Analele Universit ăţii din Oradea - Fascicula Biologie Tom. XIX, Issue: 1, 2012, pp. 35-39 35 RAPID PLANT REGENERATION FROM NODAL EXPLANTS OF Spilanthes acmella (L.) MURR. – AN ENDANGERED MEDICINAL PLANT Kuldeep YADAV * , Narender SINGH * * Plant Tissue Culture Lab., Department of Botany, Kurukshetra University, Kurukshetra, Haryana, India Corresponding author: Dr. Narender Singh, Associate Professor, Department of Botany, Kurukshetra University, Kurukshetra, 136119, Haryana, India, phone: 01744-20196(501), fax: 01744-20277, e-mail: nsheorankuk@yahoo.com Abstract. Excised nodal explants of Spilanthes acmella (L.) Murr., ‘Toothache Plant’ proliferate rapidly in vitro on MS medium containing 0.5- 2.0 mg/l of BAP. Rapid and prolific shoot proliferation occurred. Regenerated shoots vary considerably in size (3-10 cm long) and relative stage of development, with some (50%) producing adventitious roots without transferal to a separate rooting medium. With maximum possibility of adventitious roots induction was induced from middle order nodes (3 rd to 5 th node from apex) obtained from 3 months old in vivo plant on full-strength MS medium supplemented with 1.0 mg/l BAP under the photoperiod of 18-h. The possibility of adventitious roots induction directly from regenerated shoot was greatly influenced by the concentration of BAP, photoperiod, age of donor plant and nodal position on stem. Keywords: In vitro propagation, Spilanthes acmella, Endangered, Nodal segments, Photoperiod. Abbreviations: BAP – 6-Benzylaminopurine; MS – Murashige and Skoog (1962) basal medium. INTRODUCTION Spilanthes acmella (L.) Murr. (Asteraceae) is the well known as “Akarkara or Toothache plant”, is an important endangered medicinal plant widely distributed in tropics and subtropics [21]. The plant has found applications in pharmaceuticals to treat mouth aliments, stammering, stomatitis, and throat complaints. Alkylamides from Spilanthes have demonstrated strong diuretic and insecticidal properties. The major pungent compound reported in S. acmella is alkaloid spilanthol (N-isobutyl-2,6,8- decatrienamide) [13]. With the increasing worldwide demand currently, the interest in utilization and conservation of medicinal plants is rapidly increasing, and mass micropropagation of plants via in vitro culture techniques has become an alternative approach to produce large-scale rapid plant multiplication to extract valuable chemical products rather than from plants grown and harvested in the field [10]. In vitro micropropagation technique has been proved to be employed in propagation of many of medicinal plant species [6, 25, 26, 30]. Micropropagation requires not only stable shoot multiplication, but also successful rooting of microcuttings. In fact, success of the most hazardous step of propagation, i.e. transfer and acclimatization of in vitro rooted microcuttings to an ex vitro environment, depends on the quality of the root system. Thus, research on adventitious root formation is highly important from the practical point of view. In a previous studies [8, 11, 19, 23, 24, 27, 31], we found that S. acmella displays a good capacity for in vitro rooting, in the presence of auxin, mainly IBA in the medium. In vitro plants are generally considered susceptible to genetic changes due to culture stress [22]. So, the development of a direct plant regeneration system and assessment of clonal fidelity of the in vitro raised plants of S. acmella are crucial for successful commercial application of micropropagation protocol. The purpose of this work was to acquire more information on the capacity of in vitro grown shoots to simultaneously regenerate adventitious roots and shoots by treatments of different endogenous and exogenous factors. MATERIALS AND METHODS Healthy nodal explants (1.0-1.5 cm) were excised from two- to four-month-old plants growing in poly house of Botany Department, Kurukshetra University, Kurukshetra. Nodal explants were divided into three groups based on to their position along the length of the branch of the in vivo plants (2, 3 and 4 month old): distal order (containing 1 st - 2 nd node from apex), middle order (containing 3 rd - 5 th node from apex) and basal order (containing 6 th - 8 th node from apex). The explants were washed with liquid detergent under running tap water to remove dust particles. The explants were then treated with 0.1% (w/v) mercuric chloride for 3-5 minutes under aseptic conditions. After this these explants were then thoroughly washed 4-5 times with sterilized double distilled water to remove the traces of mercuric chloride. The nodal segments after trimming the ends were finally inoculated on MS medium [18] containing 30 g/l sucrose and 8 g /l agar supplemented with various concentrations (0.5 - 2.0 mg/l) of BAP alone. The pH of the medium was adjusted to 5.8 with 1N NaOH or 1N HCl prior to autoclaving at 121°C for 20 min. The cultures were incubated at a temperature of 25±2ºC and a 16-h photoperiod (intensity of 4000 lux). Effect of different durations of photoperiod, 3 light/dark cycles i.e.,14/10, 16/8 and 18/6 h on the growth of plantlets produced was also tested. When adequate number of in vitro raised adventitious roots from the surface of regenerated shoots penetrate deep in the basal media and reach the base of the culture tube, the plantlets were thoroughly washed to remove the adhering agar agar particles and transferred to pots containing sterilized soil and sand mixture (3:1) for acclimatization. The pots were covered with transparent polythene bags (to maintain humidity) with holes (to provide aeration) and kept in culture room initially for 15 days under the same light