Embryo forming cells in carrot suspension cultures.

Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic morphological stages, i.e. the globular-, heartand torpedo-stage respectively, as their zygotic counterparts. Due to the different cellular origin of somatic embryos, it is less clear to what extent the earlier pre-globular stages of somatic embryos resemble corresponding stages in zygotic embryo development. In part, this is due to a lack of a precise morphological description of this less defined stage of somatic embryo development. The current stage of these and other, more general aspects of early somatic and zygotic embryo development, are discussed in chapter 1.While the single cell origin of some carrot somatic embryos has been reported, a more elaborate morphological description of a representative number of such single embryo-forming cells and their early development has Peen lacking, so far. To describe cells that are in the process of becoming embryogenic, yet still require an externally applied stimulus, the term competent cell has been introduced. Embryogenic cells can develop into somatic embryos in the absence of an externally applied stimulus. In chapter 2 experiments are presented that show the development of many individual single competent carrot suspension cells into somatic embryos employing a cell tracking system. The capability to develop into somatic embryos appeared not to be restricted to a particular cell type distinguishable on cell morphology. In general, oval and elongated cells developed via asymmetrically shaped cell clusters while spherical cells developed via symmetrically shaped cell clusters into somatic embryos. Cells initially more variable in form developed into somatic embryos via aberrantly shaped cell clusters. These results show that the initial form of the cell and subsequent division patterns can be widely variable and yet lead to complete somatic embryos capable of developing into plants.Based upon previous findings that the monoclonal antibody JIM8 recognises a particular type of single cells only present in embryogenic carrot cell cultures, it was postulated that the JIM8 epitope could be used as a marker for competent and embryogenic cells. The cell tracking system was adapted to study the development of cells labelled with JIM8 in order to determine the reliability of this marker. In chapter 3 it is shown that only few of the single cells developing into somatic embryos reacted with the JIM8 antibody, while most of the embryos developed from cells not labeled with the JIM8 antibody. It was therefore con-cluded that the JIM8 cell wall epitope reflects embryogenic competence in a cell population rather than competence of individual cells.Stimulation of somatic embryogenesis in carrot and other species, by the addition of arabinogalactan proteins (AGPs) to the culture medium has been reported previously. In chapter 4 experiments are presented that show that carrotseed AGP fractions purified by affinity chromatography with ZUM18 monoclonal antibodies do not increase the number of somatic embryos developing in embryogenic cell cultures. An AGP fraction purified with the JIM8 antibody even decreased the number of somatic embryos. Low- embryogenic carrot suspension cultures treated with carrot-seed AGPs did show an increased frequency of embryo development after removal of vacuolated cells and enrichment for cell clusters. These results suggest that complex cell cell interactions, mediated in part by AGPs, occur in embryogenic cultures.The cell tracking system was also adapted to allow detection of the expression of bioluminescent reporter genes. In chapter 5 the expression of the firefly luciferase coding sequence under control of the Arabidopsis thaliana lipid transfer protein 1 (AtLTP1) promoter during carrot somatic embryo development is described. The carrot lipid transfer protein EP2 is expressed during protoderm formation and has been used as a molecular marker for embryogenic competence and somatic embryos. The cell tracking experiments on AtLTP1 luciferase transformed cultures showed that AtLTP1 expression is correlated with somatic embryo formation, but that not all clusters that express AtLTP1 developed into somatic embryos. AtLTP1 expression therefore is a good marker for embryogenic cell clusters, but it is not completely specific.In chapter 6 a technical description is given of the cell tracking system and the several detection systems connected to it, as applied in the preceding chapters. In chapter 7 the relevance of cell tracking to study somatic embryo development and the implications of the described results on future research are discussed