Efficient plantlet regeneration from cotyledonary node cultures of Cassia alata L. (Ringworm Bush): stimulatory effect of thidiazuron By M. R. AHMED, N. AHMAD, M. ANIS 1,2 * and I. M. AREF 2 1 Plant Biotechnology Laboratory, Department of Botany,Aligarh Muslim University, Aligarh - 202 002, India 2 Department of Plant Production, College of Food and Agriculture Science, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia (e-mail: anism1@rediffmail.com) (Accepted 13 November 2012) SUMMARY The stimulatory effect of different concentrations of thidiazuron (TDZ) on in vitro shoot proliferation in an important medicinal shrub, Cassia alata L. (Ringworm Bush or Senna) was investigated. Multiple shoots were induced from cotyledonary node (CN) explants derived from 15-d-old axenic seedlings on Murashige and Skoog (MS) medium fortified with different concentrations of TDZ. The shoot-forming capacity of CN explants (90% response) was influenced by TDZ concentration (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0 μM). The highest frequency of shoot regeneration (95%), the maximum mean number of shoots per explant (9.2 ± 0.2), and the greatest shoot length (2.6 ± 0.1 cm) were obtained on MS medium fortified with 7.5 μM TDZ after 4 weeks of culture. Prolonged exposure of CN explants to TDZ had an adverse affect on the developing shoots.To avoid this, cultures were transferred to TDZ-free MS medium, or to a medium fortified with various concentrations of 6-benzyladenine (BA) for further multiplication, proliferation, and elongation of induced shoots.The highest mean number of shoots per explant (18.9 ± 1.1) and shoot length (4.7 ± 0.1 cm) were achieved when TDZ-exposed explants were then sub-cultured for 6 weeks on MS medium containing 1.0 μM BA. In vitro-rooting of shoots was optimal on full-strength MS medium containing 0.5 μM indolebutyric acid (IBA), which gave the highest mean number of roots per shoot (7.2 ± 0.2) and the greatest root length (2.6 ± 0.1 cm). Well-rooted plantlets were hardened-off successfully and acclimatised in Thermocol TM cups containing sterile Soilrite TM . These plantlets were then transferred to earthenware pots containing garden soil and maintained in a greenhouse, with a survival rate of 85%. M edicinal and aromatic plants are a source of biologically-active compounds and are important in human healthcare. They are also an important source of medicines, and have a key role in global health (Constabel, 1990). Recently, some higher plants have attracted attention as natural sources of phytochemicals for exploitation as anti-microbials. Such plant products would also be bio-degradable (Kumar et al., 2008; Sugar et al., 2008; Krishnamurthy et al., 2008; Wang et al., 2010). In addition, in industrialised countries, increasing awareness of the use of medicinal plants can be traced to the development of several drugs and chemo- therapeutics from traditional herbs (UNESCO, 1998). Therefore, in order to meet the ever-growing demand for medicinal plants, in vitro culture of these species, using biotechnological tools and techniques, is becoming imperative. Cassia alata L. (Fabaceae), commonly known as ‘Ringworm Bush or Senna’, is an erect medicinal shrub (or small tree) that is distributed mainly in the tropics and sub-tropics. Almost all parts of the plant have medicinal uses. Decoctions of the leaves, flowers, and bark are used to treat skin diseases such as eczema or pruritis (itching), while a decoction of the wood is useful in cases of constipation. The plant is a source of a wide array of compounds such as alkaloids, lectins, glycosides, isoflavones, and phyto-estrogens (Leeuwenberg, 1987), hydroxyanthraquinones, chrysophanic acid, kampferin, and sannoxide A and B (Abo et al., 1998; Kochar, 1981). Leaf extracts from this species have shown several pharmacological, anti-microbial, and anti-fungal properties (Khan et al., 2001; Esimone et al., 2008), anti- bacterial, anti-inflammatory, analgesic effects (Palanichamy and Nagarajan, 1990), and anti- hyperglycemic activities (Palanichamy et al., 1988). The conventional method of propagation of Senna species is via seed; however, this is not reliable due to practical constraints such as low germination frequency, low seed viability, seasonal responses, and infestations by pests or pathogens which restrict propagation under natural conditions (Anonymous, 1992). Alternative methods of propagation would be beneficial to accelerate large-scale multiplication, as well as to improve and conserve this species. In vitro technology permits the rapid, mass production of plants to meet increasing demands and offers a viable tool for the large-scale propagation and multiplication of a variety of medicinal plant species (Anis et al., 2011). There have been reports of in vitro propagation of other Cassia species (Fett-Neto et al., 2000; Siddique and Anis, 2007; Perveen and Shahzad, 2010), but, so far, there have been no reports on the successful in vitro propagation of C. alata. The present study aimed to develop a rapid in vitro *Author for correspondence. Journal of Horticultural Science & Biotechnology (2013) 88 (2) 141–146