Ammonium-free medium is critical for regeneration of shoot tips of the endangered species Pogostemon yatabeanus cryopreserved using droplet-vitrification.

BACKGROUND: Pogostemon yatabeanus, synonym Dysophylla yatabeana, (Labiatae) is an endangered wild species in Korea. It has has a limited natural habitat and requires urgent conservation measures. OBJECTIVE: To develop an efficient cryopreservation protocol using in vitro shoot tips to complement traditional conservation approaches in case seeds are unavailable, or insufficient in number for conservation programs. MATERIALS AND METHODS: Node-cutting induced shoot tips of in vitro plants were produced and cryopreserved using a droplet-vitrification method following improvements in preculture, osmoprotection, vitrification solution (VS) and regrowth treatments. The starting protocol included preculture with 10% sucrose for 31 h, followed by osmoprotection with C4-35% (17.5% glycerol + 17.5% sucrose) for 40 min, and cryoprotection with A3-80% (33.3% glycerol + 13.3% DMSO + 13.3% EG + 20.1% sucrose) for 60 min on ice, cooling and warming using aluminum foil strips, and regrowth in MS hormone-free medium. RESULTS: Shoot tips of Pogostemon yatabeanus were sensitive to the osmotic stress evidenced by low survival after step-wise preculture with 17.5% sucrose and cryopreservation without osmoprotection. Among VS tested, including PVS2, PVS3 and their alternatives, A3-80% on ice for 60 min resulted in the highest post-cryopreservation survival (80%) and regeneration (20%). Post-cryopreservation regeneration significantly improved (up to 73%) by incubation of cryopreserved shoot tips on ammonium-free medium followed by GA X3-containing medium and medium without growth regulators. CONCLUSION: Cryopreservation of in vitro shoot tips using droplet-vitrification was developed as a complementary conservation approach for D. yatabeana. Adjustment of medium composition during the recovery stage was important for regeneration of healthy plants from both cryoprotected-control and cryopreserved shoot tips.