ORIGINAL PAPER Enhanced in vitro multiplication of Nothapodytes nimmoniana Graham using semisolid and liquid cultures and estimation of camptothecin in the regenerated plants V. S. Dandin • H. N. Murthy Received: 22 January 2011 / Revised: 11 January 2012 / Accepted: 13 January 2012 / Published online: 1 February 2012 Ó Franciszek Go ´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako ´w 2012 Abstract Nothapodytes nimmoniana (Icacinaceae) yields camptothecin (isoquinoline alkaloid) which is a potent anti- cancer drug. The major objectives of the present study were to develop an efficient protocol for mass propagation of N. nimmoniana using liquid medium and to compare regeneration with semisolid cultures; as also to quantify the amount of camptothecin in regenerated plants. Adventi- tious shoots were induced from the callus derived from nodal explants on semisolid and liquid Murashige and Skoog (MS) medium supplemented with 1.0, 2.0, 5.0 and 10.0 lM 6-benzylaminopurine or kinetin or 2-isopentenyl adenine (2-iP). The highest number of adventitious shoots was regenerated on medium supplemented with 2.0 lM BAP. Compared to semisolid medium (41.9 shoots per explant), liquid medium (165.9 shoots per explant) was found suitable for shoot induction and shoot multiplication. Shoots were rooted on MS semisolid medium of one-fourth strength containing IBA (2.4 lM) and IAA (5.7 lM). The plantlets were acclimatized in a growth chamber at 25°C, 60% relative humidity, with 16-h photoperiod (40 lmol m -2 s -1 ). The camptothecin content was deter- mined in ex vitro plants using HPLC. The analysis revealed that the leaves and stems of ex vitro plants had a consid- erable amount of camptothecin and these plants could be used as a raw material for camptothecin extraction. Keywords Adventitious shoots Á Camptothecin Á Liquid cultures Á Nothapodytes nimmoniana Á Propagation Introduction Nothapodytes nimmoniana Graham, formerly known as Nothapodytes foetida Sleumer and Mappia foetida Meirs (Icacinaceae), is a small tree, naturally distributed in many parts of the Western Ghats, South India, some parts of the Himalayan foot hills, Sri Lanka, Myanmar and Thailand (Suhas et al. 2007). This tree is a rich source of campto- thecin, isoquinoline alkaloid, which is currently being used for treating colorectal and ovarian cancer (Lilenbaum et al. 1995; Romanelli et al. 1998; Vladu et al. 2000). In the absence of synthetic sources, the global demand for this alkaloid is met by natural populations of N. nimmoniana. This has led to decrease in the population of this species in the Western Ghats, India, and, in fact, due to the extremely high pressure, the species has been declared as endangered (Suhas et al. 2007). The tree growth is slow, and propa- gation is usually achieved by seeds. The seeds are recal- citrant due to high sensitivity to desiccation, freezing, and have a short shelf life. Till today, a very few tissue culture methods have been developed for the propagation of this plant (Rai 2002; Thengane et al. 2001). However, these regeneration systems for N. nimmoniana produce relatively few propagules. Furthermore, multiplication rate could be enhanced by developing a liquid-based system so that the propagules could be used as a raw material for the pro- duction of camptothecin. Moreover, a liquid-based system could be used for the large scale propagation of N. nim- moniana. Therefore, the major objectives of the present work were to compare semisolid and liquid culture systems for in vitro multiplication of shoots; to ex vitro Communicated by J. Van Huylenbroeck. V. S. Dandin and H. N. Murthy contributed equally for the manuscript. V. S. Dandin Á H. N. Murthy (&) Department of Botany, Karnatak University, Dharwad 580003, India e-mail: nmurthy60@yahoo.co.in 123 Acta Physiol Plant (2012) 34:1381–1386 DOI 10.1007/s11738-012-0934-x