In vitro callus induction and seed germination protocols for the conservation of biodiversity – the case of two rare woody species with ornamental traits: Juniperus phoenicea L. and J. thurifera L.

Climate change and human actions are compromising the conservation status of natural habitats and spontaneous plant. To cope with these changes, the aims of this study were to identify both in vitro callus induction and seed germination protocols for the conservation of two rare woody species with potential ornamental traits: Juniperus phoenicea L. and J. thurifera L. Microcuttings were collected from wild adult plants. The sterilization protocols influenced the following multiplication phases. To counteract persistent fungal pollution, explants were treated for 1 h with 2 mL L-1 Enovit Metil and then 1 h in a solution containing 1% NaOCl + Tween 20. An 100 and 89% of healthy explants of J. thurifera and J. phoenicea were placed in agar both upright and recumbent. After 30 days in growth chamber (24°C with a photoperiod of 16 h light/8 h dark under 25-30 μmol m-1 s-2 under cool, fluorescent white lamps) circa 88% of recumbent J. thurifera explants started to produce callus. Two different media were then used to induce differentiation from callus: DKW + BAP 0.1 mg L-1 + 150 mg L-1 citric and ascorbic acids for J. phoenicea and WPM + BAP 0.5mg L-1 +2.4D 0.25 mg L-1 + activated charcoal + 150 mg L-1 citric and ascorbic acids for J. thurifera. In the second trial, fresh seeds or naturally digested by wild animals of both species were vernalized (5°C for three months, 20°C for one month and 5°C for one month). After 28 months in cold greenhouse, no germination was observed in J. thurifera, while in J. phoenicea was equal to 1.6 and 7.7% in fresh and digested seeds, respectively. Germinated seedlings then grew, developing lateral shoots. Taking together all these findings, these protocols could play a fundamental role for conservation, recolonization, and recovery within the genus Juniperus.