In vitro propagation of Hemidesmus indicus ( L . ) R . Br . ( Iramusu ) through nodal culture

The effects of different nodal positions and 6-benzylaminopurine (BAP) concentrations on shoot development from nodal explants of Hemidesmus indicus (L.) Br. (Iramusu) were investigated. The highest number of shoots per explant (2.57±0.97) was observed in 4 nodal position, while the longest shoot length (3.23± 0.52 cm) was achieved in 5 nodal position at 2 mg/L BAP with 0.1 mg/L Naphthalene Acetic Acid (NAA). Reduction of total shoot length and number of shoots were observed when BAP concentration was gradually increased from 5 to 15 mg/L. Half-strength semi solid MS medium with 1.5 mg/L indole-3-butyric acid (IBA) exhibited the best in vitro rooting. Hundred percent of the rooted shoots survived during the acclimatization. INTRODUCTION Hemidesmus indicus (L.) R. Br. (Iramusu), of the family Asclepiadacea is distributed in India, Bangladesh and Sri Lanka. In Sri Lanka, it thrives well only up to an elevation of 2500 m. It is common in deciduous scrubland and deciduous forest of the dry regions as well as rubber, coconut and pinus plantations (Gunatilleke et al., 2002). The plant is a perennial, semi-shrubby tawnier with a woody rootstock. The stem is very long, prostrate or ascending and slightly twining. Internodes are 1.5 7.2 cm in length. Leaves are simple, opposite and variable from oblong-oval to linear. Flowers are regular, bisexual and contain numerous bracts (Jayaweera, 1982). In ancient ayurvedic practices in India and Sri Lanka, H. indicus has been commonly utilized because of its strong fragrance, aromatic taste and medicinal properties due to presence of 2-hydroxy 4-methoxy benzoic acid in roots. It mainly acts as a blood purifier, antipyretic and antioxidant (Sreekumar et al., 2000). Roots are commonly used for the preparation of several decoctions, syrups, sherbet and medicinal mixtures to recover from venereal diseases, plumpness, asthma, bronchitis, chronic skin disorders and rheumatoid arthritis (Siddique et al., 2003). High quantities of plant materials such as stem, leaf and root extracts are used in ayurvedic, unani and homeopathic medicines in India and Bangladesh (Siddique et al., 2003). Even in such countries where the commercial level manufacturing systems exist, establishment of commercial plantations of this herb has been difficult due to unavailability of sufficient desirable quality planting materials. Therefore, wild population is exploited for the extraction purposes (Soma et al., 2003). This has been a 1 Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka. Nagahatenna & Peiris heavy threat for the existence of the plant species. In addition, field grown plants have shown high seasonal variations. To overcome these problems, in vitro propagation techniques can be applied for the rapid large-scale clonal propagation, germplasm conservation and production of multiple shoots with homogenous chemical composition of the in vitro populations. In addition to that, different in vitro protocols have been developed for the large scale production of antioxidants such as iupeol, vanillin and rutin in shoot cultures, callus cultures and root cultures (Soma et al., 2003). Even though H. indicus is a well known common ancient medicinal plant species in Sri Lanka, no sufficient attention has been given for the efficient utilization and conservation of the plant species in their natural habitat. Therefore, development of clonal propagation techniques will be an essential tool for the rapid multiplication of the plant species and to utilize in the ancient ayurvedic medicines in Sri Lanka. Furthermore, this plant species can be modified through several genetic and epigenetic techniques such as genetic transformation and mutation treatments in order to change its phenotype as an ornamental plant species and popularize among the nation. Therefore, this study was conducted with the objective of developing a suitable protocol for shoot regeneration of H. indicus through single nodal culture. MATERIALS AND METHODS Establishment of nodal cultures The nodal explants were taken from the plants grown in the plant house, two days after spraying of 0.1% Bavastin (carbendazim) solution. The nodal explants were surface sterilized by washing 3 times with Teepol, then dipping in 0.1% Bavastin solution for 30 min vacuum sterilized with 10% CloroxTM (sordium hypochlorite) and two drops of Tween 20 (Tween 20-polyoxyethelene sorbian monolaurate) for 5 min, then thoroughly shaked with 10% Clorox for 5 min. This was followed by three to four times washing with sterile distilled water to make the explant material free from chemicals. After the sterilization process 1-2 cm single nodal segments were inoculated on Murashige and Skoog (1962) (MS) basal medium supplemented with 1 mg/L, 6-benzylminopurine (BAP) and 0.5 mg/L Naphthalene Acetic Acid (NAA), 3% Sucrose, 100 mg/L Myoinositol, 15 mg/L Adenine sulphate, 0.1% streptomycin, 0.1g/L ascorbic acid and solidified with 8.0 g/L agar. Those cultures were incubated at 25±2C under the warm fluorescent light with intensity varying from 900 to 1500 lux and 16:8 hrs (day:night) photoperiod. Each treatment consists of 120 culture tubes. Performances of the plants were observed at weekly intervals. The success rate, percentage of shoot initiation and growth measurements such as shoot length and number of shoots/explant were recorded at the end of the sixth week. The effects of nodal positions of the explants on shoot induction and multiplication Surface sterilized nodal explants from five nodal positions from the apical bud (Plate 1), were inoculated on MS medium containing different combinations of BAP (0, 2, 5, 10, 15 mg/L) with 0.1 mg/L NAA. Cultures were kept under the same conditions mentioned above. Treatment combinations were replicated for six times. Performances of the plants