BackgroundRice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g−1 Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g−1 Fe in endosperm.Methodology/Principal FindingsThree populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g−1 Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g−1 Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm.ConclusionsThe OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.

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Journal articleIFPRI3; Breeding for Nutrition; ISI; HarvestPlusHarvestPlusP

This study investigated the main factors contributing to boron toxicity in plants. Growth was rapidly inhibited by internal B concentrations in the range 1-5 m M across a range of plant types that included monocot, dicot and algal species. In contrast, mature cells were able to withstand up to 60 m M B for several days. In wheat, rapid inhibition of root growth occurred if high B was applied to the root tip, but not if high B was applied to mature sections of the root. In leaves, there were gradations in B concentrations that correlated with visible symptoms of toxicity. However, there was no evidence to support the hypothesis that toxicity in leaves is due to osmotic stress induced by the accumulation of B. Analysis of the sensitivity to B of a range of metabolic processes including photosynthesis, respiration and protein synthesis leads to the conclusion that growth is not restricted by effects of B on energy supply and not directly by inhibition of protein synthesis. At higher B concentrations, many cellular activities were found to be partially inhibited and the toxicity to mature tissues was therefore considered not to arise from the disruption of a single process, but from the accumulated retardation of many cellular processes, exacerbated in light by photooxidative stress.

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.

The metalloid Se is ubiquitous in soils, but exists mainly in insoluble forms in high-Fe, low-pH and certain leached soils, and hence is often of limited availability to plants. Consequently, it is often supplied by plants to animals and human consumers at levels too low for optimum health. Se deficiency and suboptimality are manifested in populations as increased rates of thyroid dysfunction, cancer, severe viral diseases, cardiovascular disease and various inflammatory conditions. Se deficiency probably affects at least a billion individuals. Optimal cancer protection appears to require a supra-nutritional Se intake, and involves several mechanisms, which include promotion of apoptosis and inhibition of neo-angiogenesis. Evidence suggests that in some regions Se is declining in the food chain, and new strategies to increase its intake are required. These could include education to increase consumption of higher-Se foods, individual supplementation, food fortification, supplementation of livestock, Se fertilisation of crops and plant breeding for enhanced Se accumulation. Se levels in Australian residents and wheat appear to be above the global estimated mean. Wheat is estimated to supply nearly half the Se utilised by most Australians. Increasing the Se content of wheat represents a food systems approach that would increase population intake, with consequent probable improvement in public health and large health cost savings. The strategies that show most promise to achieve this are biofortification by Se fertilisation and breeding wheat varieties that are more efficient at increasing grain Se density. Research is needed in Australia to determine the most cost-effective fertilisation methods, and to determine the extent of genetic variability for grain Se accumulation. Before recommending large-scale fortification of the food supply with Se, it will be necessary to await the results of current intervention studies with Se on cancer, HIV and AIDS, and asthma.

Selenium (Se) is an essential micronutrient for humans and animals, with antioxidant, anti-cancer and anti-viral effects, and wheat is an important dietary source of this element. In this study, surveys of Se concentration in grain of ancestral and wild relatives of wheat, wheat landrace accessions, populations, and commercial cultivars grown in Mexico and Australia were conducted. Cultivars were also grown under the same conditions to assess genotypic variation in Se density. Eleven data sets were reviewed with the aim of assessing the comparative worth of breeding compared with fertilising as a strategy to improve Se intake in human populations. Surveys and field trials that included diverse wheat germplasm as well as other cereals found grain Se concentrations in the range 5-720 µg kg −1 , but much of this variation was associated with spatial variation in soil selenium. This study detected no significant genotypic variation in grain Se density among modern commercial bread or durum wheat, triticale or barley varieties. However, the diploid wheat, Aegilops tauschii and rye were 42% and 35% higher, respectively, in grain Se concentration than other cereals in separate field trials, and, in a hydroponic trial, rye was 40% higher in foliar Se content than two wheat landraces.

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