A protocol for the cryopreservation of Digitalis thapsi L-cell cultures

With a view to establishing a protocol for cryopreservation of suspension c ultures of Digitalis thapsi, we analyzed the effect of preculture duration with various compounds -mannitol (0.15 or 0.3 M), sucrose (0.3M) or proline (0.3M)- followed by treatment with a cryoprotectant solution composed of 0 .5M dimethylsulfoxide (DMSO), 0.5M glycerol and 1M sucrose, slow cooling of sample for 30 min in a -20 degrees C freezer followed by rapid immersion i n liquid nitrogen, rapid thawing and transfer of cells without washing to s tandard semi-solid medium. The highest viability rate after cryopreservatio n (60% viable cells) was obtained when cells were treated for 3d with 0.15M mannitol. After transfer in liquid medium, cultures originating from cryop reserved cells retained their capacity to accumulate cardenolides.