Nyctanthes arbor-tristis L. is an important ornamental as well as medicinal plant known for its flowers. The axillary nodes of this plant are cultured on Murashige and Skoog medium, which results in the direct organogenesis. The M.S. medium is supplemented with 3 mg/L Indole-3-acetic acid. The in-vitro regenerated shoots are sub-cultured on the MS medium supplemented with IAA and BAP and subjected to elevated level of CO₂ viz 0.5% and 2% in airtight CO₂ chamber. The comparative analysis is performed on various physical and biochemical parameters after an 8-week treatment at elevated CO₂. The results of the present study show that the enhanced CO₂. level proves beneficial for invitro growth of Nyctanthes arbor-tristis L.