Are early somatic embryos of the norway spruce (Picea abies (L.) Karst.) organised?

Background Somatic 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 Findings The 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 Significance This 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

Effects of cytokinins on in vitro mineral accumulation and bud development in Annona glabra L. Efeito de citocininas sobre o acúmulo de minerais e desenvolvimento de brotações de Annona glabra L. in vitro

Annona glabra is a tropical species that has significant agronomic potential in terms of furnishing fruits for in natura consumption and for the production of phyto-pharmaceuticals. In vitro cultivation has been considered the most promising form of propagation for this species, although large scale utilization of this technique is currently limited by high rates of leaf abscission, reduced rates of explant multiplication and slow bud growth. The present work evaluated the effects of different cytokinins on mineral accumulation in shoots of A. glabra cultivated in vitro, and their effects on growth and survival of these plants. Buds of A. glabra were cultivated in Wood Plant Medium (WPM) in the presence of 6-benzilaminopurine (BAP), thidiazuron (TDZ), kinetin (KIN), and zeatin (ZEA). KIN and BAP use resulted in the greatest growth, largest accumulation of dry mass and leaf area development, as well as the greatest survival rate during in vitro cultivation of this species. All cytokinins tested stimulated large accumulations of nitrogen and boron in shoots, but diminished levels of calcium as compared to controls.<br>Annona glabra é uma espécie frutífera tropical que apresenta elevado potencial agronômico pelo fornecimento de frutos para o consumo in natura e pela produção de fitofármacos. O cultivo in vitro tem sido preconizado como a forma mais adequada de propagação para essa espécie, embora sua utilização em larga escala ainda seja limitada pela elevada taxa de abscisão foliar, reduzida taxa de multiplicação dos explantes e crescimento lento das brotações. Objetivou-se, neste trabalho, avaliar o efeito de citocininas sobre o acúmulo de minerais nas brotações de A. glabra cultivadas in vitro e seus reflexos sobre o crescimento e sobrevivência das plantas nesse tipo de ambiente. Brotações de A. glabra foram cultivadas em meio Wood Plant Medium (WPM), na presença de 6-benzilaminopurina (BAP), thidiazuron (TDZ), cinetina (KIN) e zeatina (ZEA). A utilização de KIN e BAP induziu maior crescimento, maior acúmulo de massa seca, maior desenvolvimento da área foliar e maior taxa de sobrevivência das plantas durante o cultivo in vitro dessa espécie. Todas as fontes de citocininas testadas estimularam maiores acúmulos de nitrogênio e boro nas brotações e menores de cálcio