Development and optimization of hairy root culture systems in Withania somnifera (L.) Dunal for withaferin-A production

Abstract

Transformation of Withania somnifera was carried out by using three Agrobacterium rhizogenes strains (ATCC 15834, R1000 and K599) for hairy root induction. Induction of hairy root was carried out in leaf, petiole and internodal explants. Hairy root induction was successful only in ATCC 15834 and R1000. The highest frequency of hairy root was obtained in petiole explants (64%) infected with R1000 and it resulted in five distinct morpho-types (callus (fragile), callus (hard), callus + hairy roots, hairy roots and callusing roots). The frequency of R1000 transformation was increased up to 93.2% by the addition of acetosyringone during various steps of infection. Molecular identification through PCR analysis of rolC confirmed the presence of Ri T-DNA. The half strength Murashige and Skoog (MS) liquid medium was found to be the best medium that supports the high root biomass accumulation than the other tested medium types (MS full strength, Gamborg B5 medium (B5) full strength and B5 half strength). High performance liquid chromatography (HPLC) analysis of hairy roots revealed the accumulation of withaferin-A (72.3 mg/g dw). This study reports the influence of Agrobacterium strains, explant types and acetosyringone in the hairy root induction of W. somnifera.

Key words: Withania somnifera, hairy root, Agrobacterium rhizogenes, hairy root morpho-types.