Cryopreservation of Hyoscyamus niger adventitious roots by vitrification

Abstract

An auxin-independent adventitious root culture of Hyoscyamus niger was established, and the roots were successfully cryopreserved with a high regeneration rate of 93.3 percnt; by vitrification method. The root tips were cultured for 12 to 14 days in phytohormone-free Murashige and Skoog (MS) liquid medium, and were excised and precultured on Woody Plant (WP) solid medium supplemented with 0.3 mol/L sucrose at 25 °C in the dark. After 1 day, they were treated with MS-based loading solution for 10 min, followed by soaking in MS-based PVS2 for 10 min at 0 °C. The treated root tips were immersed in liquid nitrogen (-196 °C). For recovery, the root tips were thawed rapidly at 40 °C and washed with MS medium containing 1 mol/L sucrose prior to plating onto WP solid medium. The regenerated roots were evaluated by their growth and tropane alkaloid production. The growth and alkaloid content of regenerated roots analyzed using HPLC were found to be almost the same as those of non-treated roots.