IN VITRO PROPAGATION OF THE CHINESE MEDICINAL PLANT, DENDROBIUM CANDIDUM WALL. EX LINDL., FROM AXENIC NODAL SEGMENTS YIH-JUH SHIAU 1 , SATISH MANOHAR NALAWADE 2 , CHI-NI HSIA 1 , VANISREE MULABAGAL 1 , AND HSIN-SHENG TSAY 2 * 1 Department of Agronomy, Agricultural Research Institute, Wufong, Taichung 413, Taiwan 2 Institute of Biotechnology, Chaoyang University of Technology, Wufong, Taichung 413, Taiwan (Received 5 January 2005; accepted 11 May 2005; editor M. S. Pais) Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l 21 sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l 21 HYPONeX, 80 g l 21 potato homogenate and 2 g l 21 activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l 21 benzyladenine (BA) and 0.1 mg l 21 naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l 21 NAA and the plantlets were successfully acclimatized in soil. Key words: axenic nodal segments; Dendrobium candidum; medicinal herb; tissue culture. Introduction Dendrobium is the second largest genus in the family Orchidaceae. It exhibits a vast diversity in vegetative and floral characteristics and is of considerable interest due to its broad geographic distribution and high value of hybrids as a floricultural commodity (Hawkes, 1970; Jones et al., 1998). The stems of the Dendrobium species, also known as Shih-hu (Japanese: sekkoku; English: Dendrobium stem), are used in traditional Chinese medicine as a tonic to improve digestion and for promoting the production of body fluid, nourishing ‘yin’ and eliminating ‘evil-heat’ (Yen, 1980; Anonymous, 1999). ‘Shih’ means rock and ‘hu’ means living and signifies the plant living on rocks, referring to the saxicolous habit of the species (Hu, 1970). It was first recorded in Shen-Nung-Ben-Cao-Jing (the earliest Chinese medicine herbs book, published more than 1900 yr ago) under the ‘Inferior Category’ and was also recorded in the successive Pen-ts’aos of the subsequent dynasties (Anonymous, 1999). Shi-hu is derived from the stems of the five orchids belonging to the genus Dendrobium, namely, D. chrysanthum, D. fimbriatum, D. loddigesii, D. nobile, and D. candidum (Lau et al., 2001). The main classes of chemical compounds in Dendrobium are a group of sesquiterpene alkaloids (Tang and Eisenbrand, 1992). In the local markets in Taiwan and China, the dried drug costs about US$4000 kg 21 . D. candidum (Chinese: Tie-pi-shih-hu; 2n ¼ 38) (Withner, 1974) is an epiphyte. Its stems are terete and clustered, and about 35 cm long and 2 – 4 mm in diameter. Leaves are oblong to lanceolate, 4 – 7 cm long, 1 – 1.5 cm wide, and chartaceous. The upper flowers (usually around 2–3 in number), size about 2–4 cm in length produced from upper leafless stem are slightly fragrant, and the sepals and petals are white in color (Hawkes, 1970). The lip is egg- lanceolate, white, obscurely three-lobed, but olive-green in the wider median parts. Flowering usually occurs during the period between April and June (Hawkes, 1970; Anonymous, 1999). The growth habit is similar to that of D. aduncum Wall. (Hawkes, 1970). Its external shape is similar to D. moniliforme (L.) Sw., which is an indigenous species widely distributed in Taiwan (Lin, 1975). D. candidum is native to the regions of Southern China. In the mountain ranges of Yun-Nan, Guang-Xi, Gui-Zhou, and Fu-Jian provinces of China, this species is distributed at an elevation of 900 – 1500 m above sea level, with an annual average temperature of 12–188C and 1100 – 1500 mm annual rainfall. The plant usually requires 5–7 yr of growth before it can be used as a drug. In 1998, the production of D. candidum in Yun-Nan was estimated to be less than 1000 kg (Fu et al., 1999). Tissue culture methods have been used for conservation and vegetative propagation of many important plant species (Fay, 1992). In vitro propagation of D. antennathum and D. phalaenopsis has been reported previously (Kukulczanka and Wojciechowska, 1983) and a high rate of shoot proliferation could also be induced from shoot segments of three epiphytic orchids (Nayak et al., 1997). In D. candidum, micropropagation has been achieved using embryo, protocorm, immature seeds, and shoot tip explants (Liu et al., 1988; Zhang et al., 1992, 1993; Guo et al., 1996). Other studies on D. candidum have also been reported. They include analysis of amino acids in fresh plants (Huang and Ruan, 1997), polysacchar- ide determination from seedlings and calli (Huang and Lu, 1998; *Author to whom correspondence should be addressed: Email hstsay@ mail.cyut.edu.tw In Vitro Cell. Dev. Biol.—Plant 41:666–670, September– October 2005 DOI: 10.1079/IVP2005685 q 2005 Society for In Vitro Biology 1054-5476/05 $18.00+0.00 666