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Scientia
Horticulturae
j o u r n a l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s c i h o r t i
Somatic
embryogenesis-derived
coffee
plantlets
can
be
efficiently
propagated
by
horticultural
rooted
mini-cuttings:
A
boost
for
somatic
embryogenesis
Frédéric
Georget
a,∗,
Philippe
Courtel
b,
Eduardo
Malo
Garcia
b,
Martin
Hidalgo
c,
Edgardo
Alpizar
b,
Jean-Christophe
Breitler
a,
Benoît
Bertrand
a,
Hervé
Etienne
aaCIRAD,UMRIPME,F-34394Montpellier,France
bECOM,ExportadoraAtlanticS.A,Managua,Nicaragua,
cNICAFRANCE,Managua,Nicaragua
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received13October2016
Receivedinrevisedform
15December2016 Accepted16December2016 Keywords: Clonalpropagation F1hybrids Rejuvenation Productioncosts
a
b
s
t
r
a
c
t
Ingeneral,thecurrentindustrialsomaticembryogenesis(SE)propagationprocessesforcoffeearecostly becausetheyarenotproductiveenough.WeshowthatSE-derivedplantletsfromC.arabicahybridswere temporarily−between10and25weeksofdevelopmentinnursery−abletorootwithahighsuccessrate (upto90%)whateverthegenotypetested,beforegraduallylosingthatcapacity.Wetookadvantageof thistransientrootingcapacity,probablyduetotherejuvenationprocessoccurringduringSE,toestablish anewpropagationsystembasedonthecontinuouscultureofrejuvenatedSEplantsandontheserial rootingofcuttingsundernurseryconditions,knownashorticulturalrootedmini-cutting(HRMC).The excessivelylowSEefficiencywithanembryo-to-plantletconversionrateofonly37%canbegreatlyoffset bythemuchhigherHRMCmultiplicationrate(14insixmonths)andbetteroverallquality.Fifteen week-oldrootedmini-cuttingsprovedtobemoreuniform(2–4.5vs.1–5.5cmforplantheightdistribution)and vigorous(1.41vs.0.81mmforstemdiameter)thansame-agesomaticseedlings.Thiseffectpersisted forfiveyearsafterfieldplanting,mainlythroughaslightlygreatercollardiameter(43.3vs40.6mm), whereasatrootlevelnodifferenceswerefound.TheHRMCmethodisexpectedtodramaticallyreduce arabicahybridproductioncosts(byupto50%atUS$0.27/plantreadyforfieldplanting)andthusto promotethemassutilizationofgeneticallysuperiorhybridclonesofcoffee.
©2017TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Morethan 85% ofarabica coffee isproduced in Latin Amer-ica,fromtreescomprisingasmallnumberofso-called“American” varieties,derivedfromanarrowgeneticbase.Thesehomozygous varieties,knownas“lines”,reproducefromseed.SomeF1hybrid varietieshavebeencreatedbycrossingtraditionalAmerican vari-etieswithsomewildparentsoriginatingfromEthiopia(thecenter oforiginoftheCoffeaarabicaspecies)(Bertrandetal.,2011).The hybridvarieties produce20–40%more thanthe bestcultivated lines.Giventheirheterozygousstructure,F1hybridsmustbe veg-etativelypropagated.Thebestindividualisselectedfromthebest
Abbreviations: RH, Relative Humidity; HRMC, Horticultural Rooted
Mini-Cuttings;SES,omaticEmbryogenesis.
∗ Correspondingauthor.
E-mailaddress:frederic.georget@cirad.fr(F.Georget).
F1familiestobecloned,therebycreatinganF1hybridclone(Van derVossenetal.,2015).
Itisdifficulttopropagatearabicacoffeetreesbyconventional horticultural techniques such as cuttings, as they are rooting-recalcitrant,orbybudgrafting,whichrequirestoomuchmanpower (Etienneet al.,2002).Ininvitroconditions,micro-cutting tech-niqueshavebeendevelopedforcoffeebutcannotbeconsidered formasspropagation,asitisverydifficulttoestablishaxenic cul-turesinvitroanditinvolvestoomuchworkforlowmultiplication rates(Bertrand-Desbrunaisetal.,1991).Somaticembryogenesis (SE)hasbeeneffectivelymasteredonanindustrial levelforthe C.arabicaspecies(BobadillaLandeyetal.,2013).TheCIRAD-ECOM groupconsortiumhasbeenproducingbetweenoneandtwomillion intraspecificF1hybridplantsperannuminNicaraguaandMexico since2007(Georgetetal.,2010).Inspiteoftheseachievements, thearabica SEprocess isstill impededby twotechnical bottle-necks:anembryo-to-plantletconversionratethatistoolow,and excessiveplantlossesinthenurseryateachstepofthe acclimatiza-http://dx.doi.org/10.1016/j.scienta.2016.12.017
178 F.Georgetetal./ScientiaHorticulturae216(2017)177–185
tionandhardeningprocess,whichrendersthispropagationsystem insufficientlycost-effectiveandproductive(Etienneetal.,2013). Atpresent,theseexcessiveproductioncostsareamajorobstacle tothemassdisseminationofcoffeeF1hybridsinCentralAmerica. AsimilarsituationisfoundwiththeC.canephoracoffeespecies,but alsoinmanyotherwoodyspecies,leadingtosomaticseedling pro-ductioncoststhataretoohigh,therebyholdingbackthelarge-scale utilizationofsomaticseedlingsasplantingmaterials(Lelu-Walter etal.,2013).
Recently, we discovered that very young C. arabica plants derivedfromSEwereabletorootwithahighsuccessrate.Inthis paper,wedescribethedevelopmentofamini-cuttings propaga-tionsystemusingsomaticseedlings.Severalissuesareexamined toassessthetechnique.First,weprovideareminderofthe limi-tationsoftheSEmethodbymeasuringthebiologicalefficiencyof thefinalstagesofthispropagationsystem.Wethengoontotest theproductionofrootedmini-cuttingsbyevaluatingthegenotypic effect,theincidenceofsomaticseedlingageonthecapacityof root-ing,andthefeasibilityofsuccessivemultiplicationcycles(serial cuttings)fromasinglesomaticseedling.Weproceedtocompare thevigorandthehomogeneityofthematerialproducedbyrooted mini-cuttingscomparedtosomaticseedlingsatnurseryandfield level.Finallyweperformapilotproductiontoestimatethegainin productivityachievedandtheconsequencesforproductioncosts.
2. Materialsandmethods
2.1. Producingsomaticembryo-derivedplantletsandassessing theefficiencyofthelastSEsteps
SomaticseedlingsderivedmainlyfromsixF1hybridsobtained bycrossingtraditionaldwarfAmericanvarieties(Caturra,T5296 and17931Sarchimorlines)andwildaccessionsoriginatingfrom EthiopiaandSudan(Bertrandetal.,2005,2006)wereusedinthis study.Inthispaper,thesehybridswillbecalledH1,H3,H5,H10, H16andH17.
Theregeneration of cotyledonary somaticembryo of C. ara-bicaspecieshasalready beendescribed indetail(Etienne2005; BobadillaLandeyetal.,2013).Plantletconversionwasobtained afterdirectsowingofcotyledonarysomaticembryosinthenursery. Cotyledonaryembryosweresownverticallyontopofthesubstrate comprisingamixofPeatmoss(Pro-mix,PremierTechLtd,Canada) and sand.Somatic embryo culture density in theplastic boxes (l.w.h=30/21/10cm)wasapproximately3600persquaremeter. Thecultureswereplacedunderatransparentroofthatprovided 50%shade,andwerewateredfor2minstwicedaily.The conver-sionofthesomaticembryosintoplantswasgenerallyobserved12 weeksaftersowing,andwascharacterizedbytheemergenceofa stembearingatleasttwopairsoftrueleaves.Forthegrowthand hardeningstepinthenursery(21weeks),plantletsgrownfrom somaticembryosweretransferred to1lplastic bagscontaining soilandcoffeepulp(3/1,v/v)underconventionalnursery condi-tionsuntiltheyreachedtherequiredsizeforplantinginthefield (approx.30cm).Duringthisstage,theshade(50%light intercep-tion)andrelativehumidity(RH,90%)weregraduallyreducedover 4weeksto0%lightinterception,withnaturalRHrangingfrom40 to80%.
ThebiologicalefficiencyofthelastSEstepsinthenursery,i.e. plantletconversionaverageduration oftheacclimatizationstep wasmeasuredfromtheresultsoftheindustrialproduction,mainly withH1(SarchimorT5296xRomeSudan)andH3hybrids(Caturra xEthiopian531),of5millionembryosregeneratedinNicaragua between2010and2012.Theembryo-to-plantletconversionrate andtheaveragedurationoftheweaningphaseinthenurserywere usedasbiologicalindicators.
2.2. Experimentaldesigntoestablisharootedmini-cutting vegetativepropagationprocess
The technique known as ‘horticultural rooted mini-cuttings (HRMC)’isillustratedinFig.1andcomprisesthefollowingstages: 2.2.1. Cuttingoriginandcharacteristics
Theplantsusedtoinitiatethepropagationcycleare 15-week-oldnurseryplantletsderivedfromsomaticembryos(Etienneetal., 2012).Thesizeoftheplantletsisaround4–6cminheightwith 3pairsoffullydevelopedleaves(Fig.1A).“Tipmini-cutting”–so calledbecauseitbearstheterminalapex–isused.Itisformedof twopairsofdevelopedleaves(Fig.1B).Finesecateursareusedto makethecut,justabovethethirdpairofleaves,inordertokeep alongenoughinternode.Itisonly3–4cm long,hencetheterm “mini-cutting”tosignifytheminiaturizationoftheplantmaterial comparedtothe20cm-longconventionalcuttings.
2.2.2. Rootingandhardeningconditions
Therootingprocessconsistedofinsertingthemini-cuttingsinto trays(Fig.1C)filledwithsubstratecomposedofamixtureof30% sandand70%commercialpeatofthePeatmosstype(Pro-mix, Pre-mierTechLtd,Canada).Thetrayswerethenplaced inweaning greenhousesunderaplastictunnelwithupto95%RHanda tem-peraturerangingfrom18to20◦Catnightto25-30◦Cduringthe day.Theplasticwasremovedgradually5–6weekslater.
Aftera6-weeklongweaningperiod(Fig.1D),therooted mini-cuttingswereplacedfor4–5weeksunderhardeningconditionsat anight/daytemperatureof16–18/20–25◦Canda60–80%RH.After thehardening,therootedmini-cuttingsreachinganaverageheight of8–10cm(Figs.1E,1F)weretransferreddirectlyinto1.5lpolybags (Fig.1G)intraditionalcoffeeseedlingnurseriesfor4months–the timeneededfortheplanttobedevelopedenoughi.e.25–35cmtall tobefieldplanted(Fig.1H).
2.3. Evaluationofthegenotypiceffectontherootingcapacityof mini-cuttings
Thepilotproductionof225,000mini-cuttingsderivedfromSE (15weeksaftersomaticembryosweregerminatedandweanedin thegreenhouse)wassubjectedtotheprotocoldescribedaboveand rootingrateswerethenassessed.Rootingcapacitywasevaluated on225,000cuttingsofH1(T5296×RumeSudan),H3(Caturra×Et 531), H5 (T5296×Et06) and H10(T5296×Rume Sudan) clones (between50,000to60,000cuttingsforeachclone)byobserving thepresenceoftheadventitiousrootsatthebottomofeach mini-cutting,andcalculatingtheratebetweenthenumberofcuttings initiallysetintraysandthenumberofrootedplantstransferredto bagsinnurseriesafter10weeksoftherootingprocess.
2.4. Rootingcapacityofmini-cuttingsdependingonthe SE-derivedplantletage
Toestablishtheoptimumageforsomaticembryo-derived nurs-eryplantletstoproducerootedmini-cuttings,mini-cuttingswere producedfromsomaticseedlings atregularintervalsovera 40-weeknurseryperiodandtestedfortheirrootingcapacity.Todo this,threereplicationsof50mini-cuttingswerecarriedoutwith theH1hybridcloneforeachofthefollowingperiodsoftime:10, 15,20,25,30,35and40weeksaftersowingthesomaticembryos underexvitroconditions.Therootingresponsewasassessed10 weeksafter plantingthegeneratedmini-cuttings bynotingthe presence/absenceofrootsandmeasuringthelengthofthemain roots.Plantswithrootsupto3cminlengthwereconsideredas rooted.
Fig.1.Setting-upofanexperimentalrootedmini-cuttingvegetativepropagationprocessfromrejuvenatedsomaticembryogenesis-derivedplants.A:15-weekrejuvenated
plantsderivedfromthesomaticembryogenesisprocesssowninaplasticbox;B:Cuttingsfromsomaticseedlings;C:Plantingofthemini-cuttingsinplugtrays;D:Rooted
mini-cuttingsobtainedafter6weeksofacclimatizationinthegreenhouse;E:8-weekrootedmini-cuttingsinplotsubstrate;F:8-weekbarerootedmini-cuttings;G:3-month
rootedmini-cuttingshardeninginthenursery;H:6-monthrootedmini-cuttingsinnurserybagsreadytobeplantedinthefield.
2.5. Propagationratesachievedovera6-monthperiodthrough successivemultiplicationcycleswithrootedmini-cuttings
Aninitialpoolof26,000somaticseedlings(H1andH3hybrids) underwentsuccessiveserialmini-cuttingcycleswithinaperiodof 6months.Thenumberofcuttingsobtainedandtherootingrate ofthecuttingsweremonitoredthroughoutthisperiodaftereach mini-cuttingcycle.Inordertomaintainandtoprolongthejuvenile status,alltheplantsproducedfromtheinitialpoolwerekeptinthe greenhousewithstrongmineralnutrition,andintotalconfinement forthedurationoftheexperiment.
2.6. Qualitativecomparisonofplantsderivedfromsomatic seedlingsvsfromrootedmini-cuttingsatnurserystageand5 yearslaterinthefield
i)Inthegreenhouse:thisstudywascarriedoutundercommercial productionconditionsattheendoftheacclimatizationprocess, i.e.fifteenweeksaftersowingplantlets(somaticembryosand rootedmini-cuttingsrespectively).Thequalityoftheplantlets from batchesof somatic seedlings was compared to that of rootedmini-cuttingsundersimilargreenhouseconditions.Two hundredplantsderivedfromsomaticseedlingswerecompared
to200plantsderivedfromrootedmini-cuttingsafter15weeks’ weaninginthegreenhouse.Fourreplicationsoffiftyplantseach werecarriedoutfortheH1hybridandwererandomlychosen ineachtypeofcontainer,i.e.plasticboxesforsomaticseedlings orplugtraysforrootedmini-cuttings.Theplantbatcheswere assessedbystudyingthevariabilityinplantandrootsizes,and stemandrootdiametersandfortheiruniformgrowth. ii)Inthefield:H1hybridplantsderivedfromsomaticseedlings
and rootedmini-cuttingswereplantedin2010in Nicaragua (Cumplidafarm,Matagalpa)tocarryoutafirstagronomic eval-uationoftheconformityofthematerialproducedinbothways. Twohectaresofeachmaterialwereplantedandphenotypically observedin thefield (8000plants),five years afterplanting. Inthetwoparcels,100plantsderivedfromsomaticseedlings werecomparedto100plantsderivedfromrootedmini-cuttings andparameterssuchasplantheight,stemdiameter,internode number,primarybranchmortality,andprimarybranchlength weremeasured.Moreover,amongthestudiedplants,10plants ofeachmaterialshowingthesamevigor(withthesamestem diameter)wereuprootedtocomparetheroot systems.Root diameter,plantheight,mainrootlength,rootfreshweightand stemfreshweightwereassessed.
180 F.Georgetetal./ScientiaHorticulturae216(2017)177–185
Table1
Somaticembryo-to-plantletconversionrateandaveragedurationforplantletconversionasobtainedfromproductiondataintheframeworkofthecommercialdissemination
ofCoffeaarabicahybridsinNicaragua(ExportadoraAtlanticS.A.,ECOMgroup,datafrom2010to2012).Theplantletconversiondurationvaluesaremeans±SD.
Hybridclones No.ofembryossowed Embryo-to-plantletconversion(%) Durationforplantletregeneration(weeks)
H1(T5296×RS) 3478141 35±3.5 30±7 H3(Caturra×ET531) 1454886 36±2.8 34±10 H10(T5296×RS) 78543 56±4.9 44±12 H16(T5296×ET01A1) 142964 41±2.2 18±1 H17(Catuai×ET59A2) 264344 31±3.6 16±2 Otherhybrids 223029 33±2.8 19±4 Total/Average 5641907 37±3.5 30±11
2.7. Productioncostsofrootedmini-cuttingscomparedto
somaticseedlingsandseedlings
The production costs for plants ready to be field planted
(around30–40cmheight)regeneratedfromdifferentmaterials,i.e.
seedlings,somaticseedlingsandrootedmini-cuttings,were
calcu-latedintheproductionunitsandnurseriesofExportadoraAtlantic
S.A(ECOMgroup,Nicaragua)thencomparedwitheachother.The
costswerecalculatedin2014fromthepilotproductionof300,000
rootedmini-cuttingsderivedfromaninitialpoolof20,000somatic
seedlingsingreenhousesandtransferredtonurserybags,200,000
Caturraseedlingsinnurserybags,3millionsomaticembryosfrom
H1andH3hybridsproducedintheExportadoraAtlanticS.A.
labora-toriesand650,000somaticseedlingsofbothhybridsacclimatized
inthegreenhouseandthentransferredtonurserybags.
2.8. Statisticalanalysis
MostofdatawereanalyzedbyANOVAfollowedbytheFisher
LeastSignificanceDifferencetest(LSD).Varioussmalllettersinthe
tablesindicatesignificantdifferencesforeachparameterbetween
means(P≤0.05).Thedataarethemeansofmeasurement±SDon
independentsamples.Thebarsrepresentthestandarddeviations
(SD).
3. Resultsanddiscussion
3.1. Theembryo-to-plantletconversionstepisamajorbottleneck
inthecoffeeSEpropagationprocess
Theembryo-to-plantletconversionstepinthegreenhousefor
cotyledonaryembryosisthetrickieststageoftheSEpropagation
procedure.Atpresent,thisstageisamajorbottleneckin
indus-trialtermsas,forallthegenotypesstudied,onaverageonly37%
ofembryosregenerateplantletssuitableforsubsequenttransfer
tonurserybags(Table1).Inaddition,theaveragetimetakento
weanandacclimatizesomaticembryosisalsotoolongand con-firmstheinefficiencyofthisstage.Onaverage,ittakes30weeksto harvestalltheplantletshavingreachedtheexpectedsize.In addi-tion,plantletconversionandgrowthisasynchronouswithinthe samebatchofplantsandfourtofivesuccessiveharvestingrounds areoftenneededtoselectalltheplantletssuitableforthefollowing stagesinthenursery(datanotshown).Thisheterogeneousgrowth, whicharisesfromtheinvitrogerminationstageinabioreactor, complicatesmanagementofthetechnicalproceduresandentails extramanpower,eventhough,intermsofdevelopment,mostof theplantshavecaughtupwithseedlingsbytheendofthenursery stage(Barry-Etienneetal.,2002;Menéndez-Yuffáetal.,2010)
Industrialorpre-industrialapplicationofSEcurrentlyinvolves onlya fewwoodyspecies, suchas Douglas-fir(CellFor,Canada, http://www.cellfor.com/),whiteandSitkaspruces(Arborgen,USA, http://www.arborgen.com/),radiata,loblollyandslashpines (For-estGenetics, New Zealand, http://www.forest-genetics.com/) in thegymnosperms,alongwiththehybridyellowpoplar
(Interna-tionalPaper,USA,http://www.internationalpaper.com/),oilpalm (FelpaInc.,Malaysia,http://www.felpaglobal.com/),C.canephora (AgromodSA-deCV/Nestlé,Mexico,http://www.agromod.mx/),C. arabica(CIRAD/ECOM,Mexico,CostaRicaandNicaragua, http:// www.ecomtrading.com/) and Cocoa (Nestlé, Indonesia, http:// www.nestle.co.id/ina/) in the angiosperms. Despite these few examplesforwhichannualproductionnowexceedsafew hun-dredthousand,orevenafewmillionplants,mostofthetimethe SEpropagationproceduresforwoodyplantsarenotcost-effective astherearemanytechnicalobstacles.Thequalityofregenerated somaticembryos,theirlowconversionratesforexampleincocoa (Guillouetal.,2014)andincoffee(thispaper),andtheincidence ofsomaclonalvariationsontheconformityofthebananaandoil palmplantsproduced(Côteetal.,2000;Jaligotetal.,2011)are majordrawbacks.
3.2. Genotypeeffects
Fig.2showsthatrootingcapacityofmini-cuttingsundernursery conditionswasupto90%with15-week-oldsomaticseedlings.The genotypiceffectisoftenhighinvegetativereproductionprocesses (Lardetetal.,2009;BongaandDurzan,1987;Celestinoetal.,2013). Here,withmini-cuttingsharvestedfrom10-to25-week-oldyoung somaticseedlings,nogenotypiceffectwasfoundinthevarious hybrids’responsesforrootingcapacity.
3.3. Evidenceofrejuvenationinsomaticseedlingsthrough transientlyrecoveredrootingcapacityofArabicamini-cuttings
TheabilityofC.arabicamini-cuttingstorootwaslinkedtothe juvenilenatureoftheplantfromwhichthecuttingwastaken.In akinetic study,we tookacloserlookatthis phenomenon.The rootingcapacity of mini-cuttingstakenfrom C.arabicasomatic seedlingsdeclinedwiththeincreasingageofthesomaticseedlings fromwhichtheyhadbeencollectedinthenursery(Fig.3).Thisis particularlyobviousbetween25–35-week-oldsomaticseedlings withcorrespondingrootingratesof80–20%,respectively.When mini-cuttings were harvested from 35 to 40-week-old nursery plants,rootingcapacitywasstabilizedataround15–20%.The tran-sientrootingcapacityofmini-cuttingsderivedfromcoffeesomatic seedlingswasprobablylinkedtotherejuvenationprocessresulting fromthesuccessivecelldedifferentiation/re-differentiation mech-anismsoccurringduringtheonsetoftheSEprogramme.
Formanywoodyspecies,maturetreesarerecalcitrantto vegeta-tivepropagation.Greenwoodetal.(2001)showedthatthedecline inadventitiousrootingcapacityinforesttreecuttingsisgenerally oneofthemostdramaticeffectsofthematurationprocessandisthe mostwidelyusedmorphologicalandphysiologicalmarkertoassess maturation.Somaticembryogenesisinvitroisthemostefficient methodusedforinducingrejuvenation(Pierik1990;Carronetal., 1995;Perrinetal.,1997).Forexample,43–55%rootingfrequencies wereobtainedininvitroshootsfromtwoH.brasiliensisgenotypes regeneratedfromsomaticseedlings,whereasitwasimpossibleto initiaterootinginshootsobtainedfromthematureplantmaterial
Fig.2.Evaluationofmini-cuttingrootingcapacityofseveralC.arabicaF1hybridclones.Rootingcapacitywasevaluatedon50,000to60,000plantlets/clonebyobserving
thepresenceofrootsatthebottomofeachplanttenweeksafterrootinginduction.Rootingcapacityvaluesaremeansof50,000measurements±SD(standarddeviation).
ThebarsrepresenttheSD.
Fig.3.Evolutionofrootingcapacityinmini-cuttingsdependingontheageofthesomaticseedlings(i.e.growthdurationinnursery)fromwhichthemini-cuttingsweretaken.
Thekineticswereperformedovera10–40-weekperiodofsomaticseedlingdevelopmentinnurserybags.Eachpointrepresentsanaverageof150values(3replicationsof
50mini-cuttings)±SD.
(Lardetetal.,2009).Aginginplantswasfoundtoinducecyto-and histomorphologicalchanges andmodifycertainmetabolic path-ways inSequoiadendron giganteum(Mankessietal.,2011).DNA methylationplaysakeyroleinthevariousmaturationalchanges observedinplantsduringtheirontogeneticdevelopment–the pre-vailinghypothesisis thatduringhigher organismdevelopment, genomicDNAbecomesmoremethylated,resultinginthe modifica-tionortheswitchingonandoffofthegeneexpressionresponsible forthevariationinmaturationaltraitsfound(FinneganandKovac, 2000).Forexample,morepercentageofDNAmethylationisfound fromneedlebasesofmaturePinusradiatathanjuvenileones,to thetuneofrespectively60vs30%(Fragaetal.,2002).Recently, differencesingeneexpressionwerefoundbetweenjuvenileand adultmaterialfortheappletree(Gaoetal.,2014).Incoffee,we usedSE-inducedrejuvenationtoinitiateaHRMCpropagation pro-cess.Weshowedthatthetimingforimplementingthispropagation mustbeveryaccurateifrootingistobesuccessful,reliableand highlyefficient. The“actionwindow” is veryephemeral during earlyplantdevelopment– approx. between10–25 weeksafter plantingsomaticembryosinthenursery.
3.4. Possibleserialmultiplicationcycleswithrooted mini-cuttingsprocess
Startingfromapoolof26,000youngsomaticseedlings,335,844 rootedmini-cuttingswereproducedinthegreenhousewithina periodof6monthsofcultivation,i.e.amultiplicationrateof14 (Fig.4).Rootingcapacityduringtheserialrootedcuttingcyclesover the6-monthperioddidnotdecreaseandremainedupto90%.We observedthatthejuvenilestatusoftheyoungmini-cuttings pro-ducedcouldbeprolongedformorethan6monthswithoutanyloss ofrootingcapacityintheplantsbyinfluencingplantgrowthand forcingconditionsinthenursery,throughsuccessivecyclesof mini-cuttings,whilekeepingtheplantsconfinedwithupto90%RHand atahightemperature(rangingfrom20to25◦Catnightto30–35◦C duringtheday).Whenextrapolatedoveraperiodofoneyear,the multiplication rateshouldbearoundthirty-seven mini-cuttings fromoneSE-rejuvenatedplant.Similarcombinedtwo-step propa-gationprocesseshaverecentlybeenestablishedinconifers.Forest GeneticsLtdandArbogeninNewZealandannuallyproduceabout 50,000P.radiatasomaticseedlingswhicharesubsequentlymass
182 F.Georgetetal./ScientiaHorticulturae216(2017)177–185
Fig.4.AmplificationrateofArabicacoffeehybridsthroughserialrootedmini-cuttingprocess.Westartedfromaninitialbatchof22,614rejuvenatedsomaticseedlingsand
serialcuttingsweretakenperiodicallyfromitwithina6monthsperiod.Duringtheexperiment,plantsweremaintainedunderconfinedconditionsinthegreenhouse.The
experimentwascarriedoutin2013attheExportadoraAtlanticS.ACompany,Sebaco,Nicaragua.
Table2
Comparisonofseveralmorphologicalparametersbetween15-week-oldsomatic
seedlingsandrootedmini-cuttingsofH1Hybrid(T5296xRumeSudan)inthe
greenhousenursery.Morphologicalparametervaluesaremeansof200
measure-ments±SDonindependentsamples.Thecomparisonwasbasedonananalysis
ofvariance(ANOVA)betweensomaticseedlingsandrootedmini-cuttings.
Val-uesfollowedbydifferentlettersaresignificantlydifferentatP≤0.05(FischerLeast
SignificanceDifferencetest).
Morphologicalparameters Somatic seedling Rooted mini-cutting F Prob Plantheight(cm) 3.14±1.26a 3.34±0.92a 1.08 0.31 Stemdiameter(mm) 0.81±0.17a 1.41±0.29b 215.2 0.00 Rootlength(cm) 4.75±2.04a 4.15±1.29a 3.7 0.05 Rootdiameter(mm) 0.58±0.24a 0.94±0.28b 61.4 0.00
propagatedbyrootedcuttingstoproduce2.5–3millionplantsper
year(Bonga2015).InSitkaSpruce(Lelu-Walteretal.,2013), juve-nilesomaticseedlingstockisintensivelymanagedasoutdoorstock plants,whichareregularlyhedgedinordertostimulatethe pro-ductionofrootedaxillaryshoots.Thesesomaticseedling-derived donorplantsarerenewedevery5years,aftereachhasproduced 250cuttings(Thompson2014).
3.5. Evidenceofbettervigorandhomogeneityofrooted mini-cuttingscomparedtosomaticseedlings
Somatic seedlings and rootedmini-cuttings were compared forvigorandhomogeneity15weeksafterplantinginthe green-housenursery.Table2shows,forboth,thedifferentmorphological parametermeasurementson200plantssampled.Asregardsplant vigor,rootedmini-cuttingsweresignificantlymorevigorousthan somaticseedlingswithalargerstem(1.41vs.0.81mm)androot
diameter (0.94 vs. 0.58mm). As regards the other parameters, nostatisticaldifferenceswerefound.Whencomparedtosomatic seedlings,therootedmini-cuttingbatchesdisplayedgreater homo-geneityforplantheightandrootlengthdistribution(Fig.5).The majority ofplants derived from mini-cuttingswere groupedin a2–4.5plantsizerange,whereasmostofthesomaticseedlings werefoundinalarger1–5.5cmsizerange(Fig.6A).Similar differ-encesbetweenrootedmini-cuttingsandsomaticseedlingswere observedforrootlength(Fig.6B).Somaticseedlingsshowedthe widestdistributionoffrequenciesforrootlength.Mostoftheplants derivedfromsomaticseedlingshadrootlengthsrangingfrom3to 7cmlong,whereastherewasaclearpeakinthedistributionof rootedmini-cuttingrootlengthsataround4–6cm.Current stud-iesareaimedatanin-depthassessmentofgrowthcharacteristics underfieldconditionsinplantsderivedfromrootedmini-cuttings. Thefirstresultshaveshowedthat,overall,therearenosignificant differencesbetweensomaticseedlingsandrootedmini-cuttings, fiveyearsafterfieldplanting.Indeed,nosignificantdifferencewas foundformostoftheabovegroundparametersbetweenthetwo typesofmaterial(100plantsforeachmaterial),exceptforthestem collardiameter,whichwassignificantlybiggerinplantsderived fromrootedmini-cuttings,which denotes slightlygreater vigor (Table3).Asregardsrootsystemparameters,nosignificant dif-ferencewasobservedforanyoftheparametersstudiedforthetwo originsofplants(Table4).
3.6. Markedlyreducedpropagationcostsofrooted mini-cuttingscomparedtosomaticseedlings
InCentralAmerica,asix-month-oldnurseryplantderivedfrom aseedlingandreadyforplantinginthefieldhasaproductioncost
Fig.5.Morphologicalappearanceofsomaticseedlingsandrootedmini-cuttings.A:Heterogeneousappearanceofsomaticseedlings,15weeksafterdirectlysowingembryos
Fig.6. Comparisonoffrequencydistributionofplantheight(cm)[A]androotlength(cm)[B]parametersbetween15-week-oldnurseryplantsderivedfromsomaticseedlings
androotedmini-cuttingsfromtheH1hybrid(T5296×RumeSudan).Twohundredplantletsderivedfromsomaticseedlingswerecomparedto200plantsderivedfromrooted
mini-cuttingsthroughtheirdistributioninsizerangesforplantheight(cm)androotlength(cm).
ofaroundUS$0.20andthesellingpriceinthenurseriesranges fromUS$0.30–0.50(dependingonthecountryofCentral Amer-ica)whichamountstoatotalinvestmentofUS$1500–2500per hectarefora renovationcrop(5000plants/ha).Asix-month-old nurseryhybridplantpropagatedfromasomaticseedlingandready forthefieldhasaproductioncostofaroundUS$0.60andthe sell-ingpriceisaroundUS$0.80-1,whichrepresentsthreetimesthe priceof aseedling plant.Asix-month-oldhybrid plantderived fromarootedmini-cuttinghasaproductioncostofaroundUS$ 0.27,whichamountstoacostreductionofaround50%compared tosomaticseedlingsandrepresentsonly1.5timesthecostofa tra-ditionalseedling.GiventheearlinessofF1hybridproduction,the
lowplantationdensity(4000plants/ha)andtheirhigher produc-tivity(upto20–40%)(Bertrandetal.,2005,2011),theadditional investmentcomparedtothatoftraditionalseedlings(estimatedat US$1500)israpidlypaidbackthreetofouryearsafterplanting.A coffeeplantationhasalifecycleusuallyrangingfrom15to20years. But,thesomaticseedlingsellingpriceofUS$0.80–1forhybrids, representsanimportantbottleneckforthelarge-scale commer-cializationofF1arabicahybrids.ByusingtheHRMCmethod,we estimatedthatC.arabicahybridplantscouldbesoldbetweenUS$ 0.65–0.70,whichwouldbemoreaffordableforfarmers.
Veryfewstudieshaveprovidedinformationaboutthe produc-tioncosts ofplants derived fromSE. In anycase, based onthe
184 F.Georgetetal./ScientiaHorticulturae216(2017)177–185
Table3
Comparisoninthefieldbetweenaerialpartsofsomaticseedlingsandrootedmini-cuttingsofH1Hybrid(T5296×RumeSudan),fiveyearsafterplanting.Measurementswere
madeon200plants(100foreachmaterial)andplantswerechosenrandomlyfromthefourhectareparcel.Thecomparisonwasbasedonananalysisofvariance(ANOVA)
betweensomaticseedlingsandrootedmini-cuttings.ValuesfollowedbydifferentlettersaresignificantlydifferentatP≤0.05(FischerLeastSignificanceDifferencetest).
Morphologicalparameters Somaticseedling Rootedmini-cutting F Prob
Stemdiameteratcollar(mm) 40.6±5.3a 43.3±3.2b 6.5 0.01
Plantheight(cm) 210±2a 214±2.2a 1.54 0.21
No.ofplantinternodes 38.5±4.9a 40.4±4.9a 2.75 0.10
Primarybranchlength(cm) level10fromtheapex
45.1±8.7a 45.5±9.1a 0.03 0.85
No.ofprimarybranchinternodes level10fromtheapex
11.8±2.5a 13.0±2.8a 3.64 0.06
Primarybranchlength(cm) level15fromtheapex
59.2±6.7a 57.2±9.2a 0.92 0.34
No.ofprimarybranchinternodes level15fromtheapex
15.0±1.5a 15.8±2.2a 0.00 0.92
Primarybranchmortalitypercentage(%) 12±4a 11±5a 0.81 0.36
Table4
Comparisoninthefieldbetweenrootpartsofsomaticseedlingsandrootedmini-cuttingsofH1Hybrid(T5296×RumeSudan),fiveyearsafterplanting.Measurementswere madeon20excavatedplants(10foreachmaterial).Forthestudy,10plantsofeachmaterialwherechosenwiththesamevigor(diameterstem)andthenextractedfromthe ground.Thecomparisonwasbasedonananalysisofvariance(ANOVA)betweensomaticseedlingsandrootedmini-cuttings.Morphologicalparametervaluesaremeansof 10measurements±SDonindependentsamples.ValuesfollowedbydifferentlettersaresignificantlydifferentatP≤0.05(FischerLeastSignificanceDifferencetest).
Morphologicalparameters Somaticseedling Rootedmini-cutting F Prob
Stemdiameteratcollar(mm) 40.0±1.7a 40.8±1.4a 0.71 0.42
Plantheight(cm) 210±0.2a 197±6.7a 0.05 0.52
Rootdiameter(mm) 49.9±5.3a 51.4±4.9a 0.22 0.64
Mainrootlength(cm) 31.2±3.6a 31.2±4.4a 0.00 1
Freshbiomassstump(kg) 0.5±0.1a 0.5±0.1a 1.11 0.32
Freshbiomassaerialpart(kg) 2.6±0.2a 2.7±0.3a 0.46 0.51
limiteddataavailable,theyareallcurrentlytoohighto
commer-ciallybeacceptable.Inloblollypine,theproductioncostsofsomatic
seedlings(US$0.30–0.40)werebetween7.5–10timeshigherthan
thoseofplantsconventionallyproducedbyseedlings,i.e.US$0.05
(Bettingeretal.,2009).InSitkaspruce,somaticseedlingswere esti-matedtocostbetweenD 2–3/plantcompetingwiththeD 0.25 foraseedlingtransplant(Thompson,2014).Hence,forthesetwo species,thecostofa somaticseedlingisabout10timesthatof aseedling.Now,ifSitkasprucesomaticseedlingsaregrownfirst innurserybedstoproducecuttingsforrooting,thecostofSitka sprucerootedcuttingsdecreasestotwicethecostofaseedling. Seedlingandplantingahectareofrootedcuttingsthusbecomes economicallymorerealistic.
Asregardscoffee, Carvalhoet al.(2013)studiedthe produc-tioncostsof400,000plantletsofC.arabicaproducedbySEand reportedacostof US$0.37/plant,whichisquitedifferentfrom thecostobtainedinourstudy(US$0.60).Recently,aninteresting comparativecoststudywasdoneinC.canephorabetweenplants producedbySEandtraditionalrootedcuttingsmaturetreesfrom clonalgardensunderfieldconditions(AlvesdosSantosetal.,2015). ThecostofasomaticseedlingwasevaluatedatUS$0.23whilea rootedcuttingatUS$0.12,whichisapproximatelyhalfofthecost weobtainedfortheC.arabicaF1hybrids.
4. Conclusion
Thisstudyshows that HRMC canefficiently complement SE for producing arabica hybridsin much larger quantitiesand at cheapercost.Wedemonstratethatthelowbiological productiv-ityobservedfortheSEprocedures–particularlyduringthelate stagesofplantletconversion–canbelargelycompensatedby high-yieldinghorticulturalpropagationinthenursery.Plantsderived fromHRMCaremoreuniformandvigorousthansomaticseedlings. Weshowthatthiseffectpersistsforseveralyearsafterplanting out.Subsequently,incoffeethecommercialvegetative propaga-tionofhybridplantsshouldbeenvisagedasfollows:laboratories stillremainkeytoproducingtherejuvenatedmaterial,theexvitro
rootedmini-cuttingtechniqueisoutsourcedtothebest profession-alsinornamentalcropnurseriesandtheacclimatizationisdone bycurrentnurseriesorbyfarmersthemselves,whoareusedto doingexactlythatfromseedlings.Themethodologypresentedin thispapercouldbeextendedtotheothercropswheretheSE propa-gationprocessisstillnotcost-effectiveandthusexpandthegeneral useofSE.
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