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  • Publication
    Exploring the developmental plasticity of plant cells: somatic embryogenesis in root explants
    (University College Dublin. School of Biology and Environmental Science, 2015)
    The developmental plasticity exhibited by plant cells is well known. However, conventional ideology dictates that only specific subsets of cells within a plant are truly 'totipotent' and thus capable of regenerating embryos in the absence of sexual reproduction. This project challenges this ideology by demonstrating that embryogenic potential can be established in Daucus carota root explants when they are cultured in the presence of the synthetic auxin 2,4-D. Seven day old root explants were cultured in the presence of 2,4-D for 10d. Under these conditions, roots fail to form lateral roots but nodules appear. Following this period of culture, explants were moved to solid, hormone-free medium for a period of 8 w.When cultured in this manner, 6.8% of explants will form embryos between 5 and 8 w after the withdrawal of 2,4-D. The number of explants which form embryos can be increased 1.7-fold by the addition of conditioning factors secreted by a Agrobacterium tumefaciens cell culture. These results suggest that secreted signalling factors secreted by Agrobacterium tumefaciens cells can promote embryo formation from carrot root explants.Root explants were sectioned in order to determine the possible source of embryogenic cells. In explants cultured in the presence of 2,4-D, masses of compact, callus-like cells could be observed surrounding the xylem cells and originating from the lateral root primordia. The staining of longitudinal sections of cultured root explants with the monoclonal antibody JIM8 showed that these cells strongly express the JIM8 epitope. As such, these cells are suggested to be a possible source of embryogenic cells.The level of EP3 endochitinase and Somatic Embryogenesis Receptor-like Kinase (SERK) gene expression were quantified in response to various media in order to determine whether either gene could be used as a marker for embryogenic potential in root explants. Results suggest that the downregulation of EP3 gene expression after 10 days in culture may be negatively correlated with embryogenic potential.Additionally, the suitability of the root explant protocol for inducing somatic embryo formation from root explants of the model monocot Brachypodium distachyon was assessed. When cultured in the presence of 2,4-D and 20% Agrobacterium tumefaciens conditioned-medium for 10 d and subsequently moved to hormone-free medium for a period of 8 w, Brachypodium root explants do not form embryos. However, lateral root primordia swell and develop callus-like material. Future work will focus on developing methods for the reprogramming of these cells.
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