Modification of plant architecture of Hemidesmus indicus (L.) R. Br. (Iramusu) by in vitro colchicine treatment.

The effects of different concentrations of colchicine on shoot and root development of nodal explants and germinating somatic embryos of Hemidesmus indicus (L.)R. Br. (Iramusu) was investigated. MS medium solidified with 8 g/l agar was superior to the liquid medium in all the concentrations of colchicine. In nodal derived H. indicus plants, the highest number of shoots per explant (4.23±1.49) and the longest shoot length (6.36±4.29 cm) were achieved at 5 mg/l colchicines forming a compact bushy type plant architecture. In almost all the treatments an increment of shoot length and number of shoots per explant could be observed compared to the control plants. Furthermore, 1 mg/l colchicine was more effective in germinating somatic embryos to produce more compact plant type under in vitro condition. There was no significant difference observed in different concentrations of colchicine on root length and root number of both nodal derived plants and also in plants derived from germinating somatic embryos. INTRODUCTION Hemidesmus indicus (L.) R. Br. (Iramusu) is one of the wild plant species in Sri Lanka possessing high medicinal value. It belongs to family Asclepiadacea and widely 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). This plant species can be used as an ornamental foliage plant as well as a medicinal plant, due to its several morphological characteristics such as shape and white color margin along the mid rib of the leaves. Eventhough the medicinal properties of this plant species have been identified and used for many centuries in the ayurvedic, unani and homeopathic medicines (Siddique et al., 2003) at present, not much attention is paid to its conservation in their natural habitats. One of the reasons for less popularity as an ornamental plant is due to its vine like growth habit. Therefore, if it can be modified into a bushy type it will be easy to popularize it as an ornamental potted plant in Sri Lanka. 1 Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka. Nagahatenna and Peiris Chromosomal doubling is a common technique which is used in plant hybridization programmes. It allows induction of polyploid plants possessing superior agronomic traits over their diploid counterparts. Ployploid plants possess larger leaves and flowers, thicker stems and roots, darker green leaves, an increased width-to-length ratio of the leaves, a more compact growth habit and a higher tolerance to environmental stress (Kehr 1996; Kermani et al. 2003; Shao et al. 2003). Furthermore, autotetraploids and triploids are sterile or frequently have low fertility. It is a more important character in clonally propagated ornamental plants since it avoids contamination of the flowers by the pollen and fruit set (Guofeng et al., 2007). Colchicine is one of the pharmaceutically important alkaloids and a useful agent in the treatment of acute attacks of gout. Traditionally, colchicine was used for its antimitotic properties (Sivakumar et al., 2004) and treatment of familial Mediterranean fever. Colchicine has a well-known lipophilic drug action on tumors (Sivakumar et al., 2004). Derivatives of colchicine have shown promise as anticancer agents (Sivakumar et al., 2004). Colchicine was used for gene expression and gene amplification (Sivakumar et al., 2004). Colchicine disrupts mitosis by binding to tubulin, thus inhibiting the formation of microtubules and the polar migration of chromosomes, resulting in a cell with a doubled chromosome number. Now it is one of the most commonly used spindle inhibitors and has been used for the induction of tetraploids from diploid plants of a number of woody species including rose (Roberts et al. 1990; Kermani et al., 2003), mulberry (Chakraborti et al., 1998), rhododendron (Vainola, 2000), pear (Kadota and Niimi, 2002), and pomegranate (Shao et al., 2003). With this information, the main objective of the present study was to obtain genetic variability of H. indicus by colchicine treatment. In this research a reliable protocol is presented for obtaining in vitro cultured H. indicus plants with modified growth habit of bushy appearance. METHODOLOGY Donor plants The nodal explants were taken from 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, dipped in 0.1% Bavastin solution for 30 minutes, vacuum sterilized with 10% Clorox and two drops of Tween 20 for 5 minutes and thoroughly shake with 10% Clorox for 5 minutes. This was followed by three to four times washing with sterilized distilled water. After the sterilization process 1-2 cm nodal segments were inoculated on Murashige and Skoog (1962) (MS) basal medium supplemented with 1 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l α-naphthaleneacetic acid (NAA), 3% (w/v) Sucrose, 100 mg/l Myoinositol, 15 mg/l adenine sulphate, 0.1% streptomycin, 0.1 g/l ascorbic acid solidified with 8 g/l agar. Those cultures were incubated at 25±2 C under warm fluorescent light with intensity varying from 900 to 1500 lux and 16 h:8 h (day: night) photoperiod. Shoots were multiplied on MS basal medium supplemented with 2 mg/l BAP (Nagahatenna and Peiris, 2007).