This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have
concentration of approximately 2 μg g−1 iron (Fe) and 16 μg g−1 zinc (Zn). The HarvestPlus breeding
programs for biofortified rice target 13 μg g−1 Fe and 28 μg g−1 Zn to reach approximately 30% of
the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an
increase up to 18 μg g−1 in glasshouse settings; in contrast, under field conditions, 4 μg g−1 was the
highest reported concentration. Here, we report on selected transgenic events, field evaluated in two
countries, showing 15 μg g−1 Fe and 45.7 μg g−1 Zn in polished grain. Rigorous selection was applied
to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event
NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes
showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn
enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is
bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different
genotype backgrounds
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