title: Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells.
abstract: OBJECTIVE
  Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula.
  
  
  METHODS
  Callus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression.
  
  
  RESULTS
  Callus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature.
  
  
  CONCLUSIONS
  The ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This contrasts with other species where totipotent embryo-forming initials mostly originate from procambial cells.
authors.affiliation: Australian Research Council Centre of Excellence for Integrative Legume Research, School of Environmental and Life Sciences, The University of Newcastle, NSW 2308, Australia.
authors.forename: Xin-Ding
authors.initials: XD
authors.lastname: Wang
authors.forename: Kim E
authors.initials: KE
authors.lastname: Nolan
authors.forename: Rina R
authors.initials: RR
authors.lastname: Irwanto
authors.forename: Michael B
authors.initials: MB
authors.lastname: Sheahan
authors.forename: Ray J
authors.initials: RJ
authors.lastname: Rose
mesh_terms: D019070:Cell Lineage / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003583:Cytokinins / Q000494:pharmacology; D007210:Indoleacetic Acids / Q000494:pharmacology; D046913:Medicago truncatula / Q000166:cytology* / Q000187:drug effects / Q000196:embryology*; D018515:Plant Leaves / Q000187:drug effects / Q000196:embryology; D018517:Plant Roots / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D058526:Plant Vascular Bundle / Q000166:cytology / Q000187:drug effects; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D011523:Protoplasts / Q000166:cytology / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D039901:Totipotent Stem Cells / Q000166:cytology* / Q000187:drug effects
chemical_list: D003583:Cytokinins; D007210:Indoleacetic Acids