Characteristics of Atropa belladonna hairy roots cryopreserved by vitrification method

Atropa belladonna hairy roots (clone M8) were successfully cryopreserved by using the vitrification method. A. belladonna hairy root tips were precultured on a half strength of Murashige and Skoog (MS) solid medium with 0.1 mg/L 2,4-D or without phytohormone for 1 day, and then dehydrated with PVS2 solution for 15 minutes prior to immersion into liquid nitrogen for 1 day, 1 week, 1 month and 3 months. Hairy root tips kept in liquid nitrogen were rapidly thawed at 36 °C in a water bath. The root tips were recultured on 1/2 MS medium. The hairy root tips, precultured with 2,4-D before cryopreservation, showed a higher survival rate than those precultured without phytohormone. The hairy root tips, precultured with 2,4-D, showed an average survival rate of 83%. There was no significant difference in the viability of the hairy roots cryopreserved for different periods. The regrowth of cryopreserved hairy roots was similar to that of untreated hairy roots and tropane alkaloid productivity became stable after 4th subculture. PCR analysis of hairy roots demonstrated the conservation of the T-DNA in cryopreserved hairy roots. These results indicate that cryopreservation by vitrification method is useful to preserve A. belladonna hairy root clone M8.