Somatic embryogenesis from leaf and petiole explants of the Moroccan olive cultivar Dahbia

• A new approach for somatic embryo induction and expression in olive has been reported.
• Somatic embryogenesis has been observed on rejuvenated leaf explants taken from in vitro growing shoots.
• First report on somatic embryogenesis from leaf fragment explants of the Moroccan olive cultivar ‘Dahbia’.

Olive (Olea europaea L.) is the main fruit tree species in Morocco. In vitro regeneration from adult material, through somatic embryogenesis, has hardly been studied. Herein, we report a developmental study of olive embryogenesis using histological techniques to broaden our understanding of this process. Explants used consisted on petioles and leaf fragments, taken from in vitro rejuvenated plantlets of the Moroccan olive cultivar Dahbia. Explants were cultured on 3 different media corresponding to different stages of the embryogenic process: (a) induction phase: a liquid medium, consisting of half strength Murashige and Skoog medium (MS/2) supplemented with 30 μM thidiazuron (TDZ) and 0.5 μM 1-naphthalenacetic acid (NAA), for 2–6 days (short induction periods) or the same medium with solid texture for 2–4 weeks (long induction periods), were tested; (b) proliferation phase (8 weeks): three different media were tested, (1) Murashige and Skoog medium at half strength MS/2; (2) olive cyclic embryogenesis basal medium (ECO) supplemented with 1 g/L activated charcoal (AC) or (3) ECO basal medium supplemented with 0.25 μM indole-3-butyric acid (IBA), 0.44 μM benzyladenine (BA) and 0.5 μM 2-isopentenyladenine (2iP) and (c) expression phase: ECO medium supplemented with growth regulators as indicated in the proliferation phase, during 4 additional weeks, was used. Histological observations were carried out at different phases of the embryogenic process. Embryogenic callus was mainly obtained when MS/2 was used as proliferation medium independently of the induction treatment used; however, somatic embryos could only be recovered from leaf explants cultured for four days on liquid induction medium, then transferred to MS/2 proliferation medium for 8 weeks and finally cultured onto the expression medium over four additional weeks. Somatic embryos developed from nodular and brown callus and could be maintained on expression medium showing a high proliferation rate.