From induction to embryo proliferation: improved somatic embryogenesis protocol in cork oak (Quercus suber L.)

Cork oak (Quercus suber L.) is the dominant species in the forests ecosystems of Northern Morocco, comprising 72% of the area. It is an economically important species, producing high-quality cork, wood and acorns, and offers significant potential for further improvement. Somatic embryogenesis (SE) is the basis of clonal forestry for these species. One challenge with this approach is that SE induction in cork oak has not yet been fully optimized, especially the process of secondary somatic embryogenesis (SSE). Our study focused on developing a reproducible procedure for SSE to produce mature somatic embryos in cork oak. We evaluated the response of different PGR and physical parameters on the morphological development and somatic embryogenesis of cork oak. Darkness and a temperature of 25±2°C yielded the best results (93.30%). Additionally, incubation in complete darkness (in an oven for 2 months)) produced better responses than exposure to a 16-hour photoperiod provided by cool-white fluorescent lamps at a photon flux density of 50-60 µmol m-2s- 1. For secondary somatic embryo production, the use of Naphthalene Acetic Acid (NAA) at a concentration of 1mg/L in N30K medium blocked the germination process in cork oak somatic embryos. Regarding to secondary somatic embryogenesis, the use of NAA had no effect on the process. While the combination of ANA and BAP at 1mg/L showed better effect on stem and leaf formation (0.22±0.07) and (0.71±0.25) respectively. No significant interaction with activated charcoal was observed. In this study, we describe the conditions required for studying the process of secondary somatic embryogenesis, including darkness, temperature and other key parameters. It was concluded that the N30K medium, a temperature of 25°C and full darkness produced the best results.