Objectives Zn biofortification in wheat flour varies remarkably with regions and sites. We studied the reasons and the suitable Zn fertilizer application rates suitable for the main wheat production regions in China.

Methods This study was conducted in Yongshou of Shaanxi, Baixiang of Hebei, Zitong of Sichuan, and Shucheng of Anhui across two cropping seasons of 2022−2023 and 2023−2024. ZnSO₄·H₂O was applied in soil as basal fertilizer at rates of 0, 6.8, 13.6, 20.4, and 27.2 kg Zn/hm². Wheat yield, biomass, yield components, Zn concentration in various plant parts were analyzed, and topsoil samples were simultaneously collected to determine soil available Zn contents.

Results Zn application did not lead to significant differences in grain yield, biomass, and yield components, but significantly enhanced Zn concentration in various parts of wheat. Every 1.0 kg Zn/hm² input could lead to an average increase of 0.28, 0.15, 0.75, and 1.54 mg/kg in grain Zn in Shaanxi, Hebei, Sichuan, and Anhui, respectively. At Zn application rate of 10.5 and 0.0 kg/hm², the grain biofortified target of 40.0 mg/kg and the highest of 46.6 and 75.5 mg/kg was achieved respectively in Sichuan and Anhui, but in Shaanxi and Hebei, the grain Zn concentration only reached 31.4 and 36.5 mg/kg even at the highest Zn application rate. Every 1.0 kg Zn/hm² input could lead to an average increase of 0.14, 0.12, 0.17, and 0.50 mg/kg in grain Zn in Shaanxi, Hebei, Sichuan, and Anhui respectively, Zn concentration in flour reached 15 mg/kg in each region in the cropping season of 2023-2024. Shoot Zn use efficiency was higher in Sichuan and Anhui, with an average of 0.87% and 1.31%, which was higher than that of Shaanxi and Hebei, being correspondingly 0.25% and 0.16%. Translocation of Zn from shoot to grain was significantly decreased, and that to straw was increased with Zn application, while the distribution ratio of absorbed Zn between flour and bran in grain was not changed. Concentration of available Zn in soil was significantly increased with Zn application, with the increase in the second year being higher than that in the first year, and the highest increase was found in Anhui, followed by Sichuan and Hebei, with the lowest in Shaanxi. The average increase was 0.28, 0.16, 0.16, and 0.14 mg/kg for each 1.0 kg Zn /hm² input at the four sites, respectively.

Conclusions After continuous Zn application, available soil Zn concentration was effectively enhanced in all regions. Wheat Zn biofortification differences were mainly determined by soil pH and available Zn contents in different regions. Sichuan and Anhui could achieve the grain Zn biofortification target of 40.0 mg/kg, while Shaanxi and Hebei could not, with average grain Zn concentrations reaching 46.6, 75.5, 31.4 and 36.5 mg/kg respectively over two years. Meanwhile, flour Zn concentration in all four regions could achieve the biofortification target of 15.0 mg/kg after continuous Zn application. Overall, to achieve wheat Zn biofortification targets, the minimum Zn application rate of 10.0 kg/hm² is recommended in calcareous soils with available soil Zn lower than 0.5 mg/kg, while the Zn application rate could be less than 6.0 kg/hm² in acid soils with available soil Zn close to 1.0 mg/kg.