ORIGINAL PAPER High frequency in vitro propagation of Trichopus zeylanicus subsp. travancoricus using branch–petiole explants Kottackal Poulose Martin • A. K. Pradeep • Joseph Madassery Received: 18 April 2010 / Revised: 7 October 2010 / Accepted: 4 November 2010 / Published online: 21 November 2010 Ó Franciszek Go ´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako ´w 2010 Abstract Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 lM 2,4-dichlorophe- noxyacetic acid upon subculture to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot morphogenesis. Medium with 13.3 lM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower concentrations of BA (4.4, 6.6 and 8.8 lM) alone or in combination with lower concentration of a-naphthalene- acetic acid (NAA) or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 lM BA containing medium, but with reduced number of shoot buds. Subsequent cul- tures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived whole branch–leaf explants on MS basal medium devel- oped shoots directly from the node. On medium with 19.6 lM IBA, the whole branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium contain- ing 2.7 lM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix: a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar to that of the source plants flowered normally and set fruits. Keywords Arogyapacha Á Jeevani Á Organogenesis Á RAPD Introduction Trichopus zeylanicus subsp. travancoricus (Beddome) Burkill ex Narayanan (Trichopodaceae) known as Arogy- apacha (healthy green), is a wild plant with multifarious uses occurring in the thickets of Agasthyamalai hills of Southern Western Ghats, Kerala, India. The medicinal uses of the plant to the present day pharmacopoeia is a contri- bution of the Kani tribe, an indigenous nomadic commu- nity settled life in the forests of the Agasthymalai hills. The unripe fruits of Trichopus are highly rejuvenative and are used by the Kani tribe to ameliorate fatigue (Pushpangadan et al. 1988). The seeds are rich in saponins and the Communicated by B. Borkowska. K. P. Martin Á J. Madassery (&) Department of Biotechnology, University of Calicut, Calicut, Kerala 673 635, India e-mail: madasseryjoseph@yahoo.com A. K. Pradeep Department of Botany, University of Calicut, Calicut, Kerala 673 635, India Present Address: K. P. Martin (&) Department of Plant Sciences, University of California, One Shields Ave., Davis, CA 95616, USA e-mail: marttinet@yahoo.com 123 Acta Physiol Plant (2011) 33:1141–1148 DOI 10.1007/s11738-010-0642-3