Plant regeneration through direct shoot bud formation from leaf cultures of <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchids

Leaf explants of Paphiopedilum phiIippinense hybrids (hybrid PH59 and PH60) directly formed adventitious shoots from wound regions within 1 month, when cultured on modified Murashige and Skoog medium (1/2strength macroand full-strength micro-elements) free of plant growth regulator in darkness. The combinations of 2,4-dichlorophenoxyacetic acid ((2,4-D) acid (0, 4.52 and 45.25 μM) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) (0, 0.45, 4.54 and 22.71 μM) were used to test their effects on direct shoot bud formation from two types of explants (1.5-cm long intact leaf explants and 0.5-cm long leaf segment explants). In hybrid PH59, 4.54 μM TDZ increased mean numbers of shoots per explant with leaf segment explants. In hybrid PH60, 4.52 μM 2,4-D plus 0.45 μM TDZ promoted direct shoot bud formation from leaf segment explants. In addition, three treatments (4.52 μM 2,4-D, 22.71 μM TDZ, 4.52 μM 2,4-D plus 4.54 μM TDZ) gave a higher response than control on mean numbers of shoots per explant with intact leaf explants. Healthy plantlets each with one to three roots were obtained from leaf-derived shoots after transfer onto a hormone-free medium for 22 months. These plantlets were acclimatized in a greenhouse and grew well with 100% survival rate. Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; MS – Murashige and Skoog (1962) medium; TDZ – 1phenyl-3-(1,2,3-thiadiazol-5-yl)-urea Paphiopedilum, is a genus of terrestrial orchids with some 70 species that are native to South and Southeast Asia, and a distribution that extends from the Himalayas and Burma into Indochina and the Asean region up to Papua New Guinea (Teob, 1989). They are popularly known as Slipper Orchids because of the resemblance of the pouch-shaped lip to a lady’s slipper, and are often marketed as pot plants with high value. There are only a few reports describing aseptic culture methods for propagating Paphiopedilum orhcids, because the explants are difficult to maintain in culture (see review by Arditti and Ernst, 1993). Shoot tips have been used as explants to induce the growth of axillary shoots and regenerated plantlets (Arditti and Ernst, 1993; Huang, 1988). Huang et al. (2001) further modified their medium (Huang, 1988), and multiplied shoots taken from in vitro grown seedlings. Lin et al. (2000) established callus cultures from protocorms, and successfully regenerated plantlets through protocom-like bodies from the subcultured callus. However, poor callus formation rate, slow growth of callus and low regeneration capacity are still the major impediments limiting the utility of in vitro culture for Paphiopedilum propagation. Because of the limited success in tissue culture protocols, Paphiopedilum propagation has still been entirely by asymbiotic germination. In this communication, we established an efficient protocol for regenerating Paphiopedilum through direct shoot bud formation from leaf culture. Thirty-six-month-old in vitro grown seedlings of Paphiopedilum philippinense (hybrid PH59 and hybrid PH60) cultured on MS (Murashige and Skoog, 1962) medium free of plant growth regulators were