Multiplication Rates In Vitro and by

Stem Cuttings Propagation,, and Clonal

Development from Eucalyptus globulus
Seedlings
P.J. Wilson

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ABSTRACT. In developingnew clones of forest trees, the first main goal is to propagate enough
plantable plants for clonal field trials and a clonal archive. Models of clonal multiplicationrates in
Eucalyptusglobulusindicate that this takes a similar time (about 56 wk) from the sowingof seed,
whethermultiplicationis byaxillaryshootculturein vitroorbystemcuttingspropagation.Multiplication
of unrootedshoots is relativelyrapid in vitrobut is largelyoffset by the greatertime requiredfor
weaning, prickingout, and growth in the nursery. FOR.Sc•. 42(4):415-418.
Additional key words: Clone, micropropagation,model.

EW
CLONES
EACH
ORIGINATE
FROM
asingle
individual,perfectexponentialwithzerointercept.Forexample,when1
traditionally
aseedling
ofknown
pedigree
orplus shootgives3 shootsevery3 weeks(Hartney1981),thereare
tree,whichismultipliedbyvegetative propagation. 1, 3, 9, 27... shootsat 0, 3, 6, 9... weeks. This rate is Y =
In practice,clonesof foresttreesareoftenpropagated by eø'366x,
givingY shoots
afterX weeks,
andhasa doubling
stem cuttings, but in vitro propagation by axillary timeof 13.3days.OthershootMRspublished forE. globulus
(nonadventitious) shootcultureis claimedto give higher have similardoublingtimesof 15.4 days(Trindadeet al.
multiplicationrates(MRs) in eucalypts(Hartney1981,Le 1990) and 12.3 days(Willyams et al. 1992), giving a mean
RouxandVan Staden1991,Haines1992)andmanyother doubling
timeof 13.7days(Y= eø'354x).
species.Otherin vitropropagation techniques(adventitious These shoot MRs in vitro assume that all shoots are used
shooting andsomaticembryogenesis) givepotentiallyeven for furthershootmultiplication,
implyingthatoncea desired
higherMRs but are proneto problemssuchas genetic numberof shoots is producedperclone,all aremovedto the
instability(Henshaw1987)andlowratesofrecovery(Haines rootingphasetogether.However,rootingmustbe evaluated
1992). on more than one occasion before clones can be ranked with
In thisnote,publishedMRs for Eucalyptusglobulusby anylevelof confidence,
becausevariationbetweenoccasions
axillaryshootculturein vitroandby stemcuttings
propaga- can be considerable.In addition, when only a small or
tion are compared,andtheirpracticalimportancein clonal moderate proportion of cloneshaveacceptable rootingabil-
developmentis considered. ity, asin E. globulus(Willyamsetal. 1992),anearlyselection
for rootingis appropriate to minimizethe effort wastedon
low-rootingclones.Thus, in the comparisonof the two
Clonal Multiplication Rates
propagation systems below,it isassumedthatonce30 shoots
In Vitro havebeenproducedin vitro,20 arewithdrawnfor a nonde-
ShootMRsby axillaryshooting in vitrocanbeexpressed structiverootingtest.Theremaining10 shootsaremultiplied
as "shootsper time"sinceall (unrooted)shootsgive more to thebalanceof the desirednumberper clone,whichthen
(unrooted)shoots.Multiplicationoccursstepwise,as shoot entertherootingphasetogether(tOgivea second estimateof
culturesaresubcultured,
buttheMR canbe represented asa clonalrootingability).

The workwas donewhilethe authorwas associatedwiththe Departamentode EngenhariaFIorestai,InstitutoSuperiorde Agronomia,Tapadada

Ajuda,P-1399 Lisbon,Portugal.His currentaddressis Wye College,Universityof London,Ashford,KentTN25 5AH, UK.
Acknowledgments:The comments of an anonymousreviewerare appreciated.
ManuscriptreceivedJanuary30, 1995. AcceptedOctober 5, 1995. Copyright¸ 1996 by the Society of AmericanForesters

ForestSctence
42(4)1996 415
Stem Cuttings
In stemcuttingspropagation,unrootedcuttingscannot
be made to producenew unrootedcuttings:harvestable
shootsonly arise from rooted cuttings (mother plants).
MRs for unrootedcuttingscan be expressed"per mother
plant per time" but cannotbe exponentiated.MRs thatcan
be exponentiatedarecompositeratesof recentlyharvested
cuttings,rooted cuttings, and harvestablemother plants
(HMPs). Neitherrateis directlycomparableto shootMRs
in vitro.
To permit a comparison,the compositerate has to be
separated intoparts.This is donefor a ratepublishedfor E.
At month 0, one HMP (row 1 of Table 1) yields two
ultimatelyplantable
cuttings
(month0, row2) whichbecome
HMPs 4 monthslater (month4, row 2). Thereafter,the same
motherplant (row 1) is harvestedfor four suchcuttingsper
month(month1, row 3; month2, row 4; etc.).At month4,
eightultimatelyplantablecuttingsareharvested(row 6), four
fromtheoriginalmotherplant(row 1) andtwoeachfromthe
two motherplantsharvested for the firsttime (row 2).
Therate,overmonths
0-16 inclusive,
isY= 0.393eø'523x,
giving Y HMPs afterX months.It hasto be derivedempiri-
callybecause theexponential is inexact,beingstepped.
This
isbecause thetimestepsof therelationship correspond
to the
harvestinterval while the time requiredfor a cutting to

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globulus,in whichmotherplantsweregrownin 10 litrepots
andlightlyharvested forcuttingsevery2 to4 weeks.Cuttings becomea HMP occupiesseveralsuchintervals.
becameHMPs4 monthsaftersetting,eachyielding4 cuttings A Comparisonof theMRs of PlantablePlants,In Vitro
in the first monththen8 per HMP per monthin succeeding and by StemCuttingsPropagation
months,of which50% survivedandrootedtobecomeplantable The first main objectiveof clonaldevelopment,whichis
(Wilson, 1992). invariablein foresttrees,is to producea desirednumberof
The compositerate and its partscan be derivedempiri- plantableplantsper clone for field trials. Thus, the only
cally, eitherby spreadsheet
(Table 1) or algebraically: meaningfulMR, in anypropagation system,is of plantable
plants.
Ni = N(i_i)+ 0.5(8N(i_5)
+ 4[N(i_4)
- N(i_5)I) Thisratecanbesummarized ashavingthefollowingparts:

whichsimplifiesto 1. Time from initiationof clonaldevelopment

to the first
harvestof propagules.
Ni = N(i_i)+2[N(i_4)+N(i_5)I 2. Themultiplicationof shoots
invitro,andofmotherplants
andcuttingsin stemcuttingspropagation.
where
N/=thenumber
ofHMPs
intheithmonth,
N(i- 1)=the 3. Elongationof shootsin vitro(if required)androotingof
numberonemonthpreviously,etc.HMPs yield4 cuttingsat shootsandcuttings.
theirfirstharvest
(4[N(i 4)- NO 5)]) and8 cuttings
in 4. Weaning,includingtheprickingoutof in vitroplantlets.
subsequent
harvests
HMPsAtMonth
(8N(i-5)),
andh•]fofall
cuttings
i, inad•iontoN.HMPs,
there
become5.
are[N.... Growth,etc., in the nurseryuntil plantsare plantable.
ß t
-N/]
ultimately
plantable
cuttingsß
Ofthese,
[N•i
+1)-N/•
are 6. Losses in thepropagation
environment,
atweaningandin
thenurserydueto failureto root,mortality,andculling
atleast
3months
old,[N(i+2)-N/]areatleast
2months
old,
etc. This is illustrated in Table 1. for qualitybeforeoutplanting.

Table 1. A compositemultiplicationrate of harvestablemother plants (HMPs)and stam cuttings of variousages

in E. globulus.New cuttings/plantsare recruitedon eachrow (seatext). The cumulativenumberof HMPsat one
time is obtainedby eddingthe numbersin the part of a columnmarked (a), includingthose being harvestedfor
the first time (shown in italics).At one time there are also ($), (+), (@), and (#) cuttings at least 3, 2,1 and 0 months
old respectively.

Row Month(fromfirstharvest) Age (months)

0 1 2 3 4 5 6 7 8 9

1 1. 1. 1. 1' 1' 1' 1' 1' 1' 1'

2 2# 2@ 2+ 25 2* 2* 2* 2* 2* 2*
3 4# 46 4+ 45 4* 4* 4* 4* 4*
4 4# 46 4+ 45 4* 4* 4* 4* * > 4
5 4# 46 4+ 45 4* 4* 4* (HMPs)
6 8# 86 8+ 85 8* 8*
7 20g 206 20+ 205 20*
8 36g 366 36+ 365
9 52# 526 52+ $ >_ 3
10 76g 766 + >_ 2
11 132#
# > 0

Cumulativeharves•blemotherplants
1 1 1 1 3 7 11 15 23 43

416 ForestScience
42(4)1996
 To comparethe two propagation
systemsthe following
assumptions
areadoptedfor both:
1. Clonaldevelopment
beginswith thesowingof seed.
2. In the first propagationphaseof clonaldevelopment,
30 plantableplantsper clone are requiredfor field
trials and a further 20 propagules(shootsin vitro or
plantableplantsby cuttingspropagation) arerequired
for a clonalarchive.At thisstagemultiplicationstops
pendingtheresultsof thefield trials.(The archiveis
used to re-initiate multiplication,if desired,on the
basisof earlyfield trial results,avoidingthe sacrifice
of the trials while they continueto grow and yield
information).
3. Half of all shoots
orcuttings
becomeplantable(e.g.,70%
of propagules root,approximately
70%of thesebecome
plantable).
4. Motherplantsyieldstemcuttings
ataconstant
rate(after
the first month) and in vitro shootsgive more shoots
indefinitely.
5. In the secondpropagationphase,clonesselectedfor
goodfieldperformance aremobilizedfromthearchive
andmultipliedin preparationfor large-scaleproduc-
tion. It is assumedthat the archive propagulesgive
typicalproductivityandthat 10,000 plantableplants
are requiredto establisheachclonein a commercial
stemcuttingsnursery.
The durationof the first propagationphaseof clonal
development is foundto be verysimilarin thetwopropaga-
tion systems(Table 2).
The values in Table 2 were estimated from Blomstedt et al.
(1991), Wilson (1992 and unpubl.),and V. Hartney(1994
pers.comm.,CSIRO Division of Forestry,CollegeRoad,
Universityof Tasmania,Hobart,Australia).The observa-
tionsof thethreeauthorsstrictlyapplyto E. regnans(which
has a shootMR of x3 to x5 every3 to 5 weeks,giving a
doublingtime of 14.0 days),E. globulusand to several
temperateeucalyptsrespectively.
From sowingto first harvestrequires3 to 4 months
(3.50 month= 15.2 wk; 1 month= 30.4 days)to achievea
minimum desiredsize of nodal explant in vitro. In stem
cuttingspropagation,seedlingsgrown in the nurseryare
Table 2. The time required (in weeks), by in vitro and stem
cuttings propagation, to produce30 plantable plants per clone of
E. globulus for field trials, and a further 20 propagules per clone
for an archive.
In vitro Stemcuttings
Sowingof seedto first harvest 15.2
Multiplication 14.7 22.9
Rooting 4.0
Weaning(lab) 6.0
Weaning(nursery) 2.0
Growthandhardening 13.0
Total 54.9 56.9
transplanted
intopotsafter3 monthsandrequirea further
monthof growthbeforetheir first harvest(17.3 wk).
Atthemean
published
shoot
MR:Y=e0'354X
(Y=shoots,
X = weeks),one shootgives30 in 9.6 weeks,when 20 are
withdrawnfor a nondestructive
rootingtest.The remaining
10shoots give60 in a further11.6-6.5= 5.1 weeks,of which
20 are retainedfor the archive.By stem cuttings,at the
calculatedrate,oneHMP gives50 in 9.27 months.Thus,a
totalof 50 propagules (HMPs andultimatelyplantablecut-
tingsof variousages)are producedin 9.27-4.00 = 5.27
months= 22.9 weeks.At this time thereare 6.2 HMPs, which
are assumed to be suitable for the archive.
In principle,a timesavingof 1.1 weeksin vitroand3.3
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weeksby stemcuttings propagation
couldbeachieved if the
multiplication phasewerelimitedtothepropagules required
for field trials,leavingtheminimumnumberof shootsand
HMPs (1 and 4 respectively)for furthertime-unlimited
multiplication for thearchive.However,thiswouldinvolve
a considerable increasein managementeffort,andtheolder
plants(if relativelyvariable)wouldbe preferablefor the
archive.
Twelveweeksarerequiredto root,prickout,andweanin
vitroplantlets,butcuttingsrequireonly8 weeksbecause they
arenotprickedoutandarerelativelyhardy.Cuttingsarealso
largerandgrowingactivelyafterweaning,so2 months(8.7
wk) areallowedfor theirfurthergrowthandhardening prior
to plantingwhereas3 months(13.0 wk) are allowedfor
plantlets.
Therelativelyshortmultiplicationphasein vitro(Table2)
resultsin a time advantagefor in vitro propagationwhen
largernumbersof plantsarerequired.The secondpropaga-
tionphaseof clonaldevelopment wouldoccupy44.5 weeks
in vitro [28.0-8.5 = 19.5 wk for multiplicationfrom 20 to
20,000 shootsanda further25 wk (seeTable2) for rooting
etc.], or 51.0 weeksby stemcuttingspropagation(19.40-
7.51 = 11.89 month for multiplicationfrom 20 to 10,000
HMPs; 11.89-4.00 = 7.9 month= 34.3 wk to produce10,000
HMPs andultimatelyplantablecuttings,anda further16.7
wk for rootingetc.).
Discussion
The first propagation phaseof clonaldevelopment was
definedto lastfromthe sowingof seedto theproductionof
(a) 30 plantableplantsper clonefor field trialsand(b) 20
propagules perclonefor a clonalarchive.In E. globulusthis
phaseshouldtakeasimilartimewhether multiplication
isby
axillaryshootingin vitro or by stemcuttingspropagation
(Table 2). A secondphase,to multiplyclonesselectedfor
large-scalepropagation(from20 archivepropagules to 10,000
plantableplants)wouldtake 51 weeksby cuttingsand 6
17.3 weekslessby in vitropropagation. Essentially,themultipli-
cationphasesin vitro are relativelyshort,but are largely
6.0
__ offsetby the additionaltimerequiredfor prickingout into
2.0 nurserymedium,weaning,andgrowthpriorto planting.
8.7 The durationof the variousoperationsdependson man-
agement whichissubject to change,
atleastin stemcuttings
propagation ofE. globulus (inwhichlittledevelopment work
ForestSctence
42(4)1996 417
hasbeendone).However,overallchangeis unhkelyto be
greatbecauseshorteningone operationtendsto lengthen
another. Forexample,anearlierfirstharvest of cuttings
from
(relativelysmall)motherplantswouldbe offsetby a lower
subsequent cuttingsproductivityperplant.And lesscytoki-
nin in thein vitromultiplication
mediumwouldgivea larger
shootsize (Skirvin et al. 1986) and less severehormonal
carryovereffects(McCombandBennett1986), but a lower
shootMR. In any comparison,
time estimates
shouldbe
conservative to allow for the handlingof largenumbersof
clones,asin practice.
The assumptions usedprobablyfavorthe in vitro regime
because: (a) ShootMRs wereassumed tobeconstant whereas
importantthanmmntmmng anorderlyrouUne;and(d) clones
may be developedin the samepropagation systemas that
usedfor large-scale
propagation,irrespective
of timediffer-
ences,to avoid the risk of clone x propagationsystem
interactions.
It is oftenrepeated,andthereforeseemsvirtuallyaxiom-
atic, that in vitro propagation permitsrapidmultiplication.
However,at leastin thecontextof clonaldevelopment from
E. globulusseedlings, thisisa misleading impressioncreated
by confusionbetweenMRs for shootsand for plantable
plants.To bejustifiable,in vitropropagation mustoffersome
advantageover stem cuttingspropagation(Jones 1987).
However,in E. globulusandprobablymanyotherforesttree

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inpractice theyareinitiallyslow(Hartney1994pers.comm.); species,rapid multiplicationcan no longerbe cited as a
(b) PublishedshootMRs in vitroprobablyapplyonlyto a significantadvantage of in vitropropagationin clonaldevel-
smallminorityofreactiveclones(3.4%ofclonesinWillyams opment.
et al. 1992), adverselyaffectingbreedingobjectivesand
narrowingthegeneticbaseof thecommercial population. In Literature Cited
stemcuttingspropagation, cuttingsproductivityvariesmuch BLOMSTEDT,
C., J. CAMERON,P. WroTEMAN,AND S.F. CHANDLER.1991
lessbetweenclones;(c) Lossesareassumed to bethesamein Micropropagation
ofjuvenileEucalyptus
regnans(MountainAsh).Aust
J. Bot. 39:179-186.
the two propagation systems, but the needto prickout and
weanplantletswhichhavebeenrecentlyrootedin vitro is HAINES,
R.J. 1992.Masspropagation by cuttings,
biotechnologies,
andthe
almostcertainto resultin extramortality.Prickingoutalso capture
of geneticgain.P. 137-150inProc.of AFOCEL-IUFROSymp,
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tion phase,only two rootingtestswere assumedin vitro HARTNE¾,V.J. 1981.Vegetative
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G.G. 1987.Newtechniquesforgermplasmstorage. P. 303-313tn
of a certainage, but in both propagationsystemsmost Improvingvegetatively
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Potentialclonescouldoriginatefromgeneticengineer- Jo•r•s,L.H. 1987.Clonalpropagation
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(eds.).AcademicPress,London.
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andtissueculture
of Eucalyptus--areview.TreePhysiol.9:435-477.
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onlyatleast29.3 weekslaterby stemcuttingspropagation McCoym,J.A.,•qDI.J.BF•r•ETr.1986.Eucalypts
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418 Forest
Sctence
42(4)1996