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

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Abstract

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.

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Acknowledgments

We thank Dr. David Tepfer of Laboratoire de Biologie de la Rhizosphere, Institut National de la Rechereche Agronomique, Versailles, Cedex, France) and Dr. M. R. Davey of School of Biological Sciences, University of Nottingham, Nottingham, UK for having kindly provided the Agrobacterium rhizogenes strains. Facility for HPLC analysis was kindly provided by Dr. S. Peruncheralathan, Reader-F and the Director, National Institute of Science Education and Research (Department of Atomic Energy, Government of India), Bhubaneswar-751 005, India. Funding support by the University Grants Commission (UGC), New Delhi, India through a UGC Research Award Project to PKC is gratefully acknowledged. P. R. B. acknowledges National Medicinal Plant Board, New Delhi, India for the receipt of a research fellowship.

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Authors and Affiliations

  1. Plant Cell and Tissue Culture Facility, Post-Graduate Department of Botany, Utkal University, Vani Vihar, Bhubaneswar, Odisha, 751 004, India

    Priya Ranjan Behera, Ram Chandra Jena & Pradeep Kumar Chand

  2. Plant Biotechnology Laboratory, Bioresource Engineering Department, CSIR-Institute of Minerals and Materials Technology (Government of India), Bhubaneswar, 751013, India

    Priya Ranjan Behera & Manikkannan Thirunavoukkarasu

  3. School of Chemical Sciences, National Institute of Science Education and Research (Department of Atomic Energy, Government of India), Bhubaneswar, 751005, India

    Anuradha Das

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  1. Priya Ranjan Behera
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  5. Pradeep Kumar Chand
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Correspondence to Pradeep Kumar Chand.

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Behera, P.R., Jena, R.C., Das, A. et al. Genetic stability and coumarin content of transformed rhizoclones and regenerated plants of a multi-medicinal herb, Hybanthus enneaspermus (L.) F. Muell.. Plant Growth Regul 80, 103–114 (2016). https://doi.org/10.1007/s10725-016-0145-3

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