The aim of this study was to improve the rooting efficiency of Eucalyptus urophylla clones by in vitro reinvigoration/rejuvenation in two clones (02 and 04) from the breeding program of the V&M Florestal company. An in vitro culture began with 200 meristems of each clone, which were excised, disinfected, and inoculated in culture medium. When shoots from these first meristems inoculated reached a height of 3 cm, 100 new meristematic regions of 0.5 cm were isolated and inoculated in culture medium. The other shoots from were inoculated in a rooting medium, where they remained for 30 days. After this period, the plants were acclimatized and used as stock plants for shoot production in a commercial nursery. This process was repeated until the shoots attained an ex vitro rooting rate of more than 80%. After reinvigoration/rejuvenation of clones 02 and 04, the relationship between rooting and the presence of starch and phenolic compounds at the base of the minicuttings was histochemically analyzed. For clone 02, three in vitro subcultures were needed to increase the rooting rate, and for clone 04, only one in vitro subculture was required. In vitro reinvigoration/rejuvenation is a determining factor for greater rooting efficiency of minicuttings of 02 and 04 clones. Production of sclerenchyma fibers around the root vascular cylinder and starch and phenolic compound production are directly related to rooting efficiency.
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Abu-Abied M, Szwerdszarf D, Mordehaev I, Levy A, Rogovoy O, Belausov E, Yaniv Y, Uliel S, Katzenellenbogen M, Riov J, Ophir R, Sadot E (2012) Microarray analysis revealed upregulation of nitrate reductase in juvenile cuttings of Eucalyptus grandis, which correlated with increased nitric oxide production and adventitious root formation. Plant J 71:787–799
Alfenas AC, Zauza EAV, Mafia RG, Assis TF (eds) (2004) Clonagem e doenças do eucalipto. Viçosa, MG
Assis TF, Fett-Neto AG, Alfenas AC (2004) Current techniques and pospects for the clonal propagation of hardwood with emphasis on Eucaliptus. In: Walter C, Carson M (eds) Plantation forest biotechnology for the 21st century. Research sign Post New Delhi, India, pp 303–333
Aumond ML Jr, de Araujo AT, Jr Junkes CFO, Almeida MR, Matsuura HN, Costa F, Fett-Neto AG (2017) Events associated with early age-related decline in adventitious rooting competence of Eucalyptus globulus Labill. Front Plant Sci 8:1734
Bellini C, Pacurar DI, Perrone I (2014) Adventitious roots and lateral roots: similarities and differences. Annu Rev Plant Biol 65:639–666
Bryant P, Trueman S (2015) Stem anatomy and adventitious root formation in cuttings of Angophora, Corymbia and Eucalyptus. Forests 6:1227–1238
Bukatsch F (1972) Bemerkungen zur doppelfarbung astrablau-safranin. Mikrokosmos 61:255
Burger LM, Richter HG (eds) (1991) Anatomia da madeira. Nobel, São Paulo
Castro EM, Pereira FJ, Paiva R (eds) (2009) Histologia Vegetal: Estrutura e Função de Órgãos Vegetativos. UFLA, Lavras
Chen XW, Abdullah TL, Abdullah NAP, Hassan SA (2012) Rooting responses of miracle fruit (Synsepalum dulcificum) softwood cuttings as affected by indole butyric acid. Am J Agric Biol Sci 7:442–446
Cunha ACMCM, Paiva HN, Xavier A, Otoni WC (2009) Papel da Nutrição Mineral na Formação de Raízes Adventícias em Plantas Lenhosas. Pesqui Florest Bras 50:35–47
Da Csosta CT, de Almeida MR, Ruedell CM, Schwambach J, Maraschin FS, Fett-Neto AG (2013) When stress and development go hand in hand: main hormonal controls of adventitious rooting in cuttings. Front Plant Sci 4:1–19
De Klerk GJ, Krieken WVD, Jong JC (1999) The formation of adventitious roots: new concepts, new possibilities. In Vitro Cell Dev Biol Plant 35:189–199
de Paiva HN, Gomes JM (eds) (1993) Propagação vegetativa de espécies florestais. Imprensa Universitária, Minas Gerais
Druege U, Zerche S, Kadner R, Ernst M (2000) Relationship between nitrogen status, carbohydrate distribution and subsequent rooting of Chrysanthemum cuttings as affected by pre-harvest nitrogen supply and cold-storage. Ann Bot 85:687–701
Druege U, Franken P, Hajirezaei MR (2016) Plant hormone homeostasis, signaling, and function during adventitious root formation in cuttings. Front Plant Sci 7:381
Duarte MR, Debur MCS (2005) Stem and leaf morphoanatomy of Maytenus ilicifolia. Fitoterapia 76:41–49
Fachinello JC, Hoffmann A, Nachtigal JC (2005) Propagação de plantas frutíferas. Embrapa Informação Tecnológica, Brasília, p 221p
Ferreira DF (2011) Sisvar: a computer statistical analysis system. Ciência Agroecol 35:1039–1042
Ferreira EM, Alfenas AC, Mafia RG, Leite HG, Sartorio RC, Filho RMP (2004) Determinação do tempo ótimo do enraizamento de miniestacas de clones de Eucalyptus spp. Rev Árvore 28:183–187
George EF, Sherrington PD (eds) (1984) Plant propagation by tissue culture. Exegetics, Eversley
Hackett W (1988) Donor plant maturation and adventitious root formation. In: Davis TD, Haissig BE, Sankhla N (eds) Adventitious root formation in cuttings, vol 2. Dioscorides Press, Portland, pp 11–28
Hartmann HT, De Kester, Davies FR, Geneve RL (eds) (2002) Plant propagation: principles and practices, 7th edn. Prentice-Hall, Upper Saddle River
Huang LC, Lius S, Huang BL, Murashige T, Mahdi EFM, Gundy RV (1992) Rejuvenation of Sequoia sempervirens by repeated grafting of shoot tips onto juvenile rootstocks in vitro. Plant Physiol 98:166–173
Husen A, Pal M (2007) Metabolic changes during adventitious root primordium development in Tectona grandis linn. f. (Teak) cuttings as affected by age of donor plants and auxin (IBA and NAA) treatment. New For 33:309–323
Iritani C, Soares RV, Gomes AV (1986) Aspectos morfológicos da aplicação de reguladores de crescimento nas estacas de Ilex paraguariensis St. Hilaire. Acta Biol Parana 15:21–46
Jasik J, De Klerk GJ (1997) Anatomical and ultra-structural examination of adventitious root formation in stem slices of apple. Biol Plant 39:79–90
Jensen WA (ed) (1962) Botanical histochemistry: principles and practice. Freeman, San Francisco
Jesus MAS, Carvalho SP, Castro EM, Gomes CN (2010) Observações anatômicas em plantas de Coffea arabica L. obtidas por enraizamento de estacas. Rev Ceres 57:175–180
Johansen DA (ed) (1940) Plant microtechinique. New York, McGraw-Hill, New York. Jkertesz ZI. The pectic substances. Interscience, New York
Kratz D, Wendling I, Pires PP (2012) Miniestaquia de Eucalyptus benthamii × E. dunnii em substratos a base de casca de arroz carbonizada. Sci For 40:547–556
Li M, Leung DWM (2000) Starch accumulation is associated with adventitious root formation in hypocotyl cuttings of Pinus radiata. J Plant Growth Regul 19:423–428
Lima DM, Biasi LA, Zanette F, Zuffellato-Ribas KC, Bona C, Mayer JLS (2011) Capacidade de enraizamento de estacas de Maytenus muelleri Schwacke com a aplicação de ácido indolbutírico relacionada aos aspectos anatômicos. Revista Brasileira de Plantas Medicinais Botucatu 13:422–438
Ludwig-Müller J, Prinsen E, Rolfe SA, Scholes JD (2009) Metabolism and plant hormone action during clubroot disease. J Growth Regul 28:229–244
Maynard BOK, Bassuk NL (1988) Etiolation and banding effects on adventitious root formation. In: Davis TD, Haissig BE, Sankhla N (eds) Adventitious root formation in cuttings. Dioscorides Press, Portland, pp 29–46
McCown DD, McCown BH (1987) North American Hardwoods. In: Bonga JM, Durzan DJ (eds) Cell and tissue culture in forestry Forestry sciences, vol 24–26. Springer, Dordrecht
Mokotedi ME, Watt M, Pammenter N (2010) Analysis of differences in field performance of vegetatively and seed-propagated Eucalyptus varieties II: vertical uprooting resistance. South For 72:31–36
Murashige T, Skoog F (1962) A revised medium for rapid growth and biossays with tobacco cultures. Physiol Plant 15:473–497
Negishi N, Nakahama K, Urata N, Kojima M, Sakakibara H, Kawaoka A (2014) Hormone level analysis on adventitious root formation in Eucalyptus globulus. New For 45:577–587
Negrelle RRB, Doni ME (2001) Efeito da maturidade dos ramos na formação de mudas de guaco por meio de estaquia. Hortic Bras 19:351–365
Ono EO, Rodrigues JD (eds) (1996) Aspectos da fisiologia do enraizamento de estacas caulinares. FUNEP, Jaboticabal
Pop TI, Pamfil D, Bellini C (2011) Auxin control in the formation of adventitious rooting. Not Bot Horti Agrobot Cluj 39:307–316
Shanthi K, Bachpai VKW, Anisha S, Ganesan M, Anithaa RG, Subashini V, Chakravarthi M, Sivakumar V, Yasodha R (2015) Micropropagation of Eucalyptus camaldulensis for the production of rejuvenated stock plants for microcuttings propagation and genetic fidelity assessment. New For 46:357–371
Souza CS, Freitas MLM, Moraes MLT, Sebbenn AM (2011) Estimativas de parâmetros genéticos para caracteres quantitativos em progênies de polinização aberta de Eucalyptus urophylla. Floresta 41:847–856
Stevens ME, Pijut PM (2017) Origin of adventitious roots in black walnut (Juglans nigra) softwood cuttings rooted under optimized conditions in a fog chamber. New For 48:685–697
UTHSCSA image tool: image processing and analisis program: version 3.0. San Antonio: University of Texas, 2002. http://ddsdx.uthscsa.edu/dig/itdesc.html (30 janeiro 2012)
Vilasboas J, Da Costa CT, Fett-Neto AG (2018). Rooting of eucalypt cuttings as a problem-solving oriented model in plant biology. Prog Biophys Mol Biol (in press, corrected proof)
Wendling I, Xavier A (2005) Influência do ácido indolbutírico e da miniestaquia seriada no vigor radicular de clones de Eucalyptus grandis. Rev Árvore 29:681–689
Wendling I, Trueman SJ, Xavier A (2014) Maturation and related aspects in clonal forestry—part II: reinvigoration, rejuvenation and juvenility maintenance. New For 45:473–486
White J, Lowell PH (1984) The anatomy of root initiation in cuttings of Criselinia littoralis and Criselinia lucida. Ann Bot 54:7–20
Xavier A, Comércio J (1996) Microestaquia: uma maximização da micropropagação de Eucalyptus. Rev Árvore 20:9–16
Xavier A, Wendling I, Silva RL (eds) (2013) Silvicultura clonal—princípios e técnicas. Editora UFV, Viçosa
Zerche S, Druege U (2009) Nitrogen content determines adventitious rooting in Euphorbia pulcherrima under adequate light independently of pre-rooting carbohydrate depletion of cuttings. Sci Hortic 121:340–347
Our thanks to the V&M Florestal company for donating the eucalypt plants used in this study and to the National Research Council (CNPq), Coordination for the Improvement of Personnel in Higher Education (Capes), and the Research Support Foundation of the State of Minas Gerais (FAPEMIG) for financial support.
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Rejuvenation of clone 02 from natural Eucalyptus urophylla, 21 days after pruning. Nursery plants (1), G1 (2), G2 (3), G3 (4), and G4 (5). (a) Tubette plants. (b) Plants without tubette. Scale bar – 2 cm. (JPEG 111 kb)
Rejuvenation of clone 04 from natural Eucalyptus urophylla, 21 days after pruning. Nursery plants (1), G1 (2), G2 (3), and G3 (4). (a) Tubette plants. (b) Plants without tubette. Scale bar – 2 cm. (JPEG 107 kb)
Cross sections of the base of Eucalyptus urophylla stem minicuttings from clone 02. (a) Nursery plants. (b) First subculture generation. (c) Second subculture generation. (d) Third subculture generation stained with safrablau solution. ep – epidermis. cx - cortex. sc - sclerenchyma. ph - phloema. xl - xylem. ss - secretory structure. mp - medullary parenchyma. (JPEG 372 kb)
Cross sections of the base of Eucalyptus urophylla stem minicuttings from clone 04. (a) Nursery plants. (b) First subculture generation. (c) Second subculture generation. (d) Third subculture generation. ep – epidermis. cx - cortex. sc - sclerenchyma. ph - phloema. xl - xylem. mp - medullary parenchyma. (JPEG 372 kb)
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Mendonça, E.G., Batista, T.R., Stein, V.C. et al. In vitro serial subculture to improve rooting of Eucalyptus urophylla. New Forests 51, 801–816 (2020). https://doi.org/10.1007/s11056-019-09761-6
- Phenolic compounds
- Anatomical barriers
- Clonal forestry