67 Continuous High-Frequency Regeneration of Different Phalaenopsis Cultivars from Young Leaves of Mature Plants Pinaki Sinha 1 • M. Lokman Hakim 2 • M. Firoz Alam 1* 1 Department of Botany, Rajshahi University, Rajshahi, Bangladesh 2 Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Gonokbari. Savar, Dhaka, Bangladesh Corresponding author: * falambiotech@eudoramail.com Keywords: Orchid, Phalaenopsis, protocorm-like bodies (PLBs), continuous regeneration ABSTRACT An efficient continuous high frequency in vitro regeneration method was developed for four cultivars of Phalaenopsis amabilis from leaf segments of emerging young leaves of mature plants. Leaf segments of four cultivars of Phalaenopsis hybrids cultured on half strength Murashige and Skoog (MS) medium supplemented with N 6 -benzyladenine (BA; 8.88 M), -naphthaleneaceetic acid (NAA; 2.7 M ), 2% (w/v) sucrose, 10% (v/v) coconut water (CW), 2 gl -l peptone and 1 gl -l activated charcoal produced an average of 16-26 protocorm-like bodies (PLBs) after 12 weeks. PLB clumps were cut into four pieces and subcultured on agar-gelled ½ MS medium with 2% (w/v) sucrose + 10% (v/v) CW + 2 gl -l peptone + 150 mgl -l L-glutamine + 1 gl -l activated charcoal, where each clump of PLBs produced 198-268 PLBs, within 8 weeks. After a further four weeks of subculture the PLBs were found to be enlarged with leafy shoots. Plantlet development from leafy shoots was achieved on ½MS medium supplemented with 2 gl -l peptone, 2% (w/v) sucrose, 10% (v/v) CW and 1 gl -l activated charcoal, upon which 100% explants developed into plantlets with stout roots within 8 weeks. By repeated subculture of PLB clumps on proliferation medium and culturing leafy shoots on the plantlet regeneration medium, a continuous high frequency of plantlets could be produced. 1. INTRODUCTION Plant tissue culture is the most commercially successful aspect of plant biotechnology, which has introduced an exciting new phase into plant propagation and breeding (Prakash et al. 1996). Pot plant and cut flower production of orchids have increased greatly in recent years (Tokuhara and Mii 1993 2001, Chang and Chang 2000). Among commercially important monopodial ornamental orchids, Phalaenopsis accounts for about 40 percent from economic point of view (Debargh and Zimmerman 1991). Though not all genotypes of Phalaenopsis hybrids respond in the same manner under similar culture conditions (Reuter 1983), Tanaka and Sakanishi (1977 1980 1985), Tanaka (1992), Tokuhara and Mii (1993), and Arditti and Earnst (1993) developed protocols for in vitro clonal propagation of Phalaenopsis. However, not all of these methods can be used for commercial micropropagation because of differences in survival rate, PLB formation and plantlet regeneration. As Phalaenopsis has a high commercial value as a cut flower and as indoor pot plants throughout the world, a high frequency regeneration protocol is yet to be determined (Takuhara and Mii 2001). Recently Tokuhara and Mii (2001 2003) established a highly efficient system of micropropagation for Phalaenopsis through embryogenic callus induction and cell suspention culture, but callus mediated plant regeneration does not ensure plant homogeneity in any respect. Park et al. (2002) reported a successful in vitro propagation method for Phalaenopsis by using leaf explants derived in vitro from flower stalk nodes, although in initial studies utilizing this technique (Sinha et al. unpublished results) multiplication and conversion rates proved less than satisfactory. This paper reports an efficient and quick method for continuous high frequency clonal propagation of different cultivars of Phalaenopsis amabilis dircdtly culturing young leaf segments of mature plants as the initial starting material. 2. MATERIALS AND METHODS 2.1. Plant materials and PLBs induction Four different Phalaenopsis hybrids of high demand from the species, P. amabilis (P1: pure white flower with purple lip, P2: pure white flower with yellow lip, P3: pink flower with pink lip and P4: yellow-flower with red mottles) were selected and used as source materials for culture. Apical portion of emerging young leaf from the mature plant was used as initial explant. The explants were washed under running tap water followed by