Histocytological changes and reserve accumulation during somatic embryogenesis in Eucalyptus globulus

Abstract

We recently described a protocol for Eucalyptus globulus somatic embryogenesis (SE). For its immediate use at industrial levels, some stages of the process require better control. In particular, SE germination rates are variable, decreasing SE efficacy. As reserves may play a central role in embryogenic processes, we followed histocytological changes and reserve fluctuations, during SE. For SE induction, explants of mature zygotic embryos were grown on Murashige and Skoog (MS) medium with 3 mg l -1 a-naphthalene acetic acid and later transferred to MS without growth regulators (MS WH ). Samples of zygotic embryo cotyledons (explants), of globular and dicotyledonar somatic embryos, and of embling leaves were analysed for reserve accumulation and histocytological profiles. Cotyledon cells of zygotic embryos were rich in lipid and protein bodies, having almost no starch. After 3 weeks of induction, starch grain density increased in differentiated mesophyll regions, while in meristematic regions their occurrence was diffuse. In globular somatic embryos, starch accumulation increased with time (in amyloplasts), but protein bodies were absent. Cotyledonary somatic embryos had lower density of starch grains and absence of lipid and protein bodies. Embling leaves showed typical histological organisation. This is the first comprehensive study on histological and cytological changes during Eucalyptus SE with emphasis in reserve accumulation. With this work we demonstrate that the presently available SE protocol for E. globulus leads to reserve fluctuations during the process. Moreover, the reserves of somatic embryo cotyledons differ from those of their zygotic embryo counterparts, which reinforce the importance of reserves in the embryogenic process and suggests that manipulating external conditions, SE may be optimised giving suitable emblings production for industrial purposes.

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

“…Globular somatic embryos have been previously reported to consist of small cells with dense cytoplasm and large nuclei as were observed in species that include Daucus carota (Schiavone and Cooke 1985) and Nigella sativa (Banerjee and Gupta 1976). Both in the nodular structures that we obtained and in the structures described by Pinto et al (2008aPinto et al ( , 2010 as globular somatic embryos, the majority of the cells were parenchymatous and vacuolated. Our nodular structures that emerged from explants were anchored in subepidermal cortex.…”

2,4-Dichlorophenoxyacetic acid increases reserve compounds and spectaline contents in Senna spectabilis calli

“…Pinto et al (2010) pointed that starch begins to accumulate early during induction of Eucalyptus globulus somatic embryos with 3 mg L -1 α-naphthalene acetic acid, probably due to the sucrose present in the culture medium and in meristematic regions a lower abundance of starch were presented due consumption in mitotically active tissues that was reported earlier for other species (Barciela and Vieitez, 1993;Canhotoa and Cruz, 1996).…”

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].…”