Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells.

BACKGROUND AND AIMS: 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. KEY 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.

Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures.

We have cloned a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) gene from Medicago truncatula (MtSERK1) and examined its expression in culture using real time PCR. In the presence of the auxin 1-naphthaleneacetic acid (NAA) alone, root differentiation occurs from the proliferating calli in both the cultured highly embryogenic seed line (2HA) and a low to nonembryogenic seed line (M. truncatula cv Jemalong). Auxin stimulated MtSERK1 expression in both 2HA and M. truncatula cv Jemalong. Embryo induction in proliferating calli requires a cytokinin in M. truncatula and unlike root formation is substantively induced in 2HA, not M. truncatula cv Jemalong. On embryo induction medium containing NAA and the cytokinin 6-benzylaminopurine (BAP), expression of MtSERK1 is elevated within 2 d of initiation of culture in both M. truncatula cv Jemalong and 2HA. However, MtSERK1 expression is much higher when both NAA and BAP are in the medium. BAP potentiates the NAA induction because MtSERK1 expression is not up-regulated by BAP alone. The 2HA genotype is able to increase its embryo formation because of the way it responds to cytokinin, but not because of the cytokinin effect on MtSERK1. Although the studies with M. truncatula indicate that somatic embryogenesis is associated with high SERK expression, auxin alone does not induce somatic embryogenesis as in carrot (Daucus carota) and Arabidopsis. Auxin in M. truncatula induces roots, and there is a clear up-regulation of MtSERK1. Although our analyses suggest that MtSERK1 is orthologous to AtSERK1, which in Arabidopsis is involved in somatic embryogenesis, in legumes, MtSERK1 may have a broader role in morphogenesis in cultured tissue rather than being specific to somatic embryogenesis.