Transcriptome profiling and digital gene expression by deep sequencing in early somatic embryogenesis of endangered medicinal Eleutherococcus senticosus Maxim.

Somatic embryogenesis (SE) has been studied as a model system to understand molecular events in physiology, biochemistry, and cytology during plant embryo development. In particular, it is exceedingly difficult to access the morphological and early regulatory events in zygotic embryos. To understand the molecular mechanisms regulating early SE in Eleutherococcus senticosus Maxim., we used high-throughput RNA-Seq technology to investigate its transcriptome. We obtained 58,327,688 reads, which were assembled into 75,803 unique unigenes. To better understand their functions, the unigenes were annotated using the Clusters of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes databases. Digital gene expression libraries revealed differences in gene expression profiles at different developmental stages (embryogenic callus, yellow embryogenic callus, global embryo). We obtained a sequencing depth of >5.6 million tags per sample and identified many differentially expressed genes at various stages of SE. The initiation of SE affected gene expression in many KEGG pathways, but predominantly that in metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction. This information on the changes in the multiple pathways related to SE induction in E. senticosus Maxim. embryogenic tissue will contribute to a more comprehensive understanding of the mechanisms involved in early SE. Additionally, the differentially expressed genes may act as molecular markers and could play very important roles in the early stage of SE. The results are a comprehensive molecular biology resource for investigating SE of E. senticosus Maxim.

Pilot-scale culture of somatic embryos of Eleutherococcus senticosus in airlift bioreactors for the production of eleutherosides.

PURPOSE OF WORK: To establish pilot scale bioreactor cultures of somatic embryos of Siberian ginseng for the production of biomass and eleutherosides. Somatic embryos of Eleutherococcus senticosus were cultured in airlift bioreactors using Murashige and Skoog medium with 30 g sucrose l(-1) for the production of biomass and eleutherosides. Various parameters including the type of bioreactor, aeration volume, and inoculum density were optimized for 3 l capacity bioreactors. Balloon-type airlift bioreactors, utilizing a variable aeration volume of 0.1-0.3 vvm and an inoculum of 5 g l(-1), were suitable for biomass and eleutheroside production. In 500 l balloon-type airlift bioreactors, 11.3 g dry biomass l(-1), 220 µg eleutheroside B l(-1), 413 µg eleutheroside E l(-1), and 262 µg eleutheroside E1 l(-1) were produced.

Large-scale plantlet conversion and ex vitro transplantation efficiency of Siberian ginseng by bioreactor culture.

To achieve large-scale low-cost ex vitro acclimatization of Siberian ginseng plants, heart- and torpedo-shaped secondary somatic embryos (SEs) induced from germinated SEs on agar medium were collected and then inoculated to 10-l bubble column bioreactor, respectively. For plantlet conversion, inoculation of torpedo-shaped secondary SEs was more effective than heart-shaped SEs. TS2 (culture of torpedo-shaped SEs in a bioreactor with a 2-week subculture interval) plantlets had a higher root number and leaf number and larger leaf area than did HS3 (culture of heart-shaped SEs in a bioreactor with a 3-week subculture interval) and HS2 (culture of heart-shaped SEs in a bioreactor with a 2-week subculture interval) plantlets. Of these converted plants, TS2 plantlets had higher survival rate (83.7%) and growth characteristics after transplantation in a simple shed covered with a 50% sunshade net only for 6 months. TS2 plantlets also showed significantly lower H2O2 content and significantly increased superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione transferase (GST) expression levels as compared to HS2 plants when exposure to ex vitro conditions.

Callose deposition is required for somatic embryogenesis in plasmolyzed Eleutherococcus senticosus zygotic embryos.

Dynamic changes in callose content, which is deposited as a plant defense response to physiological changes, were analyzed during somatic embryogenesis in Eleutherococcus senticosus zygotic embryos plasmolyzed in 1.0 M mannitol. During plasmolysis, callose deposition was clearly observed inside the plasma membrane of zygotic embryo epidermal cells using confocal laser scanning microscopy. The callose content of zygotic embryos gradually increased between 0 and 12 h plasmolysis and remained stable after 24 h plasmolysis. During eight weeks induction of somatic embryogenesis, the callose content of explants plasmolyzed for 12 h was slightly higher than explants plasmolyzed for 6 or 24 h, with the largest differences observed after 6 weeks culture, which coincided with the maximum callose content and highest number of globular somatic embryos. The highest frequency of somatic embryo formation was observed in explants plasmolyzed for 12 h. The somatic embryo induction rate and number of somatic embryos per explant were markedly different in zygotic embryos pretreated with plasmolysis alone (78.0%, 43 embryos per explant) and those pretreated with plasmolysis and the callose synthase inhibitor 2-deoxy-d-glucose (11.5%, 8 embryos per explant). This study indicates that callose production is required for somatic embryogenesis in plasmolyzed explants.

Methyl jasmonate induced overproduction of eleutherosides in somatic embryos of Eleutherococcus senticosus cultured in bioreactors

This study was concentrated on the production of eleutherosides and chlorogenic acid in embryogenic suspension cultures of Eleutherococcus senticosus by exposing them to different concentrations (50-400 µM) of methyl jasmonate (MJ) during the culture period. In the bioreactor cultures, eleutheroside content increased significantly by elicitation of MJ, however, the fresh weight, dry weight and growth ratio of embryos was strongly inhibited by increasing MJ concentrations. The highest total eleutheroside (7.3 fold increment) and chlorogenic acid (3.9 fold increment) yield was obtained with 200 µM MJ treatment. There was 1.4, 3.4 and 14.9 fold increase in the eleutheroside B, E, and E1 production respectively with such elicitation treatment. These results suggest that MJ elicitation is beneficial for eleutheroside accumulation in the embryogenic cell suspension cultures.

Cellular change and callose accumulation in zygotic embryos of Eleutherococcus senticosus caused by plasmolyzing pretreatment result in high frequency of single-cell-derived somatic embryogenesis.

Eleutherococcus senticosus zygotic embryos were pretreated with 1.0 M mannitol or sucrose for 3-24 h. This pretreatment resulted in a high frequency of somatic-embryo formation on hormone-free medium. All the somatic embryos developed directly and independently from single epidermal cells on the surface of zygotic embryos after plasmolyzing pretreatment. Scanning electron microscopic observation revealed that the epidermal cells of hypocotyls rapidly became irregular and showed a random orientation before somatic-embryo development commenced. At the same time, the epidermal cells in the untreated control remained regular. Callose concentration determined by fluorometric analysis increased sharply in E. senticosus zygotic embryos after plasmolyzing pretreatment but remained low in the untreated control. Aniline blue fluorescent staining of callose showed that the plasmolyzing pretreatment of zygotic embryos resulted in heavy accumulation of callose between the plasma membrane and cell walls. On the basis of these results, we suggest that plasmolyzing pretreatment of zygotic embryos induces the accumulation of callose, and the interruption of cell-to-cell communication imposed by this might stimulate the reprogramming of epidermal cells into embryogenically competent cells and finally induce somatic-embryo development from single cells.

Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng.

The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period.

Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides.

Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.

Production of eleutherosides in in vitro regenerated embryos and plantlets of Eleutherococcus chiisanensis.

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l-1. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 microM: gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.

Enhanced post-germinative growth of encapsulated somatic embryos of Siberian ginseng by carbohydrate addition to the encapsulation matrix.

This experiment was carried out to enhance conversion and ex vitro survival of encapsulated somatic embryos of Siberian ginseng (Eleutherococcus senticosus). Cotyledonary somatic embryos were encapsulated with 3.0% sodium alginate; 96% of the encapsulated embryos converted to plantlets with well-elongated epicotyls in Perlite containing sucrose as a carbon source. However, although they germinated, post-germinative growth of encapsulated embryos was suppressed on Perlite that did not contain sucrose. Instead of sucrose addition to Perlite, addition of carbon sources to the encapsulation matrix enhanced post-germinative growth of encapsulated embryos. In the encapsulation matrix with 2% sucrose, post-germinative growth of encapsulated embryos was more than twice (23.5%) that of the control capsules without sucrose (10.0%). Embryos encapsulated with both 2% sucrose and 1% starch powder showed the highest post-germinative growth percentage (42.1%). Iodine staining and analysis of starch content in the encapsulation matrix revealed that starch in the encapsulation matrix decomposed during embryo germination. This result indicates that carbohydrate treatment in the encapsulation matrix enhanced post-germinative growth of encapsulated embryos of Siberian ginseng.