1. Poster template by ResearchPosters.co.za In vitro propagation of Aristolochia tagala (syn: Aristolochia acuminate ) – an important rare medicinal plant Himadri Sekhar M, Nilesh R Yemul, T Kalaivani. School of Biosciences and Technology, Vellore Institute of Technology Vellore-632 014, India. ABSTRACT Aristolochia tagala is a rare and endangered plant belonging to the Aristolociaceae family. In this study, an efficient regeneration protocol through micropropagation was developed using Murashige and Skoog medium (MS) with the supplementation of 5µg/l 6-benzylaminopurine (BAP) in different combination of 0.5µg/l Indole butyric acid (IBA), 2.5µg/l Napthalene Acetic Acid (NAA) and 2.5µg/l IAA (Indole Acetic Acid). Results revealed that the callus was initiated from stem and leaves but not in root explants. Callus was initiated in node and leaf at the concentration of (5µg/l BAP /0.5µg/l IBA). Thus this protocol will be efficient to conserve Aristolochia tagala in in vitro condition. INTRODUCTION Fig F TABLES FIGURES CONCLUSION Plants are an important source of medicines and play an important role in World health. Due to over exploitation of medicinal plant for their medicinal value, they are in need of conservation. A.tagala leaves and roots are widely used in the traditional tribal medicines to treat fever, snakebite, dysentery, oral problems and rheumatism [1]. Leaves are used to treat cholera and intermittent fevers in children [2]. The seeds are good for inflammation and helpful in the pharmaceutical industry. Roots can prevent inflammation, diuretic and cardiotonic disorders [3]. There is a sudden rise in demand for this herbal plant, for its alkaloid, aristolochin [4]. A. tagala is also endangered due to human interference, deforestation, jhum cultivation, poor seed propagation and due to the presence of scanty endosperm [5]. Due to this reason this plant need to be conserved and micropropagation acts as a tool for the same. Therefore, our study emphasizes an efficient protocol for the in vitro cultivation of A.tagala. Sample collection: A.tagala was collected from Kolli hills which are situated in Eastern Ghats of India and the seeds were germinated and grown shoots, leaves and roots were used as explants for further study. Surface sterilization The germinated node, stem and roots were surface sterilised using Tween 20 for 15 minutes and rinsed thoroughly with water. It was then washed with 0.1% HgCl 2 for 5 minutes, followed by 70 % ethanol wash for 30 seconds and this was finally rinsed with distilled water. The washed explants were inoculated in MS media. Media preparation The explants were inoculated in Murashige and Skoog media, 1962 (MS media) with the supplementation with 3 % (m/v) sucrose. The pH of the medium was adjusted to The results revealed that all the explants (leaf, inter node) except root showed callus initiation. Different concentration of BAP, IAA and NAA shows callus initiation in node and leaf explants but not in IBA and GA 3 plant regulators (Table 1). MS media supplemented with BAP (5µg/l) and IBA (0.2µg/l) combination showed maximum growth of 4.7 mm green colored callus for leaf explants. The same combination in node explants showed cream colored callus with the diameter of 4.8mm. The combination of BAP (4µg/l) with IBA (0.2µg/l), showed moderate growth of callus for node and leaf explants, whereas BAP (1, 2, 3 µg/l) and IBA (0.2 µg/l) combination showed very less callus initiation in both leaf and node explants around 3 weeks. (Table 2) ExplantControl Benzyl Amino Purine (BAP) Indole Butyric Acid (IBA) Indole Acetic Acid (IAA) Gibberellic Acid (GA 3 ) Napthalene Acetic Acid (NAA) Leaf-+-+-++ Node-+-++- Root-+-+-- Table 1: Effect of plant growth regulators on the regeneration of different explants of A. tagala. METHODOLOGY 5.8 before adding 0.8 % (m/v) agar medium and autoclaved at 121 °C and 1.06 kg cm -2 pressure for 20 min (5) Callus initiation The surface sterilized explants of size 1.0-1.5 cm were kept into the MS media by maintaining at aseptic condition. Cultures were incubated at 25°C with a photoperiod of 16 h at 2000 lux of cool white fluorescent light. Cultures were initiated in 25mm glass tube and sub-cultured on fresh media at four week intervals in the glass tube. Table 2: Effect of BAP with different concentration of IBA for initiation of callus in A. tagala ExplantControl BAP – 1 IBA – 0.5 (µg /l) BAP – 2 IBA - 0.5 (µg /l) BAP – 3 IBA – 0.5 (µg /l) BAP – 4 IBA – 0.5 (µg /l) BAP–5 IBA– 0.5 (µg/l) Color of callus Maximum Size of callus(mm) Percentage % Leaf-++++++++Green4.720 Node-++++++++Cream4.819 Root--------- Table 3: Effect of BAP combined with different concentration of NAA for initiation of callus in A.tagala ExplantControl BAP – 2 NAA –0.5 (µg /l) BAP –2 NAA -1 (µg /l) BAP – 2 NAA –1.5 (µg /l) BAP – 2 NAA –2 (µg /l) BAP–2 NAA–2.5 (µg /l) Color of callus Maximum Size of callus(mm) Percentage % Leaf-+++++ Green3.818 Node-++++++++Cream4.35 Root--------- Table 4: Effect of BAP combined with various concentration of IAA for initiation of selected explants. ExplantControl BAP – 2 IAA –0.5 (µg /l) BAP –2 IAA -1 (µg /l) BAP – 2 IAA –1.5 (µg /l) BAP – 2 IAA –2 (µg /l) BAP–2 IAA–2.5 (µg /l) Color of callus Maximum Size of callus(mm) Percentage % Leaf-++++++++Green423 Node-+++++ Cream3.321 Root--------- + Minimal growth, ++ Moderate growth, +++ Maximum growth, - No growth DISCUSSION Tissue culture techniques are used for the micropropagation thereby conserving the germplasm of the plant which are threatened to extinction. So we take an attempt on in vitro propagation for this endangered medicinal plant. In the present investigation, BAP is one of the most useful Cytokinin for initiation and multiplication of nodal and leaf explants. It has been observed that BAP is used for micropropagation of different plants as Sausurea obvallata, (3).From the current study, combination of BAP with NAA or IAA or IBA was found more suitable than BAP alone. Thus the combination of cytokinin and auxin produced callus. It was also depicted in Allium sativum [4]. The combination of BAP & IBA shows the higher concentration of callus from leaf and node. In many studies, this combination has proved to produce better callus from node and leaf like Jatropha curcas[2]. We have effectively produced an efficient protocol for callus initiation for this rare medicinal plant. Further shoot initiation, multiplication and root initiation study has to be progressed. a b c a: Leaf inoculation b:&c: Callus initiation from leaf (2 µg/ml BAP,1µg/ml IAA) d:Node inoculation e: Callus initiation from node ( 2µg/ml BAP&2.5µg/ml IAA) d e RESULT The initiation of callus from leaf and node explants inoculated with MS media supplemented with the concentration of BAP (2µg/l) and NAA (2.5 µg/l), maximum initiation was seen in both leaf (3.8mm) and node (4.3mm). Whereas low concentration of BAP (2µg/l) and NAA (0.5 µg/l) showed very less callus in both leaf and node explants (Table 3). Maximum callus was seen in leaf (4mm) and node (3.3mm) explants inoculated in MS media supplemented with BAP (2µg/l) and IAA (2.5µg/l). Less concentration of BAP (2 µg/l) and IAA (0.5 µg/l) showed very less callus in both leaf and node explants (Table 4). REFERENCE [1]Constabel, F., 1990. Medicinal plant biotechnology..Hybrids of family Asteraceae, 56:420-425. [2] Jose, J.K., Nimisha, K., Anu, M., Padma, N., 2012. Evaluation of Somaclonal Variation in Callus Cultures of Jatropha curcas., World J. of Agri.Sci 8:616-623. [3] Joshi, M. and Dhar, U., 2004. In vitro propagation of Saussurea obvallata (DC.) Edgew. – an endangered medicinal herb of Himalaya. Plant Cell Rep. 21: 929-939. [4] Mendelsohn, R. and Balick, M., 1994. The importance of undiscovered pharmaceuticals in tropical forests. Econ. Bot., 49: 221-228. [5] Murashige, T. and Skoog, F., 1962 A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15: 473-497. [6] Sultana, S. K. and Handique, P. J., 2004. Micropropagation of Gentiana kurroo through high shoot multiplication using nodal explants. J. Curr. Sci. 5: 447-452. Acknowledgment: We thank VIT university for constant support and encouragement. We would also like to acknowledge Dr.Rajasekaran, Dr.Siva and Ms. Abitha Benson for their coordination