Cryopreservation of small leaf squares-bearing adventitious buds of Lilium Oriental hybrid ‘Siberia’ by vitrification
2018
Pan, Chen | Liu, Jing | Bi, Wen-Lu | Chen, Haiyan | Engelmann, Florent | Wang, Qiao-Chun
We report a new cryopreservation method for Lilium Oriental hybrid ‘Siberia’. Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium (MS) supplemented with 1 mg L⁻¹ α-naphthalene acetic acid and 0.5 mg L⁻¹ thidiazuron. Small leaf squares (SLSs, 3 × 4 mm), each bearing at least one adventitious bud, were cut from leaf segments, precultured on medium with 0.5 M sucrose for 1 day, and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 for 7 h at 0 °C. Dehydrated SLSs were directly immersed in liquid nitrogen for 1 h. Cryopreserved SLSs were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, 85% survival and 72% shoot regrowth were achieved following cryopreservation. The use of SLSs bearing adventitious buds for cryopreservation reported in the present study eliminates the time-consuming and labour-intensive step of shoot tip excision, and has great potential to facilitate cryopreservation in other plant species.
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