Association of Increased Grain Iron and Zinc Concentrations with Agro-morphological Traits of Biofortified Rice

Abstract

Biofortification of rice (<i>Oryza sativa</i> L.) with micronutrients is widely recognized as a sustainable strategy to alleviate human iron (Fe) and zinc (Zn) deficiencies in developing countries where rice is the staple food. Constitutive overexpression of the rice nicotianamine synthase (<i>OsNAS</i>) genes has been successfully implemented to increase Fe and Zn concentrations in unpolished and polished rice grain. Intensive research is now needed to couple this high-micronutrient trait with high grain yields. We investigated associations of increased grain Fe and Zn concentrations with agro-morphological traits of backcross twice second filial (BC₂F₂) transgenic progeny carrying <i>OsNAS1</i> or <i>OsNAS2</i> overexpression constructs under <i>indica/japonica</i> and <i>japonica/japonica</i> genetic backgrounds. Thirteen agro-morphological traits were evaluated in BC₂F₂ transgenic progeny grown under hydroponic conditions. Concentrations of eight mineral nutrients (Fe, Zn, copper, manganese, calcium, magnesium, potassium, and phosphorus) in roots, stems/sheaths, non-flag leaves, flag leaves, panicles, and grain were also determined. A distance-based linear model (DistLM) was utilized to extract plant tissue nutrient predictors accounting for the largest variation in agro-morphological traits differing between transgenic and non-transgenic progeny. Overall, the BC₂F₂ transgenic progeny contained up to 148% higher Fe and 336% higher Zn concentrations in unpolished grain compared to non-transgenic progeny. However, unpolished grain concentrations surpassing 23 μg Fe g^-1 and 40 μg Zn g^-1 in BC₂F₂<i>indica/japonica</i> progeny, and 36 μg Fe g^-1 and 56 μg Zn g¹ in BC₂F₂<i>japonica/japonica</i> progeny, were associated with significant reductions in grain yield. DistLM analyses identified grain-Zn and panicle-magnesium as the primary nutrient predictors associated with grain yield reductions in the <i>indica/japonica</i> and <i>japonica/japonica</i> background, respectively. We subsequently produced polished grain from high-yield BC₂F₂ transgenic progeny carrying either the <i>OsNAS1</i> or <i>OsNAS2</i> overexpression constructs. The <i>OsNAS2</i> overexpressing progeny had higher percentages of Fe and Zn in polished rice grain compared to the <i>OsNAS1</i> overexpressing progeny. Results from this study demonstrate that genetic background has a major effect on the development of Fe and Zn biofortified rice. Moreover, our study shows that high-yielding rice lines with Fe and Zn biofortified polished grain can be developed by <i>OsNAS2</i> overexpression and monitoring for Zn overaccumulation in the grain.