Somatic Embryogenesis and Organogenesis from Cryopreserved Shoot Tips of Lilium Oriental Hybrid 'Siberia'

Somatic embryogenesis and organogenesis were for the first time achieved from cryopreserved shoot tips of Lilium Oriental hybrid 'Siberia'. Shoot tips (1.5-2 mm in length) including 2-3 leaf primordia were excised from 4-week-old adventitious shoots directly regenerated from basal leaf segments and precultured on MS containing 0.5 M sucrose for one day. The precultured shoot tips were treated with a loading solution composed of MS containing 0.4 M sucrose and 2 M glycerol for 20 min at room temperature and dehydrated for 4 h by Plant Vitrification Solution 2 (PVS2) at 0 degrees C. Dehydrated shoot tips were transferred onto droplets made on sterile aluminium foil (7 x 20 mm), each droplet containing 2.5 mu l PVS2 and single shoot tip, prior to a direct immersion into liquid nitrogen for 1 h. Following cryostorage, foil strips with shoot tips were incubated for 20 min in an unloading solution composed of MS containing 1.2 M sucrose at room temperature. Cryopreserved shoot tips were post-cultured on recovery media in consistent darkness for embryogenic callus formation or in the dark for three days and then transferred to light conditions for shoot regeneration. Embryogenic callus was obtained on recovery medium containing 0.1 mg/L Kinetin (KT) and 0.1 mg/L a-naphthaleneacetic acid (NAA), with embryogenic callus frequency at about 70%. Shoots were directly regenerated from cryopreserved shoot tips when post-cultured on recovery medium containing 0.2 mg/L Thidiazuron (TDZ) and 1.0 mg/L NAA, with shoot regrowth rate at about 90%. Results reported here are of significance in studies on manipulation of development of somatic embryogenesis and organogenesis from cryopreserved shoot tips. Extra commercially important Lilium species or hybrids are under investigation on their regenerative response to droplet-vitrification cryopreservation developed in the present study.