Genetic stability and coumarin content of transformed rhizoclones and regenerated plants of a multi-medicinal herb, Hybanthus enneaspermus (L.) F. Muell.

Agrobacterium rhizogenes-transformed hairy root somaclones (rhizoclones) and regenerated shoots of a rare medicinal herb, Hybanthus enneaspermus (L.) F. Muell. were investigated as a source of coumarin. Transformed nature of rhizoclones was verified by PCR amplification of rolA-B and mas2 genes. Clonal fidelity among rhizoclones was revealed by RAPD analysis exhibiting DNA monomorphism. Of a total of 104 rhizoclones of A4 origin and 76 rhizoclones of 8196 origin, a sample of 16 PCR tested clones was selected for shoot regeneration including eight clones pertaining to each strain type on the basis of sustained root growth index. Opine gene expression was demonstrated in rhizoclones and regenerated shoots. Inter-rhizoclonal variations with respect to biomass proliferation and coumarin content were evident. HPLC-tested coumarin accumulation was ca. threefold higher in the superior rhizoclone of A4 origin (A4-HRL-2B7) compared to that in natural roots. Transformed leafy shoots derived from A4-HRL-2B7 had the maximum coumarin content (3.25 mg g(-1) d. wt. extract) which was significantly higher than that of in natura aerial part samples. Genetic stability of selected fast-proliferating rhizoclones and their respective in vitro shoot regenerants in terms of RT-PCR based expression of rolB and rolC genes and coumarin content was sustained through 16 successive multiplication cycles. This investigation ushers the possibility of harnessing the genetically stable biosynthetic potential of hairy root clones and transformed shoot regenerants of H. enneaspermus in vitro towards sustainable production of coumarin and other medicinally important phytochemicals.