Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis

Abstract

Breeding methods based on genetic transformation techniques need to be implemented for Eucalyptus camaldulensis to shorten the long breeding cycles and avoid manipulation of adult trees; that requires the development of plant regeneration protocols enabling development of plants from transformed tissues. The present work aimed to optimise the regeneration process already established for the species. Cotyledonary leaves of E. camaldulensis were cultured in MS medium supplemented with naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) combinations. The most efficient treatment for bud indirect regeneration (2.7 µmol L -1 NAA and 4.44 µmol L -1 BAP) was used for further experiments. When explants were kept in the dark during the first 30 days, the percentage of explants forming calluses increased and explant necrosis was reduced in comparison with light-cultured explants. Mineral medium modifications were compared and half-strength MS mineral medium turned out to be as efficient as full-strength medium, producing 54% and 47% of explants with buds, respectively. For shoot elongation, MS medium with halfstrength nitrate and ammonium salts, and 0.2% activated charcoal yielded rooted shoots 1 to 8 cm high after one month. The procedure is an efficient protocol for E. camadulensis plant regeneration, reducing the stages necessary for the obtention of complete plants. Key words: in vitro culture, forest species, culture media, organogenesis REGENERAÇÃO DE PLANTAS DE Eucalyptus camaldulensis A PARTIR DAS EXPLANTES COTILEDONARESRESUMO: A implementação, para espécies florestais, de técnicas de melhoramento baseadas em métodos de transformação genética, permitirá reduzir os longos ciclos de melhoramento e evitar a manipulação de árvores adultas. Isto implica dispor de um protocolo de regeneração que permita o desenvolvimento de plantas a partir de tecidos transformados. Este trabalho teve como objetivo otimizar este protocolo de regeneração para Eucalyptus camaldulensis. Folhas cotiledonares foram cultivadas em meio de cultura MS suplementado com combinações de ácido naftalenoacético (ANA) e 6-benzilaminopurina (BAP). O tratamento mais eficiente em termos de regeneração indireta de gemas foi 2,7 µmol L -1 de ANA combinado com 4,44 µmol L -1 de BAP, o qual foi utilizado nos experimentos posteriores. A manutenção dos explantes no escuro durante os trinta primeiros dias elevou a porcentagem de explantes com calos e reduziu a morte dos explantes, em comparação com os que permaneceram na luz. Modificações da composição mineral do meio MS foram comparadas e mostraram que a redução de metade dos sais foi tão eficiente para a formação de gemas (54% dos explantes) quanto o meio completo (47%). O meio de cultura com a concentração de íons nitrato e amônio reduzida à metade e 0,2% de carvão ativado apresentou-se adequado para o alongamento e enraizamento das brotações que atingiram uma altura de 1 a 8 cm depois de 30 dias. O processo completo representa um protocolo eficiente para a regeneração de plantas de Eucalyptu...

Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis dehn and histological study of organogenesis in vitro

“…They were subsequently rinsed three times in sterile distilled water and sown in Petri dishes on MS/2 medium, composed of half-strength MS (Murashige and Skoog, 1962) o C. The cultures were performed in Petri dishes (10 cm diameter and 2 cm height), containing 25 mL of culture medium and sealed with PVC film, or in glass flasks (6 cm diameter and 9 height) containing 40 mL of culture medium each and sealed with rigid polypropylene caps. All media had the pH adjusted to 5.8 and were autoclaved for 20 min at 120 o C. For the plant regeneration from the cotyledonary leaves, three treatments were compared: MS, WPM (Lloyd and McCown, 1981) and JADS (Correia, 1993 Dibax et al (2005). Fifteen days after sowing, the plantlets were used as sources of explants.…”

“…The establishment and understanding of the regenerating process are essential for the success of this process. Studies on plant regeneration through organogenesis have already been reported for E. camaldulensis (Muralidharan and Mascarenhas, 1987;Mullins et al, 1997;Ho et al, 1998;Dibax et al, 2005;Quisen, 2007), E. tereticornis (Subbaiah and Minocha, 1990;Parthiban et al, 1999), E. grandis (Warrag et al, 1991;Lainé and David 1994;Hajari et al, 2006), E. urophylla (Tibok et al, 1995), E. globulus (Serrano et al 1996), E. gunnii (Hervé et al, 2001) and E. grandis x E. urophylla (Gonzales et al, 2002;Tournier et al, 2003;Alves et al, 2004). However, these mainly describe the protocol of plant regeneration and do not refer to the anatomical process.…”

Tissue Culture of Corymbia and Eucalyptus

“…Glucose at 88 or 176 mM has been used, instead of sucrose, during root induction on E. globulus and E. saligna shoots [139]. Activated charcoal at 83.3-833 mM is often incorporated into the root induction media, including for E. camaldulensis, E. globulus, E. grandis, E. grandis × E. urophylla, E. regnans F.Muell., and E. saligna [14,[82][83][84]88,[139][140][141][142][143][144]. Activated charcoal may act by adsorbing inhibitory compounds, decreasing phenolic oxidation, altering medium pH, or reducing irradiance at the base of the shoot [145,146].…”

“…Mineral adjustments have included the use of MS salts with 2.74 mM NaH 2 PO 4 , or a reduction in MS-medium strength to 1/10, for C. citriodora shoots [69,147]. They have also included the use of MS medium with half-strength macronutrients, or half-strength nitrates, for E. camaldulensis shoots [116,143], or the use of MS macronutrients with half-strength micronutrients for E. grandis × E. urophylla shoots [148]. More often, MS medium is simply reduced to half-strength during root induction, including for shoots of C. citriodora…”

“…Eucalypt shoots sometimes produce roots spontaneously in hormone-free medium or potting mix [45,46,64,88,92,96,104,113,119,143,144,150,161]. However, adventitious rooting on eucalypt shoots is usually induced with an auxin rooting hormone, typically IBA at a concentration between 0.49 and 49 µM (Table A1).…”