An effective cryobanking approach preserving the genetic, physiological and phytochemical stability of Polyscias filicifolia Bailey hairy roots

Abstract

Cryopreservation procedures can affect the physiological and genetic stability of stored samples, as well as their biosynthetic potential, which is particularly important for medicinal plants; as such, it is necessary to refine a number of factors to ensure the effective long-term storage of plant cells. The article describes an effective method of cryopreservation of Polyscias filicifolia hairy roots. Briefly, hairy roots were subjected to osmotic dehydration (OD) with or without subsequent air dehydration (AD; for three to six hours) for cryobanking in liquid nitrogen. After regeneration, genetic stability was assessed using Inter Simple Sequence Repeats (ISSRs) and Start Codon Targeted (SCoT) markers, together with nuclear DNA content, survival rate and biomass growth. In addition, chlorogenic (CGA) and oleanolic acid (OA) yields were determined using HPLC-UV-Vis, and the extract profiled by HPLC-PDA-ESI-HRMS, and the antioxidant potential of the treated root extracts was assessed. OD alone was found to be insufficient for cryopreservation; it was necessary to precede it with AD. The highest survival (93%), and biomass growth (up to 3-fold), were noted for roots subjected to OD preceded by six-hour AD; however, AD had a detrimental effect on the genetic stability of roots when applied for five or six hours. Longer dehydration times correlated with greater CGA biosynthesis. Further, CGA content was enhanced to 2.6 mg g−1 dry weight (DW) at six months post regeneration and 2.2 mg g−1 at 12 months. OA yield was not affected by the cryopreservation conditions. HPLC-PDA-ESI-HRMS analysis confirmed that the main component was CGA, accompanied by ferulic acid derivatives, 3,5-O-dicaffeoylquinic acid (isochlorogenic acid A) and 5-p-coumaroylquinic acid. Interestingly, the profile of biosynthesized compounds in the roots did not change during cultivation for up to 12 months. The highest antioxidant activity, determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assay, was noted for the extract from the OD variant, which also demonstrated the highest total phenolic content. Our findings indicate that OD followed by four-hour AD provides the best protection for hairy root meristems with regard to genetic stability, reliable growth and secondary metabolite biosynthesis capabilities.