Somatic embryogenesis in Abies nebrodensis, an endangered Sicilian fir

Somatic embryogenesis, as a promising biotechnological tool for many conifer trees, has never been applied for the Abies nebrodensis species. Although all the encouraging results previously obtained by the EU LIFE (European LIFE program) funded projects in over ten years, the critically endangered Sicilian fr remains alarmingly close to extinction. In this study, we reported the frst protocol of somatic embryogenesis obtained from mature zygotic embryos of the Abies nebrodensis. Seeds from Abies adult trees with specifc identifcation numbers (IN) were collected and full seeds were identifed by X-ray. Diferent experiments were carried out for callus initiation, from both zygotic immature and mature embryos, testing diferent culture media. The immature embryos did not give embryogenic tissue (ET). Embryogenic callus (EC) was successfully induced from mature embryos with variable frequencies (0-40%). Schenk and Hilderbrandt (SH) was the most suitable initiation medium where the obtained callus initiation rate reached up to 40% for IN7 (frst experiment). 6-benzylaminopurine (BAP) showed to be essential to induce EC (second experiment). IN8 presented the highest callus initiation rate (40%) among all tested donor trees, whereas IN13 recorded the lowest rate with 4% (third experiment). ET maturation from each singular embryo of IN7, IN8, IN10 and IN21 was successfully achieved in SH medium containing 37,83 µM abscisic acid (ABA), 8% of polyethylene glycol (PEG-4000) and 4% maltose. The encapsulation technology was assessed on the obtained ET and its proliferation was observed after encapsulation.