Somatic embryogenesis, plant regeneration, and cryopreservation for Torreya taxifolia, a highly endangered coniferous species

Torreya taxifolia Arn., an ancient evergreen tree, is on the brink of extinction from attack by a fungal disease, recently reported to be caused by a novel isolate of Fusarium. We report the development of a somatic embryogenesis tissue culture system that can be used for cryogenic storage of T. taxifolia cultures and subsequent plant regeneration. Initiation of embryogenic tissue from immature zygotic embryos occurred on a conifer tissue culture medium containing 0.25?% activated charcoal, 43.8?mM maltose, 0.5?mM 2,4-dichlorophenoxacetic acid, 0.2?mM 6-benzylaminopurine, 0.2?mM kinetin, 0.1???M brassinolide, 3.8???M abscisic acid, 20.5???M biotin, 1.13???M folic acid, 1.28?mM 2(n-morpholino)ethanesulfonic acid and 0.69?mM pyruvic acid. Embryo induction ranged from 60 to 100?% across six seed sources. Somatic embryo development occurred on a medium containing 43.8?mM maltose, 1?% activated charcoal, 37.8???M abscisic acid, 20.5???M biotin, 0.1???M brassinolide, 0.205?mM folic acid, 1.28?mM 2(n-morpholino)ethanesulfonic acid and 0.69?mM pyruvic acid. Germination of somatic embryos ranged from 64 to 82?%. Embryogenic tissue cultures from 30 genotypes representing seed from six mother trees were cryopreserved, and culture recovery was demonstrated after freezing. In contrast to many other coniferous tree seeds, the measured water potential (?MPa) of T. taxifolia megagametophyte tissue rose greatly during seed after-ripening. Duplication of this rise in vitro allowed development of somatic embryos to the cotyledonary stage.