Indian Journal of Biotechnology Vol. 4, April 2005, pp. 265-270 Micropropagation and ecorestoration of Decalepis arayalpathra (Joseph & Chandra.) Venter—An endemic and endangered ethnomedicinal plant of Western Ghats A Gangaprasad1, S William Decruse*, S Seeni and G M Nair Plant Biotechnology Division, Tropical Botanic Garden and Research Institute Karimancode P O, Thiruvananthapuram 695 562, India 1Department of Botany, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, India Received 26 August 2003; revised 27 April 2004; accepted 12 May 2004 Single nodes from young top shoots of Decalepis arayalpathra were cultured on Murashige and Skoog’s (MS) agar medium supplemented with 0.1 to 5.0 mg l-1 BAP. All the concentrations of BAP induced single axillary shoot of varying length. However, MS medium supplemented with 1.0 mg l-1 BAP supported rapid growth and produced the longest shoots (6.8 cm) in 60 days. For further multiplication, the nodes and shoot tips from in vitro derived shoots were recultured on MS medium fortified with 0.5 mg l-1 BAP, which produced the 8 cm long shoots having 5-7 nodes in 30 days period. Further, the top microshoot cuttings (3-5 cm long) with 2-3 nodes, subcultured onto MS medium supplemented with 1.5 mg l-1 IAA, produced an average of 6.3 roots in 30 days period. The rooted plants after hardening were reintroduced into their natural habitat at Kallar reserve forest, Thiruvananthapuram. After two years, 84% survival of reintroduced plants was recorded. Keywords: Decalepis arayalpathra, ecorestoration, endemic, micropropagation, Western Ghats IPC Code: Int Cl.7 A01H4/00, 5/00 Introduction constituents to that of Indian sarasaparila— Decalepis arayalpathra (Joseph & Chandra.) Hemidesmus indicus R.Br. and D. hamaltonii Weight Venter (Syn. Janakia arayalpathra Joseph & & Arn.5. Chandra.) is a monospecific genus of the family In recent years, the rapid pace of deforestation in Periplacaceae1,2. It is a leafy, perennial undershrub, the Western Ghats has depleted the wild stands of D. often found in small groups, endemic to a few forest arayalpathra and hampered its natural regeneration segments of Tirunelveli, Kanyakumari and potential. The natural regeneration as well as Thiruvanathapuram districts of Tamil Nadu and conventional propagation of this plant has also been Kerala3. The plant is rare and distributed in exposed beset with several other factors, like poor fruit set, rocky slopes up to 800-1200 m elevation. Popularly seed germination and rooting on stem cuttings5. known as ‘Amruthapala’ by the native Kani tribe, the Consequently, it has been enlisted as an endangered plant is used as an effective remedy for peptic ulcers, plant species. Therefore, there is a need to conserve cancer like affliction and as a tonic to regain the lost and propagate this undershrub on large scale for its strength and stamina. The tribal communities as a ethnomedicinal importance and potential. whole consider the tuberous roots of the plant as a The application of plant tissue culture in ex situ very important drug with wonderful curative conservation of rare and endangered plant taxa properties. Recent pharmacological studies have also including medicinal plants has long been recog- revealed that root extract of the plant had immuno- nized6-8. In vitro propagation of endangered species is modulatory and antitumour activity4. The roots of this generally undertaken to increase the number of plant are found to be similar in their chemical individuals available. The present paper describes large-scale tissue culture propagation of D. _______________ arayalpathra using nodal explant culture and *Author for correspondence: Tel: 91-472-2869626/2869226; Fax: 91-472-2869646 reintroduction of acclimatized plants into their native E-mail: [email protected] forest habitats. 266 INDIAN J BIOTECHNOL, APRIL 2005 Material and Methods Kerala Forest Department. The temperature in this Young shoot cuttings with 5-6 node length were region ranged from 12-33°C. For planting, small pits collected from actively growing branches of a wild (10×10×10 cm3) were made in the loose soil found on plant at Kallar reserve forests in Thiruvananthapuram the crevices of rocks with minimum disturbance to the district of Kerala State (Fig. 1). After removing grasses and other associated flora of the rocky area. leaves, the shoot cuttings were submerged in 2% (v/v) The nursery established plants were gently removed Teepol for 10 min and washed thoroughly in running from the pots without any damage to the growing tap water for 15 min. For surface decontamination, roots and planted carefully one in each pit. they were immersed in 10% (v/v) Steriliq (Combii Observations on the establishment of plants were Organo Chem., New Delhi) for 15 min and 0.1% made at bimonthly intervals. HgCl2 solution for 8 min with an intervening rinse in All the experiments were completely randomised sterile distilled water. After 4-5 washes in sterile and repeated at least twice. Each treatment consisted distilled water, 0.5-1.0 cm long single node explants of 20-25 replicates. were isolated and planted vertically in Murashige and 9 Skoog (MS) nutrient medium supplemented with 3% Results and Discussion (w/v) sucrose, 0.7% agar (Himedia, Bombay) and Under the conditions employed, only one of the plant growth regulators. Benzylaminopurine (BAP, 0- opposite axillary buds of each node developed into a 5.0 mg l-1) was tested individually and in combination -1 single shoot on MS agar medium with various with naphthalene acetic acid (NAA, 0.2-0.5 mg l ). combinations of plant growth regulator(s). In MS The pH of the medium was adjusted to 5.7 before basal medium, the explants remained fresh and green adding agar. The medium was dispensed in 15 ml without any sign of shoot development up to 45 days aliquots into 25×150 mm culture tubes and autoclaved -1 o -2 and thereafter showed browning. BAP (1.0 mg l ) at 121 C and 1.1 kg cm pressure for 18 min. induced axillary shoot buds (Fig. 2) within 12 days, Cultures were incubated in a culture room maintained whereas at lower concentrations (0.1-0.5mg l-1), bud at 25 ± 2oC and 55-60% RH under 8/16 hr -2 -1 emergence occurred only after 20-25 days. Hence, the photoperiod of 50-60 µmol m s light intensity MS agar medium with 1.0 mgl-1 BAP was selected for provided by cool white fluorescent tubes. Further, shoot induction. Further, the growth of shoots in this node and shoot tip explants, isolated from the in vitro combination was very prominent and the shoot length developed axillary smaller shoots, were subcultured -1 reached 6.8 cm with 7-8 nodes in a period of 60 days into medium containing BAP (0.2-2.0 mg l ) to (Table 1). None of the growth regulator combinations increase the shoot multiplication. gave multiple shoot formation, while single shoot of Shoots, 1-4 cm long, were cultured onto MS varying length was obtained in all the combinations medium containing half strength macro salts, 3% after 60 days of culture. However, further sucrose, 0.7% agar and 0-2.0 mg l-1 of indole-3-acetic multiplication was made possible in the next step by acid (IAA), indole-3-butyric acid (IBA) or NAA for reculturing in vitro shoot-derived nodes and shoot tips rooting. Shoots with roots were recovered from the on a medium containing BAP (0.2-2.0 mg l-1). culture tubes, washed thoroughly to remove agar Axillary buds have been found to be the most suitable residue and transferred to community pots (5 cm for micropropagation in several woody species10,11. In diam) filled with sand and topsoil (1:1). The plants D. arayalpathra, however, it was possible only were well irrigated and hardened for 4 weeks before through in vitro node reculture. The single axillary re-potting in 15 cm diam pots, which were eventually shoot formation, possibly due to strong apical transferred to field. dominance is also observed in Leucaena 12 13 After 6 months of establishment in community leucocephala and Acacia senegal . pots, 50 plants with an average size of 20 cm height The nodes and shoot tips of in vitro axillary shoots were transferred to the rocky area of Kallar forest that developed from the original explants were (50 sq. m) (800 m above sea level) during the onset of cultured on MS with 0.2-2.0 mg l-1 BAP for further Southwest monsoon (June). Another set of 50 plants shoot multiplication. (Table 2). During subculture, was reintroduced during Northeast monsoon axillary shoots were initiated within 5 days of culture (October) season. This area has been the original and a single shoot possessing 5-6 nodes (8 cm) was habitat of D. arayalpathra and a protected area of the obtained from node and shoot tip explants in a period GANGAPRASAD et al: MICROPROPAGATION AND ECORESTORATION OF DECALEPIS ARAYALPATHRA 267 Figs 1-6-(1) D. arayalpathra in its natural habitats at Kallar Reserve Forest in Thiruvananthapuram district of Kerala; (2) Shoot initiation from nodal segments cultured on MS + BAP 0.5 mgl' after 2 weeks; (3) Reculture of nodal segments on MS + BAP 0.5 mgl' after 4 weeks; (4) Microshoots rooted on Y2 strength MS basal medium containing 1.5 mgl' NAA. IDA. IAA [L-R). after 5 weeks of culture; (5) In vitro rooted plants established in pots after 6 months; and (6) In vitro regenerated D. arayalpathra reintroduced and established to its natural habitat 268 INDIAN J BIOTECHNOL, APRIL 2005 of 25-30 days on MS medium with BAP 0.5 mg l-1 nodes produced by both the node and shoot tip (Fig.