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Research Project: Innovations that Improve the Efficiency and Effectiveness of Managing and Preserving Ex Situ Plant Germplasm Collections

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Title: Somatic embryogenesis and organogenesis from cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’

item YIN, ZHEN-FANG - Northwest Agriculture And Forestry University
item CHEN, LONG - Northwest Agriculture And Forestry University
item BI, WEN-LU - Northwest Agriculture And Forestry University
item Volk, Gayle
item WANG, QIAO-CHUN - Northwest Agriculture And Forestry University

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2013
Publication Date: 6/22/2014
Citation: Yin, Z., Chen, L., Bi, W., Volk, G.M., Wang, Q. 2014. Somatic embryogenesis and organogenesis from cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’. Acta Horticulturae. 1039:193-200.

Interpretive Summary: Lily (Lilium sp.) shoot tips were successfully cryopreserved using droplet-vitrification techniques. The shoot tips were derived from shoots grown from cultured basal leaf segments. The cryopreservation protocol used a sucrose/glycerol preculture step, followed by a 4 h treatment with PVS2 solution, prior to plunging into liquid nitrogen within droplets on aluminum foil strips. Shoot tips were then warmed and plated onto medium. Either embryogenic callus (70%) or shoots (90%) were obtained after liquid nitrogen exposure, dependent upon the growth medium. When the results have been confirmed for additional lily cultivars or species, they can be used for both biotechnology applications and within gene banks for long-term conservation .

Technical Abstract: Somatic embryogenesis and organogenesis were achieved from cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’. Shoot tips (1.5-2 mm) were excised from adventitious shoots that were regenerated from basal leaf segments. Precultured shoot tips were then treated with MS containing 0.4 M sucrose and 2 M glycerol for 20 min at room temperature and dehydrated for 4 h using Plant Vitrification Solution 2 (PVS2) at 0 °C. Dehydrated shoot tips were transferred onto droplets made on sterile aluminium foil (7 × 20 mm), each droplet containing 2.5 µL PVS2 and single shoot tip, prior to a direct immersion into liquid nitrogen for 1 h. Following cryostorage, frozen 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 3 days and then transferred to light conditions for shoot regeneration. Embryogenic callus was recovered in 70% of the cryopreserved samples when they were cultured on medium containing 0.1 mg/L KT and 0.1 mg/L NAA. Shoots were directly regenerated (90% regrowth) from cryopreserved shoot tips when post-cultured on recovery medium containing 0.2 mg/L TDZ and 1.0 mg/L NAA. These results demonstrate that recovery medium manipulations can result in either somatic embryogenesis or organogenesis from cryopreserved shoot tips. Additional commercially important Lilium species and hybrids are currently under investigation to determine their regenerative response to the droplet-vitrification cryopreservation methods developed in the present study.