Acta Hortic. 1139. ISHS 2016. DOI 10.17660/ActaHortic.2016.1139.80 Proc. III Balkan Symposium on Fruit Growing Eds.: D. Milatović et al.

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Development and extension of walnut propagation in Iran K.VahdatiaandM.AalifarDepartmentofHorticulture,CollegeofAburaihan,UniversityofTehran,Tehran,Iran.Abstract

Persianwalnut(Juglans regiaL.)iswidelygrowninIran,asthecenteroforiginof this tree.Propagationof thisspecies isverydifficultcompared toother fruitandnut trees. Different types of grafting (epicotyl grafting, side stub grafting, omegagrafting,whipandtonguegrafting,saddleorVgrafting,patchbudding,chipbudding,shieldbuddingand topworking)and tissueculture(propagationbymicroshootsorsomaticembryogenesis)techniqueshavebeenstudiedintensivelyinthelast25yearsin Iran. In these studies,effectofgrafting typeand timeunderdifferentconditions(greenhouse, outdoor and shade house) on callus quality, graft-take, survival andgrowthofthescionshavebeenstudied.Severalexperimentshavebeenconductedonoptimizationofmicro-propagationstepssuchasestablishment,proliferation,rootingandacclimatizationofwalnut.Successfultechniqueshavebeentaughttothegrowersandagriculturalexperts.Asaresultoftheseefforts,severalwalnutgraftingnurseriesand tissueculture labshavebeenestablishedwhichpropagatewalnutcultivarsandrootstockscommerciallyforlocalandinternationalmarkets.

Keywords:Juglansregia,grafting,budding,micropropagation,topworkingINTRODUCTIONThe origin of common walnut (Juglans regia L.) is found in Persia about 2000 BC(Cociuetal.,2007).Persianwalnuts(J.regiaL.)arewidelygrowninAsiaandIran(Vahdati,etal.,2004).Iranwasrankedasthesecondlargestwalnutproducerin2012byproducing450,000t(FAO,2015).Thepreviousstudiesdemonstratedthatsexualpropagationmethodsof walnut had undesirable results. Persian walnut is propagated by seedlings in severalcountries including Iran.Vegetative propagation inwalnut ismoredifficult in comparisonwithotherfruitandnuttrees(OzkanandGumus,2001).Walnutvegetativepropagationbycuttingsisverydifficultduetotheirlowrootingability.GRAFTINGANDBUDDINGOFWALNUTDifferentmethodsofwalnutpropagationarebeinginvestigatedallaroundtheworldand one of the best methods to propagate Persian walnut is the use of graft techniques.Walnut grafting successwas reported to be affected by graft techniques (Mitrovic, 1995).Graft technique types including epicotyl grafting, side stub grafting, omega grafting,whipandtonguegrafting,saddleorVgrafting,patchbudding,chipbudding,shieldbuddingandtopworkingandthesetechniqueswereexaminedbyvariousresearchers(AchimandBotu,2001;Avanzato,2001).However,thereisnotacommonopinionworldwideonthechoiceofacertaingraftingmethod.Graftingsuccessinwalnutisaffectedbymultiplefactorsincludingtime and method of grafting (Solar et al., 2001; Tshering et al., 2006), temperature andhumidity(Barut,2001;Avanzato,2009),phenoliccompounds,hormonalcondition,choiceofcultivarsandrootstocks(Mitrovicetal.,2008),andtimeoftakingthescions(Paunovicetal.,2012).BenchgraftingisonetheclassictechniquesforpropagationortopworkingofPersianwalnuts (J. regia). Scientific literature reports different data on the efficiency of thesemethods in different countries. The most popular bench grafting methods include cleft, aE-mail: kvahdati@ut.ac.ir

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whip, omega, saddle and side grafting. The previous studies showed that different benchgraftingmethods had significant different effects on callus quality, graft take and graftingsurvival. Inastudy in Iran, theresultsdemonstratedthatomegamethodhadbestgraftingsuccessamongallcombinationsinfourwalnutcultivars(‘Hartley’, ‘Pedro’, ‘Serr’and ‘Z53’)(Dehghanetal.,2009). Inthisstudy,omegagraftingshowedthehighestcallusquality(2.5from 4), high graft take (67.77%) and grafting survival (84.33%) as well as high sciongrowth (12.9 cm) followedby side stubandwhipgrafting.Asdemonstrated in this study,percentageofgraft takeshowsahighlypositivecorrelationwithcallusqualityatdifferentgraftingmethods.Thesameresearchersinanotherstudyshowedthatomegagraftinggavethe highest callus rating (scoring 2.6 out of 4.0), the greatest number of callused plants(82%),themostgraft-take(71%),andthehighestgraftsurvivalrate(81%)incomparisonwithwhipandsidegrafting (Dehghanetal.,2010). Inanotherstudy, theeffectofgraftingtype(cleft,whipandbarkgrafting)ongraftingsuccesswasperformed(Rezaeeetal.,2008).Inthisstudy,thepercentofgrafttake,threeweeksaftergrafting,forbothgrafting(cleftandwhipgrafting)methodsusedinthepreliminarytrialwas80%.However,manyscionsfailedto continue to grow and did not survive one year after grafting. Percentages of graftingsurvivalwereabout37%incleftand0%inwhiptonguegrafting.Thisfailuremaybearesultofhighrootpressureofwalnuttreesinearlyspringorwinterfrostdamageduetoweakortoo fast growth of scions. In contrast, themodified bark grafting performed atmid-Aprilshowed the highest grafting success (100%) and survival percentage (96.3%).During therecentyears,hypocotyl grafting techniquewas introduced inpractice (VahdatiandZareie,2006;GandevandDzhuvinov,2006).Inthismethod,graftingtakesplaceduringvegetationwithgrowingtipofsoftwoodcuttingoncleft.Inthesameline,theresultshowedthatside-stubwasbetterthanhypocotylgraftsandgraftingsuccessinside-stubandhypocotylgraftswas98and70%,respectively(VahdatiandZareie,2006).Inthesamefield,Soleimanietal.(2010)evaluatedtheeffectoftwograftingmethods(omegaandsaddle)with twohotcallusingsystems includinghotcallusing inroom(HCR)and modified hot callusing cable (MHCC) on grafting success and loss. The resultsdemonstratedthatusageofMHCCsystemresultedinthehighestgraftsuccess(63.3%)andlowergraft loss(28.1%)comparingwithHCRsystemwith56.7%successand36.4%loss.Basedontheresults,Soleimanietal.(2010)announcedthatgraftsuccesswashigherusingsaddlemethod(87.7%)thanomegamethod(38.3%).Also,graftlosswaslowerwithsaddlemethodthanomegamethod(19and45.5%,respectively).Suk-Inetal.(2006)reportedanewandrapidapproachforwalnutpropagationcalledepicotyl grafting. They demonstrated that the survival rate was 78.9% in average whenapplyingthattechnique.Inthesameline,astudycarriedouttoevaluatetheeffectsofscionproperties on success ofwalnut epicotyl grafting and early growth of the grafted trees inIran (Raofi andVahdati, unpublished).Authors stated that scionsobtained fromapex andincluding two buds,with 3-6mmdiameter and 7-12 cm length produced a better callus,moregraftingsuccessandsurvivalrate,andhighershootlengthandleafnumbertree-1.BuddingisoneofthepopulartechniquesforPersianwalnutspropagationinIran.Themostimportantbuddingmethodsarepatch,I-shieldandchipbudding.Patchbuddingisoneof the oldest and popular techniques for propagation in a nursery in outdoor condition(KuniyukiandForde,1985).InastudyinNieriz,Farsprovince,Iran,Ebrahimietal.(2007)stated that the highest success rate was obtainedwith patch (91.0%) followed by shield(31.1%)andchip(19.1%)budding.Patchbuddingshowedthehighestcallusformationandscion growth followed by shield and chip budding. Results obtained in this studydemonstratedthatpercentagesof“bud-take”wereaffectedbydifferentmethodsofbuddingundergreenhouseandfieldconditions.Otherstudieswereconductedtodeterminethebestmethod for improving the quality and productivity of walnut trees through topworkingduring2001-2006,inKahrizAgriculturalResearchStationlocatedinthenorthwestofIran(Rezaeeetal.,2014).Inthisstudy,patch,andI-shapedbuddingwasdoneafterbarkslipping(April16-20)butchipbuddingwascompletedbeforebarkslipping(March25and26)usingshootswith1.5to2cmindiameter.Thepercentofgrafttake,threeweeksaftergrafting,forallofthebuddingmethods(chip,patchandI-shaped)usedinthepreliminarytrialwasvery

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high (73 to 80%) but these methods produced no scion survival. Pirkhezri et al. (2010)evaluatedtheeffectsofIndole-3-butryicacid(IBA)andgibberellicacid(GA3)onincreasingthe efficiencyof patch budding in four cultivars andone genotypeofPersianwalnut. Theresultsshowedthat ‘Chandler’hadthehighestresponsetoGA3100ppmandIBA50ppm,‘Pedro’ to IBA 50 ppm, while in ‘Z63’, IBA 100 ppm had the best effect. Based on theseresults,itwasassumedthatsuitablehormonetreatmentcouldovercomeapartofproblemsrelatingtolowlevelofgraftsuccessinPersianwalnut.TimeofgraftingisoneoftheimportantfactorsingraftingsuccessofPersianwalnut.Gandev and Arnaudov (2011) showed that time of grafting influenced the success ofpropagation and epicotyl graftingmethod. Suk-In et al. (2006) reported that the survivalrateranges from65.0to85.5%dependingonthetimeofpropagation. In thesamefield,astudybyRezaee et al. (2014)wasperformed to determine themost suitablemethodandtime for topworking of the inferiormaturewalnut trees in northwest of Iran. The resultsshowedthatthebesttime(inKahrizAgriculturalResearchStationlocated innorthwestofIran)formodifiedbarkgraftingwasmid-April,afewdaysafterbudburst.Inanotherstudy,Aminzadehetal.(2013)statedthattimeofgraftinghassignificanteffectongrafttakeandgrafting survival and they determined that the most suitable time for minigrafting isFebruary with 83.24% grafting success compared to grafting performed at other times(earlyJanuary,MarchandearlyMay).EbadiandSolagi(2002)statedthatthebesttimeforsideandsaddlegraftinginPersianwalnutislateDecembertolateJanuary.Efficiencyoftwomethodsofbuddingincludingpatchandchipbuddinginthreetimes(September,MarchandApril) were tested by Vahdati et al. (unpublished). The results of this study showed thatmaximum success for patch budding and chip budding were recorded in the late April(68.75%)andlateSeptember(55%),respectively.Kindofgraftunioncoveragecanbeanotherimportantfactorforwalnutpropagationinoutdoorcondition. In thesameway,astudywascarriedout tounderstandtheeffectofkindofgraftunioncoverage(whitepolyethylenetape,bicycletube,plaster(adhesivetape),sawdust,plaster+sawdust)ongraftingsuccess.Accordingtotheresults,thehighestrateofcallusing (4.6 from5), graftingsuccess (65%),andsciongrowth (91.25cm)wasobtainedthroughcoveringofgraftunionwithplaster+sawdust(RezaeeandVahdati,unpublished).Another study was performed to understand the effect of kind of graft union coverage(cocopeat + soil + manure and sawdust) on graft take success. The results revealed thatcoveringbysawdustwasbetterthanmixedcoverage(cocopeat+soil+manure)andcontrolintermsofcallusformation,graft-take,survivalandgrowthofthescion(SadeghiMajdandVahdati,unpublished).Rootstock and scion genotype significantly influenced the percentage of successfulbenchgraftingandbudding.Genotypes/cultivarsshoweddifferentialresponsetografttakesuccess (Ferhatoglu,1997;Erdogan,2005). Studies conducted in Iranhaveconfirmed thisentry (Ebrahimi et al., 2007; Soleimani et al., 2009; Dehghan et al., 2009; Pirkhezri et al.,2010). For example, Soleimani et al. (2009)marked a clear effect of rootstock and sciongenotypeonpropagationofPersianwalnutandthegrafttakewasalsosignificantlydifferentamongthecultivars.Theresultsofthisstudyshowedthatgrafttakewashigheronoffspringof ‘Serr’ (65.8%) in comparison with ‘Z63’ (46.7%). On the other hand, there was aninteractioneffectconsideringbothfactors(rootstocksandscions)sothatallcultivarsexcept‘Z63’,showedbetterresultson‘Serr’rootstocks.MICROPROPAGATIONOFWALNUTMicropropagation is the best method for propagation of all plants in a large scale.Micropropagationcanbeusedforlarge-scalemultiplicationofcultivarswithdesirabletraitsand theproductionof healthy anddisease-freeplants. Themicropropagationof J. regia ispossible via the cultivation of nodal segments, cotyledon, immature cotyledons, ovules,matureembryosandmeristems(Alyetal.,1992;Pijut,1993a,b;Longetal.,1995;Vahdatietal., 2004; Avıles et al., 2009). Progress in developingmethods formicro-propagation of J.regiahasbeenverysuccessfuloverthepastdecade.WalnutmicropropagationisoneofthemethodsstudiedforpropagationofwalnutgenotypeswithdesirabletraitsinIran.Astudy

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was carried out to root and acclimatize in vitro grown shoots frommaturewalnut trees.Rootingwassuccessfullydone inPersianwalnutcultivars (Figure1).Data fromthisstudyshowedthatthereisapositiverelationshipbetweenthevigorofcultivarsandrootingability(Vahdati et al., 2004). Vahdati et al. (2009) studied themicropropagation of some dwarfwalnut genotypes. The results revealed that number of axillary shoots rising from themicroshoots was the highest in dwarf and semi-dwarf genotypes compared to the highvigorousones.

Figure1.Successfulrootingofwalnutbymicropropagation.Plantletsgrowninconventionaltissueculturesystemsusuallyencounterphysiologicalandanatomicalabnormalities.Forcedventilationcanimprovethedevelopmentofleafwaxaswellasthesurvivaloftheplantsinvitroculture(Zobayedetal.,2001).Inthesameway,Hassankhahetal.(2014)reportedthatnaturalventilationhadasignificanteffectonmostofthegrowthindicesinPersianwalnut.Sucrose is frequently used as a carbon source at a concentration of 2-5% in plantculturemedia.Theimportanceofsucroseinmicropropagationasacarbonsourcehasbeenwell shown (Hazarika et al., 2004). It was reported that usage of sucrose with 15 g L-1producedhealthyplantlets(Hassankhahetal.,2014).Also,Vahdatietal.(2004)showedthatincreasingthesucroselevelintherootinductionmediumimprovedrooting.Mediummineralcompositionhasbeenreportedtoaffectmorphogenicresponses. Inanotherstudy,twomodifiedmedia(mDKW)wereformulatedbasedonthewalnutexplantmineral contents and growth rate of explants cultured onmDKWwas compared to thosecultured on DKWmedium. In this study it was found that no deficiency symptomswereobserved in explants on mDKW media. Also, explants grown on mDKW produced themaximumstem lengthandauxiliarybudnumber(NajafianAshrafietal.,2010).ThesameauthorsconductedastudytoevaluatetheeffectsofCuandmyo-inositolonshootlengthandrooting rateof explants.The resultsof this studyshowed that thehigher levelsofCuandmyo-inositolwouldbeaffectiveongrowthrateandrootingpercentageofwalnutexplants(NajafianAshrafietal.,2009).Astudywasperformedtoevaluatetheeffectsoftwochelates(FeEDDHAandFeNa2EDTA)withfourconcentrationofiron(45,91,182and360mgL-1)ongrowth and rooting. Results showed that the best treatment was 182 mg L-1 iron withEDDHA chelate. Increasing iron concentration to 182 mg L-1, caused better growth androoting,but increasingto360mgL-1decreasedthesefactors.AboutofFeNa2EDTAchelate,growthandrootingwaslowerthansameconcentrationinEDDHAchelate(NajafianAshrafiandVahdati,unpublished).MicropropagationofwalnuthasbeencommercializedinIranandthousands of plants are produced and delivered by this method (Figure 2). Commercialmodernorchardsarebeingincreasedusingown-rootedwalnutcultivarsunderconsultationoftheexpertsinIran(Figure3).

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Figure2.CommercialmasspropagationofwalnutbytissuecultureinIran.

Figure3. Establishmentof anewmodernorchardbyown-rooted tissue cultureplantsofwalnutinIran.Aboutsomaticembryogenesis,Vahdatietal.(2008)studiedtheeffectsofabscisicacid(ABA),onthematurationandgerminationofPersianwalnutsomaticembryos.Theresultsshowedthatthebesttreatmentforgerminationcontained2mgL-1ABAandresultedin41%conversion of embryos into plantlets. On the other hand, the importance of sucrose onincreaseofthenumberofsecondaryembryoshasbeendocumentedbyVahdatietal.(2008).CONCLUSIONSUndoubtedly, the results of research have markedly improved traditional walnutpropagationtechniquessuchasbudding,graftingandmicropropagationinIran,duringthelast decade. Based on the research results on budding,we recommend producing graftedwalnut plants using patch budding in right place and time of the year and/or undergreenhouseconditions.Amonggraftingtechniques,whipandtongandsaddlegraftingusingright wrapping methods and also hot callus systems are also promising. The clonalpropagation ofwalnut via rooting ofmicrocuttings fromoriginal plants is very useful notonly formass rapid propagation but also formaintaining valuablewalnut rootstocks andgenotypes. Walnut micropropagation techniques have been commercialized in Iran andtissuecultureplantsandknow-howforproducingthemareavailabletoexportandtransfertotheothercountries.

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