Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species

In this study, we investigated the possibility of using the droplet-vitrification technique for cryopreserving nodal segments of in vitro plantlets of the endangered plant species Lithodora rosmarinifolia. Among the three vitrification solutions tested, only solutions B1, containing (w/v) 50 % glycerol and 50 % sucrose, and B3, containing 40 % glycerol and 40 % sucrose, were able to induce cryotolerance in nodal explants, resulting in intermediate survival and recovery after cryopreservation. A three-step vitrification protocol, including an additional dehydration treatment with half-strength vitrification solution for 30 min before the treatment with full-strength vitrification solution, did not lead to any improvement in survival and recovery compared with the two-step protocol. The optimal protocol was the following: preculture of nodal segments in liquid medium with 0.3 M sucrose for 16 h and 0.7 M sucrose for 5 h, treatment for 20 min in loading solution containing 1.9 M glycerol + 0.5 M sucrose, dehydration with vitrification solution B1 (glycerol 50.0 %, sucrose 50.0 %, w/v) for 60 min at room temperature, rapid cooling in minute droplets of vitrification solution, and rapid rewarming by immersion of nodal segments for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 33 % recovery of cryopreserved nodal explants was achieved. Regrowth of cryopreserved samples was rapid and direct. These results indicate that long-term storage of L. rosmarinifolia by means of cryopreservation of nodal segments is possible, thereby contributing to securing the diversity of this rare and endangered plant species.