Cryopreservation of Arum palaestinum plant callus as a strategy for mitigating extinction risks

Arum palaestinum is a wild perennial plant commonly known as "Al-Loof" in Jordan. Due to overharvesting, climate change, and increasing demand, its natural populations are threatened with extinction. Cryopreservation, an effective method for conserving plant material at ultra-low temperatures, is explored for A. palaestinum calli. We investigated the applicability of encapsulation-vitrification (using different plant vitrification solutions (PVS) and incubation times), encapsulation-dehydration (using sucrose or sorbitol at different concentrations and dehydration times), and the v-cryoplate (using different pre-culture times and temperatures) techniques. In the encapsulation-vitrification experiment, a notable 82.4 % regrowth rate was achieved by desiccating calli in plant vitrification solution 2 (PVS2) for 10 minutes at 25 °C. The encapsulation-dehydration technique resulted in an 82.6 % regrowth rate by incubating calli for one day in low sucrose levels (0.1 M sucrose) following one hour of air dehydration, where the moisture content of the beads was 30 %. The moisture content of the beads decreased from 81 % before chemical and air dehydration to 71 % after 0 hours of air dehydration combined with chemical dehydration using 0.1 M sucrose or sorbitol. It further dropped to 30–34 % after one day of chemical dehydration with 0.1 M sucrose and 1 hour of air dehydration. The v-cryoplate technique successfully conserved calli, showing impressive survival and regrowth percentages (96.8 %) when the callus was pre-cultured with 0.3 M sucrose for three days at 5 °C. Temperature during pre-culture significantly influenced regrowth percentages in the v-cryoplate technique. The study establishes promising cryopreservation protocols for A. palaestinum calli, offering a means to conserve germplasm and contribute to environmental and biodiversity protection by reintroducing endangered plants to their native habitats.