Somatic Embryogenesis in Centaurium erythraea Rafn-Current Status and Perspectives: A Review

Abstract

Centaurium erythraea (centaury) is a traditionally used medicinal plant, with a spectrum of secondary metabolites with confirmed healing properties. Centaury is an emerging model in plant developmental biology due to its vigorous regenerative potential and great developmental plasticity when cultured in vitro. Hereby, we review nearly two decades of research on somatic embryogenesis (SE) in centaury. During SE, somatic cells are induced by suitable culture conditions to express their totipotency, acquire embryogenic characteristics, and eventually give rise to somatic embryos. When SE is initiated from centaury root explants, the process occurs spontaneously (on hormone-free medium), directly (without the callusing phase), and the somatic embryos are of unicellular origin. SE from leaf explants has to be induced by plant growth regulators and is indirect (preceded by callusing). Histological observations and culture conditions are compared in these two systems. The changes in antioxidative enzymes were followed during SE from the leaf explants. Special focus is given to the role of arabinogalactan proteins during SE, which were analyzed using a variety of approaches. The newest and preliminary results, including centaury transcriptome, novel potential SE markers, and novel types of arabinogalactan proteins, are discussed as perspectives of centaury research.

Keywords: Gentianaceae; antioxidative enzymes; arabinogalactan proteins; centaury; in vitro culture; morphogenesis; plant growth regulators; somatic embryo; tissue culture.

Figure 1

Direct somatic embryogenesis (SE) in Centaurium erythraea solid root culture. (a) The first response of root explant is enlargement and clear morphological changes observed five days after the culture setup on half-strength Murashige and Skoog (½MS) medium, (b) Detail of root explant with somatic embryos, (c) Cotyledonary somatic embryos developed directly from the root explant with no intervening callus phase, (d) Cross-section of root explant at the beginning of the culture. Scale bar indicates 200 μm, (e) Histological appearance of a proembryogenic structure. Scale bar indicates 100 μm, (f) Somatic embryo originated directly from the epidermal and subepidermal cells of the root tissue. Scale bar indicates 100 μm.

Figure 2

SE in Centaurium erythraea leaf culture. (a) Embryogenic callus developed at the edge of the leaf explant treated with 2,4-dichlorophenoxyacetic ac(2,4-D) and N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) in darkness, (b) and (c) Somatic embryos at different stages of development (arrows), (d–f) Micrographs showing somatic embryo development on a leaf explant, (d) Histological appearance of a meristematic center (arrow) in the subepidermal layer of the leaf explant. Scale bar indicates 200 μm, (e) Globular somatic embryo. Scale bar indicates 100 μm, (f) Cotyledonary somatic embryo with apical meristem (AM), leaf primordial (LP), and provascular bundles (PB). Scale bar indicates 200 μm.

Figure 3

Schematic overview of SE in Centaurium erythraea. Left panel: Both leaves and roots of the in vitro grown C. erythraea plants can serve as sources of explants for the induction of SE. Hairy root cultures can also be used as explants. Upper panel: SE can be induced from leaf explants on the inductive medium containing 2,4-D and CPPU, both in the light and in darkness. Somatic embryos form from differentiated somatic cells in the subepidermal layer of the leaf explant. In this case, SE is indirect and proceeds via the callusing phase. Middle panel: The obtained somatic embryos can be further used as explants for secondary or cyclic embryogenesis [40] Lower panel: SE from root or hairy root explants is spontaneous, on ½MS medium and direct. SE starts with asymmetric divisions of single totipotent cells from the epidermal or subepidermal layers of root explant. Successive divisions give rise to somatic embryos.

Figure 4

Distribution of AGPs during SE in centaury. (a) Cross-section of a root explant with somatic embryos at its surface, stained with βGlcY reagent. Scale bar indicates 80 μm (b) Indirect SE on centaury leaf explants grown on 100 μM βGlcY reagent in darkness. Somatic embryos form only on the parts of the explants that are not in direct contact with the medium. (c) Embryogenic globule developed on leaf explant and labeled with JIM15 antibody. Scale bar indicates 10 μm.

Figure 5

Arabinogalactan proteins (AGPs) with Tyr kinase domains. The first sequence is Somatic Embryogenesis Receptor-like Kinase 5 (SERK5) from A. thaliana (AT2G13800.1 or Q8LPS5 protein precursor). Four sequences below are found in the C. erythraea transcriptome, based on homology with SERK5. In addition to AG glycomodules with predicted hydroxyprolines and Tyr kinase domains, all sequences have N-terminal signal peptide and a transmembrane domain, while most have Leucine-rich repeats typical for SERK receptors. The image is generated using ragp.