Efficient Cryopreservation of Lilium spp. Shoot Tips using Droplet-vitrification

Abstract

Newly developed shoot tips from adventitious buds induced by tissue cultured bulb-scale segments of five accessions of Lilium spp. were successfully cryopreserved by a droplet-vitrification method. Bulb-scale segments cultured on Murashige and Skoog (MS) medium with 0.1 mg?L -1 IAA and 0.1 mg?L -1 zeatin were then cold-hardened at 4℃ for 7 days. The excised shoot tips were pre-cultured on solidified MS medium containing 0.3 M sucrose for 1 day at 23℃ and then soaked in a mixture of 0.7 M sucrose for a day at 23℃. Pre-cultured shoot tips were cryoprotected with two loading solutions, LD1 and LD2, which included 35% and 40% plant vitrification solution (PVS3), respectively, for 40~60 min at 23℃. The cryoprotected shoot tips were then soaked in PVS2, modified PVS2 and PVS3 for 90~120 min at 23℃. The shoot tips, frozen in microdroplets of vitrification solution, were wrapped with aluminum foil strips, which were immersed rapidly in liquid nitrogen. The shoot tips were then rapidly warmed using unloading solution, transferred to a regeneration medium, stored in the dark for two weeks at 23℃, and then cultured under white fluorescent light at an intensity of 2000 lux with a 16-h photoperiod at 23℃. The average post-cryo regeneration rates of five accessions ranged from 52.7% to 87.5%.