| Summary: | Low Zinc (Zn) availability in alkaline calcareous soil is one of the major causes of low cereal yield and quality. Conventional application of Zn sulfate (ZnSO<sub>4</sub>) fertilizer through soil application attains minimal Zn efficiency as it is readily fixed in such soils. Oxozinc nanofiber (ZnONF) was evaluated for wheat Zn biofortification using different application methods to tackle this issue. Pots in triplicate (each with 7 kg soil) were arranged in a completely randomized design with a control treatment without Zn application. The conventional ZnSO<sub>4</sub> fertilizer recommended dose (5.5 µg Zn kg<sup>−1</sup> of soil) was used for comparison and applied through soil addition, foliar spray, and seed priming, while the ZnO<sub>NF</sub> was applied through foliar spray, seed coating, and seed priming (@ 0.5 kg ha<sup>−1</sup>) either alone or in combination with ½ZnSO<sub>4</sub> applied to the soil. The application of ZnO<sub>NF</sub> significantly improved wheat plant growth as evidenced by increased plant height (14.5%), spikelets per spike (13.7%), and Zn use efficacy (611%) regardless of application methods as compared to control. The highest Zn uptake efficiency (34%) for nanofibers was obtained for theseed primed, followed by seed coating (23%) and foiar application (7%), respectively. Moreover, at the combined ZnO<sub>NF</sub> and ½ZnSO<sub>4</sub> application, further improvements for spike length, number of spikelets spike<sup>−1</sup>, grain, leaf, root, and stem Zn concentrations, as well as their respective Zn contents, were noted. These results elucidated that Zn nutrition with ZnONF was either at par with or higher than the conventional ZnSO<sub>4</sub> fertilizer application despite significantly reduced ZnO<sub>NF</sub> quantity, irrespective of the application method used. Additionally, the combined ZnO<sub>NF</sub> and ½ZnSO<sub>4</sub> (foliar spray, seed coating, or seed priming) maximized the crop Zn accumulation, wherein the ½ZnSO<sub>4</sub> + ZnO<sub>NF</sub> through foliar application exceeded grain Zn biofortification. Thus, various Oxozinc nanofibers application modes may be recommended for wheat biofortification either separately or in combination with ZnSO<sub>4</sub> in Zn deficient calcareous soils for improved Zn nourishment.
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