withforce ps/needles/razorbladesetc.) ,a thinmembraneadherestothecotyledonsevenaf terremovaloftheseedcoat, (dicotswithhardseedcoats).StainingSeedsc anbestainedinwatchglasses, '>a ruleofthumb,a concentrated( )solutioncanbeusedforlegumes,cottonandgr assesthatarenotbisectedthroughtheemrbyoa nda dilute( ) ,cOncentrationofthesolution,stainingtime at 35 C,andevaluationfora numberofimportantIndiancropsaregivenin (forfurtherdetails,referto Moore,1995).Careshouldbetakennotto overstainseedsasthiscausesdegeneration, ,seedsareplacedinthesolutionandheldat35 C periodinthesolution,thelengthofwhichvari esaccordingtotheconditionoftheseed,speci es,temperatureandconcentrationofthesolut ion.

4 Theseedis removedfromthetetrazoliumsolution,rinsed 2-3timesinwaterand. thenevaluatedaccordingto ,theseedshouldbeleftin a seedsarenotto beevaluated'immediately,theseedsin a littlewatershouldbekeptin ,2-3dropsoflactophenolclearingsolutionca nbeaddedto100seedsofsmallseededgrasses, stereomicroscope,while, (i) lesserbutlustrousinsoundtissuesthanthebr uised, gradualreductionin colourintensity fromthes~r[acctotheinterioroftheseedindi catesslowabsorptionandfirm,soundunstaine dtissuesindicatethelackofpenetrationofth etetrazoliumsolution.(ii)Weak,'iabletiss uesSuchtissuestendtostaingreyishredorbri ghterredthannormal.'[ <;alsolosetheturgidity (Hi)Weaknon-viabletissuesTissuecolouris oftenmottl~d,it ; <Jdtissueslookl1acid,liquid-logged,blurr ed, , , onlyonefractureoronesmallbrokenormissing spotin a considereddeadwhenunstainedareasincludem esocotylandseminalrootsin grassesandtipsofcolcorhizain interpretingthestainingpatterninmonocota rctheplumuletips, ,cotyledonsandintheplumbleregionindicate a thepercentageofnormalgerminableseedstobe expect~whentheseedlotis ~ ~su1ts( ).]]

5 MaybenecessarytDfirststandardiscthestain ing.'ThestandardizationoftheTZmethodsfor ragi(Eleusinecoracaneandkhesari (Lathyrussativlls)havebeenreported(Agarw alandKaur,1975; ).Ifproperlyconducted,TZtest'sresultisin closeagreementwiththegerminationpercenta ge(Agrawalet aI.,1973). ( ):(iii)(iv)'(vi)(vii)(i)Sampledifference (ii).ImpropergerminationtestingImproperT ZtestingSeeddormancy(v)Presenceofhardsee dsPresenceofseed-borneorganismsChcmia11i njury( ). (1985).216 TermsusedStained:Referstoviable(function al) :Referstonon-viable(non~functional)tissu esasestablishedbyabnormalstainingcharact eristicsand/orlackoftissueturgidity~Eval uationcodesforstainingpatternsofviablese edsofdifferentspecies( )(i) ", a istal1! , (ii) (a)Quickandfairlyacc:uratc(b)Canalsodete rminetheviabilityofa dormantseedlotshorttimc.)

6 (c)Seedsarenotdamaged(indicOls)andcanbeg ermim218I>isadvantagesofTZtest(a)Distinc tionbetweennormalandabnormalseedlingsdif ficult.(b)Cannotdifferentiatebetweendorm antandnon-dormantseeds.(c)Correctevaluat ionispossibleonlyafterprolongedexperienc e(d) , to (1968)describeda a petridishlinedwithmoistfilterpaperandkee pat25:j:1 mloftetrazoliumsolutionto Cfor4 ,formaintainingunifromity,durationshould bekeptconstantfora , to6 hourswithoccassionalstirringtilltheextra ctionofredcolouredformazanis , ,.someimportantcropspecies(adaptedfmmMoo re,1985)CropMoisteningPreparatoryStainin gStainingperiod(h)methodaftertime(h)eval uationmoistening(refertothetext)2345eel- eatsBarley6-18(a) ,B17 Oat6-18-do2-24B6 Paddy18-do-6-24B4 PearlMillel6-18-do-6-24B4 Rag!6-18-do-6-24B4orcutlaterallySorghum6 -18(a)6-24B4,817 Wheal6-18-do-3-24B6,B17-"I)ulsesChickpea 18(b)Cutthrough6-24B4,Bll,BI8theseedcoat nearmidsectionCowpea18*-do--entire18-24B 3,B13,B18seedlengthGreenGram18*-do-18-24 B3,B13,818 Lalhyrus18'"-do-&6-24B3,B13(c)Severeadis talendofcotyledonLentil18*(b)6-2483,B13, 818 Pea18-24-dO-6-2484,813,818 Pigeonpea18(b)&(C)6-2483,B13 Soybean18*(b)ornoseed6-2483,813,818coatp reparationneededOilseedsCastor18(b)&(c)6 -24A,DGroundnut18*(b)6-24B2,B8,B18 Linseed18*Treatwithalumto6-24B2,B9remove seedcoatsIiperiness& (b)12345 Mustmd18(d)Puncturethe18-2481J316seedcoa t,cutasin(b)including1 (e) , ,'eCl'OpSCotton18':' ,137.

7 Sunnhemp18(f) (c)FOJ'ageAlfaalfa()-18-do-6-24m, (Trifolium)Guineagrass6-18(g) egetahlesAmaranrhus18(b)18-24132, (g)6-2481,1312 Brinjal18 Punctureand()-24A,C(h) *(b)&(c)16-24134, (e) ,CChillies18(h)6-24A, (g)Dolichos18(f)6-24133,813,818beanLettu ce6-18(e)6-2482,817 Luffa6-18 Removethe16-2482,816wings&(d)Onion6-18*( h)6-24A,D2222345 Pumpkin6-18 Removethe6-24B2,B12wings&(g)RadishlR(d)6 -24132,816 Roundgourd6-18 Removeseedcoat6-24B2,1312sliperinessbydr ying&(g)Tomato18 Puncture&(h) ,B14 Flowel'sAster6-1f:(e)6-24B2,B14 Begonia18(e)18-24 ACannaIf:Removehardseed-24-48A,Dedness&( e)Coleus18 TreatwithalumtolR-24B2,811removeseedcoat sliperiness&(b)Dahlia6-18(e)6-2482,1314 Gladiolus18 Severethroughthe24-48A,Cdistalendofthese edRose18 Severewings&(e)24-48 OthersCitrus18 Removeseedcoat6-2482, !

8 Tea18(g)24-48A,CPine18 Cutoffcentre24-48A,Dthroughthecoat& C-Tobacco24(b)24-48A,C;Note:'"= co!.3 areasfollows:a= ,cutasin(b) ,223e= (d) ' = \)I\) ~0) ' \15227481216+-~,,--,;CJ\uco/~-i' :)<Ctj22859\ \0142293478\2."\6\5uaIJ~LJ~~,QJc:ad.~ <0~~..-c'" ,2313568959\326\ '37, \\523248\2\6qLJ"'C C.\& ~..~CJu<Cto(l)~I.'!"'"23359' \1\52344-8\2\ :,;::.'.~.,C""'"~~i:'f(4710\ \14236369\2\5' ~.,..-, 'I-~-<t237aJIt!ex).,,:,.. \ 0239u(J)(t)100)..,f"..JZ3~~().456.~.. ) ())(Q()(