Abstract:In a continuous effort to conserve Phalaenopsis species, the optimization of a previously designed defined medium was carried out to understand the effect of in vitro nitrogen nutrition on Phalaenopsis deliciosa Rchb.f. In order not to alter the nutrient balance, macronutrient level instead of solely Nitrogen (N) was adjusted. Seedlings were obtained from in vitro germination and randomly inoculated on media with various macronutrient levels (0.3×, 0.5×, 1×, 2× and 3×). Maximum shoot yield was observed at 2× macronutrient level. However, the best macronutrient level, which was 1× macronutrient level, resulted in the maximum root and seedling yield. This macronutrient level was applied on the subsequent experiment where seedlings were inoculated on media with various nitratenitrogen (NO 3 -N) to ammonium-nitrogen (NH 4 -N) ratios. Seedling yield was the highest at NO 3 -N/NH 4 -N ratio of 2.0 while Root to Shoot ratio (R/S) was the highest at NO 3 -N/NH 4 -N ratio of 5.0. NO 3 -N/NH 4 -N ratio significantly affected Water Content (WC) of seedlings and the response to NO 3 -N/NH 4 -N ratio was similar to R/S, correlating better root development to higher WC. By altering NO 3 -N/NH 4 -N ratio, plant quality in terms of yield and organ development could be encouraged.