Somatic Embryogenesis in Forestry with a Focus on Europe: State-Of-The-Art, Benefits, Challenges and Future Direction

Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Marie-Anne Lelu-Walter, David Thompson, Luc Harvengt, Leopoldo Sanchez Rodriguez, Mariano Toribio, Luc Pâques

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Marie-Anne Lelu-Walter, David Thompson, Luc Harvengt, Leopoldo Sanchez Rodriguez, Mariano Toribio, et al.. Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction.. Tree Genetics and Genomes, Springer Verlag, 2013, 9 (4), pp.883-899. 10.1007/s11295-013-0620-1. hal-01268045

HAL Id: hal-01268045 https://hal.archives-ouvertes.fr/hal-01268045 Submitted on 29 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, Forest biotechnology, clonal forestry, vegetative propagation, somatic embryogenesis, tree breeding, deployment Key words (4-6) forestry. European to relationships their Finally, a strategy to promote and to the use of regulatory) this technologythe is and proposed. and presents review acceptance programmes this public improvement addition, economic, In (biological, tree discussed. challenges and presented in are application survey this its of results and The forestry. development commercial of stage EU (under its Europe embryogenesis, across conducted somatic of was state-of-the-art the survey involved, species a the end, document to this “Treebreedex”) Action To Concerted Infrastructure Research work. ongoing the of fraction small a highlight levelofactivity ofthe research andapplication ofsomatic embryogenesis of forest trees.Publications andreports only offeranypractical methods forlarge-scale commercial use.However, verydifficult is it fully to appreciate overall the both) of combineduse embryogenesisthe somatic (and and cuttings rooted vegetativepropagation only for methods describebenefits, vegetativetypeof propagation, integration its and breedinginto programmes. different the of all Of Vegetativepropagation forestof trees offers advantages bothbreeders treeto forestandthe industry. Thisreview will Abstract Fax +33 (0)2 38 41 48 09 Phone +33 (0)2 38 41 78 00 Email: [email protected] Corresponding author: 4 IMIDRA Finca El Encin, Apdo 127, 28800 Alcalá de Henares, Madrid, Spain France Cestas, F-33610d'Arcachon, route 71 Pierroton, de Bois Forêt Campus BiotechnologyLab, and Genetics FCBA, 3 Ireland 2 Coillte Teoranta- The Irish Forestry Board, Tree Improvement Section, Kilmacurra Park, Kilbride, County Wicklow, 1 INRA, UR 588, Unité Amélioration, Génétique et Physiologie Forestières, Ardon, F-45075 Orléans Cedex 2, France Somatic embryogenesis in forestry Concise and informative title: Pâques Lelu-Walter Marie-Anne Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Title 1 883-899. DOI : 10.1007/s11295-013-0620-1 1 , David Thompson David , Comment citer cedocument:

2 , Luc Harvengt Luc , nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 1 3 , Leopoldo Sanchez Leopoldo , 1 , Mariano Toribio Mariano ,

4 and Luc E. Luc and Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, - It can produce clonal material that grows at a uniform rate, making management more predictable as well as as well as predictable more both on management effect important making an have rate, can which fibre) uniform or (timber product a quality higher and uniform at more a grows producing that material clonal produce can It - accurately can values estimateclonal general when combining ability,(e.g. cycle breeding the shorten to or plasticity, phenotypic study to genotypes, evaluate better to clones use to possibility the is there breeding, tree in evaluation genetic and studies genetic For - that clonal testing increased the efficiency of testing compared with conventional seedling progeny showed testing.(2003) al. at Isik testing.progeny Environment) in (Genotypeinteractionsby than E x G environmental and selection consecutive two between cycles.interval generation the of shortenings further allowing development, of stages propagation sexual to compared gain genetic thus and methods. intensity selection enhancing further for exploited be can accessibleand readily variationtransmissible.are non-additive are of sourcesThroughVP additivetraits only where orchard that cannot be changed. seed a in material the comparedto quickly very changed be vegetatively propagatedcan being material genetic The and produce seed.As consequence, a alsoit provides greater a flexibilityin Forest Reproductive Materialdeployment. floweringproductionproduction.seedseedorchardseedflower and delay starts in to example,noa until For is there breeding programmes which include: tree improvementprogrammes attention hasturned to VP. VP hasa number of advantagesthat make usefulit a toolin potentialthe of improvement thatshould possible.be increaseeffortan to In improvementthelevels of possible from outsideseedtheorchard reduce improvement levels. Thethatseedresultorchards is typically provide only about half genetic crops, the of potential full the deliver to fail often include: reasonsThese capableof. be shouldvery improvement they that orchards seed reasons of number a For parents. both of orchardswhere selectedparents are allowedtointerbreed and produce seed whichcombine the selectedcharacteristics What are the advantages of vegetative propagation to forestry? this that ensure to done be to needs think we what technology is developedon and comment put into commercialwill use in Europe.we Finally forestry. European to relates all ofEurope,inartSE willpresent thechallenges (biological, economic, public acceptance andregulatory) andhow this produce to largeway theamounts improved of materialrequired. is thatdiscuss Nextwill current we the state-of-the- efficient most and fastest the offer propagationsystems cuttingSE/rooted hybrid and SE that show to and available both natural and sustainable. Quercus pyrenaica in exist do trees forest 1). 2009 in wasable identify to productionthe leastat 700ofmillion vegetatively propagated forest treesper year (Table VP of survey unpublished An 1993). (Zobel angiospermcuttingssomatic(usingrootedembryogenesis,coniferandspecies andcarried out bothtree forest of SE) uses end of specific range for selected a clones of with plantations planted commercial been large have species recently More 1999). Ritchie and (Minghe years 800 last ( Chinese cuttingsfir rootedChina of In 1974).(Toda years 400 ( Japanesecedar cuttingsof Rooted idea.new a not forestry is in VP of uniquecombinationuseindividual Thegenes. each having a of resultsin handleperiodslongduring to easythey are andsowing. However, sexualresultrecombinationof the seedsare which most flexible and effective way to produce enough genetically improved material to meet future wood demands.vegetativepropagation(VP) of use required.The be will improvedmaterial of amounts large deploy and propagateefficiently to ways New needed. adaptabilityis their improve and plantations new these productivityof the maximise to trees of engineered”)“geneticallyvarietiesconventional not breeding, improvedby (produced genetically of use the require will things other among which plantations, managed intensively of use the forestry(MCPFE billion2007).thishectaresOf aboutonly 48%produces timber,fuel.Clearly increase orfibre an in hectaresaboutEurope44%or of abouttotalthisarea andnaturalof 26%are forests withonly aboutplantation as 4% industrialwoodcome havewill to from planted forests (Sedjo 2004).Europe In currently forest occupy billion over1 will be met by existing natural forests and it has been sugg and the balance as “industrial wood”. It is predicted that by 2040 that world consumption of industrial wood will will wood industrial of consumption world that increase from2040 the current 1.5 billionby m that predicted is It wood”. “industrial as balance the and Introduction - It provides a powerful testing method for genetic evaluation with a more straightforward estimation of micro- of estimation straightforward more a with evaluation genetic for method testing powerful a provides It - wellsuitedselectionearlyfor is schemes, It especially- molecular of use thosethebased markers on early aloneat reproduces It uniqueexactly genetic additive- layout non-additivea (both andin terms),unlike sexualpropagation in periodicities by affected not is it because material improved of delivery stable more and faster a provides It - seed in produced typically is world the around programmes breeding tree forest most from material Improved Theobjective ofthis review firsttois discuss theuses ofVPof forest trees, then discuss to thevarious VP methods Most plants, including forest tree species, are propagated by seed because it is easy to collect, most can be stored for Currentworld wood consumption estimated is aboutbeto 3.4 billion m Vegetatively propagated forests are not as artificial as they might seem. Naturallymightseem. occurring theyVegetatively populationsartificial clonal as as propagatedof not forestsare 2) some seed results from self-pollination, which results in poor performing progeny and performingand progenypoor self-pollination,in resultsfrom resultswhich seed some (Valbuena-Carabaña et al. 2008)and several other species, demonstrating that such populations are 883-899. DOI : 10.1007/s11295-013-0620-1 Populus tremuloidesPopulus Cryptomeriajaponica Comment citer cedocument: Prunus avium 3 /yeartoabout 2.5 billion m

(Cheliak and Dancik 1982), 1982), Dancik and (Cheliak ). ) have been used to establish forest plantations in Japan for the last last the for establishJapanplantations forest in to used been have ) nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 2 Cunninghamialanceolata 3 /year. Itis very unlikelythat this increased demand 1) not all parents consistentlyparents seed contribute all to not 3 /yearwith about halfof this usedis forfuel Picea mariana mariana Picea

in forestry is the fastest, the fastest, the is forestry in (Legere and Payette 1981), Payette and (Legere ) have been planted for the the plantedfor been have ) 3)

pollen from trees from pollen

Eucalyptus Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, et al. 1995). Not all of these potential somatic embryos will develop into plants (emblings), but even if only a a only if even but propagation (emblings), method. plants into develop will embryos somatic micro highermultiplication or macro otherfasterprocess a any possible withthan by a potential rate still is percentage SE do, these of all Not 1995). al. et ( spruce Norway of culture embryogenic of between2,000potential1,000 to somatic embryos gram showingpotential the multiplication (Pâquespossiblerate that SE with one of that copies estimated exact been of has number unlimited It theoretically genotype. a produce can it developed been has culture embryogenic Pullmanand 2007).minimisingstepsthus twoSomatic or embryosonehandlingtheformed required.are in Oncean is which stem, a and tip root a similar zygoticato bud, embryo that normallyshoot occurs theindeveloping a seed (Fig. 2), takes place of theinlaboratory consisting (Carney plant miniature complete a SE, of process the In 1997). micropropagated example For inexpensive Pinusso radiata are seedlings tree that fact the to due economic, not simply are they perhapsto 10shoots percycle, depending onthe species. requirementsand thus high perplant costs. Multiplication rateslabour from axillary shoot high formation typically in range from 2 a results which conditionsenvironmental and treatments regulator growth different to transfer manual 3) plant micropropagation In species. forest for regenerationmethod propagation by organogenesis effective involves a multi stepan process in which be can situations some in cuttings, Nevertheless,rooted species. certain with practical mostly are methods these plants, stock of ageing and seed) from from including produced plant similar a material, times 1994). more or al. two costs typicallycuttings (rooted et productioncosts higher (Lambeth to species However, clonal due tree forest of establish range a in demonstrated been to has as material of forestry volumes commercial in used is physiologicallyestablishmentthe trees,old for orchardsseedof cloneand banks. rootingThecuttings produceof however, can Grafting, propagation. large-scale shoot tips)or SE. Among the macropropagationmethods both air-layeringand grafting are too slowand expensive for “micropropagation” using How is vegetative propagation accomplished? backup while field testing is on-going. diversity genetic secure a as instance possibilities,for serving new also long-termadds cryopreservationembryos of potential of revolutionising breeding schemes, both in terms of time required and the gains achieved. The The possibilitytechniques. of VP of implementation combinationthe veryof earlyselection basedin solely genome-wideon interest marker profiles stimulate andselectedVPof embryos the has to expected is 2001) al. et (Meuwissen evaluation evaluationnewcandidates.adventbest sense, of thisthethe methods In accuracies,highvery with genome-wide like accuratelyselectionaccuracies, identifying thus high provide to able improvement and are schemestesting the when the desired assemblage of traits. and lengthymating program; a settingup of needbetween selectionno shortened,deployment lag and becauseis is there reforestationafforestation releasedprogramsfor and those improvementgenetic are phasethe fromresulting geneticimprovement directly is translated intothe commercial phase with high accuracy, because samethe genotypes genotypes foruse as field planting stock.This has three potentialbenefits oversexual multiplication whichinclude: under evaluation.components genetic the of decomposition finer provides latter the evaluation, fast allows former the the While to testing. compared progeny of complementtesting a of as implementationseen be faster should testing clonal of However, progenies. possibility of performances the the via evaluation brings VP phase, first this at Therefore, be VP. potentially can which testing, progeny of through those subjected delaysproblems toevaluated to due flowering in mimic seedproduction.and in These delays absent are testingwith that throughoften environments are of genotypes range a Elite deployment. over material commercial breeding the of stability the of assessments accurate can techniques VP productive. fully hascome the ability of providingbe multiple clonal copiesto of selected candidatesplace for testing. taken VP Multiplewhere place, takes clonal testing before copies has is canphase provide first The selection 1). improvement(Fig. scheme the of after ph then it is induced to form a root and finally finally and root a form to induced is it then P icue ehd f bt mcorpgto” (i aeig rfig otn f ctig) and cuttings) of rooting grafting, layering, (air “macropropagation” both of methods includes VP It is, however, in advanced breeding programs wherethe fullbenefits of VPcan be to befully achieved, particularly valuable of copies producing of way the provides VP where candidates, of selection after is phase second The traits containsdesiredbreedingdevelopmaterial that to objectives,breedingmainfirst programmes Tree two have 3 ) selection intensity can be substantially increased, as multiplication can be concentrated on a few genotypes with plants were reported to cost more than7 times open-pollinatedseedlings (Menzies and Aimers-Halliday 883-899. DOI : 10.1007/s11295-013-0620-1 in vitro tissue culture techniques e.g. either by organogenesis (adventitious budding, axillary Comment citer cedocument:

4) to produce a complete plant. Each of the steps typically involves a typicallya involvessteps the of Each completeplant. producea to nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1

Formost forest tree species while organogenesis possible,is 3 1) a shoot bud is initiated, Picea abies Picea 2) it is then elongated,

) contains ) 2) 2) time 1) Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, differentiating embryos. The mass production of somatic embryos has been greatly improved using synchronized using improved greatly suspension cultures,been simplesttheand procedure achieve to hasthatbeenfractionation.size has 0.5gUsing these methods, embryos somatic of production mass The embryos. differentiating cultures and temporary immersion embryos systems. somatic suspensionof namely medium, liquid numbers in embryos large somatic produce produce to used been to have techniques main needed Two profitably. is medium liquid in embryos somatic of development and growth the suggested that been has However,productionit costs. the affects handlingwhich significantof amount a Laboratory propagation options e.g. transferred to soil under (somatic emblings “acclimatised” or are emblingsSubsequently, seedlings. plantlets zygotic characteristicsas young same the to possessing plants) embryo rise giving “conversion”) called also (“germination” develop roots First embryos. zygotic are counterparts which their as way same the in develop they obtained, are plants asexual as the soon germination.As synchronise and of SE lowering the of quality the the improve to or shown been “Desiccation” also gymnosperms. has SE the the of content in moisture than effective less be to appears angiosperms woody 1992;LipavskáGutmann 1996; Konrádováal. and et 2004).However, matureABA to somatic of use theembryos of al. carbohydrateset proteins,(Attree lipids, as such reservesstorage promoting accumulationof ABA with embryos natural a plantgrowth regulator foundmaturing in major seeds,of importance is maturationfor coniferousof somatic embryossomaticthat morphologically mature and physiologically of developmentresemble normal zygotic the embryos. Theuse abscisicof to acid (ABA), lead protocols maturation Current somatic 3). allows Fig. (maturationcultures developmentembryogenic embryo the of conditions environmental the Changing 1998). al. secure et under state (Park juvenile conditions a in material the maintain to way a providing as well as conservation, resource allowing genetic (-196°C), nitrogen liquid in stored be also can cultures Embryogenic SE. “recurrent” as known process a In some woody angiosperm species new somatic embryos may form directly on previously formed somatic embryos in cycleproducing further PEMsorEMs. This ability results masses” an inunlimited “proembryogenic multiplication ofthe original plantmaterial. as continuous a known gymnosperms,enter and angiospermswoody respectively cells, for (EMs) embryogenic masses” “embryonal and initiated (PEMs) Once stage. developmental immature very a to drawback a is year been usedsuccessfully initiate to embryogenic cultures, although theirlimited availability short perioda during in the also have 2) integuments, Table ovule inner anthers, reproduction and (filamentssexual in involved tissues of range matureembryo-derivedsomatic of buds shoot fromexcised were explantsprimordial that fromshoot SE induction of the on study a describedrecently in been has fromepicormic shoots (Corredoira 2006a).al. et very Asimilar pattern thatdescribedto above fromSEfor shoot tips fromthe first leafprimordia, andhad thesame origin anddevelopmental pattern thoseas induced expandingin leaves meristemapical but the from embryosnot arose somatic severalspecies.The in initiate SE initialexplants to as used been also have tips Shoot 2003). al. (Hernández et thresholdsize certain a exceed they whenrespond to ability their embryogenic higher in results embryos initiationzygotic rates thanmature if immature zygotic embryos are cultured.of Expanding leavesculture ofcork oak usedasinitial explants, lose the species many In changes. developmental other species such as speciesas such in sourcesembryogenicof Basedthiscells. observation,on embryogenic cellswereinitiated from leavesthese of shoots from or sprouts, stump robur Quercus or root from Tissues 1978). (Olesen known well epicormicshoots (induced partial by girdling basaltrunk)of partof have been initialused explants as is induce to in SE tree the within maturity of gradient tissues. mature (Merklemore angiospermsand Nairn woody 2005) such as oaks(Vieitezwith in successful et al. 2011), than gymnospermsmore on been (Bongaetal. 2011).The existencehas later of a tissues mature and from cells embryogenicseedlings of initiation young the However, to progressed this developed, experience as and tissues cell seed with was gymnosperms promote and angiosperms woody the that both in wo n ftemi ol nteS rcs st eal opouesnhoosppltoso mroei el rproducesynchronous to populationsembryogenic able or of be cells to is process SE the in goals main the of One The majorityofSE production systems use embryogenic cultures grown on solidified a nutrient medium. requires It of onset the to related be may this and initiation the for important be may excision explant the of timing The embryogenic Establishing 3). (Fig. steps different several in factors many involving process complex a is SE angiospermswoody in SE of report first The 1950s. late the non-woody angiospermsin identifiedin first was SE Santalum album Santalum (Chalupa 2000). (Chalupa Quercussuber . In vitroIn . 883-899. DOI : 10.1007/s11295-013-0620-1 Quercus ilex in the 1960s (Rao 1965). Currently a wide range of woody angiosperms species have been been have species angiosperms woody of range wide a Currently 1965). (Rao 1960s the in . The work on conifer SE has been reviewed by Attree et al. (1993), Stasolla et al. (2002) and and (2002) al. et Stasolla(1993), al. et Attree reviewedby been has SE conifer on work The . conditionssustained allowthe maintenance proliferationand embryogenic the of cultures ex vitro conditions to be “acclimatised” to grow under normal environmental conditions. Comment citer cedocument:

, , Epicormic shoots forced to flush on crown branches have been found to be good be to found been have branches crown on flush to forced shoots Epicormic , Quercusrobur Populus ciliata

Picea glaucaPicea and and , Thevetia peruviana nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 Ulmusminor 4 (Klimaszewska et al. 2011). In addition, explants from a addition,a explants from In 2011).(Klimaszewska al. et (Table 2). Leaf explants were effective in severalexplantseffective Leafwere in 2).(Table , and Piceaabies Terminalia arjuna. (Chalupa1985;al.Hakman et Abies, Larix, Picea, Pinus, Pinus, Picea, Larix, Abies,

Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, differences in initial plants size between emblings and seedling controls because the annual height growth in both both in growth height annual to the because due controls usually seedling and is emblings between this size However, plants 2008). initial al. in differences et Niskanen 2003; 2001, al. et (Högberg seedlings control than slower Embling field performance and genetic conformity possible. is improvement geneticthat of level the increasing thus parentsselected of crossesindividuals originating from best with coupled is SE the rapid select when opportunityto the solved provide the does option mostly this case, be is any In problem 2009). might Pâques cryopreservation (Lelu-Walterand this option but species, this some of for drawbacks stocks another the clones, of ageing the physiological of above. maintenance discussed the option propagation for extra-costs bulk the the Besides to compared expense and time additional and adds testing Clonal however, clones. the selection, test to necessary is it used be to is propagation clonal If planted. be will they where site the to even and propagationprocess entire the through plant every of identity the trackingrequires This clones. synthetic a of diversity genetic the Indeed crop. occurring naturally seed stand.the a from originating population of seedling a rotation of that the than greater be during could population propagated vegetatively risks biotic and abiotic both from withstand potentialProvidedlosses to able resultingplantation field. be geneticallyshouldthe enough unrelatedused, genotypes are the in deployment and propagation during retained not is identity clonal the which in propagation” depends on both the level of genetic improvement to be captured and the regulatory as environment. or deployment) and propagation plantation). the This propagationduring tracked to thoughthe process and "clonalpropagation"identityis clonal the (wheretracked and not produced is identity either clonal be the (where can propagation" material "bulk propagated as Vegetatively planted material. produced SE of planting the about asked Deployment Options plant stock the by produced cuttings of number large the over spread throughout itsbe life. This will be discussedcan in more details later.emblings high the the approach this of In effective. costs are production and developed well are cuttings of rooting the for methods where species to plants as such stock species juvenile with well works methodas This rooting. them for cuttings provide grow and emblings use to is Ireland in Teoranta Coillte and Zealand New in Ltd. from the use of this technology. productproportionthe resulting future valuea of the based emblingsonof basedwhich “valuethepricing” is in cost a MOCASvariationa (planting of of a is biomass.materialgrownThis alternatefor of rows with planted are log saw destinedfor materialimproved“varietal” of rows which promoted“FlexStand”in Arborgenhas material. this Arborgendevelopedof uniquemarketing CellForhaveand some use encourageboth approaches the to plantationwith materialthis would uneconomicbe mostfor forest treespecies grown timberfor production, however, cost between5to 6 times thecosts of improvedseedlings of the samespecies (Sorensson 2006).To establish an entire reportedbeento have US the Emblings justify.economicallyoptionproduceemblings in difficult this to makes very to increasedcost technology, the this of use effective most the be to appears field the in emblings of planting direct ( pine produce,loblolly they species pine main the that is this (sometimes for reason Arborgen.The and 2002) (Sutton CellFor productionfield as companiesSE commercialsuch large the the of some in by used directly is planted that approach the be is This clones). will than rather “varietals” that as emblings to referred produce to is used, widely more the probably and Embling use options al. 2012). meterdependinggenotype, on whileculturessemi-solid on medium renderedm 12,000per 10,000SE to ImmersionSystems for proliferation of somatic embryoshas been estimatedatbetween 36,000 and. 85,000 per square In commercialscale. a on year seedlingsper somatic of millionsproduce as such cropswoody successfulseveral experience pre-germinatewith the follow and embryos.somaticTheymature to also embryosand more problematic (Timmis 1998; Gupta and Timmis, 2005; Salaj et al. 2007) highlyproductive, are the development conifers and maturation ofgymnosperm of somaticcultures embryo nd nd In several cases in which gymnosperm emblings have been established in the field, the initial growth is typically typically is growth initial the field, the in established been have gymnospermemblings which in cases several In Individualsphenotypically selectedfrom outstanding pollinatedopenfull-sibpropagated families, or be also canas mass or “bulk by propagated be can parents selected of crosses full-sib or families pollinated open Selected being now are past the in discussed been have that cuttings rooted deploy to best how about questions same The Thesecond approach whichis used on a large-scale by several organisations including the company Forest Genetics approach, first The emblings. large-scaleproductionof the in used been have approachesthat main two are There differentiated of clusters in SE recurrent with species with mainly used been Temporary Immersionhave Systems S eedlings) as suggested by Park (2002) as a way to reduce the per hectare plant costs. CellFor have promoted a promoteda have CellFor costs. plant hectare per the reduce to way a as (2002) Park suggestedby eedlings) as Liquidambarstyraciflua 883-899. DOI : 10.1007/s11295-013-0620-1 Coffea arabicaCoffea Comment citer cedocument: Pinus taedaPinus x x (Etienne et al. 2011) and and 2011) al. (Etienneet L. formosana L. ), does not currently lend itself to propagation by rooted cuttings. While the the While cuttings.rooted propagation by to itself lend currently not does ),

nal version ofthemanuscript published in: Liriodendron tulipiferaLiriodendron could potentiallycouldproduce4,000-6,600 6 germinable embryos in http://dx.doi.org/10.1007/s11295-013-0620-1 5 Coffea canephoraCoffea Quercus roburQuercus x Pinus radiataPinus L. chinense L. (Ducos et al. 2011) that are able to to able are that 2011) al. et (Ducos the potential of Temporary of potential the , , could potentially produce potentially could Picea sitchensis sitchensis Picea M

ixtures

. 2 2 Suspension (Mallónet O and other and f f C lones Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, Some of these companies are producing material for their own use, but there are also several commercialseveralcompanies also are there but use, own their producing materialfor companies are these of Some Commercial-Scale State-of-the-art of SE in the world other than Europe well a of lack Europe the However,in SE. species developed gymnosperm marketin for capability this improvedin R&D material identified adequate seems to befirst an a major constraint.have was we SE that demonstrates that 1985) fact al. The et (Hakman scale. larger a on SE use and develop to conclusion,necessary In the Europehavescientificappears backgroundto (knowledge, improvedskillsand material) believed that their current material (both about 15%) were also seen as an important non-technical bottlenecks. Only about 9% of the respondents appreciation by foresters of the value of the improved material (35%). Public acceptance and lack of customers for this 4 years for theprogrammesof hadalready material evaluated fieldin trials. The agetheseof trials ranged from years2 for mixtureslargeinor monoclonal blocks. Multi-clonal mixtures were the least common deployment option. About 35% programmes were working with 10 to 20 clones, but the larger programmes worked with 10 to 200 clones ( clones 200 to 10 nordmanniana with worked programmes larger the but clones, 20 to 10 with working were programmes with working programmes several whereas year, per emblings 100 than less produced they that reportedprogrammes Most scale. commercial- the at or to close “pilot-stage”6% withthe advanced to had they (27%)believed that few a while stage “experimental” the at were they that said programmes(81%)production. Most tree Christmas for clones selected of rootedfield( cuttings the to whichthengo produced are directly planted in the field (51%), whereas a few programmes (18%) use them as stock plants to produce Larix propagation( mass usedpropagation(55%),although clonal some in is used materialis the majority of the programmesvarietydeploymentGeneralCombining (47%),in for (23%)and Ability estimation conifers, (16%).For analysis of the process (28%) were seen as the major technical research needs. economic and (55%) productionImprovedprotocols(22%). embryos somatic and zygotic comparisonof and (23%) germplasmcryogenic technology(29%),physiological storagefor of biochemical SE was applicationsand studies of of species in emblings of numbers producing commercial-scale already are France) and Ireland (Denmark, programmes several although SE, of process basic the study (20%)andgermination/conversion (20%). Themajority (37%) ofrespondents said that thereason forusing wasSE to ( species several for applicationindustrial for enough well work for to deemed 21% was technology SE and improvement,current further angiosperm for 12% (about programme improvement requires still process SE tree althoughthe that programmes said the of a 46% gymnospermabout Respondentsspecies). of of part were programmes the of 20% about Only research. applied” and “basic both doing were they said 30% about while (50%) research” “basic doing were they that consideredprogrammes Most 3). (Table species angiosperm studied most the were laboratories) (10 the genus tree forest 45 total, speciesIn are beingcompanies. studied forest (27private gymnosperms and andnational 18 angiosperms). public The (19%), most(43%), studiedorganisations gymnosperm state departments species wereother some of academic or (37%) university institutes research were organisations of these stage of the majority The involved, questionnaire. species the document to Europeanorganisationscountries (including24 Belarus)43 belongingto of total A laboratoriesVP. Europe involved in to sent was in questionnaire A programmes. improvement tree SE in SE on of application the conducted and development was survey a 026076), Number Current state-of-the-art of somatic embryogenesis in forest tree species in Europe. populations could be related to adaptation to a high UV environment. et al. (2007) proposed that the family-dependent DNA instability seen in SE cultures from high altitude Norway spruce behavioural abnormalityand couldresult inincreased fitness/performance asinornamental plants and fruit trees.Burg microsatellites particularly few when conifers area involved. only mutation random 2009) non detect to sufficient al. be not may these et but used, Marum were genome the of 2007; part small very al. a covering et (Burg studies few a In changes. cellular or molecular costlywhole genome resequencing) arecurrently available powerfulbeto enough detectto significant morphological, still (outside none but variation genetic identifying for methods develop to underway presently is Work uniformity. years for Douglas fir (Dean et al. 2009; Dean 2010). Multisite growth of performance of years emblings have beentwenty reportedsummarised after seedlings 4years (2011) forand whiteemblings spruceGrossnickle between (Wahidpatterns growth etspruce, al. 2012), root afterinterior 7 and tolerance freezing dormancy,For similar showing experienceon. later same the usually is plants of types World-wideseveral companies currentlyare producing commercial amountsemblingsconiferous of of foresttrees. of lack a and (60%)produceemblings to cost the as seen were process non-technicalthe main bottlenecksof The Most of the material that is being produced as part of a tree improvement programme is used in clonal testing testing clonal in used is programme improvement tree a of part as produced being is that material the of Most “Treebreedex”(http://treebreedex.euInfrastructureResearchActionConcerted funded EU the of part As morphologicaldramatic or a necessarilymean not wouldmutation somaclonalor variation that noted be should It genetic true-to-type infer not should observed easily are which changes phenotypic pronounced of lack The ). Among the angiosperms propagation use was equally divided between mass and clonal. Most emblings Most clonal. and mass between divided equally was use propagation angiosperms the Among ). Abies, Abies Pinus, 10 years for , , Picea (8 laboratories) Picea sitchensisPicea Larix, 883-899. DOI : 10.1007/s11295-013-0620-1 , Picea, Abies

national regulations presented a serious problem. Comment citer cedocument: n and Pinus Quercus , , Picea Pinus pinasterPinus Pinus adand (18 laboratories) and and 15 years for agd bten 1,0 o 3,0 mlns poue e er Many year. per produced emblings 30,000 to 10,000 between ranged Quercus Piceasitchensis

). Most programmesMost ). monoclonaldeployedmosaics of clonesin either ). The major technical bottleneck for most species was maturation was species most for bottleneck technical major The ). nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 Picea Abies 6 ). The main use of SE in in SE of Theusemain ). Pinus , . Picea (14 laboratories) while several of the genus n and Pinus . Other than propagation, the major the propagation, than Other . Abies species is in the productionthe in species is

responded to the responded to Abies , contract , Quercus Abies Abies Abies and , Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, the programme in order to maximise interest in the vegetatively propagated material (use of tested Corsican and and Corsican tested of (use material propagated vegetatively Moroccan parentsthe crossed with Frenchin Atlantic coastinterest provenances). maximise continuously to order are in issues programme technical the the while plots commercial small first improved. In order to increasethe the financial return of plant SE material, Interprovenance to ('Interracial') crosses areowners included in forest competitive most convincethe establishdemonstration to to plots is currentstrategyTheir SE. as such material cost high sustain not could but value, genetic their whatever cost same the at material plant selling are nurseriescommercial Indeed, geneticgain. for pay reluctance highto a overallmaterial and SE plant ownersto forest of part the willingness on of (Harvengtpersonal comm.). Commercial production seenfuture ais as perspective (perhaps 10years)in lack duea to Currentlydata. about15,000 producedemblings cryo-storedyearare per a from collection 2,000moreclones than of productioncost collect to as well as trials field for materialproduce to SE propagatedusing being is parents-, value periodicity of good seed crops in Norway spruce. generationparentssamethetechnologyfrom treesthe(Lindgren SE overcome as of partly to 2009).was use The the levelimprovement, of or higher,than wouldbeexpected from seed a crop collectedin seed a orchardestablished with as “family forestry”). In this process 20 to 30 clones derived from 5 to 10 families can be expected to provide the same testedclones, interesttherealsousingwas untested in clones derived fromcrosses between tested parents (referred to Skogforskbreeding programme technology.usingSE Although originalthe objective projectworkwith the to was of project,executedSweTree initiated a by Technologies propagationbulkexplore improvedthe to of material the from ( spruce Norway Skogforsk abiesthe from material of testing clonal if see to Sweden of Institute Research Forest ( spruce Norway of the increasing quality in genetic play could it role the and SE in interested became companies forestry several 2004 in Sweden In Gymnosperms plots. werecloned (Hernández etal. 2011)and hundredsof emblingshave been established since2005 in three experimental hundred-year-oldselectedforty than More trees. trees selected thoseemblingsobtained fromwith and trees,selected of embryos zygotic half-sib from obtained emblings with tests clonal large (Spain) Extremadura in established has companyTRAGSApublicSpanish the oak, cork hundred-year-oldthe regards one As from trees.sources of range a 2006b).smallA field trialof al. et (Corredoira year following the nuts and years 3 after catkins male formingmaturity, sexual early an displayed of emblings the of In produced. been have plants of number small in as such severalcases a only 3), (Table hardwoods several for SE of protocols develop much Although present. at limited quite Woody angiosperms Applications of somatic embryogenesis of forest species in Europe companies the with collaboration in Thailand and Nigeria Coast, Michelin (France)Ivory and RRIT (Thailand).in established been have tests field of ha brasiliensis largestthe fieldemblingswith trialsof One woodyof angiosperms implementedbeenhas rubberthe with tree 2010). woody in cropsinwhich cloning by SE playsmain protocols a 2.53role millionwith clonesproduced 2009 in in Malaysia SE (Kushairietal. advanced palm, oil previouslymentioned, coffee and of case most the is countries. This other in place the takes of which of planting the Some crops woody for groups French 2011).by mostly Europe in developed Cunningham been angiospermshave and (Merkle needed are production operational the to scaling-up for required systems engineering production the develop to investments only that and these of SE by researchspecies a stillat is rather operationalthancloning an at considered is level, it Although that these protocols production. readilyare scalable biomass for clones performing superior identify to companies these with trials Field techniques. establishedcloning by yellow-poplar been have and these gum sweet hybrid of embryossomatic from derivedof plants thousandsof support the with programs breeding out carrying are ArborGen and angiosperms.universitiesUS andprivate companies TheUniversitysuch as Georgia,of International Paper Company extentwoody Americanlesseradvancedconsidered chestnutmost amongin are a those the in and gumhyb In France, maritime pine ( pine maritime France, In Among the coniferous species, the application of this technology is much further advanced. is Europe improvementgeneticin species tree forestangiospermsin woodyprogrammes implication SE of of The

successfully produce and sell gymnosperm planting stock produced by SE on the open market. These include include These market. open the on SE by produced stock planting gymnosperm sell and produce successfully ) breeding programme could accelerate the development of new improved material. In 2006, the companies the 2006, In material. improved new of development the accelerate could programme breeding ) . French researchers at CIRAD (Carron et al. 2009) have done the laboratory work on this species. Up to 65 Castanea sativa Castanea 883-899. DOI : 10.1007/s11295-013-0620-1 Quercusrobur Comment citer cedocument: Pinus pinaster Pinus Quercussuber Picea (about one hundred) that were transferred to the field survived. Many of them them of Many survived. field the to transferred were that hundred) one (about established plants are used for physiological for established used (Wilhelm,studiesAll are com).plants per.

abies work has been achieved in academic and public research institutions to to institutions research public and academic in achieved been has work

) material -mostly from full-sib families obtained from very high genetic high very from obtained families full-sib from -mostly material ) wasestablished with plants from both zygotic andsomatic embryos from ) planted in their forests. They set up a project with Skogforsk, the the Skogforsk, with project a up set They forests. their in planted ) nal version ofthemanuscript published in: Pinus taeda Pinus http://dx.doi.org/10.1007/s11295-013-0620-1 7 ) (Grossnickle and Pait 2008). Among the woody the Among 2008). Pait and (Grossnickle ) Elaeis guineensisElaeis

, one of the of one , Hevea Hevea Picea Picea Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, positive arguments in favour of VP might still not be enough to break the strong psychological barriers of forest forest of barriers psychologicalseveral are Meanwhile,challenge. there strong the landownersis the and foresters to benefitsbreak these of to idea the Sellingenough owners. be not still might VP of favour in arguments positive reduced possible lengths, these uniformity.increasedHowever productivityincreasedand rotation site), the of occupancyreduced (quicker establishmentcosts possible composition, genetic of flexibility increased use, commercial balanced be againstadvantages improvedthesethe to costsneed get All shorterwhich to materialtime include a into (Sorensson seedlings times. comparable 10 to 8 currentlyof to closerbe fact in Europeanthat whilemay2006)suggests experience pine this spruceand with times 6 to 5 between cost 2008 in pine loblolly of emblings and material be demonstrated to be practical on a commercial scale as well as effective in reducing production costs. beenproposed anotheras minimizeway to handling andreducethus production thesecosts.methods ofAll to areyet also has 1993) al. et (Lulsdorf seed” “artificial an form to embryosomatic the encapsulation of the Similarly 2012). liquidmedium allowing in transfer easywithout out handling individual of somaticembryos Egertsdotter(Aidunand process carried process is FluidicsdevelopedSE entireSystem”beenNorway which “SE the has for spruce in costs.A these the of end reduce further help to proposed been the have 2008)Krogstrup and (Find emblings of handlingat robotic and 1999) al. typically et is which handled, individually be to (maturation,embryos(Zhangsomaticqualitydesiccation, germination/conversion). computerselectionof of use The had emblings where were process SE economicanalysisprocessSE the of (Cervelli andSenaratna 1995) showed thatmostlabourthe intensive the steps in Economic Challenges major biological impediments to prevent the widespread utilisation of SE technology. in the need to develop specificmore clonesdeployment for specific environments.and propagation clonal of use the attractive.thus Clones may show greater and interaction with theenvironment testing comparedas seedling to clonal progenies. mayIt result make could this developed, be could profiles marker genome-wide of use the or parameters physiological of assessment the through evaluation early or Challenges. PublicAcceptance sectionon later discussedthe further in be population.will VP pointThis the threatsbioticto and to order in genotypes of number larger maintaina the genetic diversityof initiation of the VP population.the require may and diversity genetic the reduce can clones Recalcitrant large-scale. a on practical be not would this but genotypes, certain for protocols “customised” develop of importance maturation the has field, decreased. the in tested are plants regenerated while tissue embryogenic of storage cryogenic the and individual as it ages, was the major biological challenge facing VP by cutting. However, with the use of juvenile tissue Biological Challenges include: Challenges personal comm.). (Thompsoncuttings rooted numbersof large spreaddisappears whenitself plant stockembling the for cost the Thus hedge for 5 years. During this time the stock plant will produce more than 250 cuttings, of which about 80 % will root. grownstockas on plants outdooranin hedge orchard produce to cuttings forrooting. stock A plant willremain the in personalcomm.) an requires (which embling cm year-old(Thompson bare-root field unimproved3 the plantinga seedlingin for ready of cost the times 10 15 than more finished a of cost The clones. additional year ofgrowth theinnursery or before planting out)estimated is families costto between Eurosand3 2 perplant. is It from All solution. propagated possible a bulk as is seen is material programme breeding the from material of VP Therefore needs. current meet to improvedmaterial are yet to comeinto productionand areunlikely to supply enough improvedseed on a regular basis being is robotics of use The year. each added be will material explored as a wayof to help reduce sources production costsnew (Find and Krogstrup and 2008). testing field for produced be embryogenic 250 Currently possible. not cultures(clones) selectedis from27 have elitetreesestablishedbeen cuttings cryopreserved.and rooted Emblings fromeachclone will by species fir true most of Propagation costs. VP high the offset to help would which price high a command cou As mentioned earlier, rooted cuttings currently cost at least twice the price of comparable seedling produced produced seedling comparable of price the twice least at cost currently cuttings rooted earlier, mentioned As detailed A stock. planting SE of cost higher the is general in VP and SE about concern economic main The no currently are there thus insurmountable and are them of none limitations,biological potential these of spite In Clonal testing increases the accuracy of the selection process, but it takes time and costs money. If short-term testing abiotic both to avoidingpotential problemsdue essentialto is material VP in diversitygenetic wide Maintaining a to possible be would It lost. considered as are they result a as and VP to respond not simply genotypes will Some an propagate vegetatively to ability the of loss the or species coniferous in particularly “maturation” past the In Themain challenges for the use ofVP in forestry (including SE technology)can be divided into 4main areaswhich ( spruce Sitka Ireland, In Denmark,In Nordman ( fir 1) biological, . . Thereforeeconomicallywouldbe it emblingsimpractical field.So, directlythemare plantthe in to 883-899. DOI : 10.1007/s11295-013-0620-1 2) economic, Comment citer cedocument: Picea sitchensis Picea Abiesnordmanniana 3) public acceptance and

) is the major commercial timber species. Seed orchards to produce to orchards Seed species. timber commercial major the is ) ) is an importantan is ) Christmas species.tree Christmas trees represent a nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 8 4) regulatory challenges.

Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, to this is not simple and probably is due to several factors. andspecialised European programmes,SE therenothing is compare to with whathappening is elsewhere. The answer severalrelativelyworld?” Whileare therethe localised small, Europe of partsotherunlike in taken off not they have not possiblethrough sexual propagation.The question thenbecomes, VP “if and SEare such usefultechnologies, why geneticgain captureof the for allows also It world. the of demonstratedparts beenother in has method as VP other Challenges for the increased use of somatic embryogenesis technology in European forestry. propagated material exists. clone per site. clonalpropagation minimumclonesunrelated 30 a from10 of families requiredmaximumare plants a with50 per of clonesoriginatingtheoriginatewith abovefamiliesbutminimum seedlings from10010 as family.a from For perof full-sibfamilies with clones originating fromminimum a 100seedlingsof perfamily. For“qualified” material must it no are there material “tested” For mixtures. in needed are clones 4 of restrictions. minimum a material “tested” with and million copies (2 million for birch). For “qualified” material more than 11 clones must be used to plant clonal mixtures vegetatively propagated material in Europe: could currentsummaryregulationsthe pressure.public of a following specifically to The change is dueof coveringthis use the However, exist. currently material propagated clonally with dealing regulations specific no most, in but production.remain in to somemembercasescloneslonghow In are proportion done stateshavetheirthis, as well as However, mixtures). it is up to the individualclonal memberor states (clones to each regulatedefined (if theyare deem it necessary) clones the numberhow of clonesof and definitions are directive this In material. reproductive forest “genetically and modified”improved” “genetically in the minds terms of some of thosethe concerned of aboutconfusion the use of thissome technology. also is There relaxed. been have regulations original these then since years the regulations,strictest however, in the of some Germany set particular,and Sweden establishingregulationsstrict governing number plantedcouldclonesarea thatthebe thematerial.and thiswith of In sources different rigorousseveral for vegetatively propagatedfrom native species thanoriginate with non-native species.could regulations These material. includingcertificationforest organisations, nationalgovernments European Union.more the Regulationand this be could of use the governing regulations Regulatory Challenges concerns on could lead to increaseddepends regulation. basically these address to Failure It process. that this demonstrate in forestry. essential to communicationContinuedis addressed. forestry being clonal are clonal concerns public of about testing concerns and planning and the in fears beginning the the from stakeholders of of involvement many allay help should which (1997) from a wide range vegetativelyof populations. a produce to possible entirely is It propagatedplantation greaterthat hasa risk. genetic diversity than availableis evenat natural in forests byincluding indeed material are variation genetic restricted with monocultures geneticnaturalforestsplantations.bothdiversity of andactual levels in the proper Only understanding or term the of species failed to provide any protection against the pathogen. Similarly monocultures are usually condemned without a these naturalpopulationbothplantations the geneticfromof widediversityreduced with of genetic diversity,the yet ofcatastrophic failures suchasDutch elm disease andchestnut blight arecited asexamples ofdevastation yet tocome lossofgenetic diversity which could a in result potentially could vegetativelypropagatedmaterial that concern the specificallynature”, “manipulationof Public Acceptance Challenges such as to release different sets of a few clones over years exist but should maintained.options Technical require be regulationcould plantation adaptation.in diversitygenetic that so time same the at released and handled cost plants produced by a combined process of SE and rooted cutting technology. lower a usingunimproved andby or plants plantingSE hectaremixturesplanting of percosts,by reducethe to ways There is little doubt that SE can propagate large amounts of genetically improved planting stock faster than any any than faster stock planting improved genetically of amounts large propagate can SE that doubt little is There states member European other all For Denmark Norwayusing - bulk propagation with “tested” material theclones must originate fromminimum a 10half-sibof or Sweden Finland France Germany Europeanmemberthe All statesUnionCouncilin agreed(EU)have Directive to 1999/105/EC marketingthe on of countries several led trees forest propagated vegetatively of use widespread possible the 1980s early the In could material forest propagated vegetatively of use unrestricted seemingly the over concerns Public Goldfarb and Stelzer proposedby addressingbeen environmental forestryhas for processclonal concerns about A publicmainThe vegetatively concern of aboutuse the propagated forestbasicstemstreesconcern man’s from a of economically be could lines SE many how on question the also is challenge, cost production this to Connected - the situation is similar to Germany. - (2000)- up to 5% of the site (up to 20 ha.) can be planted with one or more clones. - material“qualified” the - in category forest of reproductive material vegetativelybecan propagated 1 to up - - only the use of clones of (2002)- only tested material can be vegetatively propagated and it must be planted in clonal mixtures. 883-899. DOI : 10.1007/s11295-013-0620-1 Comment citer cedocument:

leadtoa catastrophic failure of intensively managedforest plantations. Examples Populus

- no national regulations specifically concerning the use of clonally of use the concerning specifically regulations national no - are regulated for planting in Denmark. nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 9

result in result Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, analysis, analysis, following: the have re-enforceperhaps and to understanding improvedan of valuehelpfulmaterial.to the addition,wouldbe ofalso In it induced in tissues from adult trees allowing the clonal testing of selected phenotypes. genus the of species or some However, methods.sexual vegetativealternative by propagated cheaply or easily be cannot that species value high from material improved be Walter et al. 2008) further work would be required for these species. Among the broadleaf species there would need to workhas been done onmaritime pine ( protocols.Althoughdeveloped SE well periodicity have problemsseed and havespecies Ireland.Both Kingdomand ( spruce Norway include species therewellais developed SE techniqueavailable forthe species(Lelu-Walter and Pâques 2009). Other good candidate have trials source Seed Finally potentialarea.marketlarge providing thusEurope a orchards. of mostacross hybridsperform well best the that shownseed by provided consistently be cannot which material hybrid true for demand cherry, walnut, and many fruit trees. trees, Christmas for as characteristicsimportant such consistencyare and inter-populations or interspecifichybrids, aspen)(pines)(larch, as such means other by i material, following: Strategy for the implementation of somatic embryogenesis technology in European Forestry this of use technology the acceptthen the potentialcan public for governmentthe of regulationsmajority a can be least greatly reduced.at If accomplished. be can this how for scheme a provides correctly, done can enhance, the rather if than reduce stock, require geneticplanting propagatedvegetativelydiversity. Thewill of programme use suggested This the by Stelzer andproperly. that Goldfarb (1997) idea handled the not in public the if by confidencefactor of development limiting a become could them from develop may that issues landownersfrom considering planting vegetatively propagated material. inhibit Public acceptance andto thepossible regulatory tend may which earlier discussed been have that issues regulatory including environmentalconcerns, and the future, but this has not yet been demonstrated. cuttingsaresuccessful. Therobotsuse of andartificial seedtechnologies may alsohelp reduce productionSE costs in rooted where species some in large-scaleuse for promise hold propagation also process step two a of first the is SE such as Christmas trees, SE technology in forestry will remainmaterial, a tooluniformclonal for researchers. for demand large Hybrida productionis there unless systems seedling),or where a of cost the times 3 to (generally2 cutting played a role in the failure of the company. world-wide producedeconomic SE demandtimber. plants theofpriceforhigh recessioneffect Clearly on the its and the to due problemwas the of part failure, the for reasonsseveral company cites bankruptcy. the Whilefor filed has than a long-term investment has resulted in the loss of the long-term view. highof cost planting stock. Perhaps thefact that forestry hasbecome morebusiness ofa with short-term targets rather discourageuse may the costs front long-termadditional organisationsforestry, investmentup the for to Even used in investment an benefitfromfullytoday. made to more) yearsor 100 to rotationfull(40 the for wait to have having to problemof the is still thereresulting crop, recovered the from be materialwill this of additionalcost the if Even SE. respondentsthesurveySE to cited “Lackinterestthe of improved in material” bottleneck a as theforincreased of use the of 35% Neverthelessabout material. improved planting of costs the cover tree than from more improvementprogrammesresulting trees forest of productivity and growth the in improvements that shown have studies economic well large, no is there what typeplantingthat of stockto use. The added costofimproved be material may beseen just as another may added cost. Many Europe in problem the developed marketof for genetically improvedPart planting stock, especiallywhenhere. price becomes the majorneeded factor deciding in are efforts Further landowners. and foresters to demonstratedproven clearly and and fully been vegetativelynot propagated have improvedmaterial improved plantingmaterial,including advantages of the that problemis main the Perhapsrealised. is return a before reforest land within a specified time after harvesting that has encouraged the use of SE planting stock. be where SE technology was practisedwill on a large scale most of the land is ownedthey by the state. In this case it is the need to ) late and irregular flowering species such as for the spruces, larches, What is needed is an information campaign designed first to inform foresters about the potential benefits of using SE ( larch hybrid above mentioned species the Of the of some include might which criteria of set a meet must it useful and practical be to SE and VP For biological/technical, the perceivedas forestryconcerns clonalsuch the abouteconomic/marketing are thereFinally rooted dramaticallyproductiona seedling UnlessSE be and somewhere costscan lowereda to of betweencost the Canadianbiotechnologyforest writtenthe manuscript company CellFor was this of draft first the since time the In Forestrytends very betoa conservative business where profits are limited and costs must becarried manyfor years of areas large the unlike owned, privately are area forest total of 60% about and small are forests European Most 3) 3) 1) There needs to be a demonstrated a be Thereneedsefficient to propagation method, A detailed plan for further development and scaling-up of mass-production at the European level, level, European the mass-production at of scaling-up developmentand further for plan detailed A There needsstrongbetoa reasonuse to VPand SE, 1) 883-899. DOI : 10.1007/s11295-013-0620-1 Field demonstrations plots with vegetatively propagated material, material, propagatedvegetatively with plots demonstrations Field Comment citer cedocument: Picea abies Picea Pinuspinaster,

) for Scandinavia (Högberg et al. 1998) and Sitka spruce for the United United the for spruce Sitka and 1998) al. Scandinaviaet (Högberg for ) Larix nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 Lelu-Walter2006)etal.and Scots pine ( x 10 eurolepis Quercus Quercus 2) There needssourcebetoa ofhigh value improved ) perhaps offers the greatest potential. There is a a is There potential. greatest the offers perhaps ) currently offer high potential because SE can be be can SE because potential high offer currently ii ) valuable material that is not easy to propagate iv ) high value timber species such as wild as such species timber value high ) 4) The reasons to use VP wouldVPinclude: usereasons The to iii ) species where uniformitywhere species ) 2) A completed economiccompleted A Pinussylvestris,

Lelu- 4) A Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, Zoglauer K. C, Miguel J, Ziauka Zdravković-Korać S, E, Wilhelm M, S, Arnold von Palada J, Vitamvas K, Vitaliy A, A, Vieitez M, Vágner Nilsen I, Tsvetkov G, Naujoks J, Malá H, Lipavska MihaljevicMissonS, S, MoncaleanJ-P, GuillénRodriguezRaffinPinto A, G, Szczygieł P, Salaj T, Tretyakova R, K, I, Lee H, Häggman H, Kvaalen D, Kulagin S, J, Krajnakova Kuusiene K-A, Högberg Hazubska-PrzybylT, I, the Hakman G, Gyulai to J, Find contribution T, Fenning D, their Ewald for colleagues M, Capuana the Ćalić-Dragosavac D, S, Budimir J, Briza C, Bastien all BarsukovaA, T, Aronen questionnaire:I, Arillaga thank gratefully authors The RICA-CT-2006-026076). contract product(s), the for customer This work has been conducted under Research InfrastructureResearchconductedunder “Treebreedex”beenConcerted Action (http://treebreedex.eu has work This Acknowledgements be so much more. be to shown been have option.methods Examples and demonstrationVP of how VP andconventional SE technology can be appliedand are needed. SE combine an alsoquality Christmashigh is speciestreessuccessful.command forpriceashigh cansuch that of that trees VP The a systems hybrid of use The promising. of handlingindividual somatic embryos, liquidthe usethesuchas culture includingreducing automated selectionSE to andhandling, be seemMethods to acceptable. commercially become to VP are produce they to if cost plants the reducing SE toward especially directed material be to needs effort More future. the in years many be may which harvested is crop forest the until recovered fully be not will today cost additional The flow. cash of one is problem additional costs canberecovered an throughthe increased wood volume, in qualityand uniformity the in results VOPmaterial, the which these of some While material.material this produce to requiredincreasedlabour the VP to due propagatedmainlymaterial, seed of cost the is problem immediate greater unwillingnessThe to plant thismaterial. As discussedEurope. earlier, VP material can rangefromin 2to perhaps 10 times the cost of use its of regulation future avoid to help will which forestry VP of use the in confidence public develop help to addressed. model adequately a provided have (1997)not Goldfarb and is Stelzer attention. trees greater need as crops such VP species in diversity long-lived genetic in of narrowing VP the about Concernsof use the if one become could it but factor, main limiting factors commercially.The appear toused be be to it for sufficient is complete, not while SE, of process the understandingof basic The by the results of the survey carried out for this review, VP and the application of SE in forestry are not well developed. usedcommercially. significantEurope,the Inof spite research in levelof development and evidenced field,as this in opposition or adverse regulations. Unlesseconomicthe aspectsareaddressed, VPand SE of technologythis willremainresearch a could whentool it technologydevelopedsomeusingcases wellSE forest in trees, of and world,is VP the Currently, some partsof in Concluding Remarks 883-899. DOI : 10.1007/s11295-013-0620-1 Comment citer cedocument: 5) nidvda iln opooetebnft fS n P VP, and SE of benefits the promote to willing individual An 1) the cost, and

2) public acceptance. Public acceptance is currently not a significant nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 11 6)

No major public major No

, Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, Hernández I, Celestino C, Toribio M (2003)cultures Vegetative propagationtissue of in embryos somatic of development The (1985) T Eriksson S, Arnold von LC, Fowke I, Hakman of maturation embryo somatic on acid abscisic of Effects (1996) M-A Lelu P, Label P, Aderkas von M, Gutmann Gupta PK, Timmis R (2005) Mass propagation of conifer trees in liquid cultures - progress towards commercialization. (2008)Somaticembryogenesis GrossnickleJ Pait applyingconifer species. culturesSC, tissuevarietalforforestry to for technology GrossnickleSC(2011) visualisation Tissue culture ofconifer seedlings-20 years on: viewed through thelens seedling of and quality. In technology robot biotechnology, of Integration (2008) P Krogstrup J, Find Etienne H, Bertrand B, Ribas A, Lashermes P, Malo E, Montagnon C, Alpizar E, Bobadilla R, Simpson J, Dechamp E, R&D Nestlé at embryogenesis somatic by propagation Coffee (2011) C Lambot P, Broun E, Gibault J-P, Ducos Herold(2009)andGEWelty PerformanceDeanCA, DE geneticandparameters somaticzygoticof andprogenies of 6 at coastalwoodtraitsDouglas-firGenetic(2010) somaticgrowth, parametersand CA clones of stemDean for of sweetgum hybrid yellow-poplar and hybrid productivityof the Enhancing(2004) SA Merkle VendrameWA, J, Dai J (2006b)SamajVieitezAMSomaticembryogenesis Vieitez MujibFJ, chestnut.A, CorredoiraBallester A, In: E, in somatic developmentof and origin the Morphohistological (2006) of analysis A Vieitez ValladaresS, CorredoiraE, CheliakWM, DancikBP (1982) Genetic diversityofnatural populations clone-forming of a tree Chalupa V (2000) In vitro of embryosmature immatureand culturedregeneration plantletfrom embryogenesisSomatic (1985)and V Chalupa Cervelli R, Senaratna T (1995) Economic aspects of somatic embryogenesis. 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Hevea Hevea 1/2 . Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, Park Y-S (2002) Implementation of conifer somatic embryogenesis in clonal forestry: technical requirements and and requirements technical forestry: clonal in embryogenesis somatic conifer of Implementation (2002) Y-S Park Prospects(1995)limitationssomatic Paladaembryogenesis M and Bercetcheof PâquesM, J, of Olesen РO (1978) On cyclophysis and topophysis. 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Pinus pinaster complex) and black spruce ( . Can. J. For. Res. 28: 1536-1545 nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 13 ). Plant Cell Rep Picea mariana Picea Picea mariana Mill.). Plant Cell Rep 12:385- ) clonal population in the hemiartic zone, zone, hemiartic the in population clonal ) 28:673-682 Quercus robur Quercus ex vitro ex Picea abiesPicea

performance of performance : application of application : . In: Jain In: . Larix Pinus Pinus x Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, Zobel B (1993) Clonal forestry with the Eucalypts. In: Libby WJ, Ahuja MR (eds) Clonal Forestry,ClonalSpringer-Verlag,(eds) MR Ahuja WJ, LibbyEucalypts. In: the forestryClonal(1993) with B Zobel of embryos somatic for system and recognition pattern parameters based network neural Genetic A (1999) (2012) Hu W-S J R, Timmis Deblois C, Zhang J, Beaulieu HA, Margolis MS, Lamhamedi A, Rainville (2011) N, A Wahid Ballester N, Vidal S, Valladares C, Sánchez MC, San-José MT, Martínez E, Corredoira AM, Vieitez in study Embryogenesis. case a diversity: Somatic genetic and forests Conifer Coppice (2008) L Industry: Gil González-Martínez SC, Valbuena-Carabaña M, Forest the in Production Tree for Bioprocessing (1998) R Timmis Toda R (1974) Vegetative propagation in relation to Japanese forest tree improvement. N Z J For Sci 4:410-17 morphogenesis, conifers: embryogenesis.somatic using plantations conifer in improvementto genetic of embryos deliveryCommercial (2002) B somatic Suttton of Maturation (2002) T Thorpe EC, Yeung L, Kong C, Stasolla satellite Sorensson C (2006)SRIEG Varietal pines boomStated; in the US south. NZ United J For 51:34-40 southeastern the in forestry clonal Implementing (1997) B Goldfarb HE, Stelzer (eds) M Carson C, Walter ShelbourneCJA (1997) Genetics of addingIn: value to the end-products of radiataIn:forestry. pine. Burdon RD, Moore JM(eds), plantation in application biotechnology for Potential (2004) RA Sedjo of cultures suspension Embryogenic (2007) J Salaj A, Blehová T, Salaj Rao PS (1965) In vitro PM,PijutLawson Michler SS, (2011) CH Biotechnological efforts preservingfor enhancingand temperate hardwood forestry: clonal high-value in embryogenesis somatic Paul AD, Foster GS, Caldwell T, of McRae J (1997) Trends in genetic Application and environmental parameters (1998) for height, diameter JM Bonga JD, Barrett Y-S Park Berlin, Vol.2: pp139-48 Douglas fir. Plant. Cell Tiss Organ Cult 56:25-35 performance stability of white spruce somatic seedlings in clonal tests. For Ecol Man 270: 45-53 Application of biotechnological tools to Quercus pyrenaica Biotechnol. Prog. 14: 156-166 Ann Sc For 59:657-61 physiology, biochemistry, and molecular biology. In Vitro Cell Dev- Plant Biol 38: 93-105 workshop summary. Can J For 27:442-6 New Rotorua, December, Zealand.1-5 FRI BullConference 203:FRI-IUFRO NZ of Proceedings Pine, Radiata of Genetics 97 IUFRO Plantation Forest Biotechnology for the 21 maturation ability. Acta Physiol Plant 29:225-231 tree biodiversity, health, and productivity. In Vitro Cell Dev Biol-Plant 47:123-147 and volume in a multilocation clonal study with Loblolly pine. For Sci 43:87-98 deployment, genetic control, and stability of cryopreserved clones. In Vitro Cell Dev Biol - Plant 34: 231-239 883-899. DOI : 10.1007/s11295-013-0620-1 Willd. from Central Spain. For Ecol Man 254:225-232 induction of embryonal proliferation in pp 129–141 Comment citer cedocument: Quercus

st Century. Research Signpost, Kerala, India, pp 3-24 improvement. Eur J Forest Res 131:519-539 nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 14 Santalum album Pinus nigra Pinus L. Phytomorph 15: 175-179 Arn.: growth parameters and parameters growth Arn.:

nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 15

692 164 433 Number (million) nal version ofthemanuscript published in: 13 17 65 http://dx.doi.org/10.1007/s11295-013-0620-1 16

sp. sp., Populus sp., Ulmus nal version ofthemanuscript published in:

http://dx.doi.org/10.1007/s11295-013-0620-1 17 sp. , Terminalia Merkle and Cunningham 2011 Klimaszewska et al. 2007 Attree and Fowke 1993 Stasolla et al. 2002 Vieitez et al. 2011 Pijut et al. 2011 Review

Version postprint Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: Slovakia Serbia Russia Romania Portugal Poland Norway Ireland Germany France Finland Denmark Czech Rep Croatia Belgium Spain Serbia Romania Portugal Lithuania Italy Hungary Germany France Denmark Czech Rep Bulgaria Belarus Austria Country Table 3 state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., . Countries in Europe involved in somatic embryogenesis of forest trees (angiosperm and gymnosperm species). Gymnosperm pseudoplatanus Acer alba alba lasiocarpa nordmanniana nordmanniana, procera alba, cephalonica nordmanniana Angiosperm Abies , alba x numidica, 883-899. DOI : 10.1007/s11295-013-0620-1 hippocastanum carnea eclsCsae uaytsFgs rxnsJgasPplsPuu uru Sorbus Quercus Prunus Populus Juglans Fraxinus Fagus Eucalyptus Castanea Aesculus Comment citer cedocument: , flava

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excelsior excelsior nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 18 Picea abies, schrenkiana abies sitchensis abies abies omorika omorika abies omorika, pungens abies, breweriana, ugn cecembra , pungens nigra tremula x canescens

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heldreichii pinaster avium vu ere,rbrdomestica petraea, robur avium , peuce lx ou ue ilex, robur suber suber robur, rubra petraea robur robur spp. menziesii Pseudostuga Version postprint Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: Spain UK Sweden state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., alba x cephalonica 883-899. DOI : 10.1007/s11295-013-0620-1 Comment citer cedocument:

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, radiata Version postprint Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript Manuscrit d’auteur / Author Manuscript DIN ATI MA ON AG BR OP SS EE PR G Toribio, M.,PâquesL.E., L.(2013).Somatic embryogenesis inforestry withafocus onEurope: state-of-the-art, benefits, challenges and futuredirection. Tree Genetics andGenomes, 9(4), Lelu-Walter, M.-A.(Auteur decorrespondance), Thompson, D.,Harvengt, L.,Sanchez, L., Version définitive dumanuscrit publiédans /Fi Tree Genetics & Genomes, 2013, 9(4), 883-899 883-899 9(4), & Genomes,Tree Genetics 2013, Figure 1 Figure Seed orchards reproduction BREEDING POPULATIONBREEDING Generative Generative Evaluation Half Sib /Full Sib progenies Sib /FullSib Half Sexual Recombination 883-899. DOI : 10.1007/s11295-013-0620-1 (clones) Bulk-propagation Comment citer cedocument: Stock-plants Individuals progeny testing Multi-sites Improved planting stock Improved

Selection Emblings storage Cryogenic nal version ofthemanuscript published in:

http://dx.doi.org/10.1007/s11295-013-0620-1 A Clonal-propagation Half Sib /Full Sib progenies Sib /FullSib Half 20 testing progeny multi-sites replicated Clonally (SE) VP Clonal trials,Clonal selection Selected clones Selected (SE) (SE) VP VP Emblings Clonal testing Clonal

In vitro embryos 883-899. DOI : 10.1007/s11295-013-0620-1 culture Zygotic embryogenesis: 1seed =Zygotic 1tree Somatic embryogenesis: 1seed =Somatic embryogenesis: 1 Comment citer cedocument:

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ex vitro ex nal version ofthemanuscript published in: http://dx.doi.org/10.1007/s11295-013-0620-1 22 conditions) genetic resources) genetic of(Conservation Cryopreservation