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Often regeneration in orchids is only achieved through protocorms, i.e., the juvenile stage. In order to produce directly shoots via bud regeneration both rapidly and with a high frequency, a transverse thin cell layer (tTCL) method is extended to Rhynchostylis gigantea. Transverse thin cell layer (tTCL) explants (0.3--0.5 mm) excised along the stem from the basis to the shoot tip of one-year-old plantlets were cultured on Murashige and Skoog (1962) medium supplemented with different combinations of benzyladenine (BAP), 1-naphthaleneacetic acid (NAA), thidiazuron (TDZ) and 3% sucrose. The optimal combination for maximal bud regeneration was 3 µM BAP and 3 µM TDZ, giving rise to 11.7 buds per tTCL. Roots were obtained with 10 µM forchlofenuron (CPPU) and 1% sucrose. The in vitro plants (> 3 cm long) obtained 4 to 6 weeks after the tTCLs culture were transferred to the greenhouse; their morphology was normal. Efficient micropropagation of direct production of shoots without passing through protocorm stage of orchid species can be achieved using the thin cell layer (TCL) method. |
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