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Flowering and vegetative
propagation of pyrethrum
(Chrysanthemum cinerariaefolium Vis.)
inNflvoand invitro
NN08201.B71 Roest
S. Roest
Flowering and vegetative propagation of pyrethrum

(Chrysanthemum cinerariaefolium Vis.) in vivo and in vitro
Proefschrift
terverkrijgingvandegraadvan
doctorindelandbouwwetenschappen,
opgezagvanderectormagnificus,
dr.ir.J.P.H.vanderWant,hoogleraarindevirologie,
inhetopenbaarteverdedigen
opvrijdag,17december 1976des namiddagstevieruur
indeaulavandeLandbouwhogeschoolteWageningen
Centre for Agricultural Publishing and Documentation
Wageningen - 1976
Abstract
Roest,S.(1976).Floweringandvegetativepropagationofpyrethrum (.Chrysanthemum
oineranaefolium Vis.) invivoandinvitro.Agric.Res.Rep. (Versl.landbouwk.Onderz.)
860,ISBN9022006220,(vi)+105p.,44figs,52tables,159refs,Eng.andDutch
summaries.
Also: Doctoral thesis, Wageningen.
The influence of climatic conditions was investigated on flowering behaviour of
pyrethrum^Chrysanthemum oinerariaefolium- V i s . ) . At low temperatures high numbers of
Plants initiated high numbers of flower heads. Both the development of the i n i t i a t e d
riower heads and the vegetative development of the plants were stimulated by higher
temperatures.
The second aspect was the development of methods of vegetative propagation in vivo
ana in v i t r o . Through^ culture of peduncle explants in v i t r o detailed information was
oDtamed about the i n i t i a t i o n and development of adventitious r o o t s . With these data the
" °f adventitious root formation of shoot cuttings in vivo was optimalized. In
; , ? " , " " thls m f h o d o f vegetative propagation in vivo may be useful for a fast multi-
h6 lthy pUntS with hi h
llolToT f ? 8 y £ e l d s ° f Pyrethrins. Vegetative
acl ieved bv
clTtfSnl T l ? V i t i a t i o n and development of adventitious shoots on
d t S r ^ T.- 1 V a t 6 d i n V i t r 0 a n d subsequent adventitious root formation of
other ConôTtae P r o c e d ^ e was applicable for vegetative propagation of several
fomatt™ r ! f T a t
^"' or
8 a n ° 8 e n e s i s » adventitious, root formation, rhizogenesis, shoot
8 &K?Unt
CoôsUae ' ' P e d u n c l e > capitulum, flower head! pyrethrins, Kenya,
This thesis will also be published as Agricultural Research Reports 860.
© C e n t r e ^ Agricultural Publishing and Documentation, Wageningen, ,976.

Stellingen
1.Erwordtonvoldoendeanatomischonderzoekverrichtnaardeteonderscheidenstadiain
hetprocesvanregeneratievanadventievewortelsenscheutenbijhogereplanten.
Ditproefschrift.
2.In vitrocultuuriseenbelangrijkemethodevoorfysiologischonderzoeknaardeom-
standighedendieoptimaalzijniniederstadiumvanhetregeneratieproces.
Ditproefschrift.
3.Landbouwkundigonderzoekdatsterkgerichtisopdepraktijkvaneenontwikkelingsland,
dientinhetlandzelfterhandtewordengenomen.Slechtswanneerditonmogelijkmoet
wordengeacht,kannaeenuitgebreideorientatieinhetontwikkelingslandbeslotenwor-
denditonderzoek,ofeendeelervan,eldersuittevoeren.
4.Deresultatenvanonderzoekmetbetrekkingtotdevegetatievevermeerderinginvitro
zullenophetBedrijfslaboratoriumWeefselkweekeenpraktischetoepassingmoetenvinden.
HetsucceshiervanwordtinsterkematebepaalddoordeProefstations,welkedeschakel
zijntussenonderzoekenpraktijkenwaardezemethodenbedrijfsklaarmoetenwordenge-
maakt.
5.Menrealiseertzichindepraktijkonvoldoendedatpericlinaal-chimaeren (sporten)
viaadventievescheutvorminginvivoofinvitroniet (soort)echtvegetatiefvermeerderd
kunnenworden.
S.RoestandG.S.Bokelmann (1975).ScientiaHort.3:317-330.
6.Debetekenisvanweefselkweektechniekenvoordemutatieveredelingkomtnietalleen
totuitingindeproduktievanvolledige (niet-chimaere)mutanten,maartevensinde
snellesoortechtevegetatievevermeerderingvandemutanten.
C.Broertjes,S.RoestandG.S.Bokelmann (1976).Euphytica25:11-19.
7.DedoorMatsubaraetal.gevolgdemethode,waarbljnabestralingvolledigemutanten
wordenverkregenbij Begonia en Chrysanthemum, isomslachtigennietefficient.
H.Matsubara,K.Shigematsu,H.SudaandS.Hashimoto (1971).Proceedingsofthe
10thconferenceonradioisotopes,JapanAtomicIndustrialForum,Tokyo,p.374-376.
8.BijdestudieaandeLandbouwhogeschooldientmeeraandachttewordengeschonkenaan
hetinwoordengeschriftuitdragenvanwetenschappelijkekennis.
9. Wetenschappelijke literatuur moet a l t i j d op identieke wijze gerefereerd worden.
10. In cms land zijn wegbermen en spoordijken een belangrijk toevluchtsoord voor veel
planten en dieren. Watergangen kunnen i n d i t opzicht ook van groot belang z i j n , wanneer
ten aanzien van het hiervoor noodzakelijke mechanische onderhoud een beheersovereenkomst
tot stand komt tussen overheid en onderhoudsplichtigen.
11. Snelverkeer in woonwijken moet wijken voor wonen.
S. Roest
Flowering and vegetative propagation of n v r e n , r „ „ / rT , ... . . , ,• „. N
in vivo and in v i t r o pyretnrum (Chrysanthemum cmeranaefolium Vis.)
Voorwoord
Hetschrijvenvaneenwoordvandankdientnaarde lettergenomenalseendankbare
taaktewordenopgevat.Eenondankbaretaakishetevenwelinzoverre,dathetonmogelijk
isom iedzveen tebedanken.Velenhebbenimmerseenbijdragegeleverdaanhet totstand
komenvanditproefschriftenhenalienbenikdaarvoorbijzondererkentelijk.
Indeeersteplaatswilikmijnwaarderinguitsprekenjegensmijnoudersdiemijde
mogelijkheidhebbengebodendestudieaandeLandbouwhogeschooltevolgen.
Zonderhetinitiatiefenhetdoorzettingsvermogenvandr.R.B.Contant zouhet
onderzoekdatinditproefschriftbeschrevenisniet zijnuitgevoerd.BesteRuud,ik
hoopdatderesultatenvanditonderzoekenigepraktischebetekenis zullenhebbenvoor
depyrethrumteeltinKenya,welkgewasenwelk landjouzozeerterhartegaan.
Aangezienhetonderzoekwerduitgevoerdophet Ital,was eraanvankelijk tussen
mijnpromotorprof.J.Doorenbosenmijsprakevanincidentelecontacten.Gedurendeveel-
vuldigebezoekenisernaderhandeengoedesamenwerkingontstaan,waarbijU,doorde
snellewijzewaaropUdevelemanuscriptenvanop-enaanmerkingenhebtvoorzien,een
zeerwaardevollebijdragehebtgeleverdaanhetresultaat zoalsdatthansvoorons ligt.
Mijnco-promotordr.R.L.M.Pierikheeftdoorzijnenthousiasmeeninspiratiereeds
tijdensdeingenieursstudiemijnbelangstellingvoordeplantaardigeweefselkweekgewekt.
BesteRudolf,ookvoorjeverderebegeleidingenvoordeonbouwendekritischekant-
tekemngenwelkejemetnameplaatstebijdiegedeeltenvanhetproefschriftdie
betrekkinghebbenopdeinvitrocultuur,benikjezeererkentelijk.
Dr.D.deZeeuwendr.A.Ringoethebbenmijdemogelijkheid gebodenomhetonder-
zoekophetItaluittevoeren.Degrotematevanvrijheiddiejulliemijbijdeuit-
voermgvanhetonderzoekhebbengegeven,maartochookdezachtedwangwelkejullieop
mijhebbenuitgeoefendomhetonderzoekmeteenproefschriftafteronden,hebbenmede
totdituiteindelijkeresultaatgeleid.
IndejarengedurendewelkeTrudyBokelmannmijbijhetonderzoekassisteerde,heb
ikhaarlerenwaarderenalseenbijzonderbekwaamenervarenassistente,diehaartaak
metgrotezelfstandigheidverricht.BesteTrudy,zonderjouwdaadwerkelijkesteunzou
eenbelangrijkdeelvanhetonderzoeknietzijnuitgevoerd.TrudyenMarion,mijn
excusesvoorhetfeitdatikmijhetafgelopenjaarmindermetdedagelijksegangvan
zakenhebbemoeidendatmijnhumeur,ondanksdemooiezomer,weleensnietaltezonnig
was.
vand r 1 S r S 1 6 S" deWaard6V0lleS U ^ e s t i e s™ * » iknadelezingvanhetmanuscript

vandr.C Broertjesmochtontvangenentevensdenuttigeadviezenvandr.A.J.G.van
L o r t ; e n t ; e ^ a l g e m e n e Zake"deP — t i e betreffende,hebikzeeropprijsgesteld.
van 27lZ ien^\Adri **»«*en* » Schoutenisdenodigezorgaandeopkweek
vanpyrethrumbesteed,alhoewelmijbekendwasdatditgewasnietindeallereerste
plaatshunvoorliefdegenoot.
WilvanMarion-Noorlanderverdienteencomplimentvoordesnelleenzorgvuldige
wijzewaarop zijdedefinitieveversievanhetproefschriftheeftgetypt.
HetkwalitatiefgoedewerkvanMennoDrostenJanEikelenstamkomttotuiting in
develefoto'sengrafiekenwelke inditproefschrift zijnopgenomen.
Mededoorhetgeduldwaarmeeir.A. Keenmijnwensentenaanzienvandewiskundige
verwerkingheeftaangehoord,istenslotteeenbevredigendestatistischeanalysevaneen
aantalvandeproefresultatentotstandgekomen.
Mevr.E.M.Brounsendhr.R.J.P.Aalpolben ikerkentelijk voordeplezierigeen
deskundigebegeleidingwelkeikvanPudocondervondenhebbijdecorrectievande
Engelsetekstenbijderedactieenuitgavevanhetproefschrift.
Debeslommeringenwelkeaanhetschrijvenvaneenproefschriftverbondenzijn,
leidenonvermijdelijktoteenaantaldepressieswelkedepromovendusenmethemde
gezinsledenondergaan.Dank zijdeafleidingwelkeJolandeenYvonnemijbezorgdhebben,
maarvooraldoordesteundieJannymijgegevenheeft,heb ikditwerkkunnenvoltooien.
Curriculum vitae
Deauteurwerdgeborenop28oktober 1939teWinterswijkenbehaaldein1958het
einddiplomaHBS-BaanhetWinterswijks Lyceum.Nahetvervullenvandemilitaire dienst-
plichtbegonhijin1960metdestudieaandeLandbouwhogeschoolteWageningen,waarhij
in1969afstudeerdeinderichtingtuinbouwplantenteeltmetdekeuzevakkenplanten-
ziektenkunde (verzwaard)endierkunde.Indecember 1969werdhijaangesteld alspromotie-
assistentbijdeLandbouwhogeschoolengedetacheerdophetInstituutvoorToepassingvan
AtoomenergieindeLandbouw (ITAL)teWageningenvoorhetverrichtenvanhet onderzoek
datbeschrevenisinditproefschrift.Ditonderzoekwerd afgeslotenop1maart 1973en
vanafdiedatumishijalswetenschappelijk onderzoekerintijdelijkedienstwerkzaam
ophet ITALmetalsvoornaamstetaakhetontwikkelenvaninvitromethodenvanvegeta-
tievevermeerderingwelketoepassingkunnenvindenindemutatieveredeling.
Contents
1 General introduction 1
2 Flowering behaviour of intact plants 4

2.1 Introduction 4.
2.2 Materialandmethods 4
2.3 Factorsinfluencingflowering 5
2.3.1 Temperature 5
2.3.1.1 Constanttemperature 5
2.3.1.2 Fluctuatingtemperature 10
2.3.2 Photoperiod 14
2.4 Discussionandconclusions 16
3 Root formation of peduncle explants in vitro 18

3.1 Introduction 18
3.2.1 Material 19
3.2.2 Methods 20
3.3 Anatomicalobservations 21
3.4 Factorsinfluencingrootformation 24
3.4.1 Plantfactors 25
3.4.1.1 Genotype 25
3.4.1.2 Floweringstage 25
3.4.1.3 Explantposition 27
3.4.1.4 Explantlength 28
3.4.1.5 Wounding 30
3.4.1.6 Polarity 31
3.4.2 Nutritional/hormonalfactors 33
3.4.2.1 Agar 33
3.4.2.2 Minerals 33
3.4.2.3 Sugars 37
3.4.2.4 Auxins 41
3.4.2.5 Cytokinins 45
3.4.2.6 Gibberellicacid 47
3.4.3 Climaticfactors 49
3.4.3.1 Temperature 49
3.4.3.2 Light 52
58
4 Root formation of shoot cuttings in vivo
58
4.1 Introduction
58
4.2 Materialandmethods
4.3 Anatomicalobservations "'
63
4.4 Factorsinfluencingrootformation
63
4.4.1 Plantfactors
63
4.4.1.1 Genotype
63
4.4.1.2 Wounding
64
4.4.1.3 Shoottip
4.4.2 Nutritional/hormonal factors 65
65
4.4.2.1 Substrate
4.4.2.2 Auxins 66
4.4.3 Climaticfactors 70
4.4.3.1 Constanttemperature 70
4.4.3.2 Fluctuatingtemperature 71
5 Shoot formation of capitulum explants in vitro and -plantlet production by root

formation of detached shoots 75
5.1 Introduction 75
5.2.1 Material 76
5.2.2 Methods 77
5.3 Anatomicalobservations 77
5.4 Factors influencingshootformation 80
5.4.1 Plantfactors 80
5.4.1.1 Genotype 80
5.4.1.2 Floweringstage 81
5.4.1.3 Explantsize 82
5.4.1.4 Wounding 82
5.4.2 Nutritional/hormonal factors 83
5.4.2.1 Minerals 83
5.4.2.2 Sugars
5.4.2.3 Auxin
5.4.2.4 Cytokinin 87
5.4.2.5 Gibberellicacid 87
5.4.3 Climaticfactors
5.4.3.1 Temperature
5.4.3.2 Light 89
5.5 Plantletproductionbyrootformationofdetachedshoots 91
5.6 Vegetativepropagationofotherplantspecies 91
Summary 95
Samenvatting 97
References 100
1 General introduction
Pyrethrum {Chrysanthemum einerariaefolium Vis.)isaplantspecieswith daisy-like

capitula (flowerheads),belongingtotheCompositae. Thisperennial rosetteplantwith
deeply lobedleavesofvariable shapeandlengthiscultivatedmainlyinKenyaandother
EastAfricancountriesatanaltitudeofatleast 1900mabovesealevel.Atthis
elevationanditslowtemperature,thecapitulumisborneonabranched leafy stem,
risingfromacompactcrownoffoliage,whereasatahighertemperatureinlowerregions,
theplantsalmostexclusively developvegetatively (Ghadinger,1945;Glover, 1955).
Pyrethrumisoneofafewcommercially grownplantspecieswhichproduce compounds
usedasinsecticides.Thesixconstituentswith insecticidalproperties,collectively
calledpyrethrins,arelocatedinallplantparts,buttheovariesandachenesofthe
discandrayfloretsofthecapitulumcontainbyfarthehighestconcentrationand
largestamount (Head,1966;Brewer, 1973).Thecapitulaarepickedbyhand,driedand
ground.Thepowderandtheextractprepared therefromcontainthesepyrethrins,which
haveuniqueproperties,suchas:
-highdegreeofsuitabilityforcombinationwithsynergists (Chadwick, 1963),
-repellent,knockdownandtoxiceffectsforagreatvarietyofinsects (vanRijn, 1974),
-almostcompleteharmlessnesstomenandwarm-blooded animals (Griffin, 1973),
-rapidbreakdownandnopersistenceofresidues,
-hardlyanybuildupofresistanceininsectpopulations (Busvine,1960;Fine, 1963).
Thesepropertiespermittheuseofpyrethrins againstinsectpestsinthehouse,on
crops (evenwhenatreatmentisrequired justpriortoharvest),stored foodandlive-
stock.Becauseofanincreasingconsciousnessoftherisksassociatedwiththewide-
spreaduseofmanysynthetic insecticides,liketoxicitytomammals,persistenceof
residuesandinsectresistance,thedemandforpyrethrinsisgrowing.
Kenyaprovides about60$oftheworldproductionofpyrethrinsandthisnatural
insecticideisamongtheprincipalexportproductsofthiscountry.In1975the
productionofabout100000farmers,90$beingsmall-scaleproducers,.amountedto
approximately 15000tonsofdried flowerheads,withavalueof15millionU.S.dollars
(GlynneJones, 1973;vanRijn, 1974).
Syntheticcompoundsresembling someofthesixconstituentsofthenatural
pyrethrinshavebeenmanufactured (Elliot, 1967).Eventhoughthese synthetic
pyrethroidsaremoretoxictoinsects,itisnotlikely thattheywillreplacenatural
pyrethrinsinthenearfuture,becausethelatterareeffectiveagainstawiderrange
ofinsects,moresuitableforcombinationwithsynergists (Chadwick,1963)andless
expensive (Winney, 1973).
Theapplicationofnaturalpyrethrinsisstillrestricted,because theyaremore
expensive thanmanyotherinsecticides.Tostrengthenthepositionofthenatural
1
pyrethrinsonthemarketitisofutmost importancetodecrease theircostsby increasing
theannualproductionofpyrethrinsperacreage.Theyield isdeterminedby several
components,suchasnumber,size,percentagedrymatterandcontentofpyrethrins inthe
flowerheads.
Inpyrethrumbreedingprogrammesthemainobjectivesaretheincreaseofthefresh
floweryield (theproductofnumberandsizeoffreshflowerheads)andthepyrethrins
content.
Thefreshfloweryieldisdeterminedbymany factors,suchas:
- thegenotype (Parlevliet, 1969),
- theclimate (rainfall,temperature,etc.;Glover,1955;Parlevlietetal.,1969;
Muturietal.,1969*,Parlevliet, 1970a),
- thesoil (fertility,irrigation,ridging,,spacing,weeding,etc.;Kroll, 1962,1963;
Weiss,1966;Parlevlietetal.,1968;Mwakha, 1974),
-diseasesandpests (liketheroot-knotnematode Meloidogyne hapla; Bullock,1961;
Robinson,1963;Parlevliet&Brewer,1970,1971;Parlevliet, 1971).
Thepyrethrinscontentischiefly influencedby:
- thegenotype (Head,1967;GlynneJones,1968),
-pickingintervalandfloweringstage (Head,1966;Parlevliet, 1970b),
- theclimate (Kroll,1964;Parlevlietetal.,1969;Muturietal.,1969),
-dryingmethods (Githinji,1973;Head, 1973).
From1940-1965thepyrethrumbreedingprogramme inKenyawasprincipally directed
towardstheproductionofgoodhybrids.Hybridizationcouldeasilybeachieved,because
mostplantsareself-incompatibleandcross-compatible (Kroll,1958;Brewer,1968;
Brewer&Parlevliet,1969;Brewer&Henstra,1974).As thegeneticproperties ofthe
parentplantsarewidelydivergent,seedlingpopulations exhibitaconsiderablegeno-
typicandphenotypicvariability.Breedingand-selectionmayfurtherbecomplicatedby a
negativecorrelationbetweenfloweryieldandpyrethrinscontentsothat individual
plantswithbothahighfloweryieldandahighpyrethrinscontentareveryrare.Ina
population,however,bothgeneticcharacteristicscanbeimproved inafairlyshorttime
(Contant,1963;Parlevliet, 1974).
Thefixationofdesiredgeneticpropertiesis.oneofthegreatestproblems inthe
breedingofacross-pollinated,highlyheterozygous crop.Geneticcharacteristics of
selectedplantscanbepreservedbyvegetativepropagation.Selectionandasexual
multiplicationofsuperiorclonesmaybeanevenbettermethodofimprovementthanjust
theproductionofgoodhybrids (Brown,1965;GlynneJones,1968;Brewer&Parlevliet,
1969; Parlevliet,1969;Parlevliet&Contant,1970).
Theactualbreeding,selectionandvegetativepropagationofoutstandingclones
wasstartedbyContantinKenyain1962 (Contant,1963).Throughhis.initiativeand
thoroughnessandwithfinancialsupportoftheMinistry ofForeignAffairsofthe
Netherlands,adevelopmentalprojectwassetupinKenya.TwoDutchplantbreeders and
severalgroupsofNetherlandsvolunteerswereposted inKenyaandchargedwiththe
breedingandselectionofhighyieldingplants,andthevegetativepropagationof
selectedplantsonlarge-scalenurseries,respectively.
BecauseresearchonpyrethruminKenyalackedcontinuity andbecausethis species
wasofinteresttoseveralresearchinstitutes,fundamentalresearchonpyrethrumin
Wageningenwasstartedin1964.In1966theworkwasformallyorganizedinaresearch
projectbyContantattheAssociationEuratom-Ital.Atfirstthisresearchwas
concentratedonplantbreeding,butattheendof1969theinvestigations,whichare
describedinthisreport,wereshiftedtowardsmorepracticalaspectslikefloweringand
vegetativepropagation.Theprojectwasterminatedattheendof1972.
Avegetativepropagationofpyrethrumcanbeachievedinseveralways,like:
multiplicationfromsplits (thenormalprocedureinKenya)andmultiplicationfromshoot
cuttings.Thepracticalimportanceofpropagationbysplitshasbeenindicatedbyseveral
authors (Drain&Shuey,1934;Martin&Tattersfield,1934;Cormack,1935;Chamberlain&
Procter,1947;Osbourn,1961;Contant,1963;Brown,1965).Theuseofcuttingshasalso
beenreportedbymanyauthors (Drain&Shuey,1934;Cormack,1935;Chamberlain&Procter,
1947;Collings-Wells&Contant,1963;Canham,1968).
AsubstantialpercentageoftherootsystemsofpyrethrumplantsinKenyais
severelyattackedbytheroot-knotnematode Meloidogyne hapla sothatfloweryield
decreases.Ifaselectedstock-plantisinfected,splittingresultsinacontamined
clone,whereaswithshootcuttingsahealthycloneisbuiltup.Shootcuttingsmayalso
havetheadvantagethatamuchhighermultiplicationratemaybeachievedthanwith
splits.Hence,ahealthycloneofselectedhighyieldingplantsisprobablyobtained
morerapidlyfromshootcuttingsthanfromsplits.
Thisstudydealswithfloweringandvegetativepropagationofpyrethruminvivoand
invitro.Theinfluenceoftemperatureandphotoperiodonfloweringbehaviourofintact
plantsisdiscussedinChapter2.Chapters3,4and5describemethodsofvegetative
propagationinvivoandinvitro.InChapter3rootformationisexaminedofpeduncle
explantscultivatedinvitro.Thismethodmayprovidedetailedinformationonroot
organogenesisasaffectedbyvariousfactors.Towhatextentthisknowledgecanbe
appliedtoensureareliablerootformationofshootcuttingsinvivo,isinvestigated
inChapter4.InChapter5isdiscussedwhetheravegetativepropagationcanbe
achievedbythecultivationofcapitulumexplantsinvitro.
2 Flowering behaviour of intact plants
2.1 INTRODUCTION
Pyrethrumplantsalmostcompletely failtoproduceflowerheadsathigh temperatures

whichoccurinthelowlands (below1900mabovesealevel)oftropicalcountries.Foran
adequateproductionoflargenumbersofflowerheadspyrethrumisgrowninthehighlands
ataltitudesbetween1900and2800m,wherelowertemperaturesprevail (Martin&Tatters-
field,1934;Gnadinger,1945;Parlevliet,1969).
ThetemperaturerequirementsforfloweringundernaturalconditionsinEastAfrica
wereinvestigatedbyGlover (1955).Thefloweringbehaviourofintactplantsunder
controlledconditionsingrowthchambers,however,hasneverbeenstudied.Suchstudies
mayshowhowfactors,liketemperatureandphotoperiod,affectthevegetativeand
generativedevelopmentoftheplants.Avegetativedevelopmentisdesirableforthe
maintenanceandmultiplicationofclonalplantmaterialandagenerativedevelopmentis
essentialforthecommercialproductionofpyrethrins fromtheflowerheads.
2.2MATERIALANDMETHODS
StockplantsofClones1087,4331andMa63/1889weregrowninamoderatelyheated
greenhouse.Dependingontheweatherconditionsoutside,thetemperaturerangedfrom20-
35 insummerandfrom10-20°Cinwinter.Inthewinternatural lightwas supplemented
withhighvapourmercury lamps.Atthehighertemperaturesduringsummertheplants
almostexclusivelydevelopedvegetativelyandshootcuttingsweretakenfromstock-plants
of1-2yearsold.Shootcuttingsofthethreecloneswererootedaccordingtothe
proceduredescribedinSection4.2.Subsequently,theyoungplantswereexposedfora
fewweekstoaconstanttemperatureof20°Cunderlong-dayconditions (Philips
U0W/33RSS U l e m e n t e d
iTrTVT ^ PP **Philineaincandescent lampsataday-
engthof14h)inagrowthchamber,beforetransfertovarioustemperaturesandphoto-
periodsinthephytotron1.
n l a n t D a t 7 r C 0 U e C t e d *"* * "" * ° f f l m l i n i t i « i o n ,thenumberofflowering

thTv f t T ^ ^ "* ^ d e V e l ° P " - ^ âgeoftheflowerheads.Withrespectto
thevegetativedevelopmentattentionwaspaidtotheshapeandlengthoftheleaves.
( A
^ ~ : : : ~ ^ ~ * ~ — - —
2.3FACTORSINFLUENCINGFLOWERING
2.3.1 Temperature
2.3.1.1Constanttemperature
Experiment 1. YoungplantsofcloneMa63/1889wereexposedtoconstanttemperaturesof
9,13,17,21or25°Cforadaylengthof16handwith20plantspertemperature.
About10weeksaftertransferthefirstflowerheadsdevelopedsimultaneously at9
and13°C.Table1illustratesthesituationattheendoftheexperiment,24weeksafter
exposure.Apparently,lowtemperaturesof9and 13°Careessentialforfloralinitiation.
Thehighestnumberofflowerheadswasinitiatedat9°C.Thesubsequentdevelopmentof
W*
'uiii
Fig. 1.Theinfluenceofconstanttemperatures (9-25C)onthegenerative andvegetative

developmentofplantsofCloneMa63/1889,19weeksafterexposure.
9°C 13°C 17°C 21°C 25°C

Fig.2.Theinfluenceofconstanttemperatures (9-25C)ontheshapeandlengthofleaves
ofplantsofCloneMa63/1889,19weeksafterexposure.
Table 1.Theinfluenceofconstanttemperatures (9-25C)onthegenerativeandvegetative
developmentofplantsofCloneMa63/1889,24weeksafterexposure.
Temperature Numberofflowe- Numberof Numberofflowerheads Averageleaf

(°C) ringplants flowerheads perfloweringplant lengthincm
(outof20)
9 18 55 3.1 14.1
131 13 24 1.8 14.3
17 0 0 0 12.8
21 0 0 0 11.0
25 0 0 0 11.0
1.At 13Coneplantdidnotsurvive.
theinitiatedflowerheads,however,wasstimulatedat13°C,whichcanbeobservedin
Fig. 1,whereat9°Cflowerheadsareinthebud-stageandat13°Carealready infull
bloom.At17,21and25°Ctheplantsremainedinavegetativestate.Theappearanceof
theplantsvariedstronglywithtemperature,whichwasnotonlyduetofloweringbut
alsotothelengthoftheleaves (Table1)andtopronounceddifferences infoliage
type(Fig. 2).
Experiment 2. YoungplantsofClones1087,4331andMa63/1889wereexposedtoconstant
temperaturesof9,13,17or21°Cforadaylengthof16h.Perclone 15plantswere
exposedto9,13or17°Cand5plantsto21°C.
Thethreeclonesdevelopedthefirstflowerheadsalmostsimultaneously9weeks
aftertransfer;Clone1087firstat9and13°Candafterwardsat17°C (Fig.3a), Clone
4331atthesametimeatthefourtemperatures (Fig.3b)andCloneMa63/1889 infirst
instanceat9°Candsubsequentlyat13and17°C (Fig.3c).Clone4331yieldedthe
maximumnumberoffloweringplantsatalltemperatures,asdidClone 1087,exceptat
21C,atwhichtemperaturefloweringdidnotoccur.CloneMa63/1889hadthemaximum
numberoffloweringplantsat9°C,butthisnumberdecreasedgraduallywithincreasing
temperatureandat21°Cfloweringwasnotobserved.Thenumberofgenerativeshoots,
givingrisetotheproductionofflowerheads,wasdeterminedatregularintervalsafter
exposure (Figs4a-c).Thehighestnumbersofgenerativeshootsdevelopedat9°C;with
increasingtemperature thesenumbersdiminishedprogressively forallclones.Thedata
presentedinTable2andtheFigsSa-cindicatethatatanoptimumtemperatureof9°C
plantsofClones1087and4331formedaboutthesamenumberofflowerheads,roughly
twiceasmanyasplantsofCloneMa63/1889.Thedevelopmentoftheinitiatedflower
headswasacceleratedbyanincreasingtemperature.Thethreeclonesrespondedsimilarly
andinconformitywithExperiment1(Fig.2),withregardtoleaflengthandfoliage
type (Figs6a-c).
number of flowering plants number of flowering plants
15 • • m • •—• • • • •
15 -13-'
14 «-•—•—•—r 14
12 12
10 10
8 8
2 ® ®
0L 0L
i i i i _i i i_
6 10 14 18 22 26 30 10 14 18 22 26 30
weeks of exposure weeks of exposure
number of flowering plants
15
14
12
10
2
Figs3a-c.Theinfluenceofconstant
temperatures (9-21°C)onthenumberof
0L floweringplantsofClones1087(a), 4331
(b)andMa63/1889.(c),measuredat
i i i i i_ regularintervalsafterexposure.(15
6 10 14 18 22 26 30 plantsareexposedto9,13and17°and5
weeks of exposure plantsto21°C.)
number of generative shoots number of generative shoots
- j — i — i ' '
10 14 18 22 26 30
weeks of exposure weeks of exposure
number of generative shoots
Figs4a-c.Theinfluenceofconstant
temperatures (9-21°C)onthenumberof
generativeshootsofplantsofClones1087
(a),4331 (b)andMa 63/1889 (c),measured
atregular intervalsafterexposure.(15
18 22 26 30 plantsareexposed to9, 13and 17°and5
weeks of exposure plantsto21°C.)
©
Figs5a-c.Theinfluenceofconstanttemperatures (9-21°C)onthegenerativeand
vegetativedevelopmentofplantsofClones1087(a),4331 (b)andMa63/1889 (c) 30
weeksafterexposure. '
ft
Figs6a-c.Theinfluenceofconstant temperatures
(9-21C)ontheshapeand lengthofleavesof
plantsofClones1087 (a),4331 (b)andMa63/
9°C 13°C 17°C 21°C 1889(c),30weeksafterexposure.
deveLDment^f^l" 6 ^ 6° f r ? O M t a n ttemperatures (9-21°C)onthegenerativeandvegetative

developmentofplantsofClones1087,4331andMa 63/1889,30weeksafterexposure.
Clone Temperature Numberofflowe- Numberof Numberof Numberofflo-
(°C) ringplants generative flower werheadsper
(outof 15and shoots heads floweringplant
5*resp.)
1087 9 15 231 383 25.5
13 15 144
17 207 13.8
15 65
21* 80 5.3
0 0 0 0
4331 9 15 133 341 22.7
13 15 105 195 13.0
17 15 95 107 7.1
21* 5 12 12 2.4
Ma63/1889 9 15 120 196 13.1
13 12
17 40 49 4.1
7 11
21* 0 12 1.7
0 0 0
2.3.1.2Fluctuatingtemperature
thef W a fT S t t t W ° . e X p e r i m e n t s h a v e *ownthatwhenconstanttemperaturesaremaintained,
th intiata' " " £ a V ° U r e d ^ E l 0 W t e m p e r a t U r e «* the^sequentdevelopmentof

them m a t e d flowerheadsisstimulatedbyahighertemperature.
Experiment 3.
Inthisexperimentisexaminedwhetherthereisatemperaturecombination
10
atwhicharapidinitiationofhighnumbersoffloweringplantsandflowerheadsaswell
asarapiddevelopmentoftheinitiatedflowerheadscanbeachieved.
YoungplantsofCloneMa63/1889wereexposedtothefollowingtemperature
combinations:d9/n9(day9°C/night 9°C), d13/n9,d17/n9,d21/n9,d25/n9,d13/n13,
d17/n13,d21/n13,d25/n13ord17/n17.Ateachtemperaturecombination16plantswere
exposedtoadaylengthof16h.
Afterexposureforabout20weeksthefirstflowerheadsdeveloped.Thenumbersof
floweringplantsandflowerheadsscoredatregularintervalsafterexposureare
expressedinFigs7aand7b.Thenumbersoffloweringplantsweremaximumatthe
temperaturecombinations9/9and13/9,whileahighnumberoffloweringplantswas
observedat13/13(Table 3).However,plantsdidnotonlyflowerattheseloweraverage
temperatures,butalsoathigheraveragetemperatures,inwhichalownight-temperature
alternatedwithahigherday-temperature,someplantsbecomingreproductive.Forinstance,
atthetemperaturecombination25/13,withthehighestaveragetemperatureof21C,
floweringoccurred,whereasplantsofCloneMa63/1889neverfloweredataconstant
number offlowering plants number of flower heads

90
© 80
70
60
50
40
30
20
10
temp, combination temp, combination

i i i i—i—i—
\ % %'7/9%%2,/92,/1325/925/,3 1 1 I I I 1 1 I ' '

I i i I i I i 1 1 1 —
9.0 11.7 13.0 K3 15.7170 17018.3 19.7 21.0 9 0 11.7 130 K 3 15.7 170 17.0 ia3 197 21.0
average temp average temp.

Figs7a-b.Theinfluenceoffluctuating temperaturesonthenumberoffloweringplants
(a)andofflowerheads(b)ofplantsofCloneMa63/1889,measured 19,22,28and40
weeksafterexposure.
11
Table3.Theinfluenceoffluctuating temperaturesonthegenerativeandvegetative
developmentofplantsofCloneMa63/1889,40weeks afterexposure.
Temperature Average Numberof Numberof Numberof flower
combination tempe- flowering flower headsper flowe-
day/night rature plants heads ring plant
(°C) (°C) (outof 16)
9/9 9.0 16 90 5.6
13/9 11 16
13/13 66 4.1
13 12 23 1.9
17/9 14 5
17/13 11 2.2
15 5 5 1.0
17/17 17 3
21/9 3 1.0
17 8 9 1 1
21/13 18 2
1*1
25/9 2 1.0
19.7 4 6 1.5
25/13 21.0 5 6 1.2
temperatureof21°C (Experiments1and2).Atnighttemperaturesof9and13°C,
maintainedfor8h,flowerheadswereinitiated,eventhoughdaytemperaturesof21 and
25Cweremaintainedfor16h.Atnighttemperaturesof9and13°Ctheflowering
responsedecreasedasthedaytemperature increasedfrom9or13to25°C.Theflowering
responsealsodecreasedwhenatdaytemperaturesof13,17,21and2S°Cthenight
temperatureincreasedfrom9to13°Cor17°C.Considerablenumbersofflowerheadswere
producedwhendaytemperaturesof9or13°Cwerecombinedwithnighttemperaturesof9
and toal e s s e re x t e n t > „«,<,.^ d e v e l o p m e n t Q£ ^ . ^ . ^ ^ ^ ^ ^
acceleratedbyhighday-temperaturesof17,21and25°C.Vegetativecharacteristics,
litetheshapeandlengthoftheleaves,weredeterminedbythedaytemperatureto
whxchtheplantswereexposedfor16h.Consequently,leavesfromplantsofthe
2 Z C ° n M n a t i ° n S 17/9 '1 7 / 1 3a n d W showedasimilarappearance.Thefoliage
yp andthelengthofleavesfromplantscultivatedatdaytemperaturesof9,13,17,
withthoseofieaveswpiants atthe
Z:Z:::7:T *- ~ ™
3 Sh0W6d
-Inifft ^ 7 " * " a P 6 r i 0 d °f a C°ntinUOUS ^ m a t u r e i s
nofhi8hnmbeTSoffloweringpiantsandfi
ZT^Zul T Peri
—^
° d laSt 'b 6 f 0 r eP l a n t S ° f C 1 ° n e * " / « » - n beexposed
S i r '"^înitiat6d f l ° W^
e r deVel
a ih t l
with°Pyom
™ gP plants
" ^ 0£clone 63/1889,
^ ^ ^ J T T * YeT*"" ^ 21
- ^ e n t 16p l a T w e r e ^ o s l d I l ^ ^ T * f h^ ^ " °C-
Plantsstartedtoflower7w e L ft " t ™ f " * "&^ P e r i 0 d * * £ l r S t

lrreS ective
maintainedfor2 4 6or7 v T ' P °*Aether9°Cwas
optimumgeneratived e v e l o p u ^ ^ ^ *^ 4W e e k sW e r e inadequateforan
weeksaferJ T f e r nT h ' T ^ *" p l a n t s * » « -productiveat2 1 * .Seven
transfer,thenumberoffloweringpl antswashighestforthoseplantssub-
12
Table4.Theinfluenceofexposurefor0,2,4,6,8,10or23weekstoaconstant
temperatureof9°C,beforeexposuretoaconstanttemperatureof21C,onthegenerative
andvegetativedevelopmentofplantsofCloneMa63/1889,23weeksafterexposure.
Weeks Numberofflo- Numberof Numberofflo-

weringplants flower werheadsper
9"C- 21°C (outof16) heads floweringplant
0 23 0 0 0
2 21 5 5 1.0
4 19 7 15 2.1
6 17 16 43 2.7
8 15 16 68 4.3
10 13 16 75 4.7
23 0 16 105 6.6
jectedto9°Cfor6weeks,probablybecausetheyhadbeenexposedfor1weekto21C,
whileplantsofthegroupstobeexposedfor8,10or23weeksto9°Cwerestillat9°C,
atwhichtemperatureflowerheadsdevelopedmoreslowlythanat21°C.Sevenweeksafter
transfer,plantsthathadtobesubjectedtocoldperiodsof8,10and23weekswere
stillexposedto9°C.Theoreticallythesetreatmentsshouldnot'differ,butinreality
after7weeksinthegrouptobeexposedfor23weeksto9°Ctwomoreplantswere
number offlowering plants number of flower heads

13,23 110r
16r
100
14 |-
90
12 80
70
10
60
8 50
6 40
30
A
20
2 10
0 2 4 6 8 10 23 0 2 A 6 8 10 23
weeks at 9°C weeks at 9°C
before exposure to 21° C before exposure to 21°C
Figs8a-b.Theinfluenceofexposurefor0,2,4,6,8,10or23weekstoaconstant
temperatureof9°C,beforeexposuretoaconstanttemperatureof21C,onthenumber
offloweringplants (a)andofflowerheads (b)ofplantsofCloneMa63/1889,measured
6-23weeksafterexposure.
13
floweringthaninthegroupstobeexposedfor8or10weeksto9°C (Fig.8a).Such
slightdivergencesfromhorizontallines,whichalsooccurred 8,9and 10weeksafter
transfer,canalsobeobservedinFig.8b.Maximumnumbersoffloweringplants inthe
groupstobeexposedfor6,8,10and23weeksto9°Ccanbeobserved 8,10,11and13
weeksaftertransfer,respectively (Fig.8a). Thuschillingfor6weekswassufficient
forfloralinitiationofallplants.Initiatedflowerheadsdevelopedmostrapidlywhen
theplantswereexposedto21°Cafteracoldperiodof6weeks.Thedevelopmentofthe
flowerheadswasprogressivelyretardedwithanincreasingdurationofthecoldperiod.
Thus7and11weeksaftertransferthenumbersofflowerheadswerehighestinthose
groups,wherechillingpersistedfor6and8weeks,respectively (Fig.8b).Despitethe
delayeddevelopmentoftheflowerheads,however,15weeksaftertransferthenumberof
flowerheadswashighestinthegroupwherechillingstillpersisted (group23weeksof
9 C). Finally,23weeksaftertransfer,thenumberofinitiated flowerheads increased
withanincreasingdurationofthecoldperiod;thehighestnumberofflowerheadswas
producedataconstanttemperatureof9°C.Plantssubjectedfirstto9°Candsub-
sequentlyto2.1°C, developedtwofoliagetypes,characteristic forboth temperatures
(Fig. 2).
2.3.2 Photoperiod
Asmanyspeciesofthegenus Chrysanthemum aresensitivetodaylength,itisof

interesttocheckwhether Chrysanthemum oinerariaefoUwn alsorespondstothephotoperiod.
YoungplantsofCloneMa63/1889wereexposedtophotoperiodsof8(shortday)and
16h(longday), at9,13or17°C.Unfortunately,plantsexposedto17°C,bothunder
short-dayandlong-dayconditions,hadtobediscardedprematurelybecauseofanattack
byredspiders.
AscanbeobservedinFig.9athefirstplantsbecamereproductive5-6weeksafter
transfertolong-dayconditions,bothat9and13°C.Atadaylengthof8
hthegenerative
developmentoftheplantswasconsiderablydelayedandthefirstflowerheadsdeveloped
17-18weeksaftertransfer,at9and13°C.Floweringunderlong-dayconditionsoccurred
moregraduallythanundershort-dayconditions,wherewithin2weeksallplantsbecame
nWberS f
trr^rinit'T 1 7 ,;^ ^ °
W r e initiat6d
fl WeringPl tS
° l a
- — ° b s — * **"
™ 2'a I', ^ "*" * ^ conditionsonly,but
H i 1 M S " d d e " a P P ™ e ° f* « « âds undershort-dayconditionsw L '
- h da ! > y 'SUbStantlally" * » ™ ^ r s offlowerheadswereinitiated
71 tr A ? b T H0 0dS
"^£ 1 C WOe rr r eW« S êratures-derlong-day
re P r d U C e d 9
abT r n f *r r ^ ° ^^ ° " ^ a n t13°C
y onu l I " d h d e V e l ° P e d f a S t e r at^ * a n at9°Candfasterunderlong-
udyconditionsthanundershort-dayconditions Ata A T U r
remaineds m a i w +u J conditions.Atadaylengthof8
heads0£a 1size
htheflowerheads
^^Z^z^~^r~
long-day conditions a t 9, 13and17°C ™
conditions at 9 13^ J l ! f J ° ^ ^
^
leaVes
' " " ^ ^ ^ *"*"
developed, whereas under short-day
- -
S m l l e r b u t w i t h t h e sa
long days (Fig. \ 0 ) . " ^ ' ™ leaf-shape asfor
14
number of flowering plants number of flower heads
320 -
280 •
8h9°
240 -
200 •
8h13°
160 16h9°
120
16h 9 ° /
80 ©
16M30
40
I 16h 13° j
0
i i i i i_
13 17 21 25 29 33
,.,„„i„- ~<~ ^.,-~ weeks of exposure
r
weeks ofexposure
Figs9a-b.Theinfluenceofphotoperiodsof8and16h,atconstant temperaturesof9
and13°C,onthenumberoffloweringplants (a)andofflowerheads (b)ofplantsof
CloneMa63/1889,measuredatregularintervalsafterexposure.
A
n
16h17u 8h9" 8h 13u 8h 17L

16h9U 16h13u
Fig. 10.Theinfluenceofphotoperiodsof8and 16h,atconstanttemperaturesof9,13
and 17°C,ontheshapeandlengthofleavesofplantsofCloneMa63/1889,17weeksafter
exposure.
15
Table5.Theinfluenceofphotoperiodsof8and 16h,atconstant temperaturesof9and
13°C,onthegenerative andvegetativedevelopmentofplantsofCloneMa 63/1889,33
weeksafterexposure.
Photoperiod Temperature Numberof Numberof Numberofflower

(h) (°C) flowering flower headsperflowe-
plants heads ringplant
(outof 16)
8 9 16 248 15.5
13 16 178 11.1
16 9 16 173 10.8
13 16 82 5-1
2.4DISCUSSIONANDCONCLUSIONS
Theplanthastopassthroughdifferentstagesbeforeitflowers,namelyripeness-
to-flower,floralinductionandinitiationanddevelopmentoftheflowerheads.The
youngpyrethrumplants,whichoriginatedascuttingsfromadultplants,reachedthestage
ofripeness-to-flower.
ThevegetativeandthegenerativedevelopmentofyoungplantsofClones 1087,4331
andMa63/1889weremarkedlyaffectedbytemperature.Allclonesshowedessentiallythe
samereactiontoconstanttemperatures.Asthetemperature decreased,moreflowerheads
wereinitiated,thebestfloralinitiationwasobtainedat9°C.Itwasnotinvestigated
whetherstilllowertemperatureswouldstimulatetheinitiationofflowerheadsfurther.
Thesubsequentdevelopmentoftheinitiatedflowerheadswasstimulatedbytemperatures
of17,21and25°C.
ThetemperaturerequirementsofClones1087andMa63/1889were qualitative, sothat
at-aconstanthightemperature (21C)theplantsdidnotfloweratall,whereasthe
reactionofClone4331provedtobe quantitative, i.e.theplantsalsofloweredata
constanthightemperature (21°C).ThusplantsofClone4331mayalreadyhavebeeninduced
attheconstanttemperatureof20°C,towhichtheplantswereexposedinthegrowth
chamberbeforetransfertothephytotron,whileplantsofClones 1087andMa63/1889
cannothavebeeninducedatthistemperature.
Inconformitywithobservationsinpractice,CloneMa63/1889showedthemost
pronouncedcoldrequirementforflowering.ThisclonewasselectedintheMarindas
'districtinKenyaatanaltitudeofabout3000m,wheretheprevailingtemperaturesare
constantly low.Atloweraltitudes,withhighertemperatures,thefloweringresponseof
thisclonewasinadequate (Hoekstra,pers.commun.).
Sincesomevariationinthefloweringresponsesoftheclones.wasobserved itis
recommendedtoinvestigatethefloweringbehaviourofeachselectednewclone,tofind
outthemostsuitableregioninwhichthecloneshouldbecultivated.
Afluctuationofthetemperaturewithday C16h)andnight C8h)demonstratedthat
PlantsofCloneMa63/1889floweredatanaveragetemperatureof21°C(16hday25°and
8hnight13C),whereasfloweringwasneverobservedataconstanttemperatureof21°C.
anoptomuminitiationofflowerheadsonplantsofCloneMa63/1889,however,a
16
periodwithacontinuouslowtemperatureof9°and,toalesserextent,13°C,was
required.AllplantsofCloneMa63/1889floweredwhentheyweresubjectedto9°Cforat
least6weeks,butchillingforamoreprolongedperiod improvedtheinitiationofflower
heads.Thenumberofinitiatedflowerheadswasaboutproportionaltothedurationofthe
coldperiod.
ThesefindingscorrespondwellwiththedataofGlover (1955),whoinvestigatedthe
temperaturerequirementsforfloweringundernaturalconditionsinEastAfrica.He
observedinfieldtrialsthattheyieldofpyrethrumflowerswasdirectlyrelatedtothe
numberofdaysspentbytheplantatorbelow15°C,somethreemonthsearlier.In
vegetativeplantsflowerheadswerealreadyinitiatedafterexposureto15°Cfor10days.
Persistentchillingwasmoreeffectiveinstimulatingbudproductionthanmoresevere
chillingmaintainedatnightandalternatedwithperiodsofhigherday-temperatures.If
ameantemperatureof24°Cwasprolongedforoneweekormore,theproductionofflower
headswasinhibited.
UnderfieldconditionsinKenyaflowerheadsareproducedapproximately 10weeks
afterthestartofarainperiodwithlowtemperatures.Dependingonthedistrictthere
are2or3rainperiodsduringeachyear,alternatingwithdryperiodsofhigher
temperatures.Inanidealsituationtheplantsinitiateflowerheadsduringtherainy
seasonandtheseflowerheadsdeveloprapidlyinthedryperiod.
Inaddition,toensurealarge-scalevegetativepropagationinnurseries,itis
recommendedtocultivatepyrethrumathightemperaturesinthelowlandstostimulatethe
vegetativedevelopmentandtoavoidorgreatlyreducethegenerativedevelopmentofthe
plants.
Vegetativecharacteristics,liketheshapeandlengthoftheleaves,dependinthe
firstplaceuponthegenotype,butthedifferentclonesshowedananalogousreactionto
temperature.BothshapeandlengthofleavesofCloneMa63/1889werestronglyaffected
bytemperature,whilethephotoperiodonlyinfluencedthelengthoftheleaves.
Apartfromthetemperature,thefloweringbehaviourofplantsofCloneMa63/1889
alsorespondedtothephotoperiod.Thefirstplantsbecamereproductiveunderlong-day
conditionsanditseemedthatpyrethrumcouldberegardedasalong-dayplant.Under
long-dayconditionsthefirstflowerheadswereinitiatedrapidlyandafterwards
reasonablenumbersofflowerheadsofanormalsizedevelopedgradually.Undershort-day
conditions,however,theinitiationwasstronglyretarded,whilesuddenlyconsiderably
highernumbersofsmallerflowerheadsdeveloped.Theseresultsmaybeattributedtoa
lowerrateofphotosynthesis in8hthanin16hoflight.Inconclusion,pyrethrumhas
toberegardedasa quantitative short-dayplant,sothatbothunderlongandshortdays
floweringisachieved,butthehighestnumberofflowerheadsisproducedundershort-day
conditions.Thesephotoperiodiceffectsareofnodirectpracticalimportanceforthe
cultivationofpyrethrumintropicalcountries,wherethroughouttheyearthedaylength
isapproximately 12h.Thusthereactionofpyrethrumtothephotoperiodwasnotfurther
studied.Fromafundamentalpointofview,however,itmaybeveryinterestingtoanalyse
thisverystrangephotoperiodicresponse.
17
3 Root formation of peduncle explants in vitro
3 . 1 INTRODUCTION
Vegetativepropagationofmany cropscanbeachieved throughtheformationofroots

onbasal stemportionsofshootcuttings.Unlessrootsoriginate fromprimordiathat
already existedatthetimeofmakingtheexplant,asincertaincases,rootsresult from
a genuine initiationprocess.Thismeansadedifferentiationofcellswhich,afterhaving
revertedtotheprimarymeristematicstate,areorganizedtogivearootmeristem
(Gautheret,1966a).Whencellsoftherootmeristemdivide,differentiateandelongatea
rootprimordium,anadventitiousroot,andfinallyarootsystemdevelop.
As indicatedinChapter1vegetativepropagationofpyrethrumfromshoot cuttings
wouldbedesirable.Before developingapracticalmethod (Chapter 4 ) ,itwouldbeuseful
tohave detailed informationabouttheinfluenceofvariousfactorsonthisprocessof
adventitious rootformation.Thiscanbeachievedbycultureinvitro,thetechniqueof
growingplantsorplantpartsasepticallyonartificialnutrientmediaintesttubes.
Thefollowing advantagesofcultureinvitro,incomparisonwiththecultivationin
vivoofplantsorplantpartsinpotsorcuttingbeds (Pierik,1969),canbementioned:
-Bycultureinvitro,plantgrowthanddevelopment canbestudiedunder satisfactorily
controlled asepticconditions,bothaboveandwithinthemediuminthetesttube.A
variationofasinglegrowthfactorinvitrohardly influences otherfactors involvedin
rootformationandconsequentlytheeffectofseparatefactorscanbeexamined.Because
conditionsinvivoaremorevariable,variationofonefactorbrings aboutavariation
inother factorssothatanevaluationofasinglegrowthfactoriscomplicated.
-Substances likegrowthregulators,whichareappliedinverysmallquantitiestothe
medium,caneasilybetakenupbytheexplantinvitro,especially throughawound cut,
butnotsoeasilyunderconditionsinvivo.
-Explantsinvitrocanbemoreeasilymanipulated thanwholeplantsinvivo.
-Usingsmallplantpartsthequantityofplantmaterialrequiredforexperimentsin
vitroisrelatively small.Thecultureofexplantsrequires lessspacethanthatof
wholeplantsinvivo.
-Oneofthedifficultiesofinvestigationsinvivoisduetocorrelations,whichare
certainrelationsbetweendifferentpartsoftheplant.Thesecorrelationsmaybe
involvedinregenerationphenomena.Themethodofcultureinvitro,withsmallplant
parts,providesameansofcircumventing thesecorrelationstosomeextentandis
thereforemoreprecisethantheuseofwholeplants (Gautheret, 1966a).
-Ingeneralyoungmoreorlessundifferentiated tissuesshowthemostrapidandbest
regeneration.Plantmaterialinayoungdevelopmentalstagecanbecultivatedinvitro
underasepticconditions,butdiesoffinvivowhenattackedbymicro-organisms.
-Root formation canbeobservedinvitroatanydesiredmomentwithoutdisturbingthe
experiment.Anexaminationofrootformationatregular intervalsinvivoisimpracti-
cable.Asarule,onlyonecomprehensive observation canbemade,afterwhich theplant
materialhastobediscardedandtheexperimentisatanend.
Thefollowing disadvantagesofcultureinvitrocanbeindicated:
-More specificknowledge,skillandtechnical equipment areneededforcultureinvitro
thanforthecultivationofplantsunderconditionsinvivo.
-Oftendetaileddatafromcultureinvitrowillnotcompletely correspondwith those
foundunderconditionsinvivo.Consequently,onehastobevery carefulwiththeinter-
pretationofresultsinvitroinrelationtotheirvalidityinvivo.
Becauseofitsmany favourable aspects,themethodofcultureinvitrowasusedto
examinetheeffectofseparate factorsonrootorganogenesis (Section 3.4). Studieson
theanatomical aspectsofthisprocessofadventitious rootformationinpyrethrumare
describedinSection 3.3.Theyaremadetosupplyadditional information aboutthe
locationandkindoftissues involvedinthe initiationanddevelopmentofadventitious
rootsandhowroot formationprogressesintime.
Itwas found thatdifferentplantpartsofpyrethrum are'abletoinitiate
adventitious rootsinvitro,namely explantsofthehypocotyl,theleafblade, the
petioleandthepeduncle.Rootorganogenesis,however,wastoosporadictoaffordany
hopeofarapiddeterminationoftheunderlying conditions,exceptforexplantsofthe
peduncle.Sopeduncle explants,whichproducednumerous adventitious rootsathigh
percentages,werechosenastestmaterial.
S.2.1 Material
As explants fromplants growingoutsidecouldnotbesterilized satisfactorily,

stockplantswere growninamoderatelyheated greenhouse (Section 2.2).Atlow
temperaturesinwinter flowerheadswere initiated,whilewitharising temperaturein
spring,theinitiated flowerheadsdeveloped fastandplantsstartedblooming. Flowering
continuedforabouthalfayearandduring thisperiod theupperhalvesofpedunclesof
stock-plantsof1-2yearsoldofthethreeKenyaClones 1087,4331andMa63/1889 were
excised (Fig. 11,left). Fromtheupperportionsof20peduncles,derived from20
differentplants,thebractsandflowerheadswereexcised (Fig.11,middle).The
peduncleswere sterilizedina51(50g/1)calciumhypochlorite filtered solutionfor
20minandsubsequently rinsedseveral timeswithsterilized tapwaterfor30min.To
prevent leaching,thewaterwaspouredoffimmediately afterwashing.
Inaninoculation room,previously sterilizedbyultraviolet irradiation,the
sterile-culture techniquewasapplied (Gautheret,1959).Thetopandbaseportionsof
thepedunclewerecutoff,afterwhichitwasdivided intosegmentsofabout1.5cm,in
suchawaythatthenodal sites,wherethebractswere emplanted,wereexcluded.The
explantswere furtherwoundedatoneside,byexcisingastripofthecortexoverthe
whole lengthoftheexplant,andcutslantinglyatthemorphological baseto
19
iw^î^jpk^ywii»gj!f.liPf!IWAiiiwpi"iW'l'» P
ii
Si it.
• >±
Fig. 11.Upperportionofapeduncleinayoungfloweringstage;fromlefttoright:
- flowerheadandbractsattached,
- flowerheadandbractsexcised,
- divided into6woundedexplantsofabout 1.5cm.
distinguishitfromthehorizontally cutmorphologicaltop(Fig. 11,right). Finallythe

explantswereplacedhorizontallywiththewoundedsideontheculturemedium (Fig.13).
3.2.2 Methods
Thebasicculturemediumwascomposedofpyrex-distilledwater, 'Difco'Bacto-agar
0.6%(6g/1),thehalfstrengthofboth Knop'smajorandHeller'sminorsalts(cf.
Gautheret, 1959),sucrose 2%(20g/1)andB-indolebutyricacid (IBA)at10" 5g/ml.The
pHofthemediumwasadjustedto5.8before autoclaving.About25mlofthismediumwas
poured intopyrextesttubeswithalengthof16cmandadiameterof2.7cm.Thetubes
werepluggedwithcottonandcoveredbyaluminium caps.Themediumwas autoclavedat
115°Cfor20min;tapwaterandfilterpaperat120°Cfor30min.
Theexperiments,whicharedescribedinSection 3.4,were carriedoutbyvarying
onegrowthfactor.Generallyeachexperimentconsistedof6-9treatmentsand
consequently theupperportionsof20peduncleswereeachdivided into6-9explants
(Fig.11,right),whichweredistributed systematicallyandinabalancedwayoverthe
varioustreatments.Pertreatment20explantswereexposedfor4weeksto20°Cindark-
ness.Toestimatetherateofadventitious rootformation,about1hofilluminationper
weekwasnecessary forobservations.Theexplantsinculturewere examinedwithahand
20
lensandindoubtful caseswithastereomicroscope (WildtypeM5).Attheendofthe
experiments,4weeks after incubation,thefollowingvariableswere determined:
-rootingpercentage (over20explantspertreatment),
-theaveragenumberofroots (perrooted explant),
-theaverage lengthperrootinmm,
-theaveragedryrootweight (perrootedexplant)inmg (measured afterdryingfor4h
at 80°C).
For anatomical observations (Section 3.3) explantsofthepeduncleofClone1087
were incubatedinvitrounderconditions suitableforadventitious root formationandthe
plantmaterialwas fixated 0,1,2,...or10dafter incubationinCarnoy's fluidfor5
minandinKarpechenko'smodifiedNavashinfluid (Johansen,1940)foratleast2d. The
segmentswerewashedwith tapwateranddehydratedwith ethylandbutylalcoholand
subsequently embeddedinparaffinforsectioningwithamicrotome.Sectionsof12urn
werestainedinsafraninwith fastgreenasacounter stain.Mayer's adhesivewasusedto
preventthesections frombeingwashed offduringstainingandfinallythesections
weremountedwithartificialCanadabalsam (Rhenohistol).Picturesweremadewitha
photomicroscope (Zeisstype Standard universal).
3.3ANATOMICALOBSERVATIONS
Inmanyplantspecies adventitious rootsoriginateendogenously fromthepericycle -

ofyoung stems (Libbert, 1956/1957).Instemsof Chrysanthemum movifolivm Ram.
periclinal celldivisionsinthe interfascicular pericycle giverisetotheformationof
rootprimordia (Stangler, 1956).
Anatomical observationsofpyrethrumwerequitesimilar.Transverse sections showed
thepeduncle explant justbefore incubationinvitro (Fig. 12a)andpericlinal cell
divisionsinthe interfascicular pericycle3-4dafter incubation.Subsequent cell
divisionsintransverseandanticlinaldirectionoccurredandarootprimordiumwas
initiated6dafter incubation (Fig.12b).Thecellsoftherootprimordium divided,
enlargedanddifferentiated rapidlyduring furtherdevelopment and,growing fromthe
interfascicular pericycleinanticlinaldirectiontotheepidermis,theroot primordia
broughtaboutacollapseofcellsoftheendodermis (identifiablebyCasparian strips),
thecortexandtheepidermis8dafter incubation (Fig. 12c).Theadventitious root
penetratedtheepidermis10dafterincubation (Fig. 12d),justbetweentheribsofthe
peduncle,wherethecortexconsistedofavery loosechlorenchymoustissue,easyto
penetrate.Penetrationofthetightcollenchymouscortex tissueoftheribswould
probablybeimpossible andmoreoverthedistancetobebridgedwouldbeconsiderably
longer.Presumably,after initiationseveralpericycliccellsadjacenttothecell(s)of
theroot initialdivided,becamemeristematicandwere incorporated intotheroot
primordium.Undoubtedlyinalaterstagethecambiumalsocontributed cellstothe
growing rootprimordium,becauseaconnectiondevelopedbetweenthexylemofthe original
explantandthexylemoftheinitiatedroot.
Adventitious rootswereusually initiated duringthefirst2weeksofincubation,
whileduringthesubsequent2weeksofincubationtheroot initialsdeveloped.Theroots
21
:
: : ~ i : - v •?."-.•>':•-••:••-'•. ;<-";.- v?:"--: {j'T.r'-->:- ,"--.VJ:^'.-O
"rft£/.V. 'Kf \;- - ••'--H ^
V - . V.
500 |jm
Figs 12a-d. Transverse sections of peduncle explants:
a
t ;fore e incu b ati:n: h i C h enCl
° S e "" ^ ^ ^ ^ and t h e wounded
•«* êlow), just
6 d
c! Tnr^^ô^rWdlfarU^lLlen ^ . S ^ ^ f t "£'« ^ - ^
cells of the.endodeîs, the cortex an^Tl^JTsl f e"nLo t£ » ' C ° U a P S e °'
f t t r inluo t r S r ° 0 t P e n e t r a t e S t h e e p i d e ™ i s b ~ « , . rios o f t e ^ p l a n t , ,0d
22
'jtr-C?*. " -..•,•
;«t^' .• s- -. - -. 1 i « V - »
•A?*" •"- '•*.. •-".-•". - • i .'
-r:',,« » • ' . '. . . -• • '.'•
Or'-'
J
J =- .
r,V
,
. ! ^.-- > • • ' - . ' i'Vf
* ••'•?
74
J '.'..V,
500îm * *'""••"
.V - - ' .>.-•/•• ' 1

IOO|.-ii f ,«v ^ - , . • - ; . rij,
y.-t j; n
3
'.h •
/ / ' . ' • • ' • . ' • ^
, « # • .
* » J * •
" ' • ! •
23
IUWIJP p*mmv
// /
-./
L_
Fig. 13.Peduncleexplantplacedhorizontallywiththewounded sideontheculturemedium.
Adventitiousrootsinitiatedanddevelopedatthemorphologicalbaseandoppositethe
wounded side,4weeksafterincubation.
emergedmostlyatthemorphologicalbaseoftheexplant,opposite thewounded side (Fig.

13),andshowedapositivegeotropicresponse.Branchingofthe (main)rootsandthe
consequentformationoflateralrootswasrarelyobserved.
The suitabilityofthepericycletoinitiaterootsisexplainedbyanexisting
inequivalencebetweentheontogeneticandphysiologicalageofthetissues (Libbert,
1956/1957).Pericyclictissueisageingmoreslowlyandthereforeislessdifferentiated
andmoresuitableforasubsequentdedifferentiationandregenerationofroots,thanmore
differentiatedadjacenttissues.
3.4FACTORSINFLUENCINGROOTFORMATION
Theorganogenesisofadventitiousrootsinvitrodependsuponacomplexsystem
involvinganumberoflimitingandinteracting factors (Libbert,1956/1957;Gautheret,
1969c;Leroux,1973;Reinert,1973).Suchfactorsarerelatedtopropertiesofthe (ex)
plant,thenutritional/hormonalcompositionofthemediumandtheclimaticconditionsto
whichthe(ex)plantisexposed.Unlessotherwisestated,thechosenstandardgrowth
conditions (Section3.2)weremaintainedinallexperimentsdescribedinthissection..
24
3.4.1 Plant factors
Someoftheplant factorswhichwereevaluatedconcernthegenotypeandtheageof
theexplant,which lastcharacteristic findsexpressionintheflowering stageofthe
peduncleandthepositionoftheexplantinthepeduncle.Theotherplant factors,like
explantlength,woundingandpolarity,thatwere investigated,wererelatedtohowthe
explantwas manipulated.
3.4.1.1 Genotype
Inmostexperiments peduncle explantsofClones 1087,4331andMa63/1889wereused.

Undervarious suitable conditions,explantsoftheseclonesregeneratedhighnumbersof
adventitious rootsathighpercentages.However,rootingparametersoftheclonessome-
timesdiffered.Thesedifferentrootingresponses canbeascribedtothegenotype
influencing anatomicalandphysiologicalpropertiesofthe(ex)plant,whichintheir turn
affectadventitious root formation.
Differencesinrootformationbetweenvariousplants,withinonespeciesorwithin
onegenus,werealsofoundinvitrowithrhizomefragmentsofHelianthus tuberosum
(Gautheret,1961,1969c;Tripathi,1974)stemexplantsofParthenooissus (Leroux, 1965),
Salix (Leroux, 1966), Visum sativum (Leroux,1968ab,1973). Rhododendron (Pierik, 1969;
Pierik&Steegmans,1975c)andPrunus (Feucht&Dausend, 1976).
3.4.1.2Flowering stage
Fromthestockplantspeduncleswereexcisedinvarious stagesofdevelopment,
ranging fromyoung (upperportion just longenoughtoyield6segmentsof1.5cm)toold
(bearingoverblown flowerheads),todetermine themostsuitablestageforadventitious
rootformation.
Theyoung,softpeduncles couldeasilybedividedwithascalpel intosegments,but
thisprocedureprovedtobedifficultwithold,woodypeduncles.Incomparisonwithex-
plants fromyoungpeduncles,explantsdetached fromoldpedunclesyieldedahigher
contaminationpercentageandaretarded root formation (Fig.14).ThedataofTable6
further indicate thatsegmentsofyoungpeduncles elongated considerablymore than
segmentsofoldpeduncles.Whatever rooting characteristicwastakenasameasure, the
bestregenerationofrootswasobtainedwithexplantsexcised fromtheyoungestpeduncles,
whichmaterialwaschosenforstandardexperiments.
Ayoungdevelopmental stagewasfoundtobealsoadvantageousforrhizogenesisof
stemsegments cultivatedinvitroofRhododendron (Pierik,1969;Pierik&Steegmans,
1975c), petioleexplantsofLunaria annua (Pierik,1972)andleafmidrib fragmentsof
Gerbera jamesonii (Pierik&Segers,1973).Olieman-vanderMeeretal. (1971)reported
thatadventitious rootformationofepicotylexplantsinvitroofPhaseolus vulgaris was
notmarkedly influencedbythedevelopmentalstage.
Libbert (1956/1957)gaveanexplanationforthepositive influenceofayoung
developmental stageonadventitious rootformation.Hesuggestedthatadventitious root
25
Table6.Theinfluenceofvariousflowering stagesofthepeduncle (1isyoungto6is
old)onelongationandrootformationofpeduncleexplants.
Clone 1
averageexplantlengthinram _1
1087 23.3 21.1 17.4 16.2 16. 3
Ma63/1889 26.1 25.0 16.3 16.5 16.1 15. 5
rootingpercentage _1
1087 65 50 50 35 25
4331 80 95 85 90 90 45
Ma63/1889 100 95 40 65 75 25
numberofrootsperrootedexplant
1087 23.8 25.6 25.1 4.6 3. ,3
4331 45.6 25.1 15-.5 18.6 9.9 5,,8
Ma63/1889 38.9 26.5 8.8 9.5 6.6 2..8
averageroot lengthinmm _1
5.6 5.3 5.0 2.8 1,.9
1087
6.1 6.0 5.0 2.3 2.4 1,.7
Ma63/1889
dryrootweightperrootedexplant inmg _1
1087 2.4 1.9 1.7 0.1 0 .1
4331 3.7 3.5 1.3 1.2 0.7 0 .2
Ma63/1889 4.7 4.1 0.7 0.4 0.3 0 .0
1.Treatmentdiscarded.
rooting percentage rooting per centage

on 2 i
80| 2
_ ^ / j ^
3
70 70
4
—•—
—•—•
60 60
5
50 50 -
y —•—
—•—•
40 40
30
ijIn
30 -
3~ 6
—•—•
20 20 - 2-
775l
10 10 -
%y
0L 0 1\
i_ ' i • f
4 8 12 16 20 24 28
8 12 16 20 24 28
days of incubation days of incubation
Fig. 14.Theinfluenceofvariousflowe- Fig. 15.Theinfluenceofvarious
ringstagesofthepeduncle(1isyoungto positionsofexplantsfromtheupperhalf
6 isold)onrootformationofpeduncle
ofthepeduncle (1isapexto6isbase)
explantsofClone1087.(Stage4was
onrootformationofpeduncleexplantsof
discarded.)
Clone1087.
26
formationbecomesmoredifficultasthecellsoftheexplant,whichhavetogiveriseto
rootinitials,becomeprogressivelymoredifferentiated.Apartfromthisanatomical
aspect,whichisalsoinvolvedinpyrethrum,differences inrootregenerationbetween
variousdevelopmentalstagesofthepedunclemayalsobeattributedtodifferencesin
physiologicalproperties,likethenutritional/hormonalconditions.
3.4.1.3Explantposition
Explantscanbeisolatedfromdifferentpositionsoftheyoungpeduncle.Explants
excisedfromthelowerhalfofthepeduncleprovedtobeunsuitablefortissueculture,
becauseofhighcontaminationpercentages.Explantsdetachedfromtheupperhalfofthe
pedunclecouldbecultivatedasepticallyinasatisfactorywayandthesesegments,
locatedat6differentpositions,wereexaminedfortheirrootingability.
Fromthedistaltotheproximalpartofthisupperhalfofthepeduncle,the
segmentsbecameprogressivelymorewoody.Theyoung,softdistalpartsofthepeduncle
couldbedividedintoexplantsmoreeasilythantheproximalolderandmorewoodyparts.
ApicalexplantsofClones1087 (Fig.15)andMa63/1889initiatedadventitiousroots
morerapidlythanmorebasalexplants,whileexplantsderivedfrom6differentpositions
oftheupperpartofthepeduncleofClone4331allshowedaboutthesamerootingrate.
ExplantsexcisedfromtheapexofpedunclesofCloneMa63/1889elongatedmorethanex-
plantsfromthebasalpartoftheupperhalfofthepeduncle (Table7). Evidently,the
rootingresponseofthethreeclonesdecreasedfromtheapicaltothebasalpart.Al-
thoughtopexplantsyieldedthebestrootformation,thewholeupperhalvesofyoung
peduncleswereused,becauseotherwisetoomanypeduncleswouldhavebeenrequiredfor
eachexperiment.
Theseobservationsagreewiththosereportedbyotherscientistswhofoundthat
Table7.Theinfluenceofvariouspositionsofexplantsfromtheupperpartofthe
peduncle (1isapexto6isbase)onelongationandrootformationofpeduncleexplants
Clone 1 2 3 4 5 6
averageexplant lengthinmm
Ma63/1889 19.5 17.6 16.8 16.5 16.4 16.4
rootingpercentage
1087 80 80 70 65 50 25
4331 75 95 100 95 100 95
Ma63/1889 100 90 80 95 85 80
1087 14.9 13.1 13.0 12.0 8.7 4.8
4331 32.1 32.4 23.3 20.6 20.6 16.1
Ma63/1889 27.4 13.1 8.2 4.9 5.8 5.0
averagerootlengthinmm ,. ,,
Ma63/1889 5.1 5.0 3.6 3.5 3.2 j.i
dryrootweightperrootedexplantinmg
1087 1.8 1.6 1.5 1.3 0.8 0.7
4331 4.9 5.3 3.5 3.3 3.2 2.6
Ma63/1889 4.2 2.3 1.6 0.8 0.9 0.8
27
regeneration atdifferentregionsofastemwas influencedbyso-called gradientsof
organogenesis.Gorter (1965)observed thatwhenayoung shootof Asparagus officinalis
was cut intosegments of0.5 cmandcultured invitro,therewas agradientofrooting
capacitywhichdeclined fromthetiptowards thebaseoftheshoot.Pierik (1967)
noticed thatrootformationonexplants oftheelongatedaxisof Lunaria annua cultivated
invitrostartedfromtheterminalpartand laterfromthemedialandbasalpartofthe
axis.Thepercentageofrootedexplants attheendoftheexperimentdecreased fromthe
terminaltothebasalpartoftheplant.Theseresultspointedtotheexistence ofa
rooting gradientalongthemainaxisofaflowering Lunaria plant.Thepositionof
Rhododendron stemexplantshadno effectonrooting (Pierik&Steegmans,1975c).
Rooting gradients arecausedbydifferences inthedevelopmental stagewhichexist
betweenexplants isolated fromvariouspositions;topexplants areinayoungerstageof
development thanmorebasalexplants.Theeffectofthedevelopmental stageonroot
formationwasalready discussed inSection3.4.1.2.
3.4.1.4Explant length
Theeffectofthe initial lengthoftheexplantonrhizogenesiswasstudied on

segments oftheupperhalfofyoungpeduncleswithalengthof0.5, 1,1.5,2,2.5or3
cm.Explants ofClone4331wereturnedupsidedown,whereasexplants ofCloneMa63/1889
wereplacedhorizontally.
TherateofrootformationinClone4331decreasedwithanincreasing explant length
(Fig. 16),while inCloneMa63/1889 allexplant lengthsyieldedaboutthesamerooting
rate.Forallcharacteristics therootingresponseoftheverticallyplaced explants
decreasedwithanincreasing explantlength,whereasthehorizontallyplaced explants
showed agoodrootingforallexplantlengths (Table8 ) .
Differences inrootingresponsebetweenbothclonesmaybeascribedtothepolarity
(orientation)oftheexplants (Section 3.4.1.6).Explants ofClone4331wereturnedup-
sidedowntoabouthalftheir lengthsintheculturemedium.Hence,withanincreasing
explant lengthsubstances inthemedium,whichprovedtobeofessentialsignificance
torrooting,likesugar (Section3.4.2.3)andauxin (Section3.4.2.4),havetobe trans-
portedoveranincreasingdistancebeforeadventitious rootsareinitiated atthe
morphologicalbaseoftheexplant.Probablythistransportbecomes progressively
limitingwithanincreasingexplant length.ExplantsofCloneMa63/1889wereplaced
horizontally,withtheirwoundedsidesontheculturemedium.Underthese circumstances
theuptakeofessentialsubstances isindependent oftheexplantlength.Hence,explants
ofalllengthsyieldedagoodrootingresponse,whileadventitious rootswereformed over
thewholelengthoftheexplants.
Theeffectoftheinitialexplantsizeonrootformationvariesgreatlywith the
plantspecies.Gorter (1965)observedthatrootinitiationonexcisedshoottipsof
Asparagus officinalis, placeduprightinthemedium,decreasedwithanincreasingex-
plant length.However,Lindner (cf.Dore,1965)foundthenumberofneworgans initiated
onrootpiecesofhorseradish tobeproportionaltothelengthofthepiecesusedand
alsoNandaetal. (1968)reportedthebestrootingresponseforrelatively longstem
28
rooting percentage
90f
80
70
60
50
40
30
20
10
OL Fig.16.Theinfluenceofvariousexplant
lengths (1-3cm)onrootformationof
peduncleexplantsofClone4331,which
8 12 16 20 24 28 wereturnedupsidedown.
days of incubation
Table8.Theinfluenceofvariousexplantlengths (0.5-3cm)onrootformationof
peduncleexplants.ExplantsofClones4331andMa63/1889wereturnedupsidedownand
horizontally,respectively.
Clone 0.5 1 1.5 2 2.5 3
rootingpercentage ,
4331 J 75 85 75 65 30
Ma63/1889 70 60 50 65 60 55
numberofrootsperrooted,explant 6.0
4331 J 17.9 11.6 5.3 7.4
14.4 12.2 14.4 25.8
Ma63/1889 6.1 10.6
averagerootlengthinmm, 1.8
4.6 3.0 2.9 2.4
4331 -' 5.7
Ma63/1889 9.7 9.6 8.1 7.1 7.5
dryrootweightperrooted,explant inmg
0.2 0.1 0.1 0.1
4331 J 0.5
3.4 2.2 3.0 2.5
Ma63/1889 1.5 3.1
1.Treatmentnotincluded.
29
segmentsof Popuius nigra, placedvertically.Finally,Pierik&Steegmans (1975c)
noticedthesameamountofrootregenerationinstemsegmentsofvarious lengthsof
Rhododendron, turnedupsidedown.
Itremainsunexplainedwhyhorizontallyplacedlongexplantsofpyrethrumdonot
yieldthesamerootingresponseperunitoflengthashorizontallyplacedshortexplants,
whennutrientsandgrowthregulatorsareavailableinsufficientquantities inthe
culturemedium.Evidently,aninternallimitingfactorexistswithinlongexplants,of
whichthenatureisasyetunknown.
Explantsofpyrethrumyieldedthebestrootformationperunitoflengthwithshort
explantsof1and1.5cm.A lengthof1.5cmwaschosenasinitialexplantlengthin
standardexperiments.Ithasbeenshownthattheinitiallengthoftheexplantdoesnot
remainthesameduringthesubsequentincubationandthatexplantsbecomelonger,
dependentonthedevelopmentalstageofthepeduncle (Section3.4.1.2)andtheposition
oftheexplantinthepeduncle (Section3.4.1.3).Finally,ifrelativelyshortexplants
areused,thereislesschanceofcontaminationoftheexplantandthequantityof
experimentalplantmaterialcanbereduced.
3.4.1.5Wounding
Todeterminetheeffectofwounding,rootingwascomparedofexplantswhich,apart
fromthebasalandapicalwounds,werenotfurtherwounded,withrootingofexplants
whichwereadditionallywounded,byexcisionofthecortexatonesideoverthewhole
explantlength.Thelatterwereplacedwiththewoundedsideonthemedium.
Suchan.additionalwoundcutslightlystimulatedrootingrate,butconsiderably
enhancedtheotherrootingparameters,asexpressedinTable9.Asstandardtreatment
thepeduncleexplantswerewoundedbyexcisionofthecortexatonesideoverthewhole
explantlength.
Thefavourableeffectofwoundingonrhizogenesisisarecognizedfactandwasalso
affirmedbyPierik&Steegmans (1975c)withstemexplantsof Rhododendron cultivatedin
vitro.Libbert (1956/1957)statedthatthemostreasonableexplanationseemedtobethe
IflYaid Ma663/1889?Ce"*W ° U n d i n 8 ° nr o 0 t f o r m a t i ° " °fpeduncleexplantsofClones 1087,
1087 4331 Ma63/1889

unwounded wounded unwounded wounded unwounded wounded
rootingpercentage
65 70 75 90 40 55
numberofrootsper rooted explant
12.1 24.7 15.6 26.4 7.6 20.1
averagerootlength inmm
4.2 4.5 5.5 5.3 3.8 7.4
dryrootweightper rooted explantin mg
1.4 2.] 2.1 2.6 2.0 4.7
30
breakingofamechanicalbarrier.Suchabarriermaylimitthepenetrationand transport
ofwater,minerals,sugarandauxinwithinthe (ex)plant.Moreovertheoxygensupplymay
berestricted;theimportanceofagoodaerationforrootformationwaspointedoutby
Dore (1965).
3.4.1.6Polarity
Intheprevious sectionsitwasdeterminedwhichdevelopmental stageandwhichpart

ofthepedunclehadtobechosenastestmaterialandhowthisplantmaterialhadtobe
preparedforagoodrootingresponse.Thenextquestionconcernsthepolarity
(orientation)oftheexplants.Adventitious rootformationwas investigatedofexplants
insertedverticallyinthemediumtoabouthalftheirlengths,inuprightpositionor
upsidedown,andofexplantsplacedhorizontallywiththewoundedsideonthemedium.
Adventitiousrootswere always initiatedoppositethewoundedside.Rootssometimes
developedinthemedium,whichwere shortandthick,whereasmoreelongated thinroots
wereregeneratedabovethemedium.Adventitious rootformationofexplants inserted
uprightwasstronglyretardedincomparisonwithrootformationofupsidedownor
horizontallyplaced explants (Fig. 17).Explants inserteduprightdevelopedrootsatthe
rootingpercentage
100r
90
Fig. 17.Theinfluenceofvariouspolari-
tiesonrootformationofpeduncle explants
ofClone4331.1isexplantupright,2is
explantupsidedownand3isexplant
12 16 20 24 28 horizontal.
days of incubation
31
Table 10.Theinfluenceofvariouspolaritiesonrootformationofpeduncleexplantsof
Clones4331andMa63/1889.
4331 Ma63/ '1889
up- upside hori- up- upside hori-

right down zontal right down zontal
rootingpercentage 85 90 75 50 100 70
numberofrootsperrootedexplant 20.6 21.5 15.5 12.5 20.1 21.3
dryrootweightperrootedexplant inmg 1.4 1.5 3.3 2.2 2.3 5.4
morphologicalbaseinthemediumandjustabovethemediumatthemiddleregionofthe
explant.Onexplantsturnedupsidedownadventitiousrootswereregeneratedexclusively
atthemorphologicalbaseabovethemedium.Explantsplacedhorizontally initiatedroots
abovethemediumoverthewholelength,butespeciallyatthemorphologicalbase(Fig.
13).Noonetreatmentwaspreferabletotheotherswithrespecttorootingpercentageand
averagenumberofroots (Table 10).Dryrootweights,however,weremuchhigherfor
horizontally thanforverticallyplacedexplants.Instandardexperiments explantswere
placedhorizontallywiththewoundedsideontheculturemedium.
Theunfavourableeffectofanuprightexplantpositiononrootinginvitrohasbeen
described intheliterature.Rootformationofisolatedstemsegmentsof Rhododendron was
strongly inhibitedbyplacingtheexplantswiththeirbasalendsdown;invertedexplants,
however,rootedeasilyabovethemediumandyieldedmoreandheavierroots (Pierik,1969;
Pierik&Steegmans,1975c).Similarresultswereobtainedwithpetiolesegmentsof
Lunarla annua (Pierik,1972),leafmidribexplantsof Gerbera oamesonU (Pierik&Segers,
1973)andstemsegmentsof Helianthus tuberosus (Tripathi,1974).Inthislastcase
rootingwasevenbetterofinvertedthanofhorizontallyplacedexplants.
Substanceswhichareessentialforrooting,likesugar (Section3.4.2.3)andauxin
(Section3.4.2.4),aretransportedinapolar (basipetal)directionwithinthe(ex)plant.
Whensegmentsareturnedupsidedownthesenutrientshavetobridgethedistancefromthe
mediumtothemorphologicalbaseofthesegment,whererootingoccurs.Whenexplantsare
insertedinanuprightpositionthemorphologicalbaseisdirectlyexposedtothe
nutrientsinthemedium.Nevertheless,therootformationinthemediumwaspoor,
probablybecauseofthebadoxygensupplyinthemedium.Theimportanceofagood
aerationwasemphasizedbyDore (1965).Horizontallyplacedexplantsareassuredofa
goodaerationandagooduptakeofessentialsubstances.Consequently,inpyrethrum
adventitiousrootswereinitiatedoverthewholelengthoftheexplant.However asa
resultofbasipetaltransportmostrootswerestillinitiatedatthemorphologicalbase
oftheexplant,whichwasalsofoundwithstemsegmentsof Helianthus tuberosus
(Tripathi,1974).
32
Table 11.'The i n f l u e n c e of v a r i o u s agar c o n c e n t r a t i o n s (0-1%) on r o o t formation of
peduncle e x p l a n t s of Clone 4331.
0.2 0.4 0.6 0.8
numberofrootsperrooted explant 13.1 16.7 22.9 19.7 30.1 22.2
dryrootweightperrooted explantinmg 0.9 1.6 2.8 3.9 4.6 3.9
3.4.2 Nutritional/hormonal factors
3.4.2.1Agar
Agarisaproductmanufactured fromseaweedandusedasasolidifyingagent.The
organicandinorganiccompositionofagarisrathercomplexandvarieswiththetimeof
theyeartheproductismanufactured.Themostcriticalnutritionalworkcanbedonewhen
tissueculturesaregrowninchemicallydefinednutrientmediainwhichacomplex
componentlikeagarisabsent,i.e.inaliquidmedium.
Todeterminewhetheraliquidmediumisadequateforpyrethrum,therootingresponse
ofexplantsofClone4331wasinvestigatedatdifferentconcentrationsof 'Difco'Bacto-
agar(0,0.2,0.4,0.6,0.8 and H ) . Inaliquidmediumandatagar0.21filterpaperwas
usedtosupporttheexplants (Heller,1953).
Theobservedrootingratewasaboutthesameforalltreatments.Inaliquidmedium
rootingresponsewaspoor,whileatagar0.81rootformationwasoptimum (Table 11).
ApromotingeffectofliquidmediaonrootorganogenesiswasreportedbyGorter
(1965)forshoottipsof Asparagus officinalis andbyPierik&Steegmans(1975c)with
Rhododendron stemsegments.Thisstimulationmaybeattributedtoabetteraerationina
liquidthaninanagarmedium.AccordingtoRomberger&Tabor (1971)itmayalsobepartly
duetoincreaseddiffusionratesoflargemoleculesinliquidmedia,orpossiblyto
unknownagar-borneinhibitors.
Itremainsunexplainedwhyadetrimentaleffectofaliquidmediumonroot
formationhasbeenfoundwithrhizomefragmentsof Helianthus tuberosus (Tripathi,1974)
andinthepresentexperimentswithpeduncleexplantsofpyrethrum.Instandardexperi-
mentswiththelatteranagarconcentrationof0.61waschosen,asatthatmomentitwas
n
°tyetknownthatoptimumrootformationwouldoccurat0.8°sagar.
3.4.2.2Minerals
Fortheexaminationoftheinfluenceofthemineralnutritiononrhizogenesis,
Knop'smajorandHeller'sminorsalts (cf.Gautheret,1959)wereaddedtotheculture
mediumindifferentconcentrationsandcombinations.Theconcentrations0,i,1and2
indicatetheabsenceandthepresenceoftheminorandmajorsaltsinhalf,normaland
doublestrength,respectively.Thefollowingtreatments (withcorrespondingtreatment
numbers)wereapplied:
33
Table 12.Theinfluenceofvarious concentrationsandcombinationsofHeller's minor
saltsandKnop'smajor saltsonroot formationofpeduncle explants. (Treatments 1-6are
explained inthetext.)
Clone 1 2 3 4 5 6
rootingpercentage
1087 20 10 80 70 55 15
4331 20 5 45 65 55 60
Ma63/1889 10 10 50 65 45 25
1087 1.3 5.5 13.2 10.4 13.6 7.7
4331 3.3 4.0 13.3 11.3 16.1 14.4
Ma63/1889 5.0 5.0 17.8 7.7 14.3 10.6
averagerootlengthinmm
1087 1.4 4.9 2.8 4.9 3.0 2.0
4331 1.0 5.0 7.9 7.9 7.0 5.2
Ma63/1889 8.6 10.4 6.2 7.6 6.2 6.1
dryrootweightperrooted explantinmg
1087 0.1 0.7 0.8 1.0 0.9 0.5
4331 0.1 0.1 2.9 2.5 2.8' 1.6
Ma63/1889 0.8 0.9 3.0 2.7 2.4 1.7
Heller minor Krlopmajor

1= 0 + 0
2= i
z + 0
+ 1
3= 0 2
i + 1
4= 2 2
5= 1 + 1
6= 2 + 2
IntheabsenceofKnop'smajorsaltsandinthepresenceofthedoublestrengthof
bothmineral componentstherootingratewasconsiderably lessthanwiththeother
treatments (Fig. 18).-Ingeneraltheclonesresponded similarly (Table 12)tothe
mineralnutrition.Incomparisonwithamediumdevoidofallminerals (Treatment1) the
minorsaltsofHellerslightly stimulatedrootformation (Treatment 2). Intheabsence
ofthemajor salts,however,bothtreatments showedabadrootingresponseandfinally
theexplantsbecamenecroticanddiedoff.WithKnop'smajorsalts (Treatments3,4,5
and6)rootformationwasstimulatedandexplantsdidnotturnnecrotic.Thedouble
strengthofbothminorandmajorsaltsprovedtobesupra-optimal (Treatment 6).Onthe
wholerooting responseswerebestwithonlythemajorsalts (Treatment 3)orwithboth
componentsathalfstrength (Treatment 4).AsolutionofKnop'smajorandHeller's minor
salts,bothathalfstrength,waschosenforstandardexperiments.
AfterthedeterminationoftheimportantroleofKnop'smajorsalts,theinfluence
ofthefourseparatemineralsaltsofKnopmajorwas investigated.Inanexperimentwith
Clone4331thefourmajorsaltsweresuccessively omitted,sothatthefollowingtreat-
ments (withcorresponding treatmentnumbers)wereapplied:
34
rooting percentage
100r
rooting percentage
80r
12 16 20 24 28 16 20 24 28
Fig. 18. The i n f l u e n c e of v a r i o u s Fig. 19.TheinfluenceofKnop'smajor

c o n c e n t r a t i o n s and combinations of H e l l e r ' s saltsonroot formationofpeduncleex-
minor s a l t s and Knop's major s a l t s on r o o t plantsofClone 4331. (Treatments1-6are
formation of peduncle e x p l a n t s of Clone explained inthetext.)
1087. (Treatments 1-6 a r e e x p l a i n e d i n t h e
text.)
Knop major devoid of

1=
2= KNO,
3= MgS0 4 .7H 2 0
4=
Ca(N0 3 ) 2 - 4H 2°
5= KH 2 P0 4
6=
Alltreatments,,except1and4,resultedinahighrootingrate (Fig.19). Table13

demonstrates thattheomissionofCa(N0 3 ) 2 .4H 2 0wasmuchmoreharmfulthantheomission
ofanyoftheotherthreecomponentsofKnopmajor.TheTreatments 2,3,5and6,which
refertothepresenceofCa(N0 3 ) 2 .4H 2 0,gaveagoodrootingresponse.
Theeffectofthemineralnutrition (minorandmajorsalts)onroot formation
differsfromspeciestospecies.Ingeneralithasbeenshownthatanexogenoussupply
35
Table 13.TheinfluenceofKnop'smajorsaltsonrootformationofpeduncleexplantsof
Clone4331.(Treatments 1-6areexplained inthetext.)
averagerootlengthinmm 1.4 4.2 5.0 2.3 4.6 4.1
dryrootweightperrootedexplantinmg 0.0 2.2 3.7 0.2 1.3 2.9
ofmineralstotheculturemediumisnotessentialforrooting,probablybecause
(ex)plantscontainasufficientamountofendogenousminerals.Intheabsenceof
mineralspetiolesegmentsof Lunaria annua (Pierik,1972),leafmidribfragmentsof
Gerbera jamesonii (Pierik&'Segers,1973)andstemexplantsof Pisum sativum (Leroux,
1973), Helianthus tuberosus (Tripathi,1974)and Rhododendron (Pierik,1969;Pierik&
Steegmans,1975c)initiatedrootsinvitro,althoughsometimesthenumberoftheroot
initialswasverylimited.Thesameresultwasalsofoundforpeduncleexplantsof
pyrethrum.Justoneexamplecanbementionedinwhichrootingdidnotoccuratallona
mediumdevoidofmineralsalts,namelywithrhizomeexplantsof Helianthus tuberosus
(Tripathi,1974).
Theroleoftheminorsaltsinrhizogenesishasbeeninvestigatedsporadically.
Torrey (1956a)observedthatfragmentsofpearootsrequiredcertainmicro-elements (Zn,
Mn,Cu,MoandB)forlateralrootformation.Withstemsegmentsof Pisum sativum,
Leroux (1973)noticedadetrimentaleffect;thenumberofadventitiousrootsproduced
waslesswithonlyminorsaltsthanonamediumdevoidofallminerals.
Themostextensivestudyonrootformationasaffectedbythemineralnutritionwas
carriedoutbyTripathi (1974)withrhizomeexplantsof Helianthus tuberosus. Inthe
presenceofonlyminorsaltshardlyanyrootformationwasobtained.Whentheminorsalts
togetherwithmajorsaltswereaddedtotheculturemedium,lessrootswereregenerated
thaninthepresenceofonlythemajorsalts.Aseparateadditionofthemicro-elements
(Fe,Cu,Zn,Mn,Al,Ni,BandJ)togetherwiththemajorsalts,however,revealedthat
eachmicro-elementatacertainconcentrationenhancedrootformation.Inpyrethrum,the
organogenesisofrootsonpeduncleexplantsishardlyinfluencedbytheminorsalts.
Ontheotherhand,themajorsaltsstronglyenhancedrootformationinpyrethrum.
A favourableeffectofmajorsaltsuponrootinghasalsobeenfoundforleafmidrib
fragmentsof Gerbera jamesonii (Pierik&Segers,1973)andanalmostessentialrolewas
reportedforrhizomeexplantsof Helianthus tuberosus (Tripathi,1968,1974;Tripathi
&Gautheret,1969;Gautheret,1969c).Themajorsaltsdidnotmarkedlyinfluenceroot
formationonstemsegmentsof Pisum sativum (Leroux,1969c,1973)and Rhododendron
(Pierik,1969;Pierik&Steegmans,1975c),epicotylexplantsof Phaseolus vulgaris
(Olieman-vanderMeer&Pierik,unpublisheddata)andpetiolesectionsof Lunaria annua
(Pierik,1972).
Tripathi(1974)demonstratedthatrhizogenesisofrhizomeexplantsof Helianthus
tuberosus wascompletelyinhibitedintheabsenceofthemacro-elementsCaandN,and
thesignificanceoftheseelementswasfullyconfirmedintheexperimentswith
36
pyrethrum.Tripathifurtherreportedaslightinfluenceofthemacro-elementsK,P,Mg
andS,whichresultisalsoinconformitywiththepyrethrumexperiments.
3.4.2.3Sugars
F i r s t s i x sugars were compared to find out which was the most suitable for root
formation of peduncle explants of Clone 4331. All sugars were applied at a concentration
of 2% to the culture medium.
Adventitious roots only developed fast in the presence of glucose, maltose and
sucrose (Fig. 20). Table 14 shows that an adequate root production was achieved with
rooting percentage
80
70
60
50
40
30
20
10-
0L y _ _X^_ . L. Fig. 20. The influence of various sugars

(at 2%) on root formation of peduncle ex-
-• 1 ' ' "— plants of Clone 4331. 1 = fructose, 2=
12 16 20 24 28 galactose, 3 = glucose, 4 = lactose, 5 -
d a y s Of incubation maltose and 6 = sucrose.
Table 14. The influence of various sugars (at 2%) on root formation of peduncle explants
of Clone 4331. __—
fructose galactose glucose lactose maltose sucrose
r
°°ting percentage 60 75
60 lD
35 0
numberofrootsperrooted explant , ,_ ,Q2 15.3
] J,u
5.9 0 5.4
ryrootweightperrooted explantinmg , ,2 1.1
u-3
0.4 0 l-°
37
glucose,maltoseandsucrose.Theformationofadventitiousrootswasinadequatewith
fructoseandlactose,whileinthepresenceofgalactoserootswerenotinitiatedatall
andexplantsdiedoff.
Subsequently,inthenextexperimentswiththethreeclones,glucoseandsucrose
were addedatdifferentconcentrationstothemedium.Root formationwas completely
inhibitedwhensugarwas absent.Usually rootformationwas retardedatglucoseand
sucroseconcentrationsof1and 5%(Figs21aand21b].Obviously (Table 15)theaddition
ofsugartothemediumwasofessential significanceforrooting.Intheabsenceofsugar
rhizogenesiswascompletely inhibitedandtheexplantsbecamenecroticanddiedoff
within4weeksafter incubation.Theresultswerenotverydecisiveontheoptimumsugar
concentrationsandvaried fromclonetoclone.Intherangeof 2-4%glucoseandsucrose
mostly goodrootingresponseswere observed.
InthefollowingtwoexperimentswithClones 1087and4331,adirectcomparisonof
theeffectofglucoseandsucrose,appliedatvarious concentrationswasmade.Adelayed
rootingratewasobtainedatglucoseandsucrose 5%(Fig. 22).Table16shows that
glucoseandsucroseconcentrationsof1and S%aresuboptimalandsupra-optimal,res-
pectively.Sucroseat 3%hastobepreferredtoglucoseat 3%;instandard experimentsa
sucroseconcentrationof 2%waschosen.
rooting percentage rooting percentage

100 100
90
80
70
60
50
40
30
20
10
12 16 20 24 28 8 12 16 20 24 28
days of incubation days of incubation *
(b)ontootfo™ation e of e DPH,mM iOUSeventrations (0-5%)ofglucose (a)andsucrose

peduncleexplantsofClone4331.
38
Table 15. The i n f l u e n c e of v a r i o u s c o n c e n t r a t i o n s (0-5%) of glucose and sucrose on r o o t
formation of peduncle e x p l a n t s .
Clone Glucose Sucrose
rooting p e r c e n t a g e
45 25 25 65 70 55 45 30
1087 0 -'60 40
80 95 80 70 80 95 85 65
4331 ^ - 85 85
75 70 40 40 50 40 25 30
Ma 63/1889 0 75 70
number of r o o t s p e r r o o t e d e x p l a n t
1087 4 5 . 0 25.6 30.7 32.0 2.0 9.4 12.8 10.1 25.6 12.6
4331 1 3 2 . 2 47.7 42.9 32.1 21.9 7.9 7.6 7.6 10.4 12.0
Ma 63/1889 13.9 17.4 17.5 18.9 24.3 14.2 12.8 9.0 6.5 23.0
average r o o t l e n g t h i n mm
Ma 63/1889 0 5.9 5.2 4.8 5.5 4.5 6.4 8.5 9.8 7.7 5.3
dryrootweightperrootedexplant inmg
1087 0. 0 2.3 3.2 2.1 0.2 0 1.0 1.6 4.1 3.1 1.9
4331 -' 8 5.2 6.1 5.5 3.0 0 0.5 0.5 0.6 0.8 0.9
Ma63/1889 0 7 2.5 2.9 3.4 4.7 0 1.3 2.6 2.4 1.1 3.0
1.Treatmentnotincluded.
rooting percentage
90
80|
70
60
50
40
30
20
10
Fig.22.Theinfluenceofvariousconcen-
trationsofglucoseandsucroseonroot
0 formationofpeduncleexplantsofClone
1087.Treatments 1,2and3represent
glucose 1,3and5%andtreatments4,5
4 12 16 20 24 28 and6sucrose1,3and5%.
days of incubation
39
Table16.Theinfluenceofvariousconcentrations (1-5%)ofglucoseand sucroseonroot
formationofpeduncleexplants.
Clone Glucose Sucrose
rootingpercentage
1087 80 90 55 90 80 45
4331 100 95 95 95 95 95
1087 4.0 4.3 7.3 4.8 16.6 8.0
4331 17.4 20.2 15.6 11.7 25.0 14.0
4331
5.2 8.0 5.7 6.3 5.64.2
8
1087 0.,2 .0,.4 0..5 0,.3 0..9 0 .8
4331
0,.9 3..2 2..3 1,.0 2..8 1.5
f n ™ J 7 " T ^ i n f l u f n c e o f s u c r o s e("2%),appliedduringdifferentperiods,onroot
formationofpeduncleexplantsofClone4331.
Last2weeks First2weeks 4weeks

rootingpercentage 0
95 90
0• 6.7 15.3
averagerootlengthinmm 0
3.9 8.0
0 0.3 3.2
Finally lt was determined whether sucrose a t 1%i s e s s e n t i a l during the whole
J weeK T0t f 0 m a t i
° n °f P6dUnCle e X p l m t S
°f Clone 4331
' ^ n sucrose was addedfor
2 f l r S t 2 W6ekS f inCUbati n then
wit^ IT ^ ° ° ^ e n t i t i o u s roots were i n i t i a t e d
wi h most thesame rooting r a t e . The data o f j a b l e 17indicate t h a t theaddition of
r e q 8 Wh
u u T ^ °le Pr
°CeSS °f r ° 0 t f0raiati0n
- * » after 2weeks
deV id f SU8ar 6XPlantS
u meld" ^ T ° ° ^ — t r a n s f e - d - a medium
t T d T T ^ a M l i t y t 0 r e g 6 n e r a t e r °° tS ™ ^ - d plantsbecame
CUUUre PreS6nCe f
r t dTa ? " " ^ ° ™ for the f i r s t !weeks
8 PerCentage
iTZJLI Sh r ? ' ^ ^ ° t h 6 r r ° 0 t i n g P — t e r s were .nuch less
h e devel
The ahsmni-o • °Pment oftheroot i n i t i a l s .
»««*•. Pier* (,,,2) »it]l pe t l 0 1 „'4l„L"^! e P i C ° t y l Seg"™tS °f " " • ° ' - "
withtafIdrib£ „ _ , « J L * . * (,973)md
"~™ ™ ™d P i e r i k » S«8=" <' 9 7 3 ' (,967
-*«-».s^.rr'c^nr^r" *"->- >
effectofsugaronrootorganogenesiswasalsodemonstratedbyNandaetal. (1968)with
stemsegmentsofPopulus nigra.
Sincerootingisinhibitedbylight (Section3.4.3.2),cultureswerealwaysplaced
indarkness.However,indarknessnophotosynthesis takesplaceandthereforean
exogenoussupplyofsugarbecomesessentialforrooting.
Theinfluenceofthekindofsugaronrootregenerationwas investigatedby
Spanjersberg&Gautheret (1963ab),Paupardin (1966)andGautheret (1969c),who
reportedrhizogenesisonrhizomesegmentsof Helianthus tuberosus withfructose,
glucose,sucroseandxylose,butnoorhardlyanyrootformationwithgalactose,lactose,
maltose,mannose,raffinoseandribose.Leroux (1973)foundanoptimumrootformationon
stemsegmentsofPisum sativum aftertheadditionofsucrose.Fructose,glucose,lactose
andribosewere lessefficientandnoorhardlyanyrhizogenesisoccurredwhengalactose,
maltose,mannose,raffinoseorxylosewereaddedtotheculturemedium.Theseresults
correspondwellwith those frompyrethrumexperiments,wherealsoglucoseandsucrose
werefoundtobethemost efficientsugars.
Theimportanceofthepresenceofsugarduringthewholeprocessofrootformation,
asfoundinpyrethrum,wasalsoconfirmedbyGautheret (1969c)usingrhizomeexplants
ofHelianthus tuberosus.
Thedecreasing rootingpotentialathighsugarconcentrationscanbeexplainedbya
toohighosmoticpressure (Pierik,1969;Pierik&Steegmans,1975c).Inthepresenceof
2%glucose,theosmoticpressureofthemediumwas2.5atm.Whentheosmoticpressure
wasincreased from2.5to6atm.byaphysiological inactivesugarsuchasmannitol,
thenrootformationof Rhododendron stemsegmentsdecreased (4tsucrosecorresponds
withapproximately4atm.).
3.4.2.4Auxins
Thefollowingauxinswereexaminedwithrespecttotheinfluenceonrootformation:
2,4-dichlorophenoxyaceticacid (2,4-D),g-indoleaceticacid ( I M ) , g-indolebutyricacid
(IBA)andc-naphthaleneaceticacid (NAA).Infourexperimentstheeffectofvarious
concentrationsoftheseauxinsonroot formationofClones 1087and4331wasanalysed.
PeduncleexplantsofClone 1087onlyrootedat2,4-D10 - 7and10 g/mlandyielded
aboutthesamerootingrateatbothconcentrations.Clone4331showedthehighest
rootingrateattheconcentrations 10" 6and10" 5g/mlofI M (Fig.23),IBAandNAA.In
theabsenceofanauxin,rootsneverdevelopedandtheexplantsbecamenecroticand
diedoff (Table 18).IAAinduced rootformationinamuchwider^rangeconcentrations
than2,4-D,IBAandNAA. Intensiverhizogenesisoccurredat10" and10 g/mlof
IBAandNAA,whereas 2,4-D showedonlyaweaktendencytoformroots.
Inthenextexperimentswith thethreeclones,IAA,IBAandNAAwerecompared
M e e t l yattheconcentrations 10' 7 , 10" 6and10' 5 g/ml.At10" 5g/mlofIAA,IBAand
N M ,rateofrootingwashighestforallclones (Fig.24).Table19i n d i c a t e ^ ™
respecttoroot initiationtheoptimumconcentrationwas10 forallthreeauxinsuse .
^ e dryrootweightperrooted explant, however,washigheratlowerconcentrations,
viz- 10" 7orm - 6 ^ - m c A l P tothestroneerelongationoftherootsatthese
41
rooting percentage rooting percentage
90r- 90
80 80
70 70
60 60
50 50
40 40
30 30
20 20
10 10
0L 0L
12 16 20 24 28 12 16 20 24
Fig. 23.Theinfluenceofvariousconcen- Fig. 24.Theinfluenceofvariousconcen-
trationsofIAAonrootformationof trationsofIAA,IBAandNAAonroot
peduncleexplantsofClone4331.Treatments formationofpeduncle explantsofClone
1-61(represent0,1 0 ,10 ,1 0 ,10" and 1087. Treatments1,2and3;4,5and6;
10~ g/ml,respectively. 7, 8and9represent 10~ 7 ,10"°and 10 - 5
g/mlofIAA,IBAandNAA,respectively.
concentrations.IAAyieldedbetterresultsthanNAA,andIBAwasstillbetter thanIAA.
IBAat10 g/mlgavethebestrootingresponseandthisauxinandthisconcentration
werealsochosenforthestandardexperiments.
In.viewofthepoorrootelongationatanauxinconcentrationof10 - 5 g/ml, the
questionarosewhetherrootelongationcouldbestimulatedwhen,afterincubationfor0,
3,6,9or12donamediumcontainingIBA10 - 5 g/ml,theexplantswere transferredtoa
mediumwithout IBA.InthisexperimentwithClone4331,initiallytherooting ratewas
highestinthosetreatmentswheretheexplantsweretransferred6and9dafter
incubation,butgraduallytherootingrateofallthetreatmentsdidnotdiffermarkedly
androotingpercentageswerealmostequal39dafterincubation
Asfoundpreviously,rootswerenotinitiatedintheabsenceofauxin (Table20).
thepresenceofauxinduringthefirst \ A r.f i„ u»•
8u e t l r s t3d
.... ofincubationwassufficientforthe
initiationofaconsiderablenumberofroots.Themaximumnumberofrootsperrooted
7readledafterm a P P l i C a t i n aUxin
£
efir r
Wth inCTeaSin8
n aPPUCati0n
° °tlme « »h*i<*.Theaverageroot
2 IdTft .' "**» ^ *y ™ * -ight was
- ewZ o ^ T ^ f°r6^ M t e r ^ ^ to *« « * » ^thout a u x Lthe
grewantothemedium.Thiswashardlyevernoticedonamediumcontainingauxin.
42
Table18.Theinfluenceofvariousconcentrations (0-10 Hg/ml)of2,4-D,IAA,IBAand
NAAonrootformationofpeduncleexplants.
Clone 0 ,o" 8 ,o- 7 ,0" 6 lO"5 lO"4
rootingpercentage
1087 2,4-D 0 0 35 30 0 0
4331 IAA 0 0 30 75 90 25
4331 IBA 0 0 0 50 85 10
4331 NAA 0 0 0 80 90 0
1087 2,4-D 0 0 5.0 6.5 0 0
4331 IAA 0 0 1.2 10.2 14.6 5.2
4331 IBA 0 0 0 4.7 15.4 3.0
4331 NAA 0 0 0 4.3 16.6 0
1087 2,4-D 0 0 0.6 0.4 0 0
4331 IAA 0 0 0.2 0.7 0.7 0.0
4331 IBA 0 0 0 0.8 0.8 0.2
4331 NAA 0 0. 0 1.7 1.1 0
Table19.Theinfluenceofvariousconcentrations (10~7-10~5g/ml)ofIAA,IBAandNAA
onrootformationofpeduncleexplants.
Clone IBA NAA

IAA
lO"6 lO"5 10"7 lO'6 lO"5

10"7 lO"6 lO"5 lO"7
rootingpercentage 25 75
1087 90 5 40 85 5
40 50 90 90
4331 0 85 75 0
70 85 70 30 90
0 20 100 0
Ma63/1889 45 100 100
numberofrootsper rootedexplant 1.0 5.0 14.9
1087 23.2 1.0 9.5 31.9
3.8 7.7 0 3.5 10.5
4331 13.1 0 4.7 15.3
3.2 7.7 0 4.0 11.7
Ma63/1889 25.5 0 5.0 34.0
3.3 8.2
averagerootlength inmm 5.0 7.0 2.3
4.4 8.0 6.4 3.9
1087 28.5 9.1 0 13.1 2.8
5.2 0 18.6 4.7
Ma63/1889 29.8 8.4
dryrootweightper rootedexplantin mg 0.1 0.8 1.0
1087 0.1 1.8 2.4
3.5 1.4 1.8 0 3.2 1.7
4331 1.7 0 3.2 2.0
2.6 2.8 4.4 0 2.2 1.3
Ma63/1889 4.8 2.5 3.3 0 3.7
Tu, „„ . •j„ cn-1?dlonamediumcontainingIBA

Table20.Theinfluenceofvarious incubationperiods (012 *>°.n f d u n c l eex-
5
'0- g/ml,beforetransfer toanauxin-freemedium,onrootformation P
PlantsofClone4331,39dafterincubation.
12 Continuously
IBA I0"5g/ml
95 95 85
rootingpercentage 0 85 95
18.3 20.0 17.8
0 12.9 18.7
11.5 11.4 7.5
0 20.8 16.4
averagerootlengthinmm 4.4
7.4 5.0 4.8
dryrootweightperrooted explantinmg 0 5.7
43
Apparentlydifferentoptimaexistforroot initiationandroot elongation; root
initiationoccursathighauxinconcentrations,whereas theelongationoftheroot
initialsisstimulatedaftertransfertoanauxin-free medium.
Inthenextexperiment,explantsofClone4331were incubated for0,3,6,9or12
donamediumwithoutauxin,andsubsequently transferred tomedia containing auxin.
Theexplantsstartedrootinginthesamesequenceastheywere incubatedonmedia
containingauxin (Fig. 25).Afterincubationfor0(IBA10~ 5continuously)and3donan
auxin-freemedium,goodrootingresponseswere obtained.Withanincreasing incubation
timeonauxin-freemediatheformationofrootsdecreased (Table 21).Finally,
adventitious rootswerenotformedonamediuminwhich auxinwasabsent duringthe
wholeincubationperiod.Thisresultshowsthatauxinhastobesupplied almost fromthe
verybeginning,otherwiserootformationwillbestronglyoreven completely inhibited.
rooting percentage
60
50
40
30
20
10
30 34 3842
days of incubation
^ ^ ^ ^ ^ , ^ ^ ^ ^ 1 ^ ^ ; ^ " *™ « -in-free
peduncleexplants0£clone«„.Treatme;r-Z^^^ZrS^^LCe of
S ^ ^ I S T ^ J ^ S S ^ S ^ J S ' ( 0 - 1 2 d) ° n anauxin"freemed medium,

explantsofClone4331,42dafterincubat- o nr o o t
formationofpeduncle
0 3 6 9 No IBA
12
rootingpercentage
60 40 45 45 0
numberofrootsperrootedexplant 15
14.7 13.6 11.6 5.2 0
averagerootlengthinmm 8.3
8.5 8.8 7.1 4.7 0
dryrootweightperrootedexplantinmg 3.0
3.7 6.2 2.7 1.3 0
0.9
44
Numerouspublicationshavedealtwiththeessentialorstimulatingrolethatan
exogenousauxinsupplyplays intheformationinvitroofadventitiousrootson
differentexplantsofvariousplantspecies:shoottipsof Asparagus officinalis (Gal-
ston,1948;Gorter,1965;Andreassen&Ellison,1967),stemexplantsoftwospeciesof
Parthenooissus (Leroux,1965)andofthethreespeciesof Salix (Leroux,1966),rhizome
fragmentsandstemsegmentsof Helianthus tuberosus (Paupardin,1966;Gautheret,1969c;
Tripathi,1974),stemsegmentsof Populus nigra (Nandaetal.,1968)and Rhododendron
(Pierik,1969;Olieman-vanderMeeretal.,1971;Pierik&Steegmans,1975c),epicotyl
explantsof Phaseolus vulgaris (Olieman-vanderMeeretal.,1971),petiolesegmentsof
Lunaria annua (Pierik, 1972),leafmidribexplantsof Gerbera jamesonii (Pierik&Segers,
1973)andstemsegmentsof Pisum sativum (Leroux, 1973).
Thesuperiorityof IBAover IAA,NAAand2,4-D,whichwasfoundforpyrethrum,
cannotbeconsideredasageneralrule.Theinfluenceofdifferentconcentrationsof
differentauxinsonroot formationgreatlyvarieswiththekindofexplantandtheplant
speciesused.
Thefindingwithpyrethrumthattheoptimumauxinconcentrationforrootinitiation
isdifferentfromthatforelongationoftherootinitialsconfirms'resultsofseveral
authors(Meyer&Anderson,1952;Torrey,1956b;Thimann,1969).Theyestablishedthat
theeffectofauxinsonroot initiationshouldbeclearlydistinguishedfromtheireffect
onrootelongation.Ingeneraltheconcentrationsrequiredfortheformeraremuchhigher
thanforthelatter.Theexperimentswithpyrethrumdemonstratedthatauxinatahigh
concentrationhastobepresenttoevokerootinitiation,whilesubsequentroot
elongationispromotedby transfertoamediuminwhichauxinisabsent.Thoughroots,
afterinitiationonanauxin-containingmedium,rapidlyelongateonanauxin-freemedium,
itdoesnotnecessarily followthatauxinsarenotrequiredforrootelongation.Theex-
plantsmaycontainnaturalendogenousauxinsorsyntheticauxinstakenupfromthe
previousmedium,towhich theywereappliedexogenously.
Auxinhastobepresentfromthebeginningofthecultureinvitro;adelayofthe
auxinsupplycausesaninhibitionofrhizogenesisofpeduncleexplantsofpyrethrum.
ThiswasalsoobservedbyLeroux (1973)withstemsegmentsof Pisum sativum, whereas
Gautheret (1969c)usingrhizomeexplantsof Helianthus tuberosus noticedanoptimum
rootingwhentheauxinapplicationwasdelayedby9d.
Thepresenceofauxinduring thefirst3and6dofthecultureinvitro restate
6 1 6
ininitiationandelongationofahighnumberof-adventitiousrootson P ^ ^
atthe
ofpyrethrum.Gautheret (1969c)observedthatanapplicationofauxinfor4 ex-
beginningofculture,resulted inappreciablerhizogeneticpropertieson ^ ^ ^ ^ in
Plantsof Helianthus tuberosus, buttherootingintensityincreasedwithan mere
thedurationofapplication.
3-4.2.5Cytokinins
TheeffectofthecytokininsBA (6-benzylaminopurine)andkinetiii ^J^13™*0

Purine)onadventitiousrootformationwasanalysedwithClone4331. eseg
_Q n „ ln -8 l n _ 7 or10 g/rolt 0t n ec u i t u l c
regulatorswereappliedattheconcentrations 0,10 , w
45
mediumtogetherwithIBAat10"5g/ml.
TherootingratewasalreadyreducedbyaconcentrationofBAandkinetin (Fig.26)
_O
as low as 10 g/ml. Table 22 shows that root formation decreases byr a i s i n g the cytoki-
nin concentration. At BAand kinetin 10 g/ml root formation was completely suppressed.
Root formation was strongly delayed and adventitious roots s t i l l emerged several months
after incubation a ta concentration of 10~7 g/ml of both cytokinins. BA and kinetin a t
rooting percentage
90
80
70
60
50
40
30
20
10
01
Fig. 26. The influence of various concen-
trations of k i n e t i n on root formation of
12 peduncle explants of Clone 4331. Treatments
16 20 24 28 1-4 represent 0, 10~ 8 , ]fj~7 and 10~6 g/ml,
days of incubation respectively.
formation^ p S S S ^ L j J J S ^ C ^ ^ i r ' 1 ? ? " ( -" 1 0 " 6 ^ °f M " * ^ ^ ** ""'

perCenta es between bracketS
determined 19 weeks after incubation!) "* S " ^
10 10-7 10
rooting percentage
BA
90 90 5(25)
kinetin 90 85 25(50)
43.0 16.6 1.0
kinetin 36.3 24.0 1.8
5.3 6.2 3.0
kinetin 4.1 5.0 4.0
3.9 1.9 0.1
kinetin
2.3 2.1 0.1
46
10~8 g/ml, however, s l i g h t l y seemed to promote the elongation of roots.
In the absence of an auxin the addition of a cytokinin generally does not lead to
the formation of ( l a t e r a l ) r o o t s , as has been demonstrated in v i t r o by Torrey (1958)
with isolated roots of Convolvulus arvensis and by Gautheret (1969c) using rhizome ex-
plants of Helicmthus tuberosus. For t h a t reason mostly cytokinin is added simultaneously
with an auxin to the culture medium. I t i s quite common that high cytokinin
concentrations antagonize auxin-induced root formation in tissue cultures, as has been
observed with tobacco callus (Skoog & Miller, 1957), rhizome explants of Helianthue
tuberosus (Gautheret, 1966a, 1969c), stem segments of Visum sativum (Leroux, 1969ab,
1973), leaf midrib fragments of Gevbeva jamesonii (Pierik &Segers, 1973) and Rhododen-
dronstem segments (Pierik &Steegmans, 1975c).
Adversely, low cytokinin concentrations can promote (lateral) root formation, as
reported for tobacco c a l l u s (Skoog & Miller, 1957), isolated roots of Convolvulus
arvensis (Torrey, 1958) and pea (Torrey, 1962), carrot callus tissue (Pilet, 1961), leaf
fragments of Ciohovium intybus (Toponi, 1963) and stem segments of Visum sativum
(Leroux, 1973) and Rhododendron (Pierik &Steegmans, 1975c).
The findings with pyrethrum closely correspond with those reported in l i t e r a t u r e .
The indication that root elongation i s stimulated by a low cytokinin concentration,
suggests that the promotive effect on rooting found in other studies may be due to the
same phenomenon.
3.4.2.6 Gibberellic acid
Gibberellic acid (GAj) was added a t the concentrations 0 , 1 0 ,10 ,10 ,10 or
4 -5
10~ g/ml to the culture medium, in the presence of IBA at 10 g/ml, to determine i t s
influence on rooting of explants of Clone 4331. _g -7 ,
About the same rooting r a t e was observed a t GA., concentrations of 0, 10 , 10 an
-6 , whereas GA^ a t 10 - 5 and 10 - 4 g/ml strongly delayed rhizogenesis (Fig. 27). The
application of GA3 a t 10" 8 , 10" 7 and 10" 6 g/ml s l i g h t l y stimulated the elongation of the
initiated roots (Table 23). The other parameters'for rooting did not differ markedly at
^ 3 0, 10"8 and 10~ 7 , while root formation was s l i g h t l y inhibited by GA3 at 10 an a
strong detrimental effect was caused by GA3 at 10" 5 and 10". g/ml.
Spanjersberg &Gautheret (1964) and Gautheret (1966a, 1969c) reported that GA3>
*hen applied alone, was not able to i n i t i a t e roots on rhizome explants of HeUanthus
Census. In most studies g i b b e r e l l i c acid i s added simultaneously with an auxin to
T
! " e 23. The influence of various concentrations (O-.o" 4 g / » » of GA3 on root formation
gfj^duncle explants of'Clone 4331. — 7
r
; ~ ^ l ^ 'o-6 .0-5 to-
u t i n g percentage 100 85 90 95 70 55 ^
nu
»ber of roots p e r rooted explant 32.7 29.1 30.2 24.9 4.8 ^
p a g e root length in MH 5.0 5.9 5.8 5.8 1.^ ^
J^_r°°t weight per rooted explant in mg 2.3 2.3 2.0 • _^ .—
47
rooting percentage
100
90
80
70
60
50
40
30
20
10
0 *lg. 27,The influence ofvarious concen-

trations ofGA, onroot formation of
peduncle explants ofClone 4331. Treat-
16 20 24 28 ments 1-6 represent 0, 1 0 - 8 , 10" 7 ,10" 6 ,
days ofincubation 10 and10"^ g/ml, r e s p e c t i v e l y .
cultureêdiun,; theeffect ofGA3 o n root organogenesis greatly varied with the plant
f r e q U m i n h i M t 0 r y e f f e c t on
has b e T T^ ^ ^ auxin-induced root formation,as
Stem SegmentS
Ion P i e T l T ° f P°PUtUS ^ ^ et
- I - 1968) and Ehododen-
19?5C) 967 1968a 1973
tTat ! 1 I"™*' - ^ ^ ° ' ' ) « i t h « ™ « * * * , . noticed
^ w T 7 t0 ^ PlantS ^ WMch ^ Se ^ entS — ên, striated
T^T:iT:T-°:s:oot £ o r m t i o n ° f stems « **.*» ^ ^ erectly«
potionofiM: ; r f o ^ r o r i i r prnce of ^ d i d promote the

thecultureswerekeptin^ J ^ Z ^ Z T T ^ ^
suppressed,especiallyathigh ^ J J Z ^ ^ ^ ^ ^ ^ ™
InpyrethrumtheeffectofGAv•A •
onthestageinrootformation H i e h I T ^ C ° n C e n t r a t i ° nU S e?
d
*a l s o d e P e n d e d
3C n C e n t r a t i o n s
initiationandelongationnf ^
**«.-t . i s :i t r r ; ™ ' *reas'-™ati™sdu-*

° considerablydiminishedboththe
and hadas l i g h t l y favourable effect onroot elongation.
48
3.4.3 Climatic factors
3.4.3.1 Temperature
Explants of the three clones were exposed to constant temperatures of 9, 13, 17, 21
or 25°C in the phytotron. This experiment showed that the regeneration of roots was
accelerated by raising the temperature from 9 to 25°C. However, after an incubation for
4weeks, in the range 13-25°C rooting percentages did not differ much, while at 9 C the
development of roots was s t i l l inhibited (Fig. 28). In spite of the rapid development of
adventitious roots at high temperatures, the highest number of roots after 4 weeks was
obtained at low temperatures: 9°C (Clones 1087 and 4331) and 13°C (Clone Ma 63/1889)
(Table 24).
In Clone Ma 63/1889, the number of roots after incubation for 4 weeks at 9 Cwas
very low. Moreover, 10 explants (501) of t h i s treatment did not show macroscopically
visible roots after 4 weeks incubation. When these 10 'unrooted' explants were trans-
ferred from 9 to 20°C, one day l a t e r 6 of these explants had developed macroscopically
visible roots and after 4 days adventitious roots had emerged from a l l the explants.
After 2 weeks at 20°C these explants had developed 22.9 roots per rooted explant, the
average root length was 6.0 mmand the dry root weight per rooted explant 2.9 mg! As
anatomical observations (Section 3.3) have shown that adventitious roots did not appear
earlier than about 10 days a f t e r incubation a t 20°C, the roots on explants of CloneMa
W/1889 must have been i n i t i a t e d a t 9°C. Consequently, root i n i t i a l s almost fail to
elongate at this temperature. Hence the temperature optima for the i n i t i a t i o n and for
the elongation of adventitious roots are different, being low for initiation and higher
for elongation.
The question arose whether a more effective root formation can be obtained when
explants are f i r s t grown a t a low temperature and subsequently at a higher temperature,
^ b l e 24. The influence of constant temperatures (9-25°C) on root formation of peduncle

explants.
21 25
Clone 9 13 17
^ P ™ ^ . ^ 85 60
4331
30 60 70 90 60
"^63/1889 50 85 90 80 80
?087er ° f r ° ° t S Per ro0ted ex

Plant „ 7 „ o 14.8 9.8 3,5
4331
ill M'O '5.8 J3.8 9-'
,K2
*a 63/1889 5.1 22.1 16-3 »*•»
£ « «a g* e root length in m
» ' ' g,. 4 9.5
9.5 7.7
7.7
„ 1.0 4.5 »•* --
M«63/1889
Ma 2^ ^ 6.3 6.6 6.0
63/1889 2.0 6.1 6.3
r 0t w e i h t
lns7
uo/
° § Per rooted explant in mg nuoy 0.7 °-6
..' n 11
n. 03
0.3 '
"•? —„, 1ics
^,M, ;• ;:?- tl 5:5 -
49
rooting percentage
100
rooting percentage
90|- 90
80 80
70 70
60 60
50 50
40 40
30 30
20
20
10-
10
0.
4 8 12 16 20- 24 28 4 8 12 16 20 24 28
days ofincubation days ofincubation
Fig.28.Theinfluenceofconstanttempe- Fig.29.Theinfluenceofvarious tempe-

ratures (9-25°C)onrootformationof rature combinationsonroot formationof
peduncleexplantsofCloneMa63/1889. peduncle explantsofClone 4331.
1=28(d13DC)/0(d 21°C),2=20/8,
3=15/13,4= 10/18,5=5/23and6=0/28.
insteadofgrowing themataconstantloworhightemperature.Toanswer this question

explantsfromClone4331weresubmittedto13°Cforvariousperiods,beforebeing trans-
ferredto21°C.
Fig.29showsthatrootingspeeddecreasedastheperiod duringwhichtheexplants
werexncubatedat13°Cincreased.Table25showsthatincubationfor10and15dat13°C
eforetransferto21°Cresultsinthebestrootingresponses.The anatomical
observaxons (Section3.3)demonstrated thatataconstant temperatureof20°Cingeneral
adventitiousrootswere initiatedduringthefirst2weeksofincubationandelongation
a T k T tT00tS °CCUrredd U r l n g^ S U b S 6 q U e n t2W e e k s <*-cubation.Thus 10-15d

13Caresufficientforanadequateinitiationofadventitious roots,
ntWhlCh
I^S r w lTr
Cfor2weeks
* **?"* ° fC 1 ° n4e331* ™ «*"»*to9,13.17or
beforetransferto21°Cforthenext2weeks
Progressive!,asL t e ^ re ^ ^ ? " " "^ „

g t n e± l r s t2
weeksdecreased.Thehighestnumber
50
Table 25. The i n f l u e n c e of v a r i o u s t e m p e r a t u r e combinations (d 13 C/d 21 C) on root
formation of peduncle e x p l a n t s of Clone 4331.
28/0 20/8 15/13 10/18 5/23 0/28
rooting percentage 100 90 95 100 100 100

number of r o o t s p e r r o o t e d e x p l a n t 21.7 32.1 36.3 32.9 26.6 19.0
dry root weight p e r r o o t e d e x p l a n t i n mgimg 1.0 2.7 3.6 4.1 3.7 3.3
rooting percentage
100r
90-
80-
70[•
60
50
40
30
20
10 F i g . 30. The influence of various tempe-

r a t u r e combinations on root formation of
0 peduncle e x p l a n t s of C one 3 1. ^
weeks 9° + 2 weeks 21 C, l ' k
+ 2 weeks 21°C, 3 = 2 weeks 17
12 16 20 24 28
days of incubation 21°C and 4 = 4 we<>ks 21 0.
« ™°ts was i n i t i a t e d a t an i n i t i a l temperature of 9 and 13°C, followed by ^ ^

the last 2 weeks (Table 26). Numbers of roots as well as dry root weights were gn
2
weeks at 13°C were followed by 2 weeks a t 21°C.
^ experiments described in t h i s section clearly demonstrated that a c ^
ioM
t e ^ r a t u r e , whether low or high, does not lead to an optimum root ™'
* • M a t u r e has to be adapted to the stage reached in root formation ^ , ^
Peduncle explants of various experiments and a t different stages of root ^ ^ ^
A b a t e d simultaneously in one growth chamber, a constant temperature ot
S t a i n e d in standard experiments. rooting An
Most publications gave a constant high temperature as opti^m or '
optima of 25 °C has been found for root formation of shoot tips of Asparagus off
51
Table26.Theinfluenceofvarioustemperature combinationsonroot formationofpeduncle
explantsofClone4331.
2weeks 9+ 2weeks 13+ 2weeks 17+ 4weeks21

2weeks 21 2weeks 21 2weeks 21
rootingpercentage 90 90 100 80
numberofrootsperrootedexplant 31.9 30.8 25.4 21.5
dryrootweightperrootedexplantinmg 2.4 ng 2.4 3.6 3.9 3.8
(Gorter,1965),rhizomeexplantsofHelianthus tuberosus (Gautheret,1969c),stem

segmentsofRhododendron (Pierik,1969;Pierik&Steegmans,1975c)andPiston sativum
(Leroux,1973)andleafmidribfragments of-Gerbera jamesonii (Pierik&Segers,1973).
Thefindingswithpyrethrumshowedthat,whenfluctuating temperaturesare'
considered,alsoalowtemperaturecanplayaspecificroleintheprocessofroot
formation.Thishasalsobeenobservedinthemostdetailedstudyabouttheinfluenceof
temperatureonadventitiousrootformationofrhizomeexplantsof Helianthus tuberosus
(Gautheret,1961,1966b,1968ab,1969abc;Spanjersberg& Gautheret,1963ab;
Inpathi, 1974).Theyfoundthattheformation6fcambiawasenhancedbyahigh
temperatureandthattheorganizationofrootprimordiafromthesecambiawas
stimulatedbyalowertemperature.Infactthehightemperaturewasinvolvedinastage
priortorhizogenesisanddidnotactdirectlyuponrootformation,
was " V ' ntl ' 10US™ 0 t f ° ™ a t i o no fPîolesegmentsof Lunaria annua (Pierik,1972)
t™,r7 T " Cm C
° n t i n U 0 U S U g h t - ™ * inhibitioncouldberemovedbyacold
than^5°c. at C
'^ " d a r k n e S St h 6 r e^ "°e V M e n C e t h a t 5 ° CW a sm 0 r e e f f e C t i V e
sativtlT ° 9 7 3 )/ 6 P 0 r t e d^ 't e m P e r a t u -of5-10°Ctowhichplantsof Visum

6 X e r t e dad 6 l a y e d P
1 " rT S ' ° S i t i v e * * « onrootformationofisolated
FUrthem re he P r
12a th ° ' ° V e d t h a t 5 ° C a l s ° - r t e d adirectpositive
!™ ItSrntSW6ree X P ° S e d f ° ^
r £lrSt
d
4f
° N a t i o ntothislow
ZrJrsILi\r;r rsthisstimiation °frootM™

- can be c a ^ Z ^ ^ T j ^t e i ^ " " " ^
atiow
"" ^ ^ •,
to species and also according to t h ! + " P e r a t u r e greatly v a r i e s from species
ge reached
formation. in the process of adventitious root
3.4.3.2 Light
StockplantsofClone4331w e r e
conditions.Todeterminewhetherl i V " " " 1^ ^ g r e e n h ° U S e vaieT n a t u r a l daylight-
pedunclesofClone4^1 .v.-i. lg tmay e x e r tadelayedeffectonrootformation,
ulrc
tjoi,whichwerestill o-n- i.
attached
withblackplasticclothfor0 2 tothestockplants,were etiolated
andincubationofthepeduncle' ' '6 '8 ° r10d beforeisolationofthepeduncles
showedawiderdiameterand. V T ^ ^ V i t r ° ' P e d u n c l e s whichwereetiolatedfor10d
andalightergreencolourthanunetiolated ones.
Table27.Theinfluenceofvariousetiolationperiods (0-10d)ofpedunclesonroot
formationofpeduncleexplants ofClone4331.
0 2 4 6 8 10
averagerootlengthinmm 11.3 10.9 10.5 9.3 9.2 7.9
dryrootweightperrooted explantinmg lmg 2.8 3.0 3.1 3.2 4.1 4.2
Table28.Theinfluenceof continuous lightordarknessonrootformationofpeduncle

explantsofClones 1087,4331 andMa63/1889,29daysafterincubation.
Clone Light Darkness
rooting percentage
1087 25 40
433! 90 75
Ma63/1889 40 70
number of roots per rooted explant

5.4 19.0
1087
15.1 . 29.1
4331 25.3
12.8
Ma63/1889
average root length in mm
2.1 4.9
1087
2.8 • 6.3
4331 4.9
3.0
Ma63/1889
dry root weight per rooted explant in mg
0.4 1.2
1087 3.1
4331 1.5
2.5 3.1
Ma63/1889
The rooting rate increased with a more prolonged period of etiolation, but did not
i^rkedly differ in the various treatments 4 weeks after incubation. The number of roots
Creased and the average root length decreased by prolonging the etiolation p e n
Pable 27). Dry root weights were highest after etiolation for 8 and 10 d. Thus xt was
*own that light has a delayed inhibiting effect on adventitious root formation. ^
Thereafter, the d i r e c t influence of continuous light, and darkness on adventitious
r
°°t formation of the three clones was investigated at 21°C in the phytotron. Fig.
* * Table 28 i l l u s t r a t e t h a t , although substantial differences exist among the clones,
^ general pattern i s similar and a d i r e c t inhibiting effect of light on root formation
can be observed.
The favourable effect of darkness in comparison with light on adventitious root
formation was confirmed in two other experiments with Clone 4331. In the f i r s t
e
*Peri m e nt explants of the peduncle were exposed to various 16-h photopenods, betor
e
*Posure to darkness. When the explants were exposed to more photopenods, rooting
^ g h t l y decreased. Moreover, a l l other rooting parameters improved when the p °
P-iods were fewer and 0 (darkness) and 4 photoperiods yielded the best rooting response
fTsM„ -„^
(Table 29).
53
roo ing percentage
100 -
90 -
80
70
60 *f
50 l
40
j
30
II 1
Jr-
20
InJ
10
llrI
0
rJ Fig.31.Theinfluenceofcontinuouslight
ordarknessonrootformationofpeduncle
i — i — . i
explantsofClones 1087(1=light,2=
8 12 16 20 24 29 darkness),4331 (3=light,4=darkness)
days of incubation andMa63/1889 (5=light,6=darkness).
o
onfroot
r o formation
o f f o S L of ^ r " , 0 explants
- ^peduncle 1 a r i U S 1 6 _ h hot
T ° ofClone P 4331.
°P«i°ds (0-20),beforeexposuretodarkness,
8 12 16 20
rootingpercentage ]00
100 100 100 95 90
averagenumberofrootsperrootedexplant 15.6 13.7 12.2 13.0 11.8 10.8
averagerootlengthinmm 6#4
8.2 6.7 5.7 4,5 4.6
dryrootweightperrootedexplant inmg 3.4
3.4 2.7 2.7 2.3 2.2
Table30.Theinfluenceofvariousdarknessperiods (20-0dl W
photoperiods,onrootformsf,'™of A •, p , UU-U d),beforeexposureto 16-h
incubation. formationofpeduncleexplantsofClone4331,5weeksafter
20 16 12 8 4 0
rootingpercentage
80 95 85 80 95 45
18.9 14.8 13.4 20.3 14.7 14.0
7.2 6.1 5.3 3.8 3.9 4.0
4.3 3.3 3.0 3.7 2.2 2.8
54
rooting percentage
100r
90 r-
70
60
50 f-
40
30
20
io !-
Fig. 32.Theinfluenceofdarkness
o periodsfor20, 16,12,8,4and0
d,beforeexposureto16-hphotope-
12 16 20 24 28 32 35 riods,onrootformationofpedun-
days of incubation t cleexplantsofClone4331.
InthesecondexperimentexplantsofClone4331wereexposedfordifferentperiods
todarkness,beforeexposureto16-hphotoperiods.Alltreatmentsshowedaboutthesame
rootingrate,withtheexceptionofcontinuousexposureto16-hphotoperiods (0dof
darkness),whichtreatmentdelayedtheorganogenesisofroots(Fig.32).Somevariation
canbeobservedamongthenumbersofrootsperrootedexplant;theaveragerootlength,
however,diminishedastheexplantsbecameexposedearlierto16-hphotoperiods(Table
30).Againitwasdemonstratedthatexposureforalongperiodtodarkness (20d)
stronglyfavoursadventitiousrootformation.Instandardexperimentsdarknesswasalways
maintained.
Ashasbeenreportedinliteratureandfoundforpyrethrum,lightinhibitedor
decreasedtheformationof (lateral)roots,asdemonstratedwith:shoottipsot
^ a a u s officinalis (Galston,1948;Gorter,1 9 6 5
^ ^ 0
; ^ 7 / 6 9 ^ ; ; ^
Furuya&Torrey,1964)andstemsegments (Leroux,1967,1968ab,iswaD, J^
sativum, isolatedrootsof Convolvulus arvensis (Torrey,1958),stemexpanso
PoPulus niara (Nandaetal.,1968)and BHododendron (Pierik,1969;Olieman-vander* e r
etal.,1971;Pierik*S t e e d s , 1975c),epicotylsegmentsof Aeolus ™ ^ ™ ^
man-vanderMeeretal.,1971),petiolepiecesof Lunarta annua (Pierik, J
midribfragmentsof Gerbera jamesonU (Pierik&Segers,1973). c n 3 n i e r s-
inexperimentswith Heliantnus tuterosus (Gautheret,1961,1966a 196 Span,ers
berg& Gautheret,1963ab;Letouze»Beauchesne,1969;Kicker*Paupardm,1969,
Tripathi,1974)lightinhibitedrootformationofrhizomeexplantsofthevariety 'Violet
commun'andstemsegmentsofthevariety 'VioletdeRennes',whereasapromotingeffect
oflightonrootingwasobservedonrhizomeexplantsofthevariety 'VioletdeRennes'.
Withstemsegmentsofdifferentspeciesofboth Parthenooissus (Leroux,1965)and Salix
(Leroux,1966)itwasalsofoundthatlightinhibitedorstimulatedrhizogenesis,
dependingonthespecies.
Apromotiveeffectoflightonrootformationinisolatedtissueswasreportedfor
tobaccopithcultures (Weiss&Jaffe,1969)andexcisedcotyledonsof Sinapis alba
(Lovell&Moore,1969).
Theinhibitingeffectoflightinonegroupofplantsandthepromotingeffectin
anothergroup,makesitverydifficulttoelucidatetheactionoflight.Fourpossible
explanationsweresuggestedbyOlieman-vanderMeeretal. (1971).
Inthischapterfactorswereinvestigatedthatareinvolvedintheprocessof
adventitiousrootformationofpeduncleexplantscultivatedinvitro.
Throughthisprocedureitcouldbeestablishedthatadventitiousrootformationis
regulatedbyacomplexsysteminvolvinganumberoflimitingfactors.Thesearerelated
topropertiesofthestockplantanditsexplant,thenutritional/hormonalcomposition
oftheculturemediumandtheclimaticconditions (Section3.4).
Theanatomicaldata (Section3.3)indicatedthatduringtheprocessofadventitious
rootformation,theinitiationofadventitiousrootsandthedevelopmentoftheroot
initialstakeplaceduringthefirstandlast2weeksofincubationofpeduncleexplants,
respectively.Bothstagesdonotrespondsimilarlytothegrowthconditions,butshow
theirownoptima.
Accordingtothisstudysomenewgrowthconditionscanbementioned,thatdiffer
fromthestandardgrowthconditions (Section3.2),butwithwhichrootformationmaybe
furtheroptimalized.Theycanbesummarizedasfollows:Theupperportions,witha
lengthof1.5cm,ofpedunclesinayoungfloweringstage,havetobewoundedoverthe
wholelength (byexcisionofthecortexatonesideoftheexplant),placedhorizontally
withthewoundedsideonthemedium,whichiscomposedofpyrex-distilledwater,
supplementedwithagar0.81 themajorsaltsofKnopathalfstrengthandsucroseat21.
ThepresenceofIBAat 10"5g/mlisrequiredforthefirstweektoensureanoptimum
rootinitiation,afterwhichperiodtheexplantshavetobetransferredtoanauxin-free
mediumforanoptimumelongationofthe.rootinitials.Theexplantshavetobeexposed
todarkness;duringthefirst2weekstoabout13°Candduringthesubsequentweeksof
incubationtoapproximately20°C.Whenalltheseconditionsaremaintained,agood
initiaionaswellaselongationofadventitiousrootscanbeexpected4weeksafter
incubationofpeduncleexplantsinvitro.
been^TJT T l ^ " *° ^ ^ f a c t 0 nilwolVBdinroot ^ t i o n hasnotyet

X
t h Z , " ^ ^ i n V e S t i ^ - dearlydemonstratedthatinaddition
t Z Z I T d T S U P P l 6 m e n t ^ " " " * = * nervations areessentialfora
betterunderstandingoftheexactroleofseparatefactorsonbothrootinitiationand
56
elongation.Aspointedoutby Pierik (1969)inthiswayitbecomespossibleto
discriminatebetween factors affecting rootinitiationorrootelongation.This
distinctionwouldbe impossible onthebasis ofdataonlyaboutthephysiological
aspectofrootformation.As aconsequenceofthisapproach,optimumgrowthconditions
canbeselected andmaintained duringthedifferentstages intheprocessofroot
formation.
InChapter4 itisevaluatedwhether thisknowledge,obtained throughacultureof
peduncleexplants invitro,leads toanoptimalizationofrootformationofshoot
cuttingsinvivo.
S7
4 Rootformation ofshoot cuttingsinvivo
4 . 1 INTRODUCTION
In practice pyrethrum is propagated vegetatively by s p l i t s . This method resultsin

a low multiplication rate as one stock-plant of 1-2 years old can be divided into only
5-10 splits. Studies on alternative asexual propagation methods,, for instance from shoot
cuttings in vivo, like is described in this chapter, and by the cultivation of capitulum
explants in vitro (Chapter 5), have adirect bearing on the current attempts ofthe
pyrethrum industry to improve the rate of multiplication of selected, high yielding
clones.
Anadditionalproblemisthatthefloweryieldstronglydecreaseswhenplantsare
attackedbytheroot-knotnematode Ueloidogyne hapla (Bullock,1961;Robinson, 1963;
Parlevliet&Brewer,1970,1971;Parlevliet, 1971).Splitting infested stock-plants
resultsm acontaminatedclone,whereaswithvegetativepropagation fromshootcuttings
plantingmaterialwouldbefreeofnematodes,evenfrominfestedstock-plants.
Aprerequisitefortheproductionofaplantfromashootcuttingistheformation
ofadventitiousrootsatthebasalstemportionofthecutting.Theinfluenceofvarious
factorsontheprocessofadventitiousrootformationofpeduncleexplantsinvitrohas
alreadybeendescribedinChapter3.Althoughthecircumstancesforrootformationin
vivoandinvitroaredifferent (Section 3.1), this 'in-vitro'knowledgemayleadtothe
optimizationofadventitious rootformationofshootcuttingsinvivo.Section4.3
dealswithanatomicalobservationsandSection4.4withtheeffectofvarious factors on
rootformationofshootcuttings.
c u l t i l r / 1 " T U e S °f ^ year
" ° U S t 0 c k "P l a n t s of Clones 1087, 4331 andMa 63/1889,
0 U S e (SeCti
2TJT* T t Tf °n 2'2)> V e g e m i V e Sh00tS
* * 4-5 developing leaves
6XCiSed fr m the basal
2 e l e 7 V° ^ " " - ^ ° ™*. th cuttings
l0W6r emPl£mted leaV6S
l o Z n T T - I n t M s ^ a b a s a l *«" P - * -
was obtained, with alength nf a u , t 1 c -,
insertion Jt„ «, ' S U l t a M e £ o r t h e application of auxins and
insertion into the rooting substrate (Fig. 33 i ef1 -i TU0 „•
0 2%soini-irm nf +u r • - *' ™ e c u t t l n g s were immersed ina
«--«rr^jsrT 5 : I ~in r v " substiat - ° f —

"A at 11 („ n f l c \ * J T T I
upasfollows- f„n„„ AU •
t
fy
^ " ^ " " = i l " ^ » » P ~ t o of t h . auxin
updbroiiows.foliagedebriswascollectedandW i - *
•P e a t "P° t s - ^ e leafmouldwasmade
S
»*nureperm 3andleftto, w T ° n ey e a r 'm i x e dw i t h 2 0 0 **
andlefttodecomposeforanotheryear,thenmixedwith6kgCaC0 3perm 3
58
Tig.33.Fromlefttoright:
"prepared shootcuttingjustbeforeinsertion;thebasalstemportionwithalengthof
about1.5cmwasobtainedbyremovaloftheloweremplanted_leaves.
-rootedshootcutting in 'Jiffy'pot,4weeksafterinsertion, •noprt..-on
-rootedshootcuttingafterremovalofthe 'Jiffy'P°t,4weeksafterinsertion.
andground,subsequently leftagainforoneyear,afterwhichabout300dm of'Trio'

potsoil17+ 6kgCaCO.perm 3wereadded;finallythemixturewasgroundagainand
treatedwithmethylbromide.The 'Jiffy'potswereembeddedinaleaf ^ ^ ^ ^
a
Propagationboxes (Fig.34)inagrowthchamber (Fig.35)at15Cand80-901™ *"e
humidity.Thegrowthchamberwasilluminatedbyfluorescentlight(PhilipsTLMFn u /
33RS)supplementedwithincandescentlamps(Philinea)atadaylengthof14handalign
intensityof25000lx,measuredjustabovethepropagationboxes.Instandar
experiments,inunheatedboxes,asoiltemperatureof17°Cwasmaintained Thesubstrate
couldbeheatedupto30°Cwithheatingcoils,whichwereburiedinsandbelowthelea^
™uld layer.Thefirst2weeksafterinsertion,thepropagationboxeswerecovere
thinpolyethylenefoil (Fig.35)tokeep^ r e l a t i v ehumidityatalmost100.and
bytoreducetheevaporationoftheunrootedcuttings.
Aswasdonewithpeduncleexplantsinvitro (Chapter3),theexperimentswere
terminated4weeksafterinsertionoftheshootcuttings Thecuttingswere
CFig.33,middle)andafterremovalofthe 'Jiffy'pots(Fig.33,right),t pe
ofthecuttingswhichproducedmainandlateralroots,theaveragenumbero mai^r
Perrootedcuttingandoflateralrootspercuttingwithlateralroot« n »J *e
- a g e length (inmm)permain ^ ^ ^ ^ T t ^ ^ ^
•;q
i
i
fci~ •• — J "- ' - - mi Ilin i tJr . * _ _ » * *jrA_jll...j**...*t,_,..l.JL.»
Fig.34. Propagationbox;shootcuttingsplantedin'Jiffy'potsandembeddedin
aleaf
mouldlayer.
N^^s^w&fc&fcsêaij^^
^**MNto»»tti*fi«*
-.J
xWu.
S h t l s c o ^ d C ! : i t t e p o S y ! e n f foil!° > « * « ^ * « " ^ P o t i o n box a t the
60
number of developing shoots was also determined to obtain information about the
development of the upper p a r t of the cutting.
The most important rooting variables are those referring to formation of main roots
(percentage, number and l e n g t h ) , because t h i s i s a prerequisite for l a t e r a l root
formation. For t h a t reason, but also because l a t e r a l root formation was sometimes found
to be very sporadic, data were s t a t i s t i c a l l y analysed for variables which concern main
root formation. The r e s u l t s of the s t a t i s t i c a l analysis are presented in the form of P
values (for effects of i n t e r e s t ) and LSD values (for pairwise comparisons of a number
of treatments). These parameters are defined as follows:
P= probability of obtaining an observation about the effect of interest as extreme as
or more extreme than the one actually obtained in the experiment, where the probability
is calculated under the n u l l hypothesis (here always indicating 'no e f f e c t ' ) .
LSD= abbreviation of ' l e a s t significant difference', i . e . the smallest difference (in
absolute value) t h a t would lead to rejecting the null hypothesis of zero difference
against the a l t e r n a t i v e hypothesis that the difference is not zero with a level a
significance t e s t (a was always chosen to be 0.05).
For anatomical observations shoot c u t t i n g s of Clone 1087 with long internodes were
selected, which apart from the basal wound, were additionally wounded by excision of a
strip of the cortex a t one side of the basal internode, and then inserted into the
substrate. Basal internodal stem segments with a length of about 1.5 cm were fixated 0,
4
> 8. 11, 14, 16 or 18 d a f t e r insertion. The plant material was further prepared
according to the methods described in Section 3.2.2. Unless otherwise stated, the
standard growth conditions (Section 4.2) were maintained in a l l rooting experiments.
4-3 ANATOMICALOBSERVATIONS
When nodal stem segments are used for anatomical observations, the picture i s
confused by connections between emplanted leaves and vascular bundles and because of
^juries due to removal of the lower leaves. Therefore shoot cuttings with basal i n t e r -
n e s with a length of about 1.5 cmwere used, while a s t r i p of the cortex was excised
for a better comparison with the anatomical observations on root formation of peduncle
explants, which were wounded in a similar, way (Section 3.3).
Transverse sections of the basal internodal stem segment of a shoot cutting ( ig.
36a) and of a peduncle explant (Fig. 12a), j u s t before insertion and incubation,
respectively, look morphologically similar, though there are some differences
" Abasal stem section of a shoot cutting is almost cylindrical in outline and the cortex
consists of chlorenchymous t i s s u e , while a section of the stem on top of the pedunc
h
as a corrugated outline because in the cortex loose chlorenchymous tissue alternates
w
ith the t i g h t collenchymous t i s s u e of the r i b s . secondary
foi
- I" a peduncle segment a fascicular cambium gives r i s e to the ™tl0n
Pôem and xylem. In a basal stem portion of a shoot cutting, secondary phloem and xy!
also
originate from an interfascicular cambium.
" m the vascular bundle of a peduncle segment parenchymous P - — * ^
^ r e a s sklerenchymous fibres can be observed in the fascicular pencycle m
61
, * *
„ , ...-5*... .!> -
• ...... . ' . - •. .."••! ©
« / • • , . : : - ; • •• ,-.• "-it •;•.•
"f&r
*•• - ,~ - ••w.v.••••v-•:-;^•:•••• •'• .> • • - >

\ " i- •• ».' '•:.\':'-:'. : v - '! .••• •*• t
%-. *V;.:--^^^--V : .--- : -/
^ '•'.....•••••->^r^;-- \ ;
500urn
V.'- v-ii-1-" '•<-..' ;v'/.v,i->;-"*.•.".*/ •,-::--''•.••-;-'ia«
Tr
TllTZ TrSs i-d eSeCti0I>S
a. Note the wounded ° f b3Sal
, j u s t before
intern dal
i n s e r t i o n° " « ' • » - ' • of shoot cuttings:
iniU ed
^Zl^ZTl X 2P " ' S
C ei
l lr
s S
o f tI
h e^ J d " ^ t h e ^ " f a s c i c u l a r peri-
insertion. endodermis and thecortex, 11d after
i n S t r t i 0 r t i t i 0 U S r ° 0 t ( ° ^ ° s i t e «« — * e d side) penetrates theepidemds, ,8d after
62
stem portion of a shoot c u t t i n g .
In Figs 36b and 36c i t can be observed t h a t adventitious root formation of shoot
cuttings progresses s i m i l a r l y t o t h a t for peduncle explants (Section 3.3). Main roots
were i n i t i a t e d in the i n t e r f a s c i c u l a r p e r i c y c l e , usually within 2 weeks after i n s e r t i o n ,
and during the subsequent 2 weeks the root i n i t i a l s elongated and emerged. In contrast
with peduncle segments, r a t h e r often l a t e r a l roots branched off from main r o o t s ,
initiated and elongated on basal stem portions of shoot c u t t i n g s .
4.4 FACTORS INFLUENCING ROOTFORMATION
4.4.1 Plant faators
4.4.1.1 Genotype
InmostexperimentsdescribedinSection4.4shootcuttingsofClones1087, 4331
andMa63/1889wereusedsimultaneously.Althoughsometimessignificantdifferences
existedbetweenclones,ingeneraltheclonesreactedsimilarlyandgavegoodrooting
responsesundervariousconditions.
4-4.1.2Wounding
InSection4.2itwasdescribedthatinitially,apartfromthebasalwound,the
basalstemportionsofshootcuttingswerewoundedbyremovaloftheloweremplanted
leaves.Adventitiousrootformationofpeduncleexplantsinvitrowaspositivelyaffected
bytheexcisionofastripofthecortexatonesideandoverthewholelengthoftheex-
Plant(Section3.4.1.5).
Theeffectofanadditionalwoundonrootformationofshootcuttingswasexamined
inanexperimentwith100shootcuttingspertreatmentofClones1087,4331andMa63/
1889.Anadditionalwoundcutwasmadebyexcisionofastripofthecortexatoneside
ofthebaseofthecuttingoveralengthofabout1.5cm.
Astatisticalanalysiswasnotcarriedoutbecausethedata (Table31)indicated
thatnosignificantwoundingeffectsaretobeexpected.Evidently,theexcisionofa
stripofthecortexisnotanimportantprocedureinthepreparationofshootcuttings.
^ble 3,.Theinfluenceofwoundingonrootformationandshootdevelopmentofshoot
cuttingsofClones 1087,4331andMa63/1889. ^^ ,—.
Clone Mainrootformation Lateral_rootformation Dryroot Shoot^
% number length % number length
'087 unwounded 54 9.1 33.0 25 7.0 5.2 6.1 ^

wounded 62 8.5 28.5 24 7.3 10.4 ^^ ^
4331 unwounded 41 6.3 24.4 13 2.0 4.8 • ]Q3
wounded 36 7.4
- i / 25.4
nc/. 121? 3.63-D 4.0 ^•
Q1 82 11.0
11.0 i"106
D
Ma63/1889unwounded 71 12.0 28.3 « »-J °- ? ,06

wounded 73 10.2 31.0 43 8.9 8.5
63
However, the i n i t i a l injuries which are made by preparing shoot cuttings may stimulate
root formation. The effect of these i n i t i a l injuries on rooting could not be determined,
because a l l shoot cuttings were i n i t i a l l y wounded and unwounded shoot cuttings were not
available.
4.4.1.3 Shoot t i p
The effect of the terminal shoot t i p on root formation and shoot development was
determined m an experiment with 60 shoot cuttings per treatment of Clones 1087, 4331
and Ma 63/1889.
The data of Table 32 indicate significant promotive effects (averaged over clones)
tlP V a l
looTl 7° °n PerCentage
°f main r
°0t f
™ i 0 n «* a v e - S e » • * « • of main
C th6re nay be dif£erences
Iff ; f l ^ ^ w e e n clones with respect to the
P6rCentage
variables t ^ ^ "'** ° f "** "** f
™ i o n ^ » * » the other
1 ^ T " fOT ^ r
°° ting P6rCentage S U
^ S t e d *" ^ e effect of the
M/1889
var b es "f """" ^ "* * - «* ^ ^ ^ < * » * « 3 1 . The
t0 m i n r 0t l6ngth m d latSral r
I bv h T ° °0t £
™ i 0 n
™ « * strongly
the ther hand
Z22 oTth ; "^ * ° ^ ^ ^ « * * * — »** highe in
tlP 6ffeCt h0W6Ver n0t
tlTeZrl ' "** ' ' ^ » * ^ s t a t i s t ! ally,
llCateS Sh 0t nU er C iSeS
I f ^ t r T7 '^ ° * °^ ** » — <* ^th sho ts
^ ^ ^ r r H tipsmdfrombudsintheains °fie—^ » *-
S T L T H " ^ W 6 r e^
to n rem
° V e d '^ Sh
° 0 t™ * « has
- bereducedby
nffldllaiy
S Sh 0tS P f
1 aSaryhshoots
ts^ irarely
V d developed,
f whereashigh
° numbers ° ^ shoots
-^^ of—axillary ^ developed
«*>
Table32.Theinfluenceoftheshoot H „
shootcuttingsofClones 1087,«31 and V?£nZ*i0?ation a ds h o o t
" developmentof
3 / 1 8 M (+a n d
absenceoftheshoottip,respective?"? ~"Presentthepresenceand
Clone
^ l i f f l f f f H l f t i o n I f t e r a l _ r I o I _ ^ ™ a t i o n Dry root Shoot
% number len
8th % number l e n g t h weight number
1087
U 3 2 7
4331 + H„; - -' 2.3 75
63
H.4 34.1
67 22 10.3 9 9.9 5 4 65
15-8 415 8 ,8 7,, r -
Ma63/1889 + ,, „" " ""'*' ' "-
I8 5.77 5.7 10.9 10.9
"- 63 63
n
9 6 30
- -4 27 7s ,,,
3 2 2 8
= ^ — — — E _ _ l - 28.9 27 ' «•, - "
p — . '•' '.o 8.4 e, •* oo
st bToTXbTTî • •
stcl
cl 0.07
0.170.93
0.10 0.090.65
LSD 16
-8 6.6 18.6
st=Differencebetweenpresenceand
cl
cl- met
=Difference-
between IIZT* "* a b S e n c eo f t h e ^°ot
iaabsenceoftheshoottip.
enpresenceandabsenceofthe
64
after shoot t i p removal. The number of excised terminal shoots was j u s t compensated or
overcompensated by the number of developing axillary shoots.
4.4.2.1 Substrate
Inthefirstexperiment,with50shootcuttingsofClone1087pertreatment,the
influenceofvarioussubstratesonrootingwasanalysed.Inadditiontothesubstrates
sand,leafmouldandleafmould/sand (Section4.2),vermiculite,twogradesofperlite,
threegradesof 'Trio'potsoil,'Asef potsoil,peatlitterandpeatmouldwereusedas
rootingsubstrates.
Table33showsthatvermiculiteandsandareunsuitablerootingsubstrates.Thedata
ofbothmediawereillustratedseparately,becauseastatisticalanalysiswascarriedout
usingexclusivelythedataoftheother10rootingsubstrates.Therearesignificant
differencesbetweensubstrateswithrespecttopercentageofmainrootformationand
elongationofmainroots.AsillustratedbytheLSDvalues,thesubstratesleafmould,
peatlitterandpeatmould,havetoberegardedasunsuitableforrooting.Goodrooting
responseswereobtainedintheothersubstrates,especiallywithperlite (fineand
coarsegrained).
Inthesecondexperiment,with50shootcuttingspertreatment,theinfluenceof
foursubstratesonrootformationofshootcuttingsofClones1087,4331andMa63/1889
wasinvestigated.
Table34illustratesthatforthevariables,whichrefertomainrootformation,
therearesignificantdifferencesbetweensubstratesandbetweenclones.Thesevariables
Table33.Theinfluenceofvarioussubstratesonrootformationofshootcuttings of
Hone1087.
Substrate Mainrootformation Lateralrootformation Dryroot

weight
2 6.0 4.0 0.4

vermiculite 2 2.0 12.0
sand 7.0 8.5 4.3
36 12.5 28.5 10
ieafmould 34 15.9 16.5 9.1
74 12.7 47.3
46 28.5 13.0 16.3
leafmould/sand 84 18.4 47.4
50 24.3 13.5 16.6
Perlite (finegrained) 94 17.7 45.1
62 18.6 13.5 15.0
Perlite (coarsegrained) 96 16.9 41.8 14.7
17.5 47.4 44 22.5 16.5
Trio'potsoil17 84 15.1 13.3
Trio'potsoil26 15.3 45.0 48 22.6
84 14.6 16.7
^rio'potsoil35 18.0 47.9 40 15.8
82 24.3 15.2 14.4
Asef potsoil 18.6 46.7 36
86 34.0 14.1 13.6
Peatlitter 17.2 39.7 58
72 30.3 12.5 13.1
Peatmould 17.8 29.9 64
82
p 0.54
su ,0.03 0.73 0.00
LSD 13.7
13.7 6.2 6.8
su=Differencebetweensubstrates.
LSD
fordifferencebetweentwosubstrates.
65
Table34.Theinfluenceofvarioussubstratesonrootformationofshoot cuttingsof
Clones 1087,4331andMa63/1889.
Clone Substrate Main root formation Lateral root formation Dryroot

% weight
number length % number length
1087 sand 26 8.2 20.4 4 1.2 17.0 1.9

leaf mould 52 6.4 38.2 28 7.2 8.0 2.2
leaf mould/sand 80 10.6 35.8 46 11.7 9.3 5.1
perlite (fine grained) 74 8.8 33.3 20 14.6 8.5 3.0
4331 sand 12 4.6 16.4 4 2.2 4.0 0.7
leaf mould 40 7.7 35.1 18 2.9 10.0 2.3
leaf mould/sand 56 12.1 33.8 18 7.0 10.0 .4.2
Ma 63/1889 sand 30 13.0 26.0 16 6.3 7.9 2.7
leaf mould 82 11.0 35.6 50 13.4 9.6 5.7
leaf mould/sand 84 15.0 30.7 44 12.5 9.6 6.5
P 0.00 0.02 0.00
su
P
cl 0.00 0.00 0.29
p , 0.08 0.58 0.66
sucl
LSD 1 9.2 3.2 - 6.1
LSD 2 7. 9 2.8 5.3
su=Differencebetweensubstrates,averagedoverclones,
cl=Differencebetweenclones,averagedoversubstrates,
sucl=Differencesbetweensubstratesofthedifferencesbetweenclones.
1.LSDfordifferencebetweentwosubstrates,averagedoverclones.
2.LSDfordifferencebetweentwoclones,averagedoversubstrates.
didnotshowsignificantinteractionsbetweensubstratesandclones,sothattheeffect
ofthesubstratewasalmostindependentoftheclone.Thedataconfirmedtheresultsof
thefirstexperiment,wheresandandleafmouldwerefoundtobeunsuitableandperlite
andamixtureofleafmouldandsandtobesuitablerootingsubstrates.Leafmould/sand
yieldedthebestrootingresponseandthissubstratewasalsousedinstandard
experiments.
4.4.2.2Auxins
InanexperimentwithCloneMa63/1889variousconcentrationsofI M , IBAandNAA
(. on alebasis)weretested.Pertreatment50shootcuttingswereused,whereasthe
controlgroupconsistedof150cuttings.
a v e r a ! ! ? ? " ? 1 " ™ 3 ^ ^ ** "**"» ^ treatments

« ™ *»** *« **
r 0tS r 0 t W6ight (TaWe
LTo7 f mam rô o t f o° m a t i"*
percentage ** ^
o n were ^ ° ^ ^ ^ ^ - ^hese
^ ^ variables
^ and
^ the^
f " U v t h a n T eX7t: ^ I M " ^ ^ « * » * « * - of NAAtested were much less

m d I M Maln r 0 t e l 0 n g a t i n
r oo l o T - ° ° - d variables referring to
6 C e d
^ ^ Z ^ ^ ^ ^ * <* •**«*» * ™ * . - optimum
Vitiation of I ' h T " " el°ngati°n ^ ™ ^ ^ ^ m
^
"•am roots at high percentages ( H ) .
66
Table35.Theinfluenceofvarious concentrationsofIAA,IBAandNAAonrootformation
ofshootcuttingsofCloneMa63/1889.
Auxin% Mainrootformation Lateralrootformation Dryroot

: weight
Control 61 4.2 53.8 47 33.7 11.8 8.23
IAA 0.25 64 4.2 49.4 56 35.6 13.8 9.0
0.5 78 6.0 59.1 70 47.6 12.6 16.0
1.0 86 14.4 56.7 82 47.6 11.4 25.0
IBA 0.25 72 5.9 55.3 62 47.9 13.2 15.0
0.5 80 5.3 59.0 70 44.5 13.8 16.0
1.0 86 7.6 58.0 82 47.6 12.4 21.1
NAA 0.05 62 4.3 55.6 60 34.0 13.2 10.2
0.1 78 3.9 57.7 64 38.6 13.9 12.8
0.2 70 4.8 54.5 66 37.2 12.2 11.7
P 0. 07 0.00 0.58 0.00
a
LSD1 22. 3 2.7 9.5 11.7
LSD2 18. 3 2.2 7.8 9.6
a=DifferencebetweenvariousconcentrationsofIAA,IBAandNAA. (Controlisdesignated
asauxinconcentration 0.)
1.LSDfordifferencebetweentwoauxinconcentrations.(Controlexcluded.)
2.LSDfordifferencebetweencontrolandauxintreatment.
3
-Dryrootweightof50shootcuttings.
Mainroot initiationmayfurtherbeimprovedathigherauxinconcentrations.This
wasexaminedinthree experimentswith50shootcuttingspertreatmentofCloneMa63/
1889.Various concentrationsofIAA,IBAandNAAwereappliedaspowders (basalstem
Portionsdipped in.auxinpowdersontalcbasis)aswellasliquidsolutions (basalstem
portionsimmersedinauxinsolutionsfor24 h ) .
Thevariables referringtomain,,rootformationwerestatistically analysedandal-
mostalwaysauxin applicationhadasignificantpositiveeffect (Table 36). Becausethe
auxinNAAhadaless favourable effectonmainroot initiationthan IAAand IBA, the
influenceofNAAisnotfurther discussed.
Theoptimum concentrationofIAAandIBAvariedwith thevariable involved.In
generalhighpercentagesofshootcuttings initiatedhighnumbersofmainrootsathigh
auxinconcentrations,whereasthelengthoftheinitiatedm a mrootsandlateralroot
formationwere enhancedbylowauxinconcentrations.Thedryrootweightsincreased
substantially aftertheapplicationofboth IAAand IBA,buttendedtodecreaseagainat
thehighestconcentrations used.Incomparisonwiththecontroltreatmenttheshoot
numberwashigheratlowconcentrationsofIAA,appliedinpowderorsolution, and
lightlyhigheratlowconcentrationsofIBA,appliedinpowder.Theshoot™ * « ^ ^
Progressively decreasedatincreasing concentrationsofIAAandIBA.Itis ™P°$S1
^dicateanauxinconcentration thatisoptimumforallthevariables.Onthe ^ ' ^
goodrootingresponseandshootdevelopment canbeobservedafterapplication
ofIAAorIBAataconcentrationofU . ,.
Todeterminewhether these findingsarealsovalidforClones 1087 f ^ ' ^
effectofIAAandIBAatvariousconcentrations (»ontalcbasis)onrootformationand
67
Table36.Theinfluenceofvariousconcentrationsofpowders (%) andsolutions(mg/1)
ofIAA,IBAandNAAonrootformationandshootdevelopmentofshootcuttingsofClone
Ma63/1889.
Auxincone. Main rootformation Lateralroot formation Dryroot Shoot

weight number
Control 80 9.3 33.7 40 11.8 9.8 6.4 113

IAA 0.1% 86 11.0 28.5 30 18.6 7.5 5.4 156
0.25 96 10.9 33.3 64 13.9 8.0 8.0 121
0.5 100 12.5 33.4 60 13.9 8.0 10.3 133
1.0 100 17.4 37.7 54 27.4 7.8 15.0 136
2.5 98 27.7 33.5 28 13.1 7.8 16.2 126
5.0 94 38.7 25.9 4 6.0 2.0 16.1 62
10mg/1 88 11.4 37.2 60 22.2 9.5 10.8 154
25 96 17.2 43.6 56 30.0 8.4 19.9 155
50 100 26.0 35.9 12 9.5 4.9 17.1 99
100 100 48.3 27.7 4 3.0 3.0 23.7 50
250 96 56.4 22.3 2 5.0 1.0 23.0 25
0.00 0.00 0.00
LSD' 9.5 7.1 5.6
Control 80 .6 29.7 28 15.7 8.0 3.9 102

IBA 0.1% 82 9.3 34.4 42 11.3 13.5 5.9 112
0.25 88 12.9 41.7 56 33.6 12.7 10.8 117
0.5 96 12.7 37.3 46 20.5 10.6 9.9 113
1.0 98 18.9 39.3 20 18.6 8.4 16.9 118
2.5 98 25.0 35.1 14 20.3 10.0 14.7 60
5.0 90 23.7 30.7 6 13.0 4.0 12.0 50
10mg/1 88 14.2 39.6 34 25.5 11.3 11.4 100
25 90 18.5 36.0 16 8.5 6.5 10.2 66
50 94 24.1 34.5 26 37.0 14.2 14.9 56
100 90 24.9 35.9 4 7.0 4.0 14.0 30
250 84 23.3 28.9 4 4.0 4.0 7.5 16
P 0.07 0.00 0.03
a
2
LSD 13.1 5.0 7.7
Control 56 6.2 27.0 14 11.6 14.3 1.8 104

NAA 0.1% 64 5.2 34.5 32 11.3 16.9 2.5 101
0.25 76 5.7 29.4 26 10.5 10.5 2.4 82
0.5 70 5.7 26.7 22 7.8 10.6 2.1 80
1.0 84 5.3 23.7 22 8.5 15.4 3.1 57
2.5 78 5.0 15.4 14 4.6
5.0 62 7.8 2.7 45
5.3 6.9 0 0 0 0.7 39
10mg/1 46 5.3 23.0 10
25 3.3 5.1 1.0 77
40 5.1 31.0 12 4.3
50 7.0 1.2 63
48 4.Z 21.0 8 6.7
100 4.3 1.2 54
56 5.5 25.7 14 5.1
250 10.3 2.0 46
58 3.7 15.7 10 6.2 7.0 0.8 30
a 0.00 0.80 0.00
3
LSD 22.1 2.4 7.3
a=DifferencebetweenvariousconcentrationsofpowdersandsolutionsofIAA (1)»1"A
(2)andNAA (3). (Controlisdesignatedasauxinconcentration 0.)
LSDfordifferencebetweentwoconcentrationsofIAA (1),IBA (2)andNAA(3).
68
shootdevelopmentofshootcuttingsofbothcloneswasinvestigated.Perhormonetreat-
ment30shootcuttingswereused,whereasthecontroltreatmentsconsistedof60shoot
cuttings.
Foranunknownreasonlowrootingresponseswereobtainedinthisexperiment (Table
37).Theauxinconcentration (averagedoverauxinsandclones)hadasignificant
positiveeffectonpercentageofmainrootsformedandtheshootnumber.Inaddition
thislastvariablewasinfluencedbyclones (averagedoverauxinsandconcentrations).
Ofallthecalculated interactionsbetweenauxins,concentrationsandclones,the
interactionbetweenconcentrationsandclonesispresented,becauseonlythisinter-
actionwasfoundtoinfluencesignificantlythenumberofshoots.Incomparisonwiththe
controltreatmenttheapplicationofI M orIBAat1and2.5°sresultedinhigherrooting
percentagesandnumbersofmainroots.MainrootelongationofClone1087seemedtobe
enhancedby IAAand IBAat0.51,whereasmainrootelongationofClone4331was
Table37.TheinfluenceofvariousconcentrationsofIAAandIBAonrootformationand
shootdevelopment ofshootcuttingsofClones 1087and4331• ^ ^
Clone Auxin Mainrootformation Lateralrootformation Dryroot Shoot

weight
'* number
% % number length % number length
1087 3.1 26.3 7 3.3 5.6 0.32 42'

Control 18
17 2.8 10.5 0.7 37
IAA0.5 40 7.6 35.6
10 2.2 2.5 1.3 31
1.0 53 12.6 23.7
17 8.4 10.6 1.5 34
2.5 53 9.9 32.8
10 6.0 8.0 0.4 34
5.0 37 9.2 24.8
4.3 14.2 1.5 38
IBA0.5 50 4.8 32.7 27
1.7 5.0 0.9 33
1.0 37 10.9 31.0 7
6.2 11.7 0.5 36
2.5 33 8.8 27.4 13 30
0 0 0 0.3
5.0 10 5.0 8.9
7.8 11.7 0.42 3.2
433! Control 10 4.9 29.0 10
10.0 8.3 0.5 30
IAA0.5 10 7.0 20.0 7 30
10 6.0 6.7 1.1
1.0 30 6.5 24.4 31
7 2.5 3.3 0.6
2.5 27 6.3 21.0 32
3 3.3 3.3 0.9
5.0 27 13.6 21.0
6.7 0.4 30
IBA0.5 23 6.3 25.6 10 6.7
3.3 2.0 30
1.0 23 13.4 21.5 7 5.0
0 0.9 31
2.5 13 11.5 21.7 0 0
0 0.4 30
5.0 13 6.8 26.2 0 0
0.27
P 0.24 0.86 0.92
a 0.02
P 0.04 0.17 0.54
co 0.02
P
cl 0.19 0.76 0.58
0.01
p 0.44 0.93 0.37
cocl 1.1
LSD1 15.1 5.8 10.4
a=DifferencebetweenIAAandIBA,averagedoverc o n c ^ " " ° n ^ r auxinsandclones.

co=Differencebetweenvariousauxinconcentrations,averaged
(Controlisdesignated asauxinconcentration0.)_ concentrations.
cl=Differencefetweenclones,averagedoverauxin - ^ f ^ ^ c l o n e s .
cocl=Differencesbetweenconcentrationsottne Q U I auxinsandclones.
1-LSDfordifferencebetweentwoconcentrations,averaged
2-Dryrootweightsandshootnumbersof30shootcuttings.
69
inhibitedatallauxinconcentrations.Theinfluenceofauxinsonlateral root formation
wasslightandnotclear,withtheexceptionofIRAat2.5and5%,atwhich
concentrations lateralrootformationwas almostcompletely suppressed.Afterthe
applicationofauxinshigherdryrootweightsweremeasured thaninthecontrol treat-
ments,withoptimaatabout H ofI M and IBA.TheshootnumberofClone 1087decreased
withanincreasing auxinconcentration,whileinClone 4331 aboutthesamenumberof
shootsdevelopedinalltreatments.
Hencebothauxinsyielded aboutthesamerooting response.Aswas foundinpre-
cedingexperiments,optimumconcentrationsareapproximately 14ofIAAorIBA(ontalc
basis);theformerwaschosenasstandard treatment.
4.4.3 Climatic factors
4.4.3.1Constant temperature
Inthephytotronintwoexperimentstheinfluenceofconstantairtemperaturesof
9, 13,17,21or25°ConrootformationofshootcuttingsofClones 1087andMa63/1889
wasexamined.Pertreatment60shootcuttingswere exposedtoadaylengthof16h.
Because therewerenoreplicates,thedataofTable38couldnotbeanalysed
statistically.However,shootcuttingsofbothclonesresponded similarlytotemperature.
ShootcuttingsofClone 1087yieldedhighrootingpercentagesat13and17°C,while
shootcuttingsofCloneMa63/1889 regenerated rootsathighpercentagesatall
temperatures.Onshootcuttingsofbothclones thehighestnumberofmainrootswas
initiatedat13°C;anoptimumelongationoftheinitiatedmainroots,however,was
observedat21and25C.Inviewofthepoorelongationofmainroots initiatedat9°C
onshootcuttingsofClone 1087,probablyapartoftheinitiated rootsdidnot become
macroscopicallyvisible,asoccurredinvitro (Section 3.4.3.1).Hence,ahigher
percentageofshootcuttingsofClone 1087mayhave initiatedahighernumberofmain
rootsthanisexpressedinTable38.Becauseofthepoormainrootelongation, lateral
rootsdidnot format9(and13°C);anoptimum lateralroot formationwas realizedat21
Table 38.Theinfluenceofconstant temperatures (9-25°C)onroot formationofshoot

cuttingsofClones 1087and11a63/1889.
Clone Tempera- Main root formation Lateral root formation Dry root
ture (°C)
% weight
number length % number length
1087 9 50 14.4 1.9 0 0 0 1.6
13 78 21.6 12.2 0 0 0 7.8
17 82 17.3 20.0 27 8.8 5.4 1.7.6
21 62 10.7 26.4 50 10.8 8.9 7.9
25 43 9.7 26.2 38 11.4 9.6 4.7
Ma 63/1889 9 85 14.5 8.8 0 0 0 1.8
13 91 20.2 18.4 0 0 0 5.8
17 90 14.5 19.2 23 9.6 11.5 4.5
21 83 17.3 24.3 35 10.3 13.1 4.6
25 80 7.1 22.7 55 18.4 12.2 2.9
70
Table 39. The influence of constant s o i l temperatures (17-30 C) on root formation of
shoot cuttings of Clone 1087.
Soil . t e m p e - Mairt r o o t f o r m a t i o n L a t e r a l r o o t formation Dry r o o t

rati;ire (°C) % number l e n g t h % number l e n g t h
17 81 25.0 39.0 32 22.2 12.4 28.0

22 96 15.6 49.4 69 34.8 13.5 38.8
26 82 15.8 44.3 67 43.6 15.9 40.6
30 13 4.3 17.8 10 6.6 9.2 1.7
P 0. 00 0.02 0.00 0.02
r
LSD 18. 3 10.7 11.0 5.7
t = Difference between s o i l t e m p e r a t u r e s .
LSD for d i f f e r e n c e between two s o i l t e m p e r a t u r e s .
and 2S°C. The highest dry root weights were obtained a t 13, 17 and 21 C.
Subsequently, in a growth chamber at a constant a i r temperature of 15 C, the
influence of constant s o i l temperatures of 17, 22, 26 or 30°C on root formation of
shoot cuttings of Clone 1087 was examined. The effect of the temperature on root
formation was investigated in four r e p l i c a t e s , to make sure that a s t a t i s t i c a l analysis
could be carried out. Per temperature 100 shoot cuttings were inserted into the
substrate
Thevariables referringtomainrootformationanddryrootweightwere
-gnificantly influencedbytemperature.Asoiltemperatureof30°Calwaysyieldedthe
worstresults (Table 39).At-17,22and26°Crootingpercentageswerehigh.Thenumber
ofmainroots increasedwithdecreasingtemperaturetoanoptimumat17C,andthe
elongationoftheinitiatedmainrootswas improvedathighertemperaturesof22and
26°C.Anoptimumlateralrootformationandthehighestdryrootweightscouldalsobe
observedatsoiltemperaturesof22and26°C.
Frombothexperimentsitcanbeconcludedthattheinitiationofmainrootsis
favouredbyalowtemperatureandthatahightemperatureissuitableform a m root
elongationandlateralrootformation.Anatomicalobservations (Section4.3)haveshown
thatmainrootsareusually initiatedduringthefirst2weeksafterinsertionandthat
therootinitialselongateandlateralrootsareformedduringthesubsequent2weeks
b e f o r e , thequestion ariseswhethertheprocessofrootformationcanbestimulated
b
yafluctuating soiltemperature.
4
-4.3.2Fluctuating temperature
TheeffectofatemperaturecombinationwasexaminedinanexperimentwithClones
1087,4331andMa63/1889.Duringthefirst2weeksalowsoil t * ^ ™ * * ^
S t a i n e d andduringthesubsequentperiodahighsoiltemperatureo •
temperaturecombination (17/25)wascomparedwiththecombination25/17andcons
temperaturesof17and25°C.Percloneandateachtemperature ^ ^ j ]
s t i n g s were inserted.ThenumbersofTable40aretheaverageofdatascor
« * 4weeksafter insertion.Astatisticalanalysiswasnotcarriedout,because
71
Table40.Theinfluenceofvarious soiltemperature combinationsonroot formationof
shoot cuttingsofClones 1087,4331 andMa63/1889.
Clone Soiltempe- Main rootformation Lateralroot formation Dryroot

rature (°o weight
Z number length % number length
1087 17 86.7 19.1 20.9 1.7 5.0 5.0 8.0

17/25 78.3 15.2 28.3 30.0 13.7 8.0 7.0
25/17 60.0 14.9 30.8 21.7 19.3 14.5 7.1
25 65.0 10.5 38.2 48.3 20.2 13.8 9.8
4331 17 35.0 16.0 14.8 1.7 3.3 1.7 2.5
17/25 63.3 12.0 29.0 21.7 7.2 4.6 4.4
25/17 20.0 7.3 19.1 3.3 2.5 2.8 0.6
25 16.7 3.4 30.7 5.0 5.3 6.7 0.9
Ma63/1889 17 98.3 29.3 21.3 15.0 9.7 6.9 22.2
17/25 98.3 21.8 24.3 20.0 13.8 7.0 13.5
25/17 95.0 14.1 22.7 58.3 12.5 8.8 10.7
25 85.0 17.9 35.2 43.3 12.9 12.1 13.5
werenoreplicates.
Highrootingpercentagescouldbeobservedatalltemperature combinations,withthe
exceptionofClone4331,especiallyat25/17and25°C (Table 40). Rootingpercentagesand
numbersofmainrootswerehighestat17and17/25andingeneralmainroot elongation
andsubsequent lateralroot formationwerefavouredat25and17/25°C.Theunsuitability
ofthetemperaturecombination25/17canbeseenfromthedryrootweights.Thehighest
dryrootweightsweremeasuredat17and17/250C.
Itcanbeconcludedthattheinitiationofmainrootsandtheelongationofroot
initials (andlateralrootformation)arestimulatedbylowandhightemperatures,
respectively.Agoodrootingresponseisobtainedatafluctuating temperature:alow
temperatureforthefirst2weeksandahightemperatureforthesubsequent2weeks.
Theexperiments m Section4.4showedthatadventitiousroot formationonbasal

stemportionsofshootcuttingsaswellasthedevelopmentoftheupperpartofshoot
cuttingscanbeaffectedinvariousways.
Anatomical observations (Section4.3)haverevealed thatthe processof
adventitiousrootformationprogresses similarlyashasbeenreported forpeduncleex-
plants (Section 3.3).Mainrootsare usually initiatedduringthefirst2weeks after
insertionoftheshootcuttingsintothe rooting substrateandmainroot elongation
(andlateralrootformation)occurduringthesubsequent2weeks.
Consequently,avariationofafactorjustbefore insertionorduringthefirst2
weeksafterinsertiondirectlyaffectsmainroot initiation.Suchavariationhasa
delayedeffectonsubsequentmainrootelongationandlateralrootformation,which
aredirectly regulatedbyfactorsvariedduringthenext2weeks•
Theinfluenceofauxinandtemperatureonadventitiousrootformationofshoot
cuttingswasidenticaltotheeffectofthesefactorsonrootformationofpeduncleex-
72
Inconformitywith investigations invitro (Section3.4.2.4)theapplicationof
exogenousauxinswas favourable foradventitiousrootformationofshootcuttings.The
initiationofmainrootswas enhancedbyhighauxinconcentrations andsubsequentmain
rootelongation (and lateralroot formation)bylowerauxinconcentrations.Theauxins
IAAandIBAweremore,suitable forroot formationthanNAA;optimumconcentrationofIAA
orIBAwasapproximately H (ontalcbasis).
Furthermore,inagreementwithexperiments invitro (Section3.4.3.1),ithasbeen
shownthatmainroot initiation ispromotedby lowtemperaturesandrootelongation (and
lateralrootformation)byhighertemperatures.Ofthetemperaturecombinationstested,
thebestwas 17°forthe first 2weeks afterinsertionfollowedby25Cforthenext2
weeks;at17°shootcuttings athighpercentages initiatedhighnumbersofmainroots,
whichelongated andbranched rapidlyat25°C.
Incontrastwith experiments invitro (Section3.4.1.5),however,excisionofa
stripofthecortexhardly influencedrootformationofshootcuttings,probablybecause
thisisanadditionalwoundcut.
Theshoottip turnedouttobeofgreat importanceandadventitiousrootformation
"assubstantiallypromotedby itsremoval.Thismaybeduetoendogenoussubstances,
likeauxin,which are synthesizedby shootsdeveloping fromaxillarybuds (Meyer&
Anderson,1952).
Goodrooting responseswere achieved inthesubstratesleafmould/sandand
perlite.Sinceasubstrateof leafmould/sand ispreparedaccordingtoaspecial
procedure (Section4.2) and isnotwidelyavailable,themanufacturedproductperlite,
whichisusedallover theworld,has tobepreferredasrootingsubstrate.
Someofthe factors affecting rootformationalsodemonstratedaneffectonthe
developmentofshoots ontheupperpartofshootcuttings.
Inthepresence oftheshoottipaxillaryshootsrarelydeveloped,aphenomenon
called 'apicaldominance1. Breakingofapicaldominanceandthedevelopmentof
axillarybudswas considerably stimulatedbyshoottipremoval.
Axillary shootdevelopmentwasalsopromotedbyanapplicationwithexogenous
auxinsatlowconcentrations,whereas itwas inhibitedbyhigherauxinconcentrations,
™ r e soby theapplicationofauxins insolutionsthaninpowders.Axillaryshoot
development isprobably inhibitedbyauxinsolutions (appliedfor24h)becauseauxins
aredirectlyaccessible totheshootcuttingandthenahigherauxinconcentrationmay
b"ildupwithinthe shootcutting.Whencuttingsaredippedinauxinpowders,much
auxinmay getlostasaresultofinsertionoftheshootcuttingsintotherooting
substrateandrinsingoffafterwateringand,moreover,auxinshavetobedissolved
foretheyareaccessible totheshootcutting. ^
Bothrootformationandshootdevelopment,processeswhichareessenia
vegetativedevelopmentofshootcuttings,areinfluencedbyanapplication " ^
oogenous auxins, ingeneralroot f o ^ t i o n iss t i l t e d byh i êrau,inconcentrati^
thanshootdevelopment.A compromisecanbereachedatU ofIAA i
atwhichconcentrationrootingresponseandshootdevelopmentaregood.
«henshootcuttings arestruckunderthestandardconditions SecU n ) ^
addition,theteiminalshoottip isremovedandthebasalstemportionsofthecutt
73
areexposed toafluctuating soiltemperature (initially alowtemperature,which is
gradually raised),itisexpected thatshootcuttingsofallclonesproduce high
numbers ofrootsquiterapidly andathighpercentages.Onanaverage 25-100 shoot
cuttings canbeobtained fromonestock-plant of1-2yearsold,instead of5-10splits,
whichmeans thatthemultiplication ratecanbe increased considerably.
Tobeassuredoftheavailability ofvegetative shoots throughout theseason,the
plants shouldbekeptinavegetative state,whichcanbe achieved athigh temperatures
inthelowlandsoftropical countries (Chapter2 ) .
74
5 Shoot formation of capitulum explants in vitro and
plantlet production by root formation of detached shoots
5.1 INTRODUCTION
Vegetative propagation can be achieved in several ways, for instance through the
formation of adventitious shoots. By definition such shoots do not arise from pre-exis-
ting (terminal or a x i l l a r y ) meristems, but originate, like adventitious roots (Section
3.3), from a more or less differentiated t i s s u e , which after dedifferentiation, gives
rise to the formation of a shoot meristem and a shoot primordium (Venverloo, 1973). The
development of a shoot primordium leads to the formation of an adventitious shoot and
when, ultimately, adventitious roots are i n i t i a t e d on the basal end of a detached shoot,
a complete p l a n t l e t i s obtained.
Adventitious shoots can emerge in vivo from different plant p a r t s , like roots,
stems and leaves. Broertjes et a l . (1968) l i s t e d over 350 species, reported in the
literature, which can be propagated through adventitious shoot formation on detached
leaves. However, attempts to produce adventitious shoots on detached leaves of
pyrethrum in vivo f a i l e d .
Although shoots do not regenerate in v i t r o so commonly as adventitious r o o t s , t h i s
Phenomenon has been reported in l i t e r a t u r e with various plant parts cultivated m v i t r o .
Since cultures in v i t r o are increasingly used for the rapid vegetative propagation of
n«ny plant species, i t seemed worthwhile to examine the regeneration ability of
Pyrethrum explants excised from various plant p a r t s .
I t has been shown (Chapter 3) that peduncle explants cultivated in v i t r o rapidly
Produced high numbers of adventitious roots at high percentages. Therefore the abi i y
°f this plant material to regenerate shoots was f i r s t examined. Even though shoots were.
V i t i a t e d on peduncle explants of Clone 4331, a satisfactory development of the shoot
i n i t i a l s was not r e a l i z e d . , , rlnupr
Thereafter attempts were made to propagate pyrethrum vegetatively through £low r
(head) explants cultivated in v i t r o , as has been described for s*™**™?*" ar
e , .„ rT„ D,, 0 lQd?1 Phlox drwmondir (Konar
-g. Nemeaia stvwnosa and Kalanchoe globulvfera (La Kue, i s ^ j , ,WHM
» Konar, 1966), Kalanchoe pinnata (Mohan Ram » Wadhi, 1966, 1968), ^ t T e t Z ^
( J ohri &Ganapathy, 1967), Chrysanthemummorifolium (Hill, 1968, * * * * * " Ja
R°est » Bokelmann, 1975), Eanunculus eoeleratus (Konar & " j a , .0rMdaMM
oleraceae ( P o w , 1 9 6 9 } ) Beta âris (Margara, 1970), Lvltm ^ ^ ' ^
ira
ssn~ i:;r:"^
« al.,1 9 7 5 ,Inprelimin?ryexperimentsitwas ^ - J ^
-?:r::r
™ ^ r f
obtained throughthecultureinvitroofsectionsofthecapituluml
Pyrethrum (Roest&Bokelmann, 1973).
75
Section5.3dealswithanatomicalobservationsontheorigin,initiationand
developmentoftheshoots.InSection5.4-theinfluenceofvariousfactorsinthis
processofshootformationisexamined.Theproductionofplantletsbyrootformation
ofdetachedshootsisdescribedinSection5.5andfinallyinSection5.6is
investigatedwhetherotherplantspeciescanbepropagatedvegetativelyinvitro.
5.2.1Material
Clone4331isoneofthemostimportantclonesinKenya,becauseofitsveryhigh
floweryields.Thereforethisclonewaschosenandstockplantsweregrowninthe
greenhouse.Inwinteratlowtemperaturesflowerheadswereinitiatedandinspringand
summer,witharisingtemperature,theinitiatedflowerheadsdevelopedrapidlyand
plantsstartedflowering (Section2.2).Capitulain
ayoungfloweringstage (stages10,
11and12,Fig.40)weredetached,eachwiththetopportionofthepeduncle (Fig. 37a).
a S% calciumhypochloritefilteredsolutionfor30min
Flowerheadsweresterilizedin
;>v
\ f
ifcte: ft
• •1flrf
• tM
cuftiv^dWttr °f P l 3 n t l e t P r o d u c t i ° » fron, a capitulun, explant of Clone 433.

a. Capitulum inayoung flowering stage.
c!Sorâinhr 1 ^ T 1 ^ * JUSt bef ° re Nation.
5.eZ Tflll tZtltlH °ne t a U Sh
°0t l n i t i a t e d and d
^ ° P e d onacapitulun, explant,
d-.Plantlet production by r o o t N a t i o n ofadetached shoot, 3weeks after transfer to
76
and subsequently rinsed several times with s t e r i l i z e d tap water for 30 min. In an
inoculation room under a stereomicroscope, -the capitulum was divided aseptically and
as accurately as possible into 2 equal sections. In each section the upper bracts of the
involucrum were almost completely cut off and the florets were excised above the bottom
half of the ovary (Fig. 37b). Finally, the explants were placed in a v e r t i c a l position,
with the portion of the, peduncle in the medium, and distributed systematically and in a
balanced way over the various treatments.
5.2.2 Methods
Thebasicculturemediumwascomposedofpyrex-distilledwater, 'Difco'Bacto-agar
0.61,Knop'smajorandHeller'sminorsalts (bothathalfstrength),sucrose0.5%and
6-benzylaminopurine (BA)at10~ 6 g/ml.ThepHofthemediumwasadjustedto5.8before
autoclaving.Pertreatment20explantswereexposedinagrowthchambertoaphotoperiod
of14h(fluorescent tubes (PhilipsTLMF 140W/33RS)supplementedwithincandescent
light(Philinea))andatemperatureof18°duringthedayand14°Cduringthenight.
Theformationofthefirstshootswasobservedabout3weeksafterincubation.The
developmentoftheshootsvariedwiththetreatmentandwasalmostcomplete1J-3months
afterincubation (Section 5.3). Inthisfinalstagetheexperimentswereterminatedand
thepercentageofshoots formedwasmeasuredover20explantspertreatment.Mostlythe
meannumberofshootspershoot-formingexplantwasalsocalculatedandthelengthof
theseshootswasmeasured.Accordingtolength,shootsweredividedintosmallshoots
(«0.2cm), thenumberifwhichwasestimated,intermediateshoots (0.2-0.6 cm),which
werecountedratherprecisely,andtallshoots (>0.6cm),whichnumbercouldbecounted
veryexactly.
Foranatomicalobservations (Section5.3)capitulumexplantsofClone4331were
incubatedinvitrounderthestandardconditionsdescribedinthissection,whichare
suitableforshootdevelopment.Theplantmaterialwasfixated0,3,6,9,12,14, 16,
19or23dafter incubation.Foradetailed'descriptionhowtheplantmaterialwas
preparedfurther,seeSection3.2,wheremore informationisalsopresentedaboutthe
sterile-culturetechniques.
5.3ANATOMICALOBSERVATIONS
Figs 38a-cshowthemorphologyofaninflorescence,arayandadiscfloret.The
capitulumisborneonthepeduncleandbuiltupasfollows:ontopofaslightlycon
receptacle,sheathedbygreeninvolucralbracts,whitepetalledrayfloretsare
situatedonthemarginandyellowdiscfloretsoccupythecentre.The «*™»*"*
floretshaveastrap-shapedcorolla,thebaseofthecorollaistubularandenvelopesa
bi-lobedcylindricalstyle. T,e styleisemplantedinthecentreofthefloreton P f
theinferiorovary.Asmallgreen,irregularlyshapedcalyxisattachedtoa p h o n a l
ovary.Thebisexualdiscfloretshaveatubularyellowcorollaandfivestamensin
additiontothestyle. . t h eb o t t o m
Thecapitulumexplantswerepreparedbycuttingofftheflorets
77
B C
"c)S(Br:;:;,T"968):Ph0l°8y° f'"i n f l o r e s c - " <*>>*rayfloret (b)andadiscfloret
ca: calyx pollen
co: corolla po: st: stalk
d: discfloret r: ray floret . sta: stamens
inv: involucre re: rest anther sti: stigma
ov: ovary rec: receptacle sty: style
half of the ovary (Fig. 39a). Microscopical observations showed the f i r s t c e l l

divisions in the epidennal cell layer of the bottom halves of the ovaries of disc
f l o r e t s , 6 d after incubation (Fig. 39b). An i n i t i a t e d shoot meristem was observed
after an incubation for 9 d (Fig. 39c) and a shoot primordium was i n i t i a t e d 15 d after
R a t i o n (Fig. 39d). Most shoots originated adventitiously from the epidennal c e l l
1 6 S fOT
I mtime V T ' " ™ ^ ^ « * * * CAsahira et a l . , „ 7 S ) . In pyrethrum
W6re n t i C e d emerging a l r e C U y frOT the
mr " 1 J ° * « * " * e and occasionally
0bSerV6d aXU f inV 1U al b
ritlT? ^
the shoots may be of an axillary * ^
origin. ° » ° - — . Therefore someof
A variable number of shoots fi-^n ,,-;+i,
snoots (1 50, with an average of about 25 per section)
78
<"->:~-S^rf*J?&If&f--•''
0*\* w-s **•*v* »** *-*^jy**v i»â-*w*>i /it.
i^W^vf*^*"X-••?»
«^«^^?'^
Figs39a-d.
a.Receptaclebearingthebottomhalvesoftheovariesofdiscflorets,justbefore
" t u X i s i o n intheepidermalcelllayeroftheovaryofadiscfloret (arrow),6d

afterincubation.
c-Initiated shootmeristem (arrow),9dafterincubation.
d.Initiated shootprimordium (arrow), 15dafterincubation.
79
developedafteranincubationfor3weeks.Duringfurtherincubationafew (tall)shoots
elongatedtoalengthofatleast0.6 cm(Fig.37c);themajorityoftheshoots,however,
attainedalengthofonlyafewmillimetersorless (intermediateandsmallshoots).The
developmentoftheshootswasalmostcompleteafteranincubationperiodfor1J-3months.
5.4FACTORSINFLUENCINGSHOOTFORMATION
Thefactorsinvolvedintheprocessofshootformationcanbedistinguishedinto
factorswhichareassociatedwiththe(ex)plant,thenutritional/hormonal composition
ofthemediumandtheclimaticconditions.Unlessotherwisestated,thestandardgrowth
conditions(Section5.2)havebeenmaintainedinallexperiments.
5.4.1 Plant factors
5.4.1.1Genotype
TheformationofshootswasinvestigatedwithcapitulumexplantsofClones1087,
4331andMa63/1889.Boththepercentageshootformationandthetotalnumberof
initiatedshootsweresimilarforeachofthethre _iones(Table41).Mostinitiated
shootsdidnotdevelopandremainedsmall,dueundoubtedlytocompetitionamong
developingshoots.OncapitulumexplantsofClones1087and4331highernumbersof
intermediateandtallshootsdevelopedthanoncapitulumexplantsofCloneMa63/1889.
CapitulumexplantsofClone4331werechosenforstandardexperiments.
Table41.ShootformationofcapitulumexplantsofClones 1087,4331andMa 63/1889,12

weeksafterincubation.
Clone Percentage Numberofshootspershoot-forming explant

shoot
formation tall intermediate small total
1087 95 1.3 3.3 15.6 20.2
4331 100 1.0 2.3 18.3 21.6
Ma63/1889 100 0.0 0.2 18.3 18.5
;f^.:.%ssn,i1r;^,:inss r i ^ f t o t a -°~—'*-
Flowering Percentage Numbero fshootsper shoot-•forming exp lant
stage shoot
0 0 0 0 0
5 0 0
0 0 0
8 40 0
10 0.8 4.5 0.8
50 6.1
12 1.2 5.0 4.3'
90 10.5
13 1.5 6.2 4.3 12.0
85 3.5 6.8 2.3 12.6
80
»• I 4 M W I U •.9V?•**"•?
a
I -ft
•'8"l l' 9 'f ;"o"f

•J™ -
iu\
r
l'~l I
fig-40.CapitulaofClone 4331 invarious floweringstages.
5
-4.1.2Flowering stage »
Sectionsofcapitulainvarious flowering stages (Fig.40)wereusedtodetermine

themostsuitable stageforshoot formation.Explantsofflowerheadsintheoldest
developmental stagesdidnotproduce shootsatall(Table42;stages0and5).Asthe
ageoftheflowerheaddecreased,shootformation increasedandexplants fromcapitula
intheyoungest flowering stagesyieldedthebestinitiationanddevelopmentof
adventitious shoots.Instandardexperiments sectionswereexcisedfromflowerheadsin
stages 10-12.
Thesignificanceofayoungdevelopmentalstageoftheexplantonshootformation
"asalsofoundbyMohanRam&Wadhi (1966,1968)whoreportedthatyoung flowerbudsof
Kalanchoe pinnata developed shootsmorerapidlyandatahigherpercentagethanthose
ofanolderdevelopmental stage,andbyBajaj (1972)whonoticedabettershoot
formationonyoung excised leavesofTorenia foumievi ascomparedwitholderleaves.
IncontrastPieriketal. (1975)foundnodifferencesinshootdevelopmentbetweenwide
openandolder flowerheadsof Gerbera janesonii, whileclosedflowerheadsandvery
youngflowerheadswere inferior.Foramoredetaileddiscussionabouttheinfluenceo
thedevelopmental stageonorganogenesisseeSection3.4.1.2.
Table43.Theinfluenceofvariousexplantsizesonshootformationofcapitulum
explantsofClone4331,8weeksafterincubation.
Sectionspercapitulum Percentageshootformation
2 88
3 56
4 44
5 19
6 25
5.4.1.3Explantsize
Asintactflowerheadsyieldedonlyaverylimitednumberofshoots,flowerheads
weresectionedinto2,3,4,5or6segments.Pertreatment16explantswereused
to
determinetheexplantsizethatwouldresultinthebestinitiationanddevelopment
of
shoots.
Table43showsthatthepercentageshootformationincreased.withanincreasing
explantsize.However,whenthepercentageshootformationwascalculatedpercapitulum,
itbecameclearthatitdoesnotmakemuchdifferencewhetherthecapitulumissectioned
into2,3,4,5or6segments.AlthoughitisnotillustratedinTable43,itwasfound
thatsectioningofthecapitulainto2segmentsresultedinthebestdevelopmentofthe
initiatedshoots,andthispreparationmethodwasalsochoseninstandardexperiments.
|Inconformitywithpyrethrumitwasfoundthatthenumberofregeneratedbulblets
onbulbscalesegmentsof Hyaointhua oriental-is (Pierik&Woets,1971;Pierik&Ruibing,
1973;Pierik&Post,1975)increasedalmostlinearlybyincreasingthelengthorwidth
oft#ieexplants,whilealsothedevelopmentofthebulbletswaspromotedwith
an
increasingexplantsize.Anoptimumdevelopmentofshootsoncapitulaof Gerbera
iamdsonU (Pieriketal.,1973,1975)wasachievedafterdivisioninto4or6explants;
shootdevelopmentdecreasedwhenthecapitulawerecutinto8,10or12explants.With
pedicelexplantsof Chrysanthemum mortfoUum (Roest&Bokelmann,1975)afavourable
effectofanincreasingexplantlengthonshootformationwasobserved.
Probablyexplantsderivedaftersectioningtheflowerheadinto2segmentsyielda
bettershootformationthansmallerexplantsbecausethetotalamountofendogenous
nutritional/hormonalsubstancesislargerwithinthebiggerexplants.Flowerheadsthat
werenotsectioned,however,regeneratedasmallernumierofshootsthanexplantsthat
were!obtainedaftersectioningthecapituluminto2segments.Thisresultmay
be
attributedtotheadditionalwoundcutthatismadebysectioningthecapitulum.Such
a
woundmayassureabetteraerationoruptakeofexogenousappliednutritional/hormonal
substancesfromthemedium(Section3.4.1.5)andsostimulateshootregeneration.
5.4.1.4Wounding
when W e T e T 1
^ 6XPeriment
* W
^ ^ ^ Sh
° 0 t *>™*ian did not occur
JUSt S e C t i n e d S6Cti0nS
« rT ^ ° "* ^ « » A b a t e d without further
wounding. To detenmne the effect of additional wounding onshoot formation, various
82
woundingprocedureswerecompared.Thefollowingpartsoftheflowerhead(Figs38a-c)
werecutoff:
1=Thefloretsabovethecalyx.
2=Thefloretsabovetheovary.
3=Thefloretsabovethebottomhalfoftheovary (Fig.39a).
4=Theentirefloretsandtheuppercelllayersofthereceptacle.
Shootformationdidnotoccurwhentheovarieswerenotwoundedatall(Treatments1
and2)orwhentheovarieswerecompletelyexcised (Treatment4),whereas401ofthe
flowerheadsproducedshootswhentheovariesofthefloretswerewoundedaccordingto
thestandardpreparationprocedure (Treatment 3).Theimportantroleoftheovariesin
theprocessofshootformationwasfullyconfirmedbyanatomicalobservations (Section
5.3).
Astimulatingeffectofwoundingonshootformationwasalsoreportedforleafex-
plantsof Dendrophthoe faleata (Nag&Johri,1970)andof Tovenia fournieri (Bajaj,1972).
Theeffectofwoundingontheregenerationofrootsinvitrowasalreadydiscussedin
Section3.4.1.5.
5.4.2.1Minerals
Theinfluenceofmineralnutritionwasexaminedinanexperimentwithvarious
combinationsoftheminorsaltsofHellerandthemajorsaltsofKnop(bothathalf
strength)intheculturemedium.
AscanbeobservedinTable44additionofmineralstotheculturemediumisnot
necessaryforshootinitiation.WiththeminorsaltsaccordingtoHellerthe
developmentoftheshootwasnotstimulated,buttheadditionofthemajorsalts
accordingtoKnopseemedtobeimportantforshootdevelopment.Instandardexperiments
* emajorsaltsofKnopandtheminorsaltsofHeller(bothathalfstrength)wereadded
totheculturemedium.
Withtheexceptionofcallustissue.of AnthuHum andreanu* (Pierik,1976)where
a
%^z thighconcentrationshadfoundtorepressandatlowconcentrationstobe
... s a l t s and Knop's major

Tabl
•able44.Theinfluenceofvariouscombinationsoff^ " ' ^ m l g " weeks a f t e r i n c u b a t i o n .
s!altsonshootformationofcapitulumexplantsofCloneOJJ ,
Numberofshootspershoot-formingexplant
Percentage
shoot intermediate small total
formation tall
16.4 17.0
0.0 0.6
devoid of m i n e r a l s 95 16.4
0.2 16.2
He
l l e r minor \ 85 0.0
16.9 18.8
Kn 0.4 1.5
°P major { 85 19.1
2.7 16.1
He
llerminor k 95 0.3
+K
nopmajor \
83
promotiveforshootformation,saltscontainingN0 3orNH 4 ionshavebeenreportedto
stimulateshootformation,e.g.stemsegmentsoftobacco (Miller&Skoog,1953),leaf
discsof Cardamine pratensis (Paulet&Nitsch,1959),peduncleexplantsof Brassica
oleraceae andpedicelsegmentsof Chrysanthemum morifolivm
(Margara,1969) (Roest&
Bokelmann,1975).
Theimportantroleofnitrogeninrootregenerationwasalreadypointedoutin
Section3.4.2.2andwhethernitrogenisalsoinvolvedinshootformationofpyrethrum
shouldbeinvestigated.
5.4.2.2Sugars
'Toexaminetheeffectofsugaronshootformation,sucrosewasaddedatvarious
concentrationstotheculturemedium.Intheabsenceofsucroseshootswerenotformed
(Table45)andexplantsbecamenecroticanddiedoff.Intherangeof0.5-2.5%sucrose
alltreatmentsyieldedthemaximumpercentageshootformation,butthetotalnumberand
lengthoftheshootsdiminishedasthesucroseconcentrationincreased.Inthe
concentrationrangetestedoptimumresultswereobtainedaftertheadditionof0.5'»
sucrosetothemedium (Fig. 41).
Althoughvegetativeshootswereusuallyproduced,thedevelopmentof'floral
primordial,whichlooklike'discflorets',occurred6-7weeksafterincubation.Atthe
endoftheexperimentthenumberoftheseprimordiawaspositivelycorrelatedwiththe
sucroseconcentration.These 'discflorets'with5-10stamensandanequalnumberof
Table45.Theinfluenceofvariousconcentrations (0-2.5%)ofsucroseonshootformation
of"capitulumexplantsofClone4331,10weeksafterincubation.
Sucrose% Percentage Number per shoot-formingexplantof

shoot
formation vegetative shoots discflorets
tall intermediatesmall total
0.0 0 0 0 0 0 0
0.5 100 6.6 11.6 10.3 28.5 0.0
1.0 100 3.1 3.9 21.1 28.1 0.1
1.5 100 1.1 1.6 22.6 25.3 1.3
2.0 100 0.4 0.8 26.7 27.9 3.6
2.5 100 0.4 1.4 20.0 21.8 4.4
c nce at
!apituLTexpiantsenferif ™i™* ° ^ ^^ ofsucroseonshootformationof
i n C u b a t i
i X uT, M J S ? ' " ^ ^'^ -F r
- lefttoright:0.5,
84
P^.AIlM^WJJIi^m^
-N
Fig.42.'Discflorets'initiatedand
developedonacapitulumexplantof
Clone4331at2.5%sucrose,10weeks
afterincubation.
_ .-•„„= Cn5-2 5%)ofsucroseandglucoseon

Table46.Theinfluenceofvarious concentrations 0.5 i.>£ incubation,
shootformationofcapitulumexplantsofClone4331,8weeksalter __
Sugar X Percentage Number p e r shoot-forming e x p l a n t of

shoot • • discflorets
formation vegetativeshoots
intermediate small total
tall
16.3 30.1 0.0
Sucrose0.5 100 4.4 9.4 23.8 1.8
0.9 22.5
1.5 100 0.4 19.6 21.8 3.9
2.5 100 0.0 2.2
29.4 0.0
9.4 15.0 1.6
Glucose0.5 100 5.0 24.2 25.0
1.5 100 0.1 0.7 22.9 2.7
0.8 22.1
2.5 95 0.0
corollateeth,canbeobservedinmoredetailinFig.42. concentrations.
Inasecondexperimentsucroseandglucosewere« * » * " B o t hs u g a r s ,
Table46showsthattheinfluenceofthetwosugarsdidnot £ormti n.
o
atallconcentrationstested,yieldedalmostmaximumpercentages o f *o^ ^ ^
Sucroseandglucoseat0.5%favouredthedevelopmentofahign^ ^ ^ ^ £ o n n a tion
vegetativeshoots.Moreoveralowsugarconcentrationis ^ " ^ b e^ f o r a
of 'discflorets'isprevented.Unlikevegetativeshoots,they
directvegetativepropagation. p s s e n tial roleofsugarinshootorgano-
Therearenumerouspublicationsontheessei ^ ^ ^ ^ optifflumf orshoot

genesis.Mostlyahighsugarconcentration^ ^ ^ ^ t 0b e o p t i ] M „forshoot
formation.However,lowsugarconcentrationsn eXplantsof Gerbera jamesonii

formationinpyrethrumandshootdevelopmentofcapitulum xp
(Pieriketal., 1973,1975).
85
Athighsugarconcentrationsfloralprimordiawereformedinpyrethrumexplants.
Suchadetermininginfluenceofthesugarlevelontheorientationofthebudstowards
thevegetativeorthefloweringstatewasalsoreportedforstemsegmentsof Niootiana
tabasum (Chouard&Aghion,1961;Aghion-Prat,1965),petioleorroottissue (Paulet&
Nitsch,1964)andpeduncleexplants (Margara,1965)of Cichorium intybus, stemsegments
of Plumbago indioa (Nitsch&Nitsch,1967ab)andpeduncleexplantsof Lunaria annua
(Pierik,1967).
5.4.2.3Auxin
Inpreliminaryexperimentsitwasobservedthatshootformationdidnotoccurwhen
onlyIAA,atvariousconcentrations,wasaddedtothemedium.
ThereforetheinfluenceofvariousIAAconcentrationswasinvestigatedinthepresence
ofthecytokininBAat10 g/mlinthemedium.
IncombinationwithBA,allconcentrationsofIAAinhibitedshootformation(Table
47),thisinhibitionbeingmorepronouncedathigherIAAconcentrations.
Sometimeswithoutcytokinininthemedium,exogenouslyappliedauxinstimulated
shootformation,ashasbeenreportedforflowerbudsof Kalanchoe pinnata (MohanRam&
Wadhi,1968),leafdiscsof Streptocarpus (Appelgren&Heide,1972)andbulbscale
segmentsof Hyaointhus orientalis (Pierik&Steegmans,1975b).
Ingeneralitcanbestatedthatthecombinationofalowauxinconcentrationwith
ahighcytokininconcentrationisfavourableforshootformationandthatahighauxin
concentrationcombinedwithalowcytokininconcentrationhasadetrimentaleffecton
shootformation,whichforinstancewasfoundfortobaccotissue (Miller&Skoog,1953;
Skoog&Miller,1957),leafexplantsof Ciahorium intybus (Toponi,1963;Bouriquet&
Vasseur,1966),stemsegmentsof Plumbago indica (Nitsch&Nitsch,1967ab),leafdiscs
of Begonia (Ringe&Nitsch,1968),endospermculturesof Sourrula pulverulenta (Bhojwani
&Johri,1970),excisedleavesof Atropa belladonna (Zenkteler,J971),pedicelexplants
of Chrysanthemum morifolium (Roest&Bokelmann,1975)andrachisexplantsof Solanum
tuberosum (Roest&Bokelmann,1976).
Table47.Theinfluenceofvariousconcentrations (0-10-4g/ml)0 fIAAonshootformation

ofcapitulumexplantsofClone4331,6weeksafterincubation.
gMl ^hoot111386 N
"êr °fshootspershoot-formingexplant
formation tall intermediate sma ll total
l 2K0
»0~I 60 00 ?'? V°
8 3 I0
10"! 25 06 A 2
- 3
-°
I0"f 10 00 n'n -° -°
10-4 5
J:! 0°:0° •»:» j:;
86
Table 48. The i n f l u e n c e of v a r i o u s c o n c e n t r a t i o n s ( 0 - 1 0 _ 4 g/ml) of BA on shoot formation
of capitulum e x p l a n t s of Clone 4 3 3 1 , 6j weeks a f t e r i n c u b a t i o n .
BA Percentage Number o f shoots per shoot-•forming explant

g/ml shoot
intermediate small total
formation tall
0„ 0 0 0
0 0
l(f8 0 0 0 0 0
10" 75 0.0 1.3 3.7 5.0
10" 4.0 12.0 17.0
I0 90 1.0
1
10"4
90
80
0.1
0.1
18.0
13.5
23.6
17.8
41.7
31.4
5-4.2.4Cytokinin
Thecytokinin BAwasadded atvarious concentrations tothemediumtodetermine its

effectonshoot formation.Table 48illustrates thatBAwasrequired forshoot formation.
Foranadequate shootproduction BAconcentrations of 10" 8 and10" g/mlweretoolow
^ d suboptimal,respectively. High numbersofshootswere initiatedanddevelopedatBA
10~6, 10" 5 and 10" 4 g/ml.Although atBA10" 6 thehighestnumberoftallshootswas
âsured,BAat 10" 5 g/mlhastobepreferred,becauseatthisconcentrationaveryhigh
nunfcerofintermediate shootshasdeveloped. Instandard experimentsBAwasappliedat
,„-6 .
g/ml to
g/mi tothe
theculture
culture medium.
medium.
Thefinding inpyrethrumthat theadditionofcytokininisessentialforshoot
formation,hasalsobeen reported formanyplant species cultivated m vitroe.g.tor
tobaccotissue (Miller&Skoog, 1953; Skoog »Miller, 1957),stemsegmentsof « * * * '
indica (Nitsch &Nitsch, 1967ab), peduncle explants of B r a s s * * oleraceae (Margara, ,,
excised leaves of Dendropthoe f a l c a t a (Nag&Johri, 1970),pedicelexplantsoi
<**U*<mfhmm mortfolivm (Roest&Bokelmann,1975)andrachisexplantsof S o l a n s
tuberosum (Roest&Bokelmann, 1976). .
As already pointed outinSection 5.4.2.3shootfonnationisoftenoptimumwhenan
a
« i n isadded simultaneously withacytokinin totheculturemedium.
5-4.2.5Gibberellic acid
Inprevious experiments itwasnoticed that althoughahighnumberof ^ ^

ûally initiated, thedevelopment oftheshoot initials isg e n e r a J ^ J ^ ^
R o w i n g twoexperiments,with 10explants^ " ^ enhancethe d e v e l o p
theadditionofGA 3 ,inthepresenceofBAat1U g/""-.
°ftheinitiatedshoots. n 1 i e d atvariousconcentrationsto
InthefirstexperimentautoclavedGA3wasappn influencedbyGA,
^ e culturemedium.Thepercentageof^ . f ^ 1 " , n^ development^
(Table 49).Ataconcentration ofGA3at10 ^ ^ ^ shootd e v e i 0 F m e n twas
*oots were suppressed. Incomparisonwiththeother treatments
s t r i a t e d by ^ at 10" 5 g/ml. ^ ^ t0^ culture
Inthesecond experiment autoclaved UVjat w &
Table49.Theinfluenceofvariousconcentrations (0-10 g/ml)ofGA,onshootformation
ofcapitulumexplantsofClone4331,7weeksafterincubation.
GA3 Percentage Numberof shootsper shoot-formingex] plant

g/ml shoot
90 2.4 7.5 10.0 19.9

10 100 3.3 7.9 9.3 20.5
10" 100 4.0 12.5 2.5 19.0
10'-4 80 0.0 1.1 8.3 9.4
Table50.TheinfluenceofGA.at10 g/mlappliedatvariousperiods (0-4Jweeksafter

incubation)onshootformationofcapitulumexplantsofClone4331,7weeksafter
incubation.
Application Percentage Numberofshootspershoot-forming explant

time shoot
(weeksafter formation tall intermediate small total
incubation)
0 100 4.0 12.5 2.5 19.0

11 90 3.8 11.9 10.9 26.6
3 90 3.3 9.4 10.6 23.3
4J 70 1.2 12.5 12.5 26.2
medium0,1i,3or41weeksafterincubation,todetermineatwhichtimeafter
incubation,additionofGAjwouldresultinthebestdevelopmentoftheinitiatedshoots.
Alltreatmentsshowedmoreorlessthesamenumberofinitiatedshoots (Table50).
AdditionofGAj4Jweeksafterincubation,however,hadanunfavourableeffectonthe
percentageshootformationandthedevelopmentoftheinitiatedshootsincomparison
withearlierapplications.Thoughdifferencesweresmallbetweenthetreatmentswith
additionsofGAj0,1Jor3weeksafterincubation,thedevelopmentoftheshootswas
stimulatedwhenGAjwasappliedduringthefirstweeksafterincubation.
Theinhibitingeffectofgibberellicacid,especiallyathighconcentrations,was
alsofoundforshootregenerationonleafdiscsof Begonia (Schraudolf&Reinert,1959),
stemsegmentsoftobacco(Aghion-Prat,1965),leaffragmentsof Ciahorium intybus
(Bouriquet&Vasseur,1966),peduncleexplantsof Lunarta annua (Pierik,1967),stem
segmentsof Plumbago indioa (Nitsch&Nitsch,1967ab),leafdiscsof Streptoearpus
(Appelgren&Heide,1972)andbulbscalesegmentsof Hyaainthus orientalis (Pierik&
Steegmans,1975b).
Gibberellicacid,mostlyatlowconcentrations,hasalsobeenreportedtobeoptimum
forshootformationonleafdiscsof Cardamine pratensis (Paulet&Nitsch,1959)and
CichoHm intybus (Bouriquet&Vasseur,1966),pedunclesegmentsof Brassioa oleraceae
(Margara,1969)andrachisexplantsof Solanum tuberosum (Roest&Bokelmann,1976).
58
Table51.Theinfluenceofconstant temperatures (9-25C)onshootformationofcapitulum
explantsofClone 4331,6weeks afterincubation.
Temperature Percentage Number of shoots per shoot-•forming exp lant

C shoot
9 80 0.0 0.1 6.9 7.0

13 95 0.1 4.4 17.1 21.6
17 95 0.7 3.8 17.9 22.4
0.6 4.3 8.8 13.7
21 80 28.2
25 85 0.6 3.3 24.3
5.4.3 Climatic faators
5.4.3.1Temperature
Incontinuous lightinthephytotron,theeffectofvariousconstanttemperatures
onshootformationwasexamined.Alltreatmentsshowedaboutthesamepercentageshoot
formation (Table 51).Thetotalnumberofinitiatedshootsaswellasthedevelopment
oftheseshootswassuboptimalat9°C.Presumably,thelowtotalnumberofshoots
initiatedat21°Cwasduetosomeexperimentalerror.Thisalsoresultedinalownumber
ofsmallshoots,unsuitableforplantletproduction.Thenumbersofintermediateand
tallshoots,whichcanbeusedforvegetativepropagation,wereaboutthesameat13,
17,21and25°C.Apparently,almost independentofthenumberofinitiatedshoots,a
certainnumberofshootsdeveloped,whichcanbeascribedtocompetitionamong
developingshoots.Becausetherewasadecreaseinthenumberoftallshootsat13 L, a
temperatureintherange 17-25°Cisbestforanoptimumdevelopmentofinitiatedshoots.
Mostlyahightemperaturehasbeenreportedtobeoptimumforshoot f ° ™ t l 0 I \
Thismaybeduetoafastdevelopmentofshootsatahightemperature,ashasbeenfound
forleafdiscsof Streptooarpus (Appelgren&Heide,1972),bulbscalesegmentsof
Uyaeinthus orientalis CPierik&Ruibing,1973)andcapitulumexplantsof Gerbera
iamesonii (Pieriketal., 1975). ..t.ti_,n n
Alowtemperature,however,wasdescribedtobepromotiveforshootinitiation
leafdiscsof Streptooarpus (Appelgren*Heide,1972)andbulbscalesegments
Uyaeinthus orientalis (Pierik&Ruibing, 1973).
5.4.3.2Light
inpreliminaiyexperimentsaperiodofcontinuouslightju,tafter * ^ ™
unfavourableforl o fformation.Tnustheinfluenceofvariousperiodsoflightand
darknesswasexaminedat21°Cinthephytotron. formationas
* — inT«e*an— . - * - * ' ^ . " I shoot
»=Uo» te «e,u,!to,.!— . ofm « - 27ZX the— - -
•Mti,!,depended.„thetreatment;a*£*«££ „ „jouncedasthe
experimentpromoted shootdevelopment.Thispromoziui
Table52.Theinfluenceofvariousperiodsoflightanddarknessonshootformationof
capitulumexplantsofClone4331,4weeksafterincubation.
Percentage Number of shoots persho ot-formingexp lant

shoot
4weekslight 90 0.3 1.7 18.0 20.0

1week darkness+ ) 85 0.4 3.3 20.7 24.4
3weekslight.
2weeksdarkness+
2weekslight ) 90 0.4 5.6 17.0 23.0
4weeksdarkness 95 0.4 8.7 15.0 24.1
durationofthedarknessperiodincreasedandthebestshootdevelopmentwasobserved
after4weeksofdarkness.Whenanetiolatedpaleyellowcolouredshoot,resultingfrom
incubationindarkness,wasexposedtolight,itturnedgreenwithinafewdays
(Fig.43).
Theinfluenceoflightanddarknessonshootformationgreatlydifferedfrom
speciestospecies.Bygrowingexplantsfirstindarknessandsubsequently inlightthe
formationafadventitiousshootswasalsopromoted inleafexplantsof Ciahorium intybus
(Legrand,1972)andflowerbudsof Freesia (Pierik&Steegmans,1975a),whileaperiod
ofdarknessalsoenhancedshootdevelopmentoncapitulumexplantsof Gerbera jamesonii
(Pieriketal.,1973,1975).Adversely,animmediateexposuretolightwasfavourable
forshootformationonstemsegmentsof Plumbago indiaa (Nitsch&Nitsch,1967ab),
peduncleexplantsof Brassica oleraeeae (Margara,1969)andincallustissueof
Anthurium andreanum (Pieriketal.,1974).Finally,noeffectoflightordarknesswas
.A-
x«b£$£»£.
t ••'*•'*
^»».«
*M
jP» . •'-:'L=.V-
•7,, '-•- Fig.43.Shootformationofacapitulumex-
1
•:«8^.-» «---" K * plantofClone4331,9weeksafterincuba-
v/ tion.Theexplantwasexposed todarkness
for8Jweeks,afterwhichperiod theetio-
latedshootturnedgreenwithinafewdays
m light.
90
found onshoot regeneration of bulb scale segments of Hyacinthus orientalis (Pierik &
Ruibing, 1973).
5.5 PLANTLET PRODUCTION BY ROOT FORMATION OF DETACHED SHOOTS
Attheterminationoftheexperiments 1J-3monthsaftertheincubationofthe
capitulumexplants,tallshootswithalengthofatleast0.6cm(Fig.37c),were
detachedfromtheexplant.Thedetached shootsweresubculturedinvitroonamedium
containing auxinforroot formationandsubsequentplantletproduction.However,
adventitious root formationwasnever observed.
A goodrootingwas achieved aftertreating thebasalendoftheexcisedshootwith
U IAA(ontalcbasis)andsubsequently transferring theshoottoanunstenlizedsoil
mixtureofleafmouldandsand.Bymaintaininga.relativehumidityofapproximately
100%,adventitious rootswere initiatedatthebasalendoftheshoot,within3weeks
aftertransfertosoil (Fig.37d).Thusplantletswereobtainedfrommostshoots,about
3monthsaftertheincubationofthecapitulumexplantsinvitro.
Furthercultivationoftheplantletsinagrowthchamberand,subsequently,
outsideonthe field,yieldeduniformplantsthatlooked identicaltothestockplants
andstartedtoflower abouthalfayearaftertheincubationofthecapitulumsections
invitro.
5.6 VEGETATIVE PROPAGATION OF OTHER PLANT SPECIES
In t h i s section i ti s investigated whether other Composite can be propagated

vegetatively according t othe same procedure as described for pyrethrum. ^
capitula were sectioned into 2segments, additionally wounded by excising the flo e
above thebottom half oftheovary andthe explants were incubated ona cuUur «*m
in thepresence of p y r e x - d i s t i l l e d water, • « * • , Bacto-agar 0 , ôp nând
Heller's minor s a l t s (both a t half strength), sucrose HandBAat lu g/
exposure to 20°C anda daylength of14h shoot development was alsc, observedon
• , fn i(J 44a), Calendula officinalis L . ,
capitulum explants of Anthems arvensis L. (Hg- U chrysanthemum
Chrysanthemum leucanthemum L. (Fig.44b), Chrysanthen^ monfoUum Ram hry
i,i u 0 f M rhrusanthemum segetum L., i,eii«««
Parthenivm Bernh., Chrysanthemum roseum Web. e t N., u Mc) Hupochaeris
•, T MiSracivm umbellatum L. (Hg- **<-)> "Vf
Oamesonii Hook., Helenium autwmale L . , Hieracivm w ^ Taraxaom officinale Web.
radieata L . , Leontodon autumnali's L . , Matricaria marituna •» ^ Rudbeakia.
(Fig. 44d), Tagetes patula L. andof unidentified species o ^ ^ ^ . ^ ^
Accordingto the s ^ ^ ™ ^ X T ^ ^ ° *"«~ «*' "
lowerexplantsofHypericum V^fora^L ^ RaWncuUceae, respectively.
Zucc, which belong tothe families ofthe Hypencace unsterilized soil

Atransfer of isolated shoots ofsomeof^ ' f ^ Z Z s roots ^
fixture ofleaf mould andsand, lead to ^ ^ e m u m leucanthemumL . , Chrysanthemum
consequently, p l a n t l e t s were produced of Chrysa ^ ^ ^ ^ ^ ^ ^
morifoUum Ram., Chrysanthemum parthenium Bernn., ^ ^ ^
Perforatum L . , Matricaria maritima L., Gazanea sp.
91
o
•A v-'r. '
A» »; /-*/ ' •A
v'4
IWHHUIJ»JUPijiMMiijimMw.
m
\
*t
f
/ /
fn8:it4r4r Sh
° 0 t — ^ - n t onHowerCnead)explants o f
otherplant species cultivated
a.Anthemis arvensis L.
f'^ a r ^ « ^ offi0imie Web.
c Hzdraeium umbellatumL. e. Hypericum perforatum L.
92
Because i t was not examined anatomically whether shoots developed adventitiously
or axillarily on explants of the plant species mentioned before, t h e i r origin remains
obscure.
5.7 DISCUSSION AND CONCLUSIONS
Anatomical observations (Section 5.3) have shown that almost a l l shoots developing
oncapitulum explants of pyrethrum do not a r i s e from pre-existing shoot i n i t i a l s , but
that they emerge from shoot meristems formed adventitiously in the epidermal c e l l layer
of the ovaries of f l o r e t s . Several successive stages had to be passed before an
adventitious shoot has been produced, such as the i n i t i a t i o n of a shoot meristem and
the subsequent development (elongation) of the i n i t i a t e d shoot.
The experimental r e s u l t s (Section 5.4) have demonstrated that many factors are
involved in shoot organogenesis; the optimum conditions for,the separate stages in the
process of adventitious shoot formation were different.
Since a l l three clones t e s t e d , namely 1087, 4331 and Ma 63/1889, i n i t i a t e d and
developed adventitious shoots, shoot formation on capitulum explants of pyrethrum
seems to be a rather common phenomenon. However, i t has to be emphasized that despite
the i n i t i a t i o n of high numbers,of shoots (on average approximately 25), the development
of the small shoot i n i t i a l s into intermediate and t a l l shoots (which can be used for
vegetative propagation) was commonly found to be too variable and too sporadic (a few
shoots per explant). . ,
It has already been shown t h a t the development of i n i t i a t e d shoots can be stimulated
by the addition of GA_ a t 10 - 5 g/ml to the medium (Section 5.4.2.5). Nevertheless,
additional research has to be concentrated on factors which can effect a better develop-
ment of the shoots.
Low sugar concentrations were optimum for shoot formation in pyrethrum (Sectio^
S
-4-2.2) and shoot development on capitulum explants of Gerbera jcmesomi ^
" - . 1973. 1,75). in both p l . t species an o p t i m ^ o w sugar ™ ^ j £ £ ~
^ t h a stimulating effect of a period of darkness during the f i r s t weexs
(Section 5 . 4 . 3 . 2 ) . The coincidence of the inhibiting effect of a high sugar
concentration and of a period of l i g h t during the f i r s t weeks of incubation, may
to the synthesis of a supra-optimum amount of carbohydrates in light.
P l a n t l e t s were produced by root formation of detached t a l l s oots » J ,
* i c h had previously been developed on a medium containing BAat 1 g ^ ^ ^
shoot formation on capitulum explants, however, was realized in th P
1°- 5 gAnl (Section 5 . 4 . 2 . 4 ) . Difficulties m»y arise with r e s p e c t ^ o ^ - ^ ^
isolated shoots, ™
ated shoot,. previously
™ , s ! v grown « » u - supplemented
erown on a medium - ^ with BA , t s a t BA
^ found in Section 3.4.2.5 that peduncle explants ^ ^ ^ . ^ ^ ^ of

^ and no roots a t a l l a t higher BAconcentrations * t * ^ £ * be
s ^ o t s , which had developed a t a BA concentration o ^ ^ ^ ^ ^
Prevented or greatly reduced because of an excess of the e J ^ . ^ ^ ^ ^
shoot. As long as i t i s uncertain whether good rooting ^ r e c o m r a e n d e d for
cultivated previously a t BA 1 0 - 5 , a concentration of BA
shootformationtoensureareliablesubsequentrootformationof isolatedshoots.
Shootdevelopmentwas alsoobservedoncapitulumexplantsofmany otherspeciesof
the Compositae, andonflowerexplantsoftwospeciesbelonging tootherfamilies,
cultivated invitro.Insomeofthesespecies,byrootformationofisolatedshoots,
plantletswereproduced (Section5.6).
Apartfromtheaspectofvegetativepropagation,themethod invitromayalsobeof
significance tokeepdisease-freeplantmaterial instock foralongperiod,ashasbeen
foundin Chrysanthemum morifoliwn (Roest&Bokelmann,1975).
Moreover,anadventitiousbudtechnique invitromaybeapowerful tool inmutation
breeding. Invivo ithasbeendemonstrated inanumberofornamentals,like Strepto-
aarpus (Broertjes,1969), Aohimenes (Broertjes,1972a), Saintpaulia (Broertjes, 1972b),
Kalanehoe (Broertjes&Leffring,1972)and Begonia (Doorenbos&Karper,1975),that
afterirradiationofdetached leavesandthroughsubsequentadventitious shoot formation
andplantletproduction,solid (non-chimeral)mutantscanbeobtained.This findingis
basedonthephenomenonthat (theapexof)adventitious shoots,formed atthebaseof
thepetiole,ultimatelyoriginate(s)fromasingle (epidermal)cell.Recently,after
irradiationofpedicelexplantsandthroughadventitious shootformation invitro,solid
mutantshavebeenproduced in Chrysanthemem morifoliwn (Broertjes etal., 1976).
After irradiationofcapitulumexplantsofpyrethrumandby theregenerationof
adventitious shootsinvitro,solidmutantsmightbeproducedandchimera formation
wouldbeavoidedoratleastgreatlyreduced.Mutationbreedingmaybeauseful
approachbecauseClone4331 isavailable,whichhasveryhighfloweryields,butis
characterizedbyanundesirableratioofthesixpyrethrinconstituents.
94
Summary
Chapter 1 describes why flowering and vegetative propagation of pyrethrum (Chrysanthemum

ainerariaefolium Vis.) were studied.
Flowering (Chapter 2 ) , root formation of peduncle explants (Chapter 3) and of shoot
cuttings (Chapter 4 ) , as well as shoot formation of capitulum explants (Chapter 5), are
processes which are controlled by various factors. These factors are related to proper-
ties of the (ex)plant, the nutritional/hormonal composition of the medium or substrate
and the climatic conditions to which the (ex)plant is exposed. Anatomical observations
have revealed t h a t in these processes successive stages can be distinguished, such as
initiation and development, before flower heads, roots or shoots are produced. Some
factors had an effect during i n i t i a t i o n , which differed from that during the stage of
development. Hence, an optimum course of these processes can only occur when specific
conditions are provided in every stage.
Chapter 2 The effect of temperature and photoperiod on the flowering behaviour of

plants of Clone Ma 63/1889 was examined.
Of the various temperatures studied, 9, 13, 17, 21 and 25°C, high numbers of plants
initiated high numbers of flower heads after being at 9°C for at least 6 weeks. Both
development of i n i t i a t e d flower heads and vegetative development of the plants were
stimulated a t 17, 21 and 25°C. . ,_._. of
For an adequate flowering, which is a prerequisite for the commercial p«duc of
natural i n s e c t i c i d e (pyrethrins) from the flower heads, plants have to be cultivated
low temperatures in the highlands of tropical countries. For an optimum "***"
development of p l a n t s , desirable for vegetative propagation by s p l i t s or shoot cuttings,
cultivation a t higher temperatures in the lowlands is to be preferre •
Flowering was a f f e c t by the photoperiod as well andpyrethr u . t o » £ £ £ * *
as a quantitative short-day plant. This finding, however, is not of^d P
importance for the c u l t i v a t i o n of pyrethrum in tropical countries, where through
year the photoperiod amounts to approximately 12 h.
• , e r f , of adventitious root formation were
Chapter 3 Anatomical and physiological aspects of adven
studied by the c u l t i v a t i o n of peduncle explants m v i t r o . ^ ^ ^ initiated in the
Anatomical observations have shown that ^ ^ ^ T ^ ^ . ^ ^ whereas root i n i t i a l s
interfascicular pericycle during the f i r s t two weeks o ^ . ^ ^ ' ^ s t a i n e d during
developed during the subsequent two weeks. When °P t ™™ °° ^ ^ 63/1889 y i e lded
these separate s t a g e s , peduncle explants of Clones
good rooting responses.
9S
Chapter 4 Subsequently,itwasexaminedwhethervegetativepropagationcanberealized
byadventitiousrootformationofshootcuttings invivo.
Anatomicalobservationsshowedthattheprocessofadventitious rootformationon
basalstemportionsofshootcuttingsprogressed inanalmostidenticalway tothatof
peduncleexplants.Withtheinformationobtainedfromexperimentswithpeduncle explants
invitro (Chapter 3), goodrootingresponsesofshootcuttings ofClones 1087,4331and
Ma63/1889wereachieved.
Onevegetativestock-plantof 1-2yearsoldcanbedividedinto5-10 splits,instead
of25-100shootcuttings.Hence,vegetativepropagationbyroot formationofshoot
cuttingscanconsiderably improvetherateofmultiplicationandmay further leadtothe
buildupofahealthycloneofselected,highyieldingplants.
Chapter 5 Finally,itwas investigatedwhetherplantletscanbeobtained through

adventitiousshootformationoncapitulumexplantscultivated invitro.
Anatomicalobservationshaverevealed thatadventitiousshootswere initiated in
theepidermalcelllayeroftheovariesofdiscfloretsduringthefirstweeks after
incubation,whereastheinitiatedshootsdevelopedduringthesubsequentweeks.Byroot
formationofdetachedshootsplantletswereproducedofClones 1087,4331 andMa63/1889.
Thismethodofvegetativepropagationinvitroisnotyetavailablefor large-scale
multiplicationbecausedespitetheinitiationofnumerousshoots,theirdevelopment into
intermediateandtallshoots,which areused forplantletproduction,was still
inadequate.
Initiallythetechniqueinvitromaybeappliedforafastpropagationofselected
genotypes,whereas forfurtherclonalmultiplicationavegetativepropagationinvivoby
splitsorshootcuttingshaspresumablytobepreferred.
Thedevelopmentofshootswasalsoachievedoncapitulumexplants ofother
Compositae cultivated invitro.Thesimpleprocedureofplantletproductionseemsto
becomeattractiveforafastvegetativepropagationofvarious Compositae.
Apart frompropagation,thistechnique invitromaybeusefulforthemaintenance
ofselected,highyieldingandhealthyplantmaterial instock,whileitmay alsobeof
significanceformutationbreedingofpyrethrum.
96
Samenvatting
Pyrethrum (Chrysanthemum oinevaviaefolium Vis.,behorendetotdefamilievande

Compositae) wordtgeteeldvanwegedeindebloemhoofdjesvoorkomendeinsekticiden,welke
gezamenlijkwordenaangeduidalspyrethrinen.Gunstigeeigenschappenvandezepyrethrinen
zijn:
-degeringegiftigheidvoormensenenzoogdieren,
-desnelleafbraak in'hetmilieu,
-ervindtnauwelijksopbouwplaatsvanresistentieininsektenpopulaties.
Vanwegedezeeigenschappen is'devraagnaarditnatuurlijkinsekticidedelaatste
jarengeleidelijktoegenomen.Voordepr^duktievanpyrethrinenuitdebloemhoofdjesis
bloeiessentieel.Omdieredenisdeinvloedvanklimatologischeomstandighedenopde
bloeionderzocht (hoofdstuk2 ) .
PyrethrumwordtvooralgeteeldinOostafrikaanselanden,waarvanKenyadegrootste
producentisvanpyrethrinenmet601vandewereldproduktie.InKenyakomtmenonder
meeraandetoegenomenvraagnaarpyrethrinentegemoetdoorveredeling,selectieop
Plantenmeteenhogeopbrengstaanpyrethrinenenvegetatievevermeerderingvangeselec-
teerdeplanten.Indepraktijkwordendeplantenklonaal-vermeerderddoorscheuren,aan
welkemethodeevenweltweenadelenverbondenzijn:
-eenmoederplantlevertmaareenbeperktaantalnakomelingen,
-hetwortelknobbelaaltje Metoidogyne hapla, datalgemeenwordtaangetroffenennam -
fectievanworteldeleneensterkeverminderingvandebloemopbrengstveroorzaakt,wordt
van'geinfecteerdemoederplantenoverdescheurlingenverdeeld.
Hetiswenselijkdataltematievemethodenvanvegetatievevermeerderxngwordenont-
wikkeld,welkedezenadelennietvertonen (hoofdstukken3,4en5).Onderzoeknaareen
vegetatievevenneerderingdoorndddelvanscheutstekkenverdienthieibij ^ J J ^ Z
o^datviaeenstekmethodedevermeerderingssnelheidaanzienlijkverhoogdkanworden^n
uitgaandevangeinfecteerdemoederplantengezondenakomelingenwordenverkregen,omdat
bijscheutstekkende(geinfecteerde)worteldelenwordenweggesneden
VooreenvegetatievevermeerderingviascheutstekkenishetnoodzakeUk d a adven
tievewortelswordengevormdaandebasisvandestekken.Onxnzxcht ^ Z n
factorenwelkev-xinvloed zijnopdewortelvormingwerdgebruxk^ ™ ^ ^
invitro,waarbijbloem,teel-e*plantatenonder ^ ^ T ^ ^ . ^
voedingsbodemindecultuurbuiswerdenopgekweekt (hoofdstuk3.ve g scheutstekken
1
zochtofmetbehulpvandezekenniseengoedeadventieve^ " ™ ^ ^ ver.
gerealiseerdkonworden (hoofdstuk 4). Tenslottewerd ^ ^ ^ J ^ Z

o r d e r i n g konwordenverkregenviadevormingvanadventxevescheutjesaan
gekweektebloemhoofd-explantaten (hoofdstuk5).
97
Devormingvanbloemhoofdjes,wortelsenscheutenzijnprocessenwelkemoeten
wordenonderscheidenineenaantalopeenvolgendestadia,zoalseenstadiumvan initiatie
(aanleg)enontwikkeling (uitgroei).Dezeprocessenwordenbeinvloeddoorfactorenwelke
samenhangenmetdeplantofhetexplantaat,desamenstellingvanhetsubstraatofde
voedingsbodemendeklimatologischeomstandigheden.Eenaantalvandeze factorenver-
toondeeeneffectgedurendehetstadiumvaninitiatiewelkeduidelijkafweekvandat
gedurendehetstadiumvanontwikkeling.Vooreenoptimaalverloopvandezeprocessenis
hetdusnoodzakelijkdatinbeidestadiadeomstandighedenoptimaalzijn.
Deresultatenzullenvervolgensperhoofdstukgedetailleerdwordenbeschreven.
Inhoofdstuk2wordtdeinvloedbesprokenvantemperatuurendaglengteopdebloei
vanplantenvankloonMa63/1889.
Wanneerplantenwerdengeplaatstbijtemperaturenvan9, 13,17,21of25C,kwamen
demeesteplanteninbloeienwerdhetgrootsteaantalbloemhoofdjes geinitieerdbij9C,
welketemperatuurgedurendetenminste6wekenmoestwordengehandhaafd.Zoweldeont-
wikkelingvandegeinitieerdebloemhoofdjesalseenvegetatieveontwikkelingvande
plantenwerdengestimuleerdbij 17,21en25C.
Eengoedebloei,essentieelvoordecommercieleproduktievanpyrethrinen,kandus
alleenverwachtwordenbijde lagetemperatureninhethooglandvantropischegebieden.
Vooreenoptimalevegetatieveontwikkelingvandeplanten,welkedevoorkeurverdient
wanneerdeplantenvegetatiefwordenvermeerderddoorscheurlingenofscheutstekken,
moetpyrethrumechterwordengeteeldbijdehogeretemperatureninhet laagland.
Debloeiwerdookbeinvloeddoordedaglengteenpyrethrummoetbeschouwdworden
alseenkwantitatievekorte-dag-plant.Diteffectvandedaglengte isnietvandirecte
praktischebetekenisvoordeteeltvanpyrethrum intropischegebieden,waar dedag-
lengtehetgehelejaardoorongeveer12uurbedraagt.
Inhoofdstuk 3wordthetprocesvanadventievewortelvorminganatomischenfysiolo-
gischonderzochtaaninvitrogekweektebloemsteel-explantaten.
Anatomischonderzoektoondeaandatadventievewortelsvoornamelijkwerdengeini-
tieerdgedurendedeeerstetweewekenvanincubatieinhet interfasciculairepericambium,
terwijldegeinitieerdewortelstotontwikkelingkwamengedurendededaaropvolgendetwee
weken.Wanneeroptimaleomstandighedenwerdengehandhaafd gedurendebeidestadia,werd
eengoedebewortelingverkregenaanbloemsteel-explantatenvandeklonen 1087,4331 en
Ma63/1889.
Hoofdstuk4beschrijftonderzoekbetreffendeadventievewortelvormingvanscheut-
stekkenwaardooreenvegetatievevermeerderinginvivokanwordengerealiseerd.
Anatomischonderzoekheeftuitgewezendathetprocesvanadventievewortelvorming
aanbasalestengeldelenvanscheutstekkenopeenvrijwelidentiekewijzeverlooptalsis
waargenomenbijbloemsteel-explantaten.Doorgebruiktemakenvandekennisverkregenvia
.deinvitrocultuurvanbloemsteel-explantaten (hoofdstuk 3),werd eengoedewortel-
vorminggerealiseerdaanscheutstekkenvandeklonen 1087,4331 enMa63/1889.
Eenvegetatiefontwikkeldemoederplantvan 1-2 jaaroudkanwordenverdeeld in 5-10
98
scheurlingen, t e r w i j l 25-100 scheutstekken van een dergelijke plant kunnen worden ver-
kregen. Een vegetatieve vermeerdering in vivo via scheutstekken kan de vermeerderings-
snelheid dan ook aanzienlijk verhogen en tevens resulteren in de opbouw van een gezonde
kloon, bestaande u i t geselecteerd, hoogwaardig plantmateriaal.
Hoofdstuk 5 behandelt onderzoek betreffende vegetatieve vermeerdering via de vorming

van adventieve scheuten aan in v i t r o gekweekte bloemhoofd-explantaten.
Anatomisch onderzoek heeft aangetoond dat adventieve scheutjes gedurende de eerste
weken na incubatie werden geinitieerd in de epidermis van de vruchtbeginsels van buis-
bloemen en dat de scheutjes t o t ontwikkeling kwamen gedurende de daaropvolgende weken.
Na wortelvorming van geisoleerde scheutjes werden plantjes verkregen van de klonen 1087,
4331 en Ma 63/1889.
Deze in v i t r o methode van vegetatieve vermeerdering kan nog niet op grote schaal
worden toegepast omdat, ondanks de i n i t i a t i e van een groot aantal scheutjes, de ont-
wikkeling hiervan t o t middelgrote en grote scheutjes (welke voor de vegetatieve vermeer-
dering worden gebruikt) nog onvoldoende i s . Aanvullend onderzoek zal noodzakelijk zijn
omde scheutontwikkeling t e verbeteren.
In de p r a k t i j k zal de in v i t r o techniek aanvankelijk van betekenis kunnen zi 3 n voor
een snelle vermeerdering van geselecteerde genotypen, terwijl een verdere klonale ver-
meerdering vemoedelijk het beste gerealiseerd kan worden via scheurlingen of scheut-
stekken in vivo.
ScheutvorMng werd ook waargenomen aan in v i t r o gekweekte bloemhoofd-explantaten
van andere Composite. De eenvoudige wijze waarop na beworteling van geisoleerde
scheutjes plantjes worden geproduceerd, l i j k t ook perspectief te bieden voor een
vegetatieve vermeerdering van diverse composieten.
Tenslotte kan een in v i t r o vermeerderingsmethode nuttig zijn voor het op laan van
geselecteerd, gezond en hoogwaardig plantmateriaal in de cultuurbuis en bovendien
belangrijk zijn voor de mutatieveredeling van pyrethrum.
99
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1A[ r

Flowering and vegetative propagation of pyrethrum

Centre for Agricultural Publishing and Documentation

This thesis will also be published as Agricultural Research Reports 860.

© C e n t r e ^ Agricultural Publishing and Documentation, Wageningen, ,976.

11. Snelverkeer in woonwijken moet wijken voor wonen.

vand r 1 S r S 1 6 S" deWaard6V0lleS U ^ e s t i e s™ * » iknadelezingvanhetmanuscript

2 Flowering behaviour of intact plants 4

3 Root formation of peduncle explants in vitro 18

5 Shoot formation of capitulum explants in vitro and -plantlet production by root

Pyrethrum {Chrysanthemum einerariaefolium Vis.)isaplantspecieswith daisy-like

Pyrethrumplantsalmostcompletely failtoproduceflowerheadsathigh temperatures

n l a n t D a t 7 r C 0 U e C t e d *"* * "" * ° f f l m l i n i t i « i o n ,thenumberofflowering

Fig. 1.Theinfluenceofconstanttemperatures (9-25C)onthegenerative andvegetative

9°C 13°C 17°C 21°C 25°C

Temperature Numberofflowe- Numberof Numberofflowerheads Averageleaf

number of flowering plants

number of generative shoots

deveLDment^f^l" 6 ^ 6° f r ? O M t a n ttemperatures (9-21°C)onthegenerativeandvegetative

th intiata' " " £ a V ° U r e d ^ E l 0 W t e m p e r a t U r e «* the^sequentdevelopmentof

number offlowering plants number of flower heads

temp, combination temp, combination

\ % %'7/9%%2,/92,/1325/925/,3 1 1 I I I 1 1 I ' '

average temp average temp.

Plantsstartedtoflower7w e L ft " t ™ f " * "&^ P e r i 0 d * * £ l r S t

Weeks Numberofflo- Numberof Numberofflo-

number offlowering plants number of flower heads

Asmanyspeciesofthegenus Chrysanthemum aresensitivetodaylength,itisof

16h17u 8h9" 8h 13u 8h 17L

Photoperiod Temperature Numberof Numberof Numberofflower

Vegetativepropagationofmany cropscanbeachieved throughtheformationofroots

As explants fromplants growingoutsidecouldnotbesterilized satisfactorily,

distinguishitfromthehorizontally cutmorphologicaltop(Fig. 11,right). Finallythe

Inmanyplantspecies adventitious rootsoriginateendogenously fromthepericycle -

"rft£/.V. 'Kf \;- - ••'--H ^

Figs 12a-d. Transverse sections of peduncle explants:

.V - - ' .>.-•/•• ' 1

/ / ' . ' • • ' • . ' • ^

emergedmostlyatthemorphologicalbaseoftheexplant,opposite thewounded side (Fig.

Inmostexperiments peduncle explantsofClones 1087,4331andMa63/1889wereused.

rooting percentage rooting per centage

Theeffectofthe initial lengthoftheexplantonrhizogenesiswasstudied on

Clone 0.5 1 1.5 2 2.5 3

IflYaid Ma663/1889?Ce"*W ° U n d i n 8 ° nr o 0 t f o r m a t i ° " °fpeduncleexplantsofClones 1087,

1087 4331 Ma63/1889

Intheprevious sectionsitwasdeterminedwhichdevelopmental stageandwhichpart

4331 Ma63/ '1889

up- upside hori- up- upside hori-

0.2 0.4 0.6 0.8

3.4.2 Nutritional/hormonal factors

Heller minor Krlopmajor

Fig. 18. The i n f l u e n c e of v a r i o u s Fig. 19.TheinfluenceofKnop'smajor

Knop major devoid of

Alltreatments,,except1and4,resultedinahighrootingrate (Fig.19). Table13

0L y _ _X^_ . L. Fig. 20. The influence of various sugars

rooting percentage rooting percentage

(b)ontootfo™ation e of e DPH,mM iOUSeventrations (0-5%)ofglucose (a)andsucrose

Clone Glucose Sucrose

Clone Glucose Sucrose

Last2weeks First2weeks 4weeks

Finally lt was determined whether sucrose a t 1%i s e s s e n t i a l during the whole

Clone 0 ,o" 8 ,o- 7 ,0" 6 lO"5 lO"4

Clone IBA NAA

n l a n t D a t 7 r C 0 U e C t e d " * "" * ° f f l m l i n i t i « i o n ,thenumberofflowering

**«.-t . i s :i t r r ; ™ ' reas'-™ati™sdu-

V.'- v-ii-1-" '•<-..' ;v'/.v,i->;-".•."./ •,-::--''•.••-;-'ia«

a v e r a ! ! ? ? " ? 1 " ™ 3 ^ ^ ""» ^ treatments