Factors affecting maintenance, proliferation, and germination of secondary somatic embryos of Eucalyptus globulus Labill

Abstract

The described protocol for repetitive somatic embryogenesis (SE) in Eucalyptus globulus produced more somatic embryos than the primary SE protocol. Primary somatic embryos (induced on MS 3NAA ) were transferred to the same medium, leading to new cycles of somatic embryos, for at least 2 years. The influence of medium (MS and B5), plant growth regulators (auxins and cytokinins), and light on secondary SE was tested. The MS medium without growth regulators (MS WH ) was more efficient for cotyledonary embryo formation and germination than the B5 medium. Reducing auxin (NAA) levels increased the proliferation of globular somatic embryos and allowed SE competence to be maintained on medium free of plant growth regulators. The addition of two cytokinins (BAP and KIN) to the MS medium did not improve proliferation of globular secondary embryos, but was crucial during later stages of the SE process (germination and conversion). Data also show that light may influence the quality of the process, depending on its stage. Darkness should be maintained until the cotyledonary stage is reached, after which exposure to light is recommended.

Tissue Culture of Corymbia and Eucalyptus

“…Other surface sterilants such as hydrogen peroxide (H 2 O 2 ), combinations of H 2 O 2 and ethanol, and alkyldimethylbenzalkonium chloride have been used occasionally for decontaminating eucalypt explants [47,[69][70][71][72][73][74][75][76]. A drop of detergent or wetting agent such as Tween 20 ® is often added to the solution to improve contact between the sterilant and the explant surface, which is often hairy in the case of eucalypt shoots or leaves.…”

“…NAA at concentrations between 10.8 and 81.0 µM has been used for somatic embryogenesis from C. citriodora, E. camaldulensis, E. dunnii, and E. globulus zygotic embryos, cotyledons, or germinating seedlings, sometimes with the addition of 100 mL L −1 coconut water or 0.5-1 g L −1 casein hydrolysate [73][74][75][76]81,[132][133][134]. NAA at 5.4 µM has been used in combination with 2.22 µM BA or 4.52 µM 2,4-D for inducing embryogenic callus from zygotic embryos, hypocotyls, or cotyledons of E. globulus and E. nitens [134,135], with coconut water at 100 mL L −1 incorporated into the callogenesis medium for E. nitens [135].…”

“…Somatic embryos can then be germinated on hormone-free MS medium [73][74][75][76]132,134], MS medium with 0.89 µM BA, and 1.08 µM NAA [74], MS medium with 1.24 µM BA, 2.46 µM kinetin, and 2.48 µM NAA [74], or half-strength MS medium with 2.22 µM BA and 0.54 µM NAA [133]. Embryo germination has also been performed on filter paper suspended over liquid MS medium containing 0.44 µM BA [138].…”

Histological analysis reveals the formation of shoots rather than embryos in regenerating cultures of Eucalyptus globulus

“…However, the majority of studies mainly describe protocols for plant regeneration but do not concern the histological characteristics of the process. Therefore the aim of the current study was to characterize structures from explants of E. globulus in culture conditions described previously as effective in SE regeneration (Pinto et al 2002(Pinto et al , 2008a(Pinto et al , b, 2010. From histological analysis we conclude that the developing shoots are predominantly of organogenic, rather than embryogenic, origin.…”

“…Cultures were established following the protocol described by Pinto et al (2008a). Briefly, seeds were surface sterilized with a mixture of 1:1 absolute ethanol: hydrogen peroxide 30 % (v/v) for 15 min.…”