Long-Term Conservation for the Safeguard of : An Endemic and Endangered Species of Sicily.

The combined approaches between ex situ and in situ conservation are of great importance for threatened species in urgent need of protection. This study aims to develop concrete actions to preserve the relic of 30 adult trees of the Sicilian fir () from extinction using long-term germplasm conservation in liquid nitrogen (LN, -196 °C). Pollen grains were collected, and their moisture content (MC) was measured. Then, viability (2,3,5-tryphenyl tetrazolium chloride, TTC), in vitro germinability, and enzymatic antioxidant activity (ascorbate peroxidase, APX; catalase, CAT) were evaluated before and after cryopreservation. Seeds collected from mature cones underwent X-ray analysis, and only full seeds were used to excise the zygotic embryos (ZEs) for cryopreservation. The MC percentage of ZEs was determined, and then they were plunged in LN with (+PVS2) or without (-PVS2) Plant Vitrification Solution 2; untreated ZEs were used as a control. Viability (TTC test) and in vitro germination were assessed for all ZEs (+PVS2, -PVS2, and control). Embryogenic callus (EC) lines obtained from mature ZEs were cryopreserved applying the 'encapsulation-dehydration' technique. This study has allowed, after optimizing cryopreservation protocols for pollen, ZEs, and EC of , to establish the first cryobank of this endangered species in Polizzi Generosa (Palermo, Italy), inside the 'Madonie Regional Park'. The strategy developed for Sicilian fir conservation will pave the way for similar initiatives for other critically endangered conifer species.