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Advancement in nanotechnology has improved ways for large-scale production and characterization of nanoparticles of physiologically important metals. The current study explores the impact of Zinc Oxide Nanoparticles (ZnO-NP) and Chitosan-Zinc oxide nano-bioformulation (CH-ZnO) in tissue culture raised callus of Nicotiana benthamiana. Results indicated augmented biomass in CH-ZnO treated callus, while a reduced biomass was observed in ZnO-NP treated callus, at all the concentrations tested. Higher chlorophyll and carotenoid content were recorded in callus treated with 800 ppm CH-ZnO as compared to ZnO-NP treated callus. A higher accumulation of proline was observed in CH-ZnO treated callus when compared to ZnO-NP treatment, which was significantly higher at 50, 200 and 400 ppm CH-ZnO treatment. A maximum reduction in malondialdehyde (MDA) content was recorded at 800 ppm, for both the nano-formulations tested. Likewise, a significant reduction in the H 2 O 2 levels was observed in all the treatments, while the callus treated with 400 ppm ZnO-NP and 800 ppm CH-ZnO recorded the highest reduction. Phenylalanine Ammonia-Lyase (PAL), activity increased significantly in callus treated with 400 ppm concentration for both ZnO-NP and CH-ZnO with respect to control. An increased level of tannin and nicotine were recorded in callus supplemented with 50, 200 and 400 ppm CH-ZnO. Notably, a significant decline of 94 and 52% in tannin content and 25 and 50% in nicotine content was recorded in the callus treated with 800 ppm CH-ZnO and ZnO-NP, respectively. The findings of this study suggest that an optimized dosage of these nano-bioformulations could be utilized to regulate the nicotine content and stress tolerance level.

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