Propagation and cryopreservation of Ulleungdo hemlock (Tsuga ulleungensis) via somatic embryogenesis

Key messageThis study describes a method for the regeneration of Ulleungdo hemlock through somatic embryogenesis and a protocol for the cryopreservation of embryogenic cultures.AbstractUlleungdo hemlock (Tsuga ulleungensis) is a rare endemic forest species growing on Ulleung Island in Korea. Climate change and other factors such as insect pests and a low seed germination rate further endanger this species. Here, we established an in vitro propagation and cryopreservation system for Ulleungdo hemlock via somatic embryogenesis. Whole megagametophytes with zygotic embryos from immature Ulleungdo hemlock seeds were cultured on a modified Litvay medium supplemented with 9 mu M 2,4-dichlorophenoxyacetic acid and 4.5 mu M 6-benzylaminopurine. The cone collection date had a significant effect on the induction of embryogenic tissue (P<0.001). Maturation medium containing Litvay salts, 50g l(-1) polyethylene glycol 4000, 30g l(-1) maltose, and abscisic acid (ABA) was used to test the effects of three different concentrations (30, 60, and 120 mu M) of ABA on somatic embryo production. There were statistically significant effects of ABA concentration on somatic embryo production per 80mg of embryogenic tissue (P<0.001), and the most productive ABA concentration was 60 mu M. We also conducted experiments to determine the effect of cryopreservation treatment on the maturation capacity of somatic embryos. Although the highest mean number of somatic embryo production per 80mg of embryogenic tissue was obtained from a non-cryopreserved cell line (36.3 +/- 3.5), no significant differences between the cryopreservation and non-cryopreservation treatment were observed (P=0.093). Our results described here have great potential to contribute to the propagation and conservation of Ulleungdo hemlock in its native habitat.