{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T09:12:11Z","timestamp":1778317931838,"version":"3.51.4"},"reference-count":40,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2020,7,29]],"date-time":"2020-07-29T00:00:00Z","timestamp":1595980800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/00239\/2020 (CEF),"],"award-info":[{"award-number":["UIDB\/00239\/2020 (CEF),"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Forests"],"abstract":"<jats:p>There is, at the present time, a great demand for chestnut rootstocks with improved resistance to Phytophthora cinnamomi Rands in the nurseries. New genotypes are emerging from European chestnut breeding programs and the production of thriving plants to restore old orchards with low yields due to a high incidence of diseases, namely root rot, is necessary. Micropropagation is a useful technique for clonal propagation. Nevertheless, in vitro culture propagation is genotype-dependent. Consequently, the existing protocols may demonstrate poor reproducibility and low efficacy. Thus, the need to contribute to the development of new micropropagation protocols suitable for large production of emerging genotypes. As a contribution to fill this gap, a three-step protocol was developed by using new combinations of Murashige &amp; Skoog, Woody Plant, and adapted modified Melin-Norkrans media in different stages of the propagation process. About 90% of shoots were rooted, and after three months of acclimatization, 85% of these plants survived and were capable of continuous growth in the field. Currently, this protocol is being used in the production of several hybrid genotypes (with improved resistance to P. cinnamomi), selected from our ongoing breeding program and also in Castanea sativa Mill. and Castanea crenata Siebold and Zucc. species.<\/jats:p>","DOI":"10.3390\/f11080826","type":"journal-article","created":{"date-parts":[[2020,7,30]],"date-time":"2020-07-30T03:36:38Z","timestamp":1596080198000},"page":"826","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["A New Clonal Propagation Protocol Develops Quality Root Systems in Chestnut"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2685-6347","authenticated-orcid":false,"given":"Patr\u00edcia","family":"Fernandes","sequence":"first","affiliation":[{"name":"Instituto Nacional de Investiga\u00e7\u00e3o Agr\u00e1ria e Veterin\u00e1ria I.P., Avenida da Rep\u00fablica, 2780-159 Oeiras, Portugal"},{"name":"Green-It Bioresources for Sustainability, ITQB NOVA, Av. Da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7380-5983","authenticated-orcid":false,"given":"Sara","family":"Tedesco","sequence":"additional","affiliation":[{"name":"Instituto Nacional de Investiga\u00e7\u00e3o Agr\u00e1ria e Veterin\u00e1ria I.P., Avenida da Rep\u00fablica, 2780-159 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. Da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2916-4848","authenticated-orcid":false,"given":"In\u00eas","family":"Vieira da Silva","sequence":"additional","affiliation":[{"name":"Instituto Nacional de Investiga\u00e7\u00e3o Agr\u00e1ria e Veterin\u00e1ria I.P., Avenida da Rep\u00fablica, 2780-159 Oeiras, Portugal"},{"name":"Instituto de Investiga\u00e7\u00e3o do Medicamento (iMed.ULisboa), Faculdade de Farm\u00e1cia, Universidade de Lisboa, 1649-003 Lisboa, Portugal"},{"name":"Departamento de Bioqu\u00edmica e Biologia Humana, Faculdade de Farm\u00e1cia, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3743-7591","authenticated-orcid":false,"given":"Carmen","family":"Santos","sequence":"additional","affiliation":[{"name":"Instituto Nacional de Investiga\u00e7\u00e3o Agr\u00e1ria e Veterin\u00e1ria I.P., Avenida da Rep\u00fablica, 2780-159 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. Da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Helena","family":"Machado","sequence":"additional","affiliation":[{"name":"Instituto Nacional de Investiga\u00e7\u00e3o Agr\u00e1ria e Veterin\u00e1ria I.P., Avenida da Rep\u00fablica, 2780-159 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9503-6126","authenticated-orcid":false,"given":"Rita","family":"Louren\u00e7o Costa","sequence":"additional","affiliation":[{"name":"Instituto Nacional de Investiga\u00e7\u00e3o Agr\u00e1ria e Veterin\u00e1ria I.P., Avenida da Rep\u00fablica, 2780-159 Oeiras, Portugal"},{"name":"Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2020,7,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1093\/treephys\/3.3.247","article-title":"Identification of two compounds correlated with lack of rooting capacity of chestnut cuttings","volume":"3","author":"Vieitez","year":"1987","journal-title":"Tree Physiol."},{"key":"ref_2","unstructured":"Double, M.L., and MacDonald, W.L. (1992). The Lack of Rootability of Chestnut Cuttings. Proceedings of the Proceedings International Chestnut Conference, West Virginia University Press."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1080\/14620316.1997.11515565","article-title":"Propagation of chestnut cultivars by grafting: Methods, rootstocks and plant quality","volume":"72","year":"1997","journal-title":"J. Hortic. Sci. Biotechnol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1111\/j.1364-3703.2005.00308.x","article-title":"Phytophthora\u00a0cinnamomi","volume":"6","author":"Hardham","year":"2005","journal-title":"Mol. Plant Pathol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0304-4238(97)00136-2","article-title":"In vitro propagation of chestnut (Castanea sativa \u00d7 C. crenata): Effects of rooting treatments on plant survival, peroxidase activity and anatomical changes during adventitious root formation","volume":"72","author":"Diogo","year":"1998","journal-title":"Sci. Hortic. (Amsterdam)"},{"key":"ref_6","unstructured":"Breisch, H., Boutitie, A., Reyne, J., Salesses, G., and Vaysse, P. (1995). Ch\u00e2taignes et Marrons."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1661","DOI":"10.1111\/gcb.13492","article-title":"Current and projected global distribution of Phytophthora cinnamomi, one of the world\u2019s worst plant pathogens","volume":"23","author":"Burgess","year":"2017","journal-title":"Glob. Chang. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Costa, R., Santos, C., Tavares, F., Machado, H., Gomes-Laranjo, J., Kubisiak, T., and Nelson, C. (2011). Mapping and transcriptomic approches implemented for understanding disease resistance to Phytophthora cinammomi in Castanea sp.. BMC Proc., 5.","DOI":"10.1186\/1753-6561-5-S7-O18"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"901","DOI":"10.1111\/ppa.12313","article-title":"Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance","volume":"64","author":"Santos","year":"2015","journal-title":"Plant Pathol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1051\/forest:19970404","article-title":"Reinvigoration treatments for the micropropagation of mature chestnut trees","volume":"54","author":"Ballester","year":"1997","journal-title":"Ann. Sci. For."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1243","DOI":"10.1093\/treephys\/20.18.1243","article-title":"Reinvigoration of mature chestnut (Castanea sativa) by repeated graftings and micropropagation","volume":"20","author":"Giovannelli","year":"2000","journal-title":"Tree Physiol."},{"key":"ref_12","first-page":"460","article-title":"Improving chestnut micropropagation through axillary shoot development and somatic embryogenesis","volume":"76","author":"Ballester","year":"2001","journal-title":"For. Snow Landsc. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1684","DOI":"10.21273\/HORTSCI.39.7.1684","article-title":"Micropropagation of Japanese chestnut (Castanea crenata Sieb. et Zucc.) seedlings","volume":"39","author":"Tetsumura","year":"2004","journal-title":"HortScience"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"171","DOI":"10.21273\/HORTSCI.51.2.171","article-title":"Improving Rooting and Shoot Tip Survival of Micropropagated Transgenic American Chestnut Shoots","volume":"51","author":"Oakes","year":"2016","journal-title":"HortScience"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/0304-4238(89)90059-9","article-title":"Prevention of shoot-tip necrosis in shoot cultures of chestnut and oak","volume":"41","author":"Vieitez","year":"1989","journal-title":"Sci. Hortic. (Amsterdam)"},{"key":"ref_16","unstructured":"Lumdsen, P.J., Nicholas, J.R., and Davies, W.J. (1994). Rooting and acclimatization of chestnut by in vitro propagation. Physiology, Growth and Development of Plants in Culture, Kluwer Acad. Pub."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Vieitez, E., and Vieitez, A. (1976). Juvenility factors related to the rootability of chestnut cuttings. Acta Hortic., 269\u2013274.","DOI":"10.17660\/ActaHortic.1976.56.31"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1006\/anbo.1999.0865","article-title":"Anatomical and Biochemical Events during in vitro Rooting of Microcuttings from Juvenile and Mature Phases of Chestnut","volume":"83","author":"Ballester","year":"1999","journal-title":"Ann. Bot."},{"key":"ref_19","first-page":"468","article-title":"Factors influencing in vitro rooting of chestnut","volume":"76","year":"2001","journal-title":"For. Snow Landsc. Res."},{"key":"ref_20","first-page":"77","article-title":"Doubling Acclimatization Survival of Micropropagated American Chestnuts with Darkness and Shortened Rooting Induction Time","volume":"31","author":"Oakes","year":"2013","journal-title":"J. Environ. Hort."},{"key":"ref_21","first-page":"169","article-title":"Micropropaga\u00e7\u00e3o de Clones de Castanheiro (Castanea sativa Mill.) Resistentes \u00e0 doen\u00e7a da tinta","volume":"1","author":"Seabra","year":"1993","journal-title":"Silva Lusit."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"515","DOI":"10.3389\/fpls.2017.00515","article-title":"Expression profiling of Castanea genes during resistant and susceptible interactions with the oomycete pathogen Phytophthora cinnamomi reveal possible mechanisms of immunity","volume":"8","author":"Santos","year":"2017","journal-title":"Front. Plant Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1111\/j.1399-3054.1962.tb08052.x","article-title":"A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures","volume":"15","author":"Murashige","year":"1962","journal-title":"Physiol. Plant."},{"key":"ref_24","first-page":"421","article-title":"Commercially feasible micropropagation of mountain laurel, (Kalmia latifolia) by use of shoot tip culture","volume":"30","author":"Lloyd","year":"1980","journal-title":"Comb. Proc. Int. Plant Propagators\u2019 Soc."},{"key":"ref_25","first-page":"411","article-title":"The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. II. Production, identification, and biological activity of antibiotics produced by Leucopaxillus cerealis var. piceina","volume":"59","author":"Marx","year":"1969","journal-title":"Phytopathology"},{"key":"ref_26","unstructured":"Rodrigues, R., S\u00e1nchez Tam\u00e9s, R., and Durzan, D. (1990). Rejuvenation of Adult Specimens of Castanea Sativa Mill: Through In Vitro Micropropagation. Plant Aging: Basic and Applied Approaches., Plenum Press."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1111\/j.1399-3054.1989.tb05659.x","article-title":"Etiolation as a pretreatment for in vitro establishment and multiplication of mature chestnut","volume":"77","author":"Ballester","year":"1989","journal-title":"Physiol. Plant."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1016\/j.biotechadv.2008.08.003","article-title":"The role of activated charcoal in plant tissue culture","volume":"26","author":"Thomas","year":"2008","journal-title":"Biotechnol. Adv."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Beyl, C.A., and Trigiano, R.N. (2015). Getting Started with Tissue Culture: Media Preparation, Sterile Technique, and Laboratory Equipment. Plant Propagation Concepts and Laboratory Exercises, CRC Press.","DOI":"10.1201\/b17340"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1007\/BF00045910","article-title":"Micropropagation of the silk tassel bush, Garrya elliptica Dougl","volume":"44","author":"Woodward","year":"1996","journal-title":"Plant Cell. Tissue Organ Cult."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1007\/s11240-007-9305-5","article-title":"An efficient system for the production of clonal plantlets of the medicinally important aromatic plant: Salvia africana-lutea L.","volume":"92","author":"Makunga","year":"2008","journal-title":"Plant Cell. Tissue Organ Cult."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1080\/14620316.1997.11515531","article-title":"Improving micropropagation conditions for adult- phase shoots of chestnut","volume":"72","author":"Sanchez","year":"1997","journal-title":"J. Hortic. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1007\/s11627-997-0039-1","article-title":"Micropropagation of american chestnut: Increasing rooting rate and preventing shoot\u2014Tip necrosis","volume":"33","author":"Xing","year":"1997","journal-title":"Vitr. Cell. Dev. Biol.-Plant"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1007\/s11240-017-1285-5","article-title":"Micropropagation of axillary shoots of hybrid chestnut (Castanea sativa \u00d7 C. crenata) in liquid medium in a continuous immersion system","volume":"131","author":"Cuenca","year":"2017","journal-title":"Plant Cell Tissue Organ Cult."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1016\/0304-4238(92)90132-V","article-title":"Root development of in vitro hybrid walnut microcuttings in a vermiculite-containing gelrite medium","volume":"51","author":"Capelli","year":"1992","journal-title":"Sci. Hortic. (Amsterdam)"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1023\/A:1025072922054","article-title":"The influence of medium aeration on in vitro rooting of Australian plant microcuttings","volume":"75","author":"Newell","year":"2003","journal-title":"Plant Cell. Tissue Organ Cult."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Corredoira, E., Mart\u00ednez, M., Cernadas, M., and San Jos\u00e9, M. (2017). Application of Biotechnology in the Conservation of the Genus Castanea. Forests, 8.","DOI":"10.3390\/f8100394"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Vieitez, A.M., S\u00e4nchez, M.C., Garc\u00eda-Nimo, M.L., and Ballester, A. (2007). Protocol for Micropropagation of Castanea sativa. Protocols for Micropropagation of Woody Trees and Fruits, Springer.","DOI":"10.1007\/978-1-4020-6352-7_28"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/S0304-4238(02)00037-7","article-title":"Effect of ex vitro conditions on growth and acquisition of autotrophic behaviour during the acclimatisation of chestnut regenerated in vitro","volume":"95","author":"Carvalho","year":"2002","journal-title":"Sci. Hortic. (Amsterdam)"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1007\/s10725-014-9965-1","article-title":"Influence of in vitro growth conditions on the photosynthesis and survival of Castanea sativa plantlets during ex vitro transfer","volume":"75","author":"Bravo","year":"2015","journal-title":"Plant Growth Regul."}],"container-title":["Forests"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4907\/11\/8\/826\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:52:40Z","timestamp":1760176360000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4907\/11\/8\/826"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,7,29]]},"references-count":40,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2020,8]]}},"alternative-id":["f11080826"],"URL":"https:\/\/doi.org\/10.3390\/f11080826","relation":{},"ISSN":["1999-4907"],"issn-type":[{"value":"1999-4907","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,7,29]]}}}