Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

Abstract

BackgroundSomatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters.Methodology/Principal FindingsThe main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster’s three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth.Conclusions and SignificanceThis work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.

Somatic Embryogenesis Induction in Woody Species: The Future After OMICs Data Assessment

“…(Shamouti orange) (Kochba et al., 1977); Pinus taeda (Becwar et al., 1990); conifers (Bonga, 1995), Camellia sinensis (Nakamura, 1988; Mukhopadhyay et al., 2015); Coffea sp. (Nakamura et al., 1994), Camellia japonica (Barciela and Vieitez, 1993); (Pedroso and Pais, 1992, 1993, 1995); Laurus nobilis (Canhoto et al., 1999); Chestnut (Corredoira et al., 2006, 2006a); Picea abies (Chalupa, 1985; Hakman et al., 1985); Picea glauca (Park et al., 1993); Theobroma cacao (Alemanno et al., 2008); Cyphomandra betacea (Correia et al., 2011); Quercus suber (El Maâtaoui et al., 1990; Bueno et al., 1992); Quercus robur (Corredoira et al., 2006); Pinus species (Häggman et al., 1999; Montalbán et al., 2015); Alnus glutinosa (Corredoira et al., 2013); Fraxinus mandshurica (Kong et al., 2012); Larix kaempferi (Kim, 2015), P. abies (Petrek et al., 2015), woody plants in general (Yuan et al., 2016), among many others.…”

Suspension Culture and Somatic Embryogenesis of Korean Pine

“…From the perspective of plant SE yield, the formation of larger aggregates from ET is an important factor to reduce SE yield [13]. Decomposing ET into smaller aggregates with liquid medium can increase the growth rate of ET and promote the development of SE [10].…”

“…Among them, only early SEs in PEMIII stage can respond to maturation, thus limiting the yield of maturation SEs. In conifers, the metabolic activity of the internal tissues of ET decreases as the proliferation time increases, and only a few early SEs on the surface can respond to maturation, resulting in a decrease in ET utilization [13]. The liquid culture medium can effectively reduce the aggregation of cells by shaking, and make the distribution of nutrients and metabolites uniform.…”