MICROPROPAGATION OF A RARE ORCHID DENDROBIUM GRATIOSISSIMUM USING THIN CELL LAYERS

Abstract

Dendrobium gratiosissimum Rchb. f. is a very graceful wild orchid of Thailand and is a rare species. Establishment of an efficient method for micropropagation is urgently needed because of its conservation and commercial requirements. An efficient method of rapid and mass propagation of D. gratiosissimum using thin cell layers (TCLs) had been developed. Four-week-old protocorms were segmented into two halves and each half was considered as a TCLs explant. The shoots, each with 1 to 2 nodes were cut into approximately 0.3 to 0.5 mm thickness from the base to the shoot tip. The TCLs explants from both protocorms and young stems were cultured on MS (Murashige and Skoog, 1962) medium supplemented with different combinations of N 6 -benzyladenine (BA), kinetin (Kn), 1-naphthaleneacetic acid (NAA) and 20 mg/L sucrose. MS medium without growth regulators failed to induce protocorm-like bodies (PLBs) formation from stem explants. The TCLs explants from both protocorm and stem developed PLBs within 3 to 4 weeks on the MS containing growth regulators. Of the two different TCLs explants types used, the highest percentage of PLBs formation (83%) and highest number of PLBs (18) per explants were observed on the protocorm sections, whereas only 66% of stem sections were able to produce PLBs with an average 9 PLBs per explants. MS containing 2 mg/L Kn was found to be the optimum concentration for PLBs development from thin protocorm section. In contrast, the highest PLBs production of stem sections was obtained from explants on 5 mg/L Kn and 1.0 mg/L NAA. Plantlet conversion from PLBs was successfully achieved on regulator-free growth medium.