High Iron Rice Research Articles

The effect of iron absorption in ferrous gluconate form from enriched rice flour using an in vitro digestion model and a Caco-2 cell model.

Iron deficiency can cause serious diseases in infants and young children such as indigestion, anemia, and nervous system dysplasia. Consumption of high-iron rice flour can prevent iron deficiency. The objective of this study was to evaluate the potential application of ferrous gluconate as an iron source in high-iron rice flour used as a type of accessory food for infants and young children. In this study, the differences in iron absorption ability between ferrous gluconate and ferrous fumarate in rice flour with the same ingredients in both high and low phytic acid systems were evaluated. The results showed that there was no significant difference in the bioaccessibility/bioavailability between ferrous gluconate and ferrous fumarate at both low and high phytic acid contents. In low phytic acid and high phytic acid systems, the iron absorption rate of ferrous gluconate is 11.53% and 13.45% higher than that of ferrous fumarate, respectively (p < 0.05). In summary, the iron absorption rate of ferrous gluconate was higher than that of ferrous fumarate in the rice flour system. Additionally, the low phytic acid environment is more conducive to iron uptake and utilization. Therefore, ferrous gluconate can be used as an alternative source of iron in accessory foods for infants and young children.

Insights into the recent approaches for rice (Oryza sativa L.) biofortification

Rice is the most essential source of calories for humans among the cereals and over half of the world's population is fed on rice. As part of a complete food systems approach, biofortification is an effective technique for nutrition enrichment which refers to the development of micronutrient-rich diet by utilising traditional breeding practises and sophisticated biotechnological tools. To enhance the profile of rice grain for biofortification-related properties, researchers must first understand the genetics of critical biofortification characteristics. Significant increases in micronutrients like iron and zinc, as well as many other important minerals and provitamins are acquired in rice grain using the biofortification strategies. Most indica and japonica rice types have been biofortified over the world, giving them the titles of high iron rice, low phytate rice, high zinc rice, and high carotenoid rice or golden rice. Some of the recent approaches towards rice biofortification, as well as their effects, have been explored in this article.

Effect of High-iron Rice Intervention on VO2max of Female Students of Al-Falak Bogor Islamic Boarding School

Female adolescents are at risk of iron deficiency which is known can negatively affect fitness. High-iron rice has been developed through biofortification but there is still lack of evidence regarding its benefit in improving VO2max as a physical fitness indicator. This study aimed to determine impact of high-iron rice intervention towards VO2max value. A total of 18 female students from Al-Falak Bogor Islamic boarding school aged 12-18 years were selected purposively in this quasi-experimental study. High-iron rice contained 50.4 ppm iron provided thrice daily for 4 weeks. A 20 m shuttle run test was conducted to obtain VO2max value. Other data such as food intake, BMI for age, hemoglobin, and serum ferritin were also evaluated. Paired t-test or Wilcoxon Ranks test was performed to analyze difference in data value before and after the intervention. Most nutrient intakes experienced a significant decline (p&lt;0.05) after the intervention, except for iron intake. Hemoglobin concentration also became lower significantly, whereas serum ferritin did not alter significantly. Nevertheless, VO2max improved significantly after the intervention. To summarize, there was an increase in fitness after high-iron rice intervention for 4 weeks in female students.

Rice Biofortification: High Iron, Zinc, and Vitamin-A to Fight against “Hidden Hunger”

One out of three humans suffer from micronutrient deficiencies called “hidden hunger”. Underprivileged people, including preschool children and women, suffer most from deficiency diseases and other health-related issues. Rice (Oryza sativa), a staple food, is their source of nutrients, contributing up to 70% of daily calories for more than half of the world’s population. Solving “hidden hunger” through rice biofortification would be a sustainable approach for those people who mainly consume rice and have limited access to diversified food. White milled rice grains lose essential nutrients through polishing. Therefore, seed-specific higher accumulation of essential nutrients is a necessity. Through the method of biofortification (via genetic engineering/molecular breeding), significant increases in iron and zinc with other essential minerals and provitamin-A (β-carotene) was achieved in rice grain. Many indica and japonica rice cultivars have been biofortified worldwide, being popularly known as ‘high iron rice’, ‘low phytate rice’, ‘high zinc rice’, and ‘high carotenoid rice’ (golden rice) varieties. Market availability of such varieties could reduce “hidden hunger”, and a large population of the world could be cured from iron deficiency anemia (IDA), zinc deficiency, and vitamin-A deficiency (VAD). In this review, different approaches of rice biofortification with their outcomes have been elaborated and discussed. Future strategies of nutrition improvement using genome editing (CRISPR/Cas9) and the need of policy support have been highlighted.

Enhanced Grain Iron Levels in Rice Expressing an IRON-REGULATED METAL TRANSPORTER, NICOTIANAMINE SYNTHASE, and FERRITIN Gene Cassette.

Micronutrient malnutrition is widespread, especially in poor populations across the globe, and iron deficiency anemia is one of the most prevalent forms of micronutrient deficiencies. Iron deficiency anemia has severe consequences for human health, working ability, and quality of life. Several interventions including iron supplementation and food fortification have been attempted and met with varied degrees of success. Rice, which is a staple food for over half of the world’s population, is an important target crop for iron biofortification. The genetic variability of iron content in the rice germplasm is very narrow, and thus, conventional breeding has not been successful in developing high iron rice varieties. Therefore, genetic engineering approaches have targeted at increasing iron uptake, translocation, and storage in the rice endosperm. We previously reported that AtIRT1, when expressed together with AtNAS1 and PvFERRITIN (PvFER) in high-iron (NFP) rice, has a synergistic effect of further increasing the iron concentration of polished rice grains. We have now engineered rice expressing AtIRT1, AtNAS1, and PvFER as a single locus gene cassette and compared the resulting lines with transgenic lines expressing AtIRT1 and PvFER gene cassettes. We also evaluated the efficacies of the MsENOD12B and native AtIRT1 promoters for the expression of AtIRT1 in rice in both types of gene cassettes, and found the native AtIRT1 promoter to be a better choice for driving the AtIRT1 expression in our biofortification strategy. All the single insertion transgenic lines have significant increases of iron concentration, both in polished and unpolished grains, but the concerted expression of AtIRT1, AtNAS1, and PvFER resulted to be a more effective strategy in achieving the highest iron increases of up to 10.46 μg/g dry weight. Furthermore, the transformed high iron lines grew better under iron deficiency growth conditions and also have significantly increased grain zinc concentration. Together, these rice lines have nutritionally relevant increases in polished grain iron and zinc concentration necessary to support human health.

Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots.

The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants . Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds.

Nicotianamine synthase overexpression positively modulates iron homeostasis-related genes in high iron rice.

Nearly one-third of the world population, mostly women and children, suffer from iron malnutrition and its consequences, such as anemia or impaired mental development. Biofortification of rice, which is a staple crop for nearly half of the world's population, can significantly contribute in alleviating iron deficiency. NFP rice (transgenic rice expressing nicotianamine synthase, ferritin and phytase genes) has a more than six-fold increase in iron content in polished rice grains, resulting from the synergistic action of nicotianamine synthase (NAS) and ferritin transgenes. We investigated iron homeostasis in NFP plants by analyzing the expression of 28 endogenous rice genes known to be involved in the homeostasis of iron and other metals, in iron-deficient and iron-sufficient conditions. RNA was collected from different tissues (roots, flag leaves, grains) and at three developmental stages during grain filling. NFP plants showed increased sensitivity to iron-deficiency conditions and changes in the expression of endogenous genes involved in nicotianamine (NA) metabolism, in comparison to their non-transgenic siblings (NTS). Elevated transcript levels were detected in NFP plants for several iron transporters. In contrast, expression of OsYSL2, which encodes a member of yellow stripe like protein family, and a transporter of the NA-Fe(II) complex was reduced in NFP plants under low iron conditions, indicating that expression of OsYSL2 is regulated by the endogenous iron status. Expression of the transgenes did not significantly affect overall iron homeostasis in NFP plants, which establishes the engineered push-pull mechanism as a suitable strategy to increase rice endosperm iron content.

Comparative analysis of nutritional compositions of transgenic high iron rice with its non-transgenic counterpart

Iron is an essential micronutrient for human nutrition and polished rice contains very low amount of iron. Rice with high iron content in seed endosperm has been developed by insertion of soybean ferritin gene under the control of the endosperm specific glutelin promoter into the genome of indica rice line IR68144. The nutritional composition of the brown and milled rice grain has been compared with that of the non-transgenic rice of the same variety. In this study, the nutritional components, as well as the anti-nutrient levels, were measured. Our studies established that apart from the increased level of iron and zinc in transgenic seeds, the nutritional quality of both the brown and milled rice grains from the transgenic line was substantially equivalent to that of the non-transgenic rice plants. The result clearly shows that the measured amounts of the nutritional components are well within the range of values reported for other commercial lines.

Molecular breeding of Osfer2 gene to increase iron nutrition in rice grain

Rice being a staple food, contains little iron in the edible grain. To increase the iron nutrition in rice grains, our present study highlights the first time development of high iron rice grain by exploring the endosperm specific overexpression of endogenous ferritin gene. The gene has been cloned from rice and overexpressed under the control of endosperm specific GlutelinA2 (OsGluA2) promoter. After genetic transformation of aromatic indica rice cultivar, Pusa-sugandhi II, the milled seeds of resulting T3 transgenics exhibited 7.8-fold of ferritin overexpression, which contributed to 2.09- and 1.37-fold of iron and zinc accumulation respectively. T3 seeds demonstrated endosperm specific localization of iron that confirms the tissue specific activity of GluA2 promoter. Transgenic and non-transgenic plants showed no difference in their agronomic traits. Our study suggested that overexpression of rice endogenous ferritin gene is a step ahead toward cisgenic approach and can act as an effective tool for iron biofortification.

Iron content and availability studies in some Sri Lankan rice varieties

SummaryThirty‐eight rice varieties, grown in Low Country Wet Zone, during Yala (2006) and Maha (2006/2007) seasons were screened for stable high iron rice varieties and were statistically analysed. Iron contents ranged 1.89–3.73 mg 100 g−1 and varied significantly (P &lt; 0.05) with variety and season. Selected fifteen high iron rice varieties, at degree of polishing 8–10% were analysed for iron, zinc and phytate. High iron contents in endosperm were observed in Suduru Samba (0.47 mg 100 g−1), Basmati 370 (0.37 mg 100 g−1), Kalu Heenati (0.42 mg 100 g−1), Rathu Heenati (0.44 mg 100 g−1) and Sudu Heenati (0.37 mg 100 g−1). Phytate contents of polished rice varieties ranged from 200–300 mg 100 g−1. Large reduction in iron content (84.5–93.6%) was observed in polished rice while the reduction in phytate content (18.9–40.8%) was low. Percentage dialyzability of iron in selected endosperm high iron rice varieties ranged from 1.73 to 8.71. Dialysability of iron in cooked polished rice did not show a relation to the phytate content in raw rice.

A Robust and Cost-Effective SGOC Method for Testing Rice Iron Concentration in Biofortified Breeding

Iron is an important micro-nutrient to human health. Malnutrition of iron is a serious problem associated with resource poor population in many developing countries. Development and consumption of iron-rich rice ( Oryza sativa L.) varieties are considered one of the ways to solve this problem. To facilitate large-scale screening of breeding materials for iron concentration in the rice iron-biofortified breeding program of China, a new method “surging and grind-milling of orthophenanthroline colorimetry testing (SGOC)” was developed. Based on the testing results of 3 sets of 84 diverse rice genotypes that differ greatly in grain iron concentration, the correlation coefficient was as high as 0.87 between the SGOC method and the standard ICP-MS testing method. The per sample cost of the SGOC method was about 0.1 US dollar or 50 times less the ICP-MS method, indicating that the SGOC method is robust, fast, and cost-effective, particularly useful for preliminary screening of the iron concentration of large numbers of early generation breeding materials. The iron concentration of processed rice that tended to be significantly increased when milling and polishing with iron-made equipment was also eliminated in the SGOC method. Based on the analysis of iron concentration in 59 backcross progenies, the introgression of genes/quantitative trait loci for high iron concentration from high iron rice germplasm into elite local rice varieties is an efficient way to develop high yielding rice varieties with significantly improved rice iron concentration in future rice biofortified breeding.

Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin

Nearly one-third of the world's population, mostly women and children, suffer from iron malnutrition and its consequences, such as anaemia or impaired mental development. Iron fortification of food is difficult because soluble iron is either unstable or unpalatable, and non-soluble iron is not bioavailable. Genetic engineering of crop plants to increase iron content has therefore emerged as an alternative for iron biofortification. To date, strategies to increase iron content have relied on single genes, with limited success. Our work focuses on rice as a model plant, because it feeds one-half of the world's population, including the majority of the iron-malnourished population. Using the targeted expression of two transgenes, nicotianamine synthase and ferritin, we increased the iron content of rice endosperm by more than six-fold. Analysis of transgenic rice lines confirmed that, in combination, they provide a synergistic effect on iron uptake and storage. Laser ablation-inductively coupled plasma-mass spectrometry showed that the iron in the endosperm of the transgenic rice lines accumulated in spots, most probably as a consequence of spatially restricted ferritin accumulation. Agronomic evaluation of the high-iron rice lines did not reveal a yield penalty or significant changes in trait characters, except for a tendency to earlier flowering. Overall, we have demonstrated that rice can be engineered with a small number of genes to achieve iron biofortification at a dietary significant level.

Distribution of Protein Bodies and Phytate‐Rich Inclusions in Grain Tissues of Low and High Iron Rice Genotypes

ABSTRACTThe present study aims to understand whether genotypic differences in grain iron (Fe) concentration in four rice genotypes are related to its association with protein bodies containing phytate‐rich inclusions. Rice genotypes with high and low grain Fe concentrations in unpolished brown rice were grown in a greenhouse at Chiang Mai, Thailand, and grains were harvested at maturity. The presence of protein bodies and phytate‐rich inclusions in rice grain tissues were examined by means of light and transmission electron microscopy (TEM). The composition of mineral elements in different grain tissues was examined using energy dispersive X‐ray microanalysis (EDX) and chemical analysis. The relative distribution pattern of protein bodies in the tissues was similar among the four rice genotypes, which resembled the pattern of grain N concentrations in these tissues. The high grain Fe genotypes (based on brown rice Fe concentration) had more protein bodies containing phytate‐rich inclusions in the embryo and aleurone layer tissues than the low Fe genotypes. Phytate‐rich inclusions were not detected in the endosperm tissues in all genotypes. In conclusion, the presence of protein bodies with phytate‐rich inclusions predominantly in the embryo and aleurone regions of the grain is an important parameter contributing to the variation in brown rice Fe concentration among the genotypes, but not in the white rice (the endosperm). Iron associated with the phytate‐rich inclusions present in the embryo and aleurone layer tissues are largely lost during the polishing process to produce white rice.

Iron Absorption: Comparison of Prediction Equations and Reality. Results from a Feeding Trial in the Philippines

A number of algorithms have been developed that seek to predict the bioavailability of iron from mixed meals and diets, but their direct validity in predicting change in iron status remains questionable. Throughout the course of conducting a large feeding trial in convents in Manila, we collected weighed food-intake data and have the opportunity to directly compare the performance of these prediction equations. The specific aims of this particular analysis are as follows: (a) to determine habitual intakes of macro- and micronutrients in religious sisters in Manila, (b) to determine the predicted efficiency of iron absorption from each of the published bioavailability algorithms (Monsen, Hallberg, Reddy, Tseng, Barghava, and Du), and (c) to determine which of these equations best predicts the actual "gain" in iron in religious sisters over the duration of the trial. The efficacy of consuming high-iron rice was tested during a nine-month feeding trial with a double-blinded dietary intervention; these results have been published [7]. Religious sisters living in 10 convents around metropolitan Manila, the Philippines were randomly assigned to consume either high-iron rice (3.21 mg/kg Fe) or a local variety of control rice (0.57 mg/kg Fe) within each convent. Religious sisters in convents consumed a diet that appeared to be typical of habitual intakes of macro-and micronutrients in this part of the Philippines. The analysis of the six equations revealed highly significant differences in predicted efficiency of iron absorption. The Hallberg, Monsen, and Reddy equations all predicted similar median efficiency (6.88, 7.92, and 6.42%). In contrast, Bhargava (4.68%), Tseng (3.23%), and Du (2.92%) were significantly lower. The correlation (r = 0.98) of Monsen to Hallberg was highly significant and the slope was not different than unity. The median efficiency of absorption based on the gain in body iron in 114 subjects over nine months, combined with an estimate of daily iron requirements, was 17.2%. Thus, none of these equations approximated the computed iron absorption based on improvement in serum ferritin and suggests alternative approaches to predicting iron accumulation from diet need to be formulated. Inhibitory factors in the prediction equations either had little effect or had too large of an effect on apparent bioavailability as compared to median absorption over a nine-month period. The causes of the lack of agreement between computed iron gain and predicted absorption are open to discussion and will need to be resolved.

Iron-Biofortified Rice Improves the Iron Stores of Nonanemic Filipino Women

Iron deficiency is endemic in much of the world, and food system-based approaches to eradication may be viable with new plant breeding approaches to increase the micronutrient content in staple crops. It is thought that conventional plant breeding approaches provide varieties of rice that have 400-500% higher iron contents than varieties commonly consumed in much of Asia. The efficacy of consuming high-iron rice was tested during a 9-mo feeding trial with a double-blind dietary intervention in 192 religious sisters living in 10 convents around metro Manila, the Philippines. Subjects were randomly assigned to consume either high-iron rice (3.21 mg/kg Fe) or a local variety of control rice (0.57 mg/kg Fe), and daily food consumption was monitored. The high-iron rice contributed 1.79 mg Fe/d to the diet in contrast to 0.37 mg Fe/d from the control rice. The 17% difference in total dietary iron consumption compared with controls (10.16 +/- 1.06 vs. 8.44 +/- 1.82 mg/d) resulted in a modest increase in serum ferritin (P = 0.10) and total body iron (P = 0.06) and no increase in hemoglobin (P = 0.59). However, the response was greater in nonanemic subjects for ferritin (P = 0.02) and body iron (P = 0.05), representing a 20% increase after controlling for baseline values and daily rice consumption. The greatest improvements in iron status were seen in those nonanemic women who had the lowest baseline iron status and in those who consumed the most iron from rice. Consumption of biofortified rice, without any other changes in diet, is efficacious in improving iron stores of women with iron-poor diets in the developing world.

Molecular characterization of marker-free transgenic lines of indica rice that accumulate carotenoids in seed endosperm

A single Agrobacterium strain harbouring two binary plasmids was successfully used for the first time to develop a marker-free transgenic rice of improved nutritional value. Sixty-eight T0 co-transformants were obtained in three indica rice cultivars--two popular high-yielding Bangladeshi varieties (BR28 and BR29), and one high-iron rice cultivar (IR68144). Marker-free lines were obtained from 14 out of 24 selected co-transformants screened in the T1 generation. The accumulation of total carotenoids in polished T2 rice seeds of the primary transgenic VPBR29-17-37 reached levels of up to 3.0 microg/g, with the level of beta-carotene reaching 1.8 microg/g. In the cultivars BR28 and IR68144, total carotenoid levels in the transformants reached 2.0 microg/g of polished rice seeds. The levels of lutein and other carotenoids in the seeds were also significantly enhanced. T1 plants obtained from primary transgenics with simple gene-integration patterns tended to have a lower carotenoid content than the original parental lines. This study describes the development of marker-free transgenic rice lines containing high levels of carotenoids, and addresses the relationship between the rearrangement of transgenes and the presence of metabolic end products in transgenic rice.

Impact of Feeding High-Iron Rice on Plasma Iron, Hemoglobin and Red Blood Cell Variables of Early-Weaned Piglets

Severe iron deficiency is a major nutritional problem encountered throughout the world. We assessed the effect of a conventionally bred, high-iron rice variety on plasma iron, hemoglobin, and red blood cell variables of early-weaned piglets during a 33-day feeding trial. 26-day-old male piglets were assigned to 3 treatment groups: group 1 = low-iron rice + low-iron supplementary feed; group 2 = high-iron rice + low-iron supplementary feed, and group 3 = low-iron rice + high-iron supplementary feed. Plasma iron, hemoglobin and red blood cell variables were measured on days 8, 16, 23, 30, and 33. Feed intake and weight gain were not significantly different between study groups. No significant differences in the iron-related parameters analyzed were found between the piglets of groups 1 and 2, except in red blood cells. Modifications regarding study design, study duration and subject’s growth rate are recommended to increase the possibility of detecting changes in the iron status triggered by diets having small differences in dietary iron.