Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the T(L) T-DNA, and 90% of them carried the T(R) T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the T(L) and the T(R)-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the T(R) T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the T(L) T-DNA genes, while all the callusing lines induced by strain A4 contained the T(L) DNA. Four of these callus lines produced both withaferin A (0.15-0.21% dry weight) and withanolide D (0.08-0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.