Time-lapse cell tracking reveals morphohistological features in somatic embryogenesis of Araucaria angustifolia (Bert) O. Kuntze

Abstract

Key message Somatic embryogenesis in A. angustifolia is dependent on using smaller cell aggregates and a high osmotic potential culture medium followed by transfers to maturation culture medium with ABA. Abstract Araucaria angustifolia has been extensively studied as a model system for somatic embryogenesis (SE). This protocol is characterized by the development of embryogenic cultures (EC), which are multiplied in pro- embryogenic masses (PEM). However, it hampers in the maturation of somatic embryos from PEM. The building of a SE fate map, allowing the analysis of individual stages of embryonic development, may help identify the morpho- histological features that are causing failure in the matu- ration process. In this sense, the aim of this work was to characterize by means of time-lapse cell tracking the pro- cess of proliferation and maturation of A. angustifolia EC. We also performed transmission electron microscopy in EC and light microscopy analysis in early somatic embryos obtained. The TEM analysis showed a novel characteri- zation of the different cell types that constitute the PEMs and the identification of intriguing mitochondrial structural morphology. In the cell tracking results, smaller cell aggregates showed to be more suitable for transfer to maturation step. Accordingly, the use of smaller cell aggregates together with a high osmotic potential culture medium during maturation phase I (ABA-free), and sub- sequently transfers to the maturation phase II (with ABA) proved to be more suitable for early somatic embryos obtainment. Moreover, the direct transfer of EC from proliferation to maturation with ABA seems to inhibit the further development of somatic embryos. Finally, the early somatic embryos characterized by light microscopy revealed the presence of intercellular spaces, which tended to develop poorly organized shoot apical meristems and abnormal embryos.