Investigating the cryopreservation of nodalexplants of Lithodora rosmarinifolia (Ten.)Johnst., a rare, endemic Mediterraneanspecies

In this study, we investigated the possibility ofusing the droplet-vitrification technique for cryopreservingnodal segments of in vitro plantlets of the endangered plantspecies Lithodora rosmarinifolia. Among the three vitrificationsolutions tested, only solutions B1, containing (w/v)50 % glycerol and 50 % sucrose, and B3, containing 40 %glycerol and 40 % sucrose, were able to induce cryotolerancein nodal explants, resulting in intermediate survivaland recovery after cryopreservation. A three-step vitrificationprotocol, including an additional dehydration treatmentwith half-strength vitrification solution for 30 minbefore the treatment with full-strength vitrification solution,did not lead to any improvement in survival andrecovery compared with the two-step protocol. The optimalprotocol was the following: preculture of nodal segments inliquid medium with 0.3 M sucrose for 16 h and 0.7 Msucrose for 5 h, treatment for 20 min in loading solutioncontaining 1.9 M glycerol ? 0.5 M sucrose, dehydrationwith vitrification solution B1 (glycerol 50.0 %, sucrose50.0 %, w/v) for 60 min at room temperature, rapid coolingin minute droplets of vitrification solution, and rapidrewarming by immersion of nodal segments for 20 min inunloading solution containing 1.2 M sucrose. Under theseconditions, 33 % recovery of cryopreserved nodal explantswas achieved. Regrowth of cryopreserved samples wasrapid and direct. These results indicate that long-termstorage of L. rosmarinifolia by means of cryopreservationof nodal segments is possible, thereby contributing tosecuring the diversity of this rare and endangered plantspecies.