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Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation of Picrorhiza kurroa: an endangered medicinal herb of the alpine Himalayas

  • Biotechnology/Genetic Transformation/Functional Genomics
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Abstract

Picrorhiza kurroa Royle ex. Benth. is a medicinal herb of immense therapeutic value with restricted geographic distribution. Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation was developed for this plant. Multiple shoot bud induction was achieved from leaf explants cultured in Gamborg’s B5 medium containing 3 % (w/v) sucrose, 3 mg/l kinetin and 1 mg/l indole-3-butyric acid. More than 90 % of leaf explants formed shoot buds leading to whole plant regeneration. An Agrobacterium-mediated genetic transformation protocol was developed using A. tumefaciens strain GV3101 harboring binary vector pCAMBIA1302 containing the green fluorescent protein and hygromycin phosphotransferase genes. Leaf explants precultured for 2 d were the most suitable for co-cultivation with Agrobacterium and transformation efficiency was enhanced with 200 μM acetosyringone. Putative transformants were selected using media containing 15 mg/l hygromycin. Transformation was verified by detection of the green fluorescent protein using fluorescence microscopy and by polymerase chain reaction. Approximately 56 % of the explants were transformed with an average of 3.4 ± 0.4 transgenic plantlets per explant. An efficient regeneration and transformation protocol thus developed enabling a fresh perspective of metabolic engineering in P. kurroa using an Agrobacterium-mediated transformation. This is the first report of direct organogenesis from leaf explants and genetic transformation of P. kurroa.

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Acknowledgments

We gratefully acknowledge a financial grant from the Council of Scientific and Industrial Research, Government of India, New Delhi, under Network Project NWP 0008.

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Authors and Affiliations

  1. Plant Biotechnology, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi, 180001, India

    Wajid Waheed Bhat, Surrinder K. Lattoo, Satiander Rana, Sumeer Razdan, Niha Dhar & Rekha S. Dhar

  2. Medicinal Chemistry, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi, 180001, India

    Ram A. Vishwakarma

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  1. Wajid Waheed Bhat
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  2. Surrinder K. Lattoo
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  3. Satiander Rana
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  4. Sumeer Razdan
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  5. Niha Dhar
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  6. Rekha S. Dhar
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  7. Ram A. Vishwakarma
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Correspondence to Surrinder K. Lattoo.

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Editor: M. Nakano

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Bhat, W.W., Lattoo, S.K., Rana, S. et al. Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation of Picrorhiza kurroa: an endangered medicinal herb of the alpine Himalayas. In Vitro Cell.Dev.Biol.-Plant 48, 295–303 (2012). https://doi.org/10.1007/s11627-012-9434-3

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  • DOI: https://doi.org/10.1007/s11627-012-9434-3

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