Plant regeneration from shoot regrowth and de novo embryo-like structures from cryopreserved shoot tips of Lilium spp.

Plant regeneration via both shoot regrowth and de novo embryo-like structures was obtained from cryopreserved shoot tips of Lilium spp. Shoot tips (1.0-3.0 mm in length) of Lilium Oriental hybrid 'Siberia' containing two- to four-leaf primordia were excised from 4-wk-old adventitious shoots and cryopreserved by droplet vitrification. When post-cultured for 6 wk on recovery medium containing 0.1 mg/l alpha-naphthaleneacetic acid (NAA) in combination with different kinetin (KT) concentrations (0-0.4 mg/l), three types of regenerants were produced: (1) only embryo-like structures, (2) shoot regrowth with embryo-like structures, and (3) only shoot regrowth. The highest frequencies (a parts per thousand yen75.0%) of total embryo-like structures were obtained from cryopreserved shoot tips post-cultured on recovery medium containing 0.1 mg/l NAA and 0.1-0.2 mg/l KT. Embryo-like structures proliferated within 4 wk of culture on medium containing 1.0 mg/l NAA and 0.05 mg/l thidiazuron (TDZ). Somatic embryo-like structures matured and converted into whole plantlets after 8 wk of culture on Murashige and Skoog (MS) medium without any plant growth regulators. The highest frequencies (a parts per thousand yen25.0%) of total shoot regrowth were produced from cryopreserved shoot tips post-cultured on recovery medium containing 0.1 mg/l NAA and 0.05-0.4 mg/l KT. With the protocols reported here, frequencies of total embryo-like structures and shoot regrowth ranged from 45.0 to 90.0% and 25.0 to 52.5%, respectively, from six lily varieties belonging to five Lilium species or hybrids, representing diverse Lilium germplasm. The results reported here have potential applications to long-term conservation of genetic resources and genetic transformation in Lilium spp.