The in vitro vegetative propagation technique of the Paphiopedilum glaucophyllum (callus propagation) still have problems to date. The aim of this study is to determine the effect of the media and plant growth regulator (PGR) on seed germination and callus induction with shoot tip as an explant and use the planlet from elongation treatment result. The seed germination uses modified Knudson C (KC medium) with the addition of 3 concentrations of NAA (0; 5; 10 mg/L). The second study is the elongation treatment of the explants uses four modified Murashige and Skoog (MS) medium (1/2 P, ½ P5, MP and MP5). And for the callus induction study consisted of two methods, callus induction with direct planting to the treatment medium by using SH medium with addition of 2,4-Dichlorophenoxyacetic acid (2,4-D) 1 mg/L and Thidiazuron (TDZ) (0, 0.5, 1, 1.5 dan 2 mg/L). The second method used the explants resulted from elongation treatment. It used half-strength Wattanawikkit medium with addition of TDZ (0; 0.5; 1; 1.5; 2 mg/L) and 2,4-D (0; 1; 5; 10 mg/L). All callus then subcultured in the half-strength MS (½ MS) medium containing 2,4-D 1 mg/L and TDZ (0; 1; 1.5 mg/L). All the experiments used a completely randomized design with 3 repetitions. The results showed that the seeds germinated 2 months after planting (MAP) for all media. The optimal media for explants elongation of P. glaucophyllum was MP5 media. Meanwhile, for the first callus induction experiments, explants had more callus in basic SH media with 2.4-D 1 mg/L and TDZ 0.5-1.5 mg/L that grew in the dark. The second experiment, the optimal medium for callus induction was half MS with Thidiazuron 1.5 mg/L and 2.4-D 1 mg/L (T1.5D1). The the suitable medium for callus development is ½ MS) medium with 2,4-D 1 mg/L.

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