An optimized protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Plumbago zeylanica L. was developed through selection of suitable explant type and the bacterial strain. The infection of internodal explants from an in vivo plant and leaves of in vitro origin with the A4 strain resulted in the emergence of hairy roots at a transformation frequency of 86.33 and 42.33 %, respectively. Independent transformed root somaclones (rhizoclones) capable of sustained growth were maintained under a low illumination in auxin-free agar-solidified Murashige and Skoog (MS) medium through subcultures at periodic intervals. The presence of pRi TL-DNA rolB or rolC genes and pRi TR-DNA mas2 gene in the transformed rhizoclone genome was ascertained by PCR amplification. Concentrations and type of carbon source, auxin and media strength were optimized for root biomass growth. Five independent rhizoclones each from A4- and LBA9402-transformed root lines were studied for their plumbagin accumulation at different growth phases, using HPLC analysis. The potential for plumbagin biosynthesis was expressed in all the tested rhizoclones, although distinct inter-clonal variations were noted. It was evident that maturation of hairy roots was more important for plumbagin accumulation; slow-growing and early-maturing rhizoclones accumulated more plumbagin compared to fast-growing and late-maturing rhizoclones. A4-induced rhizoclone HRA2B5 was identified as the most superior clone with a higher plumbagin yield potential in comparison with other tested hairy root clones, in vitro-grown non-transformed roots and in vivo roots of naturally occurring P. zeylanica. © 2014, Springer Science+Business Media New York.