BIOLOGIA PLANTARUM 55 (1): 16-20, 2011 16 Micropropagation of Zingiber rubens and assessment of genetic stability through RAPD and ISSR markers S. MOHANTY 1 , M.K. PANDA 1 , S. SAHOO 2 and S. NAYAK 1 * Centre of Biotechnology, Siksha O Anusandhan University, Khandagiri, Bhubaneswar-30, Orissa, India 1 P.G. Department of Botany, Utkal University, Vanivihar, Bhubaneswar, Orissa, India 2 Abstract Protocol was developed for high frequency in vitro multiplication of an endemic species, Zingiber rubens Roxb. The sprouted buds of the rhizomes were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 0.5 - 5.0 mg dm -3 ), indole-3-acetic acid (IAA; 0.5 -2.0 mg dm -3 ), kinetin (KIN; 1.0 - 3.0 mg dm -3 ), naphthaleneacetic acid (NAA; 0.5 - 1.0 mg dm -3 ) and adenine sulphate (ADS; 80 - 100 mg dm -3 ). MS basal medium supplemented with 3 mg dm -3 BA and 0.5 mg dm -3 IAA was optimum for shoot elongation. The elongated shoots (1 - 2 cm) were transferred to multiplication medium containing 2 mg dm -3 BA, 1 mg dm -3 IAA and 100 mg dm -3 ADS. The multiplication rate remained unchanged in subsequent subcultures. Upon ex vitro transfer, 85 % of plants survived. Genetic stability of micropropagated clones were periodically evaluated at an interval of 6 months up to 30 months in culture using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis and genetic uniformity in all regenerants was confirmed. Additional key words: auxins, cytokinins, ex vitro transfer, genetic integrity, red ginger. Introduction Red ginger (Zingiber rubens Roxb.) is an endemic species of family Zingiberaceae (Jain and Prakash 1994) restricted to northeast India and to eastern Himalayas. The plant is used as an ornamental or for medicinal purposes. The plant propagates slowly producing maximum of 8 plants per rhizome in a year in the field. The endemic nature and slow natural propagation rate insists its conservation and propagation through tissue culture technique. In vitro technique can be used in propagation and conservation of endemic species either to produce new plants or as intermediate or long term storage system (Munoz 1995). Plant regeneration in vitro and reintroduction into natural habitat is one strategy for conservation of important plant species (Rout and Das 2002). Periodic monitoring of the degree of genetic stability of in vitro conserved plants is of utmost importance for commercial utilization of true-to-type plants of the desired genotype. The assessment of the genetic integrity of in vitro grown regenerants in regular intervals can significantly reduce or eliminate the chance of occurrence of somaclonal variation (Larkins and Scowcroft 1981) at the early or late phase of culture. Of the various molecular markers random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analysis are the simplest and quickest method for genetic stability assessment of in vitro grown plants. In vitro cultivation has been reported for many species of genus Zingiber, i.e., Z. officinale (Bhagyalakshmi and Singh 1988, Balachandran et al. 1990, Kacker et al. 1993, Sharma and Singh 1997, Rout and Das 2002, Mohanty et al. 2008), Z. cassumunar (Poonasapaya and Kraisintu 1993), Z. petiolatum (Chang and Criley 1993), Z. spectabile (Faria and Illg 1995), Z. wighitianum Z. montanum, and Z. zerumbet (Tyagi et al.. 2006). In the present paper we report micropropagation, in vitro conservation and genetic stability of in vitro regenerated plantlets of ethnobotanically important Zingiber rubens. ⎯⎯⎯⎯ Received 27 July 2009, accepted 3 March 2010. Abbreviations: ADS - adenine sulphate; BA - benzyladenine; IAA - indoleacetic acid; MS - Murashige and Skoog medium; NAA - napthaleneacetic acid; KIN - kinetin; RAPD - random amplified polymorphic DNA; ISSR - inter simple sequence repeats. Acknowledgements: The authors are grateful to Dr. S.C. Si, Centre of Biotechnology and Dr. M.R. Nayak, Anusandhan University for providing facilities and encouraging throughout. Financial assistance from Department of Biotechnology, New Delhi, India, is also duly acknowledged. * Author for correspondence; fax: (+91) 674 2350642, e-mail: sanghamitran@yahoo.com