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

Gene Expression Profiling of Shoot-Derived Calli from Adult Radiata Pine and Zygotic Embryo-Derived Embryonal Masses

<div><p>Background</p><p>Although somatic embryogenesis has an unprecedented potential for large-scale clonal propagation of conifers, the ability to efficiently induce the embryonal cultures required for somatic embryo production has long been a challenge. Furthermore, because early stage zygotic embryos remain the only responsive explants for pines, it is not possible to clone individual trees from vegetative explants at a commercial scale. This is of particular interest for adult trees because many elite characteristics only become apparent following sexual maturation.</p><p>Findings</p><p>Shoot explants collected from adult radiata pine trees were cultured on four induction media differing in plant growth regulator composition, either directly after collection or from <i>in vitro</i>-generated axillary shoots. Six callus lines were selected for microscopic examination, which failed to reveal any embryonal masses (EM). qPCR expression profiling of five of these lines indicated that explant type influenced the absolute level of gene expression, but not the type of genes that were expressed. The analysis, which also included three EM lines induced from immature zygotic embryos, encompassed five categories of genes reflective of metabolic, mitotic and meristematic activity, along with putative markers of embryogenicity. Culture medium was found to have no significant impact on gene expression, although differences specific to the explant’s origin were apparent. Expression of transcriptional factors associated with vegetative meristems further suggested that all of the callus lines possessed a substantive vegetative character. Most notable, however, was that they all also expressed a putative embryogenic marker (LEC1).</p><p>Conclusions</p><p>While limited in scope, these results illustrate the utility of expression profiling for characterizing tissues in culture. For example, although the biological significance of LEC1 expression is unclear, it does present the possibility that these callus lines possess some level of embryogenic character. Additionally, expression of vegetative meristem markers is consistent with their vegetative origin, as are differences in expression patterns as compared with EM.</p></div

Embryogenic marker expression within primordial- and axillary-shoot derived tissues of radiata pine.

<p>Expressed as the number of mRNA transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates significant differences based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s003" target="_blank">S3 File</a>). Note that the level of LEC1 expression within the three axillary shoot-derived genotypes is comparable to that seen in EM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.g005" target="_blank">Fig 5</a>), albeit of greater variability.</p

Expression of embryogenic markers within three genotypes of EM induced from immature zygotic embryos of radiata pine.

<p>Average of three biological replicates expressed as the number of transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates no significant differences based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s001" target="_blank">S1 File</a>).</p

WOX4 gene expression within cultures derived from primordial- and axillary-shoot explants of radiata pine.

<p>Expressed as the number of mRNA transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates significant differences based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s003" target="_blank">S3 File</a>).</p

Induction medium composition.

<p>Induction medium composition.</p

Cell division gene expression within three genotypes of EM induced from immature zygotic embryos of radiata pine.

<p>Average of three biological replicates expressed as the number of transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates significant differences in histone 4 expression based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s001" target="_blank">S1 File</a>).</p

List of qPCR primers.

<p>*Average LRE-derived amplification efficiency (n = 32)</p><p>List of qPCR primers.</p

YLS8 expression within primordial and axillary shoot-derived tissues of radiata pine.

<p>Expressed as the number of YLS8 transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates no significant differences based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s003" target="_blank">S3 File</a>).</p