Developmental pathway of somatic embryogenesis in Picea abies as revealed by time‐lapse tracking

Abstract

Several coniferous species can be propagated via somatic embryogenesis. This is a useful method for clonal propagation, but it can also be used for studying how embryo development is regulated in conifers. However, in conifers it is not known to what extent somatic and zygotic embryos develop similarly, because there has been little research on the origin and development of somatic embryos. A time-lapse tracking technique has been set up, and the development of more than 2000 single cells and few-celled aggregates isolated from embryogenic suspension cultures of Norway spruce (Picea abies L. Karst.) and embedded in thin layers of agarose has been traced. Experiments have shown that somatic embryos develop from proembryogenic masses which pass through a series of three characteristic stages distinguished by cellular organization and cell number (stages I, II and III) to transdifferentiate to somatic embryos. Microscopic inspection of different types of structures has revealed that proembryogenic masses are characterized by high interclonal variation of shape and cellular constitution. In contrast, somatic embryos are morphologically conservative structures, possessing a distinct protoderm-like cell layer as well as embryonal tube cells and suspensor. The lack of staining of the arabinogalactan protein epitope recognized by the monoclonal antibody JIM13 was shown to be an efficient marker for distinguishing proembryogenic masses from somatic embryos. The vast majority of cells in proembryogenic masses expressed this epitope and none of cells in the early somatic embryos. The conditions that promote cell proliferation (i.e. the presence of exogenous auxin and cytokinin), inhibit somatic embryo formation; instead, continuous multiplication of stage I proembryogenic masses by unequal division of embryogenic cells with dense cytoplasm is the prevailing process. Once somatic embryos have formed, their further development to mature forms requires abscisic acid and shares a common histodifferentiation pattern with zygotic embryos. Although the earliest stages of somatic embryo development comparable to proembryogeny could not be characterized, the subsequent developmental processes correspond closely to what occurs in the course of early and late zygotic embryogeny. A model for somatic embryogenesis pathways in Picea abies is presented.

VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation

“…20 Using time-lapse tracking technique, we have characterised previously all the successive stages in this developmental pathway, including the stages of proembryogenic mass, proembryogenic mass-to-embryo transition, early embryogeny and late embryogeny. 21 We found that somatic embryo development of Norway spruce is similar to zygotic embryogeny of Pinaceae, even though embryo origin is different in each case (i.e., somatic cells in proembryogenic mass versus zygote). 21,22 Our more recent studies suggest that autophagic programmed cell death regulates active shape remodeling during both somatic and zygotic embryo development, 23,24 and that dysregulation of this cell death leads to embryonic aberrations.…”

“…21 We found that somatic embryo development of Norway spruce is similar to zygotic embryogeny of Pinaceae, even though embryo origin is different in each case (i.e., somatic cells in proembryogenic mass versus zygote). 21,22 Our more recent studies suggest that autophagic programmed cell death regulates active shape remodeling during both somatic and zygotic embryo development, 23,24 and that dysregulation of this cell death leads to embryonic aberrations. 25 Using somatic embryos of Norway spruce, we characterized the cell biological pathway of autophagic death in the suspensor, a temporal structure, which is composed of the cells at successive steps of the death pathway and is finally eliminated during late embryogeny.…”

“…On the top of each bar, a schematic drawing of the corresponding developmental stage is shown (not drawn to scale), including proliferating proembryogenic mass (3d þ PGR), proembryogenic mass competent to form embryos (7d þ PGR), the beginning of early embryogeny (1d ÀPGR), active growth of the embryo-suspensor (4d ÀPGR), embryo at the end of early embryogeny with the fully elongated suspensor (7d ÀPGR), embryo in the beginning of late embryogeny showing elimination of the suspensor (7d ABA) and mature embryo (35d ABA). 21,23,25 Blue colour corresponds to vacuolated cells, which undergo programmed cell death and stain in vivo blue with Evan's blue. The meristematic living cells are shown in red, which stain in vivo red with acetocarmine.…”

A transcriptional view on somatic embryogenesis

“…They can be distinguished from the surrounding cells/callus by their relatively high nucleus to cytoplasm ratio, a nucleus with a single large nucleolus and relatively low heterochromatin levels, the presence of fragmented vacuoles, and by their callose‐containing cell walls (Verdeil et al., 2007). Although we have a good idea about the types of cells that form somatic embryos in vitro (Filonova, Bozhkov, & Arnold, 2000; Toonen et al., 1994) and the cellular characteristics of these cells (Emons, 1994; Verdeil et al., 2007; Yeung, 1995), the process remains largely undescribed at the molecular level. High throughput expression analyses have been used to identify characteristics of embryogenic explants (Salvo, Hirsch, Buell, Kaeppler, & Kaeppler, 2014; Trontin, Klimaszewska, Morel, Hargreaves, & Lelu‐Walter, 2016; Wickramasuriya & Dunwell, 2015; Yang et al., 2012), but most studies use whole explants, which contain a complex mixture of tissues and cell types, making it difficult to specifically assign molecular identities to embryogenic cells.…”

Somatic embryogenesis of selected coniferous tree species of the genera Picea, Abies and Larix

“…For initiation of embryogenic callus on explants of all spruce and larch species are generally used the basal medium supplemented with an auxin (9 µM 2.4-D) and a cytokinin (4.4 µM BAP) (Hakman et al 1985;Becwar et al 1987;Bozhkov and von Arnold 1998;Filonova et al 2000a). In this experiment, those hormones also usually proved to be sufficient for embryogenic callus initiation in those species.…”