Cryopreservation and post freeze molecular and biosynthetic stability in transformed roots of Beta vulgaris and Nicotiana rustica

Abstract

Crypopreservation methods were firstly developed for root-tips from hairy root cultures of Beta vulgaris, established after transformation by Agrobacterium rhizogenes. The effects of culture age, pre-growth, cryoprotection, freezing rate and post-freeze culture conditions were determined. The resulting freezing protocol was then used to cryopreserve transformed root cultures of Nicotiana rustica. Both species were viable after freezing (ca. 80%), according to fluorescein diacetate vital staining. However, on average the regeneration of proliferating roots from surviving root-tips was low (<20%). Growth rates, secondary metabolite production and T-DNA structure of a number of hairy root lines were examined and found to be unchanged after cryopreservation.