Micronutrient deficiency poses a threat to human health worldwide. An estimated 161 million children under 5 years of age are stunted, partly because of hidden hunger, which occurs when foodstuffs lack essential vitamins and minerals1. In Nigeria, 75% of preschool children and 67% of pregnant women are anemic2, and 20% of children under 5 years of age have zinc deficiency3. Iron-deficiency anemia affects the immune system, stunts growth and impairs cognitive development in children4. Zinc deficiency causes increased risk of death from diarrhea, stunting and hindered cognitive development4. Biofortification of staple food crops through biotechnology is one of several strategies for improving essential micronutrients in foods for at-risk populations5. Approximately 800 million people worldwide consume the tropical root crop cassava, and one-third of the sub-Saharan African population relies on cassava for more than 50% of their caloric intake6. Although cassava is an excellent source of carbohydrate, the storage roots provide inadequate levels of bioavailable iron and zinc5,7. A lack of genetic variation for mineral traits within the cassava germplasm8 makes breeding new lines with improved mineral content challenging. A genetic engineering strategy has been undertaken to increase iron and zinc concentrations in cassava storage roots9.

Genetic engineering has been successfully applied to increase mineral concentrations in cereal crops, including rice. Iron concentrations in polished rice grains have been increased by overexpressing the soybean or rice storage protein ferritin10 and coexpressing Arabidopsis nicotianamine synthase, common bean ferritin and Aspergillus phytase11. Overexpression of AtIRT1, AtNAS1 and bean FERRITIN in rice resulted in 3.8-fold higher iron and 1.8-fold higher zinc concentrations than in the wild-type control12. Recently, overexpression of the soybean ferritin SFER-H1 and rice nicotianamine synthase OsNAS2 has achieved dietary targets for both iron and zinc nutrition in rice grains13. Despite successes in rice, reports of engineering-improved mineral biofortification in dicotyledonous plants are rare and are mainly restricted to the model plant A. thaliana. Nongrass plants use a reduction-based mechanism for iron acquisition14 mediated by the plasma-membrane-bound oxidoreductase FRO2, and the ZIP-family transporter IRT1. Transgenic overexpression of the algal iron assimilatory protein FEA1 has resulted in a threefold increase in storage-root iron concentrations in greenhouse-grown cassava15, but these promising results were not maintained in field trials. Increased zinc concentrations in storage roots have been achieved by overexpression of the A. thaliana (At) zinc transporters AtZIP1 and AtMTP1, but shoot development in transgenic plants is impaired16.

We previously found that overexpression of the A. thaliana vacuolar iron transporter VIT1 in cassava results in a three- to four-times increase in iron concentration in storage roots compared with the concentrations in nontransgenic controls under greenhouse conditions17. Here, we report that coexpression of a mutant A. thaliana iron transporter (IRT1)18 and ferritin (FER1) generates transgenic cassava plants that accumulate iron and zinc in storage roots to substantial levels in the human diet. Data from field-grown VIT1 and IRT1 + FER1 transgenic lines in Puerto Rico field trials (2014–2017) indicate that both technologies result in cassava storage roots and foodstuffs with elevated iron and zinc levels that may beneficially affect the nutritional status of consumers.

We designed IRT1 and FER1 expression cassettes to improve mineral uptake, by placing AtIRT1 (ref. 18) under control of the A14 promoter, and to store iron in plastids, by expressing AtFER1 (ref. 19) under control of the patatin type 1 promoter (Supplementary Fig. 1a). Transgenic cassava plants of cultivar TME 204 coexpressing IRT1 and FER1 mRNA were established in the greenhouse. The shoot and storage-root growth phenotypes were similar for all transgenic and nontransgenic control lines during 16 weeks of growth (Supplementary Fig. 1b–g). The presence of IRT1 and FER1 transgenes in the leaves of 4-week-old plants was confirmed by PCR (Supplementary Fig. 2a). Southern blot analyses verified the integration of the IRT1 and FER1 transgenes at one or two copies of the transfer DNA (T-DNA; Supplementary Fig. 2b), and mRNA expression of IRT1 and FER1 was confirmed by RT–qPCR in leaves, fibrous roots and storage roots of transgenic plants (Supplementary Fig. 2c–h). Inductively coupled plasma optical emission spectroscopy (ICP–OES) analysis revealed that the storage roots of IRT1 + FER1 transgenic plants had five- to six-times-higher iron and zinc concentrations than the storage roots of nontransgenic controls (Supplementary Fig. 1j,k). The maximum iron accumulation reached 55 ± 13 µg/g dry weight (DW; mean ± s.d.) compared with 10 ± 2 µg/g DW for storage roots of nontransgenic plants (Supplementary Fig. 1j), and 26 ± 12 µg/g DW zinc compared with 5 ± 1 µg/g DW zinc in the nontransgenic controls (Supplementary Fig. 1k). IRT1 + FER1 transgenic plants had leaf iron concentrations two to three times higher than those of nontransgenic controls (Supplementary Fig. 1h), but no increase in zinc concentration was observed in foliar tissues (Supplementary Fig. 1i). The total iron and zinc content was determined in leaves, petioles, stem, fibrous roots and storage-root peels to assess whether the elevated mineral levels in the storage-root parenchyma resulted from depletion in other organs. When assessed as whole plants, IRT1 + FER1 transgenic lines, compared with nontransgenic controls, showed significantly higher (P ≤ 0.01) total iron and zinc content, by up to five and two times, respectively (Supplementary Fig. 3a,b). The maximum total iron accumulation reached 7,059 ± 204 µg iron in transgenic line 8023-14 compared with 1,311 ± 38 µg iron in nontransgenic plants. The iron content increased in all organs except fibrous roots, and the greatest increase occurred in storage roots (Supplementary Fig. 3a). The zinc content was increased in storage roots, root peels and stems, but not in leaves, petioles or fibrous roots of IRT1 + FER1 plants (Supplementary Fig. 3b).

VIT1 and IRT1 + FER1 transgenic lines were evaluated in confined field trials at Isabela field station, University of Puerto Rico (Supplementary Fig. 4). Over a 12-month trial period, no significant differences were found between VIT1 transgenic plants and nontransgenic controls for root or shoot biomass, storage-root dry-matter content, number of roots, harvest index or linamarin concentration (Fig. 1 and Supplementary Fig. 5). For IRT1 + FER1 transgenic plants, 12 of the 17 lines tested in the field generated storage-root yields (Fig. 1g) and shoot yields (Supplementary Fig. 6a) comparable to those of nontransgenic controls, and there were no significant differences in the number of storage roots, harvest index, dry matter or total linamarin concentration (Supplementary Fig. 6).

Fig. 1: Agronomic yield of nontransgenic, VIT1 and IRT1 + FER1 transgenic cassava plants. a–e, Shoot phenotypes of 12-month-old nontransgenic control TME 204 (a), VIT1 (b) and IRT1 + FER1 (c) plants. Waxed storage roots of nontransgenic control and transgenic VIT1 (d) and IRT1 + FER1 (e) plants. f,g, Storage-root yields for VIT1 (f) and IRT1 + FER1 (g) transgenic plants, along with nontransgenic control. Box-and-whisker plots were constructed with the R package ggplot2. The upper whisker extends from the hinge to the largest value, no further than 1.5× the interquartile range (IQR, distance between the first and third quartiles) from the hinge. The lower whisker extends from the hinge to the smallest value, at most 1.5× the IQR of the hinge. Data beyond the ends of the whiskers are considered outlying points and are plotted individually. For VIT1, n = 9 biologically independent plants (3 plants/replicate); for IRT1 + FER1, n = 4 biologically independent plants. Statistical tests were performed with two-sided Student’s t test, relative to nontransgenic control. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. WT, wild-type plants; EV control, empty-vector control plants. Full size image

Mineral accumulation was determined in the storage roots of field-grown plants. We previously reported that cassava plants overexpressing AtVIT1 accumulated up to 48 µg/g DW iron in storage roots under greenhouse conditions, a level three to four times higher than that in nontransgenic controls17. Similar results were seen in field-grown materials, in which 10 of the 15 VIT1 lines had iron concentrations three to seven times higher, reaching a maximum of 60 ± 7 µg/g DW (Fig. 2a). No elevation in zinc concentration was observed in the storage roots produced by VIT1 transgenic plants (Fig. 2b). All 17 IRT1 + FER1 transgenic lines grown in the field accumulated significantly elevated levels of iron (P ≤ 0.001) in their storage roots, which reached 130 ± 39 µg/g DW, an 18-fold increase over the 7.2 ± 3 µg/g DW in nontransgenic controls (Fig. 2c). Fifteen IRT1 + FER1 transgenic lines also had significantly elevated levels of storage-root zinc (P ≤ 0.001), and line 8023-19 reached a maximum of 103 ± 30 µg/g DW, a level ten times higher than that in nontransgenic controls (Fig. 2d). The elevated iron in these storage roots was positively correlated with an elevation of zinc concentration (r = 0.64; Fig. 2c,d). VIT1 transgenic plants showed a minor but significant elevation in copper, manganese and nickel concentrations (Supplementary Fig. 7), but the cadmium concentrations were below the detection limits. Likewise, IRT1 + FER1 plants had elevated copper and manganese concentrations (Supplementary Fig. 8), but the nickel and cadmium concentrations were below the detection limits (data not shown).

Fig. 2: Storage-root mineral concentrations of VIT1 and IRT1 + FER1 transgenic cassava at harvest 12 months after planting under field conditions. a,b, Iron (a) and zinc (b) concentrations in storage roots of VIT1 transgenic cassava plants. c,d, Iron (c) and zinc (d) concentrations instorage roots of IRT1 + FER1 transgenic cassava plants. For VIT1, n = 9 biologically independent plants (3 plants/replicate); for IRT1 + FER1, n = 4 biologically independent plants. Box-and-whisker plots were constructed with the R package ggplot2. The upper whisker extends from the hinge to the largest value, no further than 1.5× the IQR from the hinge. The lower whisker extends from the hinge to the smallest value, at most 1.5× the IQR of the hinge. Data beyond the ends of the whiskers are considered outlying points and are plotted individually. Statistical tests were performed with two-sided Student’s t test, relative to nontransgenic control. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Full size image

The IRT1 transporter would be expected to drive iron and zinc uptake from the soil, and FER1 would be expected to provide a sink for iron storage19. Native IRT1 is a high-affinity ferrous-iron transporter necessary for metal uptake and is upregulated under low-iron conditions20. The mutant version of IRT1 (IRT1 K146R K171R) that we used maintains an upregulated state under iron-abundant conditions, and efficacy has been demonstrated in A. thaliana18. We found that overexpression of mutant IRT1 drove elevated iron and zinc accumulation in a crop plant under iron-abundant field conditions (83 µg/g DW iron; Supplementary Fig. 4a). IRT1 can transport manganese, cadmium and cobalt in addition to iron and zinc21. Elevated concentrations of toxic heavy metals in biofortified foods is a safety concern. Field-grown cassava plants coexpressing IRT1 + FER1 accumulated elevated manganese and copper levels, but not to toxic levels (Supplementary Fig. 8a,b).

The cadmium concentrations in greenhouse growth medium and field soil were below detectable levels, a result that may explain why cadmium was not detected in the storage roots of transgenic cassava plants. The potential for IRT1 + FER1 plants to accumulate cadmium was further tested by growing the high-iron- and high-zinc-accumulating line 8023-19 (Fig. 2c,d) in potting medium spiked with 10 µM cadmium sulfate. Nontransgenic control plants accumulated undetectable cadmium in the control medium, with levels increasing to 0.8 ± 0.3 µg/g DW in leaves and 0.64 ± 0.2 µg/g DW in storage roots, when grown in medium supplemented with cadmium sulfate. Transgenic plants also accumulated cadmium when grown in high-cadmium medium but did so at levels two to five times lower than those in the nontransgenic controls (Supplementary Fig. 9a,b). In the presence of high cadmium, transgenic plants accumulated less iron and zinc in their storage roots than when grown in medium without supplemental cadmium (Supplementary Fig. 9d,f), thus indicating possible competition among cadmium, iron and zinc transport in cassava. Cultivation of cassava plants on medium artificially supplemented with cadmium indicated that IRT1 + FER1 transgenic plants accumulated cadmium at levels lower than those in nontransgenic controls (Supplementary Fig. 9a,b), thus suggesting that IRT1 + FER1 transgenic plants would not pose a higher risk of cadmium toxicity than nonmodified plants if grown in high-cadmium soils.

We planted stake cuttings from nontransgenic controls, three VIT1 (8012-4, 8012-11 and 8012-18) and three IRT1 + FER1 (8023-8, 8023-10 and 8023-17) transgenic lines in the field to assess the stability of mineral enhancement across the vegetative cropping cycle (Supplementary Fig. 10). After 12 months of growth, the storage roots were harvested and analyzed. All VIT1 lines showed a significant (P ≤ 0.001) six- to seven-times-higher iron concentration than that in nontransgenic controls, reaching a maximum of 62 ± 14 µg/g DW (Supplementary Fig. 11a), levels equivalent to those obtained in the first planting cycle (Fig. 2a). VIT1 transgenic plants also showed a minor but significant increase in zinc concentration (Supplementary Fig. 11b). Likewise, IRT1 + FER1 lines established from stake cuttings accumulated iron and zinc, reaching 80 µg/g DW and 60 µg/g DW, respectively, in their storage roots (Supplementary Fig. 11g,h), levels equivalent to the concentrations measured in the first cropping cycle (Fig. 2c,d). At the end of the second 12-month growing period, there were no significant differences between shoot and storage-root yields in two of the VIT1 transgenic lines (Supplementary Fig. 11c–f). Lower shoot and root yields were observed in IRT1 + FER1 transgenic plants than in nontransgenic controls over the second cropping cycle (Supplementary Fig. 11i–l). However, the storage-root yields observed for all three transgenic lines remained equivalent to historical averages achieved for the control cultivar TME 204, as measured across five confined field trials previously performed at the Isabela field station, Puerto Rico (Supplementary Fig. 12).

We analyzed the localization of iron and zinc in the stems and storage roots of transgenic plants by using elemental mapping through synchrotron X-ray fluorescence microscopy (XRF)22. On the basis of variations in tissue-section thickness and hydration, elemental and Compton-scattering XRF maps were obtained to compare and report elemental distributions (Supplementary Fig. 13a,b). The maps revealed that accumulated iron was associated with vascular tissues of the stem and storage roots in VIT1 and IRT1 + FER1 plants (Supplementary Fig. 14). VIT1 stems and storage roots showed strong localization of iron but minimal localization of zinc (Supplementary Fig. 14b,e), whereas strong colocalization of iron and zinc was seen in IRT1 + FER1 plants within the same tissue types (Supplementary Fig. 14c,f). In stems, this colocalization was associated with the stele and in the storage root with xylem vessels of the storage parenchyma (Supplementary Fig. 14c,f).

To be nutritionally useful, the increased mineral concentrations in transgenic plants must be retained in foodstuffs after processing. Therefore, we assessed the retention and bioaccessibility of iron and zinc in foods prepared from biofortified storage roots. Peeling and boiling of cassava is performed by many communities in East Africa23. VIT1 and IRT1 + FER1 transgenic storage-root parenchyma tissues showed no significant decrease in iron or zinc content after boiling (Supplementary Fig. 15a–c). Processing to produce the West African cassava foodstuffs gari and fufu23 is a more complex process involving chopping, soaking, fermenting, pressing and roasting. Iron retention in gari and fufu reached a minimum of 60% in both VIT1 and IRT1 + FER1 storage parenchyma compared with raw roots from the same plants (Fig. 3a,b), thus indicating the release of iron from the food matrix during processing. Zinc concentrations were 25–45% lower in gari and 55–60% lower in fufu than in unprocessed storage roots harvested from IRT1 + FER1 transgenic lines (Fig. 3c). Importantly, however, equal rates of mineral loss were also found in gari and fufu prepared from storage roots of nontransgenic controls (Fig. 3a–c), thus indicating that the minerals in transgenic plants were retained at levels similar to the baseline levels present in nonmodified tissues. The steps in processing resulting in loss of iron and zinc are unknown, but a similar loss of iron has been reported during milling of rice, millet and wheat24, and in cooked cowpea meal25.

Fig. 3: Mineral retention and bioaccessibility of processed VIT1 and IRT1 + FER1 storage roots. a, Iron concentrations after processing of gari and fufu from the storage roots of VIT1 plants. b,c, Iron (b) and zinc (c) concentrations after processing of gari and fufu from storage roots of IRT1 + FER1 plants. d,e, Iron (d) and zinc (e) bioaccessibility of after processing of gari and fufu from storage roots of VIT1 plants. f,g, Iron (f) and zinc (g) bioaccessibility after processing of gari and fufu from storage roots of IRT1 + FER1 plants. For VIT1, n = 3 biologically independent plants; for IRT1 + FER1, n = 4 biologically independent plants (2 technical replicates/plant). Box-and-whisker plots were constructed with the R package ggplot2. The upper whisker extends from the hinge to the largest value, no further than 1.5× the IQR from the hinge. The lower whisker extends from the hinge to the smallest value, at most 1.5× the IQR of the hinge. Data beyond the ends of the whiskers are considered outlying points and are plotted individually. Statistical tests were performed with two-sided Student’s t test, relative to raw storage roots within each line (a–c) or to nontransgenic control (d). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Full size image

Bioaccessibility was assessed to determine the potential of availability of iron and zinc present within cassava foods for absorption in the gut after digestion. VIT1 transgenic storage roots had significantly higher iron bioaccessibility in uncooked roots (up to 1.5 times higher), processed gari (up to 2.8 times higher) and processed fufu (up to 3 times higher) than that of foods processed from the nontransgenic control (Fig. 3d). No differences were observed in zinc bioaccessibility in raw and processed fufu in the VIT1 transgenic storage roots and nontransgenic controls (Fig. 3e), whereas processed gari had significantly higher levels in three events and lower levels in two transgenic events, as compared with nontransgenic controls (Fig. 3e). No significant differences in iron or zinc bioaccessibility were detected in processed foods from IRT1 + FER1 and nontransgenic control plants (Fig. 3f,g). Interestingly, VIT1 transgenic storage roots had significantly lower levels of both iron (20–65%) and zinc (15–65%) bioaccessibility in processed gari and fufu than in uncooked samples from the same plants (Fig. 3d,e). IRT1 + FER1 transgenic storage roots showed significantly higher levels (27–54%) of iron bioacessibility in processed fufu (Fig. 3f) and significantly lower levels (18–36%) of zinc bioaccessibility in processed gari than in uncooked samples (Fig. 3g). The significantly higher iron bioaccessibility in VIT1 transgenic plants than in nontransgenic controls (Fig. 3d) may have been due to an association of stored iron with soluble organic acids within the vacuole26, whereas the lack of differences in bioaccessibility in IRT1 + FER1 transgenic plants relative to nontransgenic controls (Fig. 3f,g) may have resulted from iron stored as ferritin being less available for release from the storage-root tissues19.

The nutritional effects of consuming cassava storage roots biofortified by overexpression of VIT1 and IRT1 + FER1 was assessed by calculating their potential contribution to the EAR27 for iron and zinc. On the basis of consumption patterns in West Africa28, iron and zinc present in nonmodified cassava storage roots provide only 5–8% and 13–14% of the EAR for iron and zinc, respectively, for children 1–3 years old (Fig. 4a,d). Our data on mineral accumulation and retention enabled us to predict that IRT1 + FER1 transgenic plants of TME 204 contributed up to 40–50% of the EAR for iron for children (1–3 years old) and nonlactating, nonpregnant women, and 65–75% of the EAR for iron for children (4–6 years old) (Fig. 4a,c). In addition, IRT1 + FER1 plants provided 60–70% of the EAR for zinc for children (1–3 years old), children (4–6 years old) and nonlactating, nonpregnant women (Fig. 4b,d).

Fig. 4: Contribution of biofortified transgenic cassava to EARs for iron and zinc. a,b, Graphical plots of iron (a) and zinc (b) concentrations from the transgenic cassava plants against the percentage EAR for children (1–3 years of age), children (4–6 years of age) and nonpregnant nonlactating women. Blue dashed line, percentage EAR calculated from processed food of baseline wild-type storage roots; red dashed line, percentage EAR calculated from processed food of transgenic cassava storage roots. c, Iron-biofortified VIT1 (containing an additional 40–50 µg/g iron) or IRT1 + FER1 (containing an additional 80–90 µg/g iron) cassava lines, showing the potential nutritional contribution toward the EARs for different demographic groups relative to baseline WT-TME 204 (10 µg/g iron). d, Zinc-biofortified IRT1 + FER1 (containing an additional 90–100 µg/g zinc) cassava lines showing the potential nutritional contribution toward the EARs for different demographic groups relative to baseline WT-TME 204 (10 µg/g zinc). Full size image

Caco-2 studies were not undertaken on the iron- and zinc-biofortified foods reported here, because it can assay for only iron bioavailability but does not generate data for zinc bioavailability or provide quantifiable values for mineral release from a digested food. Instead, an in vitro bioaccessibility assay was used to assess the release of iron and zinc from gari and fufu in transgenic versus nontransgenic derived foods, thus enabling calculation of the EAR for both minerals (Fig. 4).

In summary, of the 18 transgenic cassava plant lines coexpressing IRT1 + FER1 mRNA, 17 (94%) attained nutritionally meaningful levels of iron and zinc that were able to provide 30–50% of the EAR for iron, and 15 attained levels were able to provide 40–70% of the EAR for zinc for children and nonlactating, nonpregnant women (Figs. 2 and 4). The success of our method surpasses that previously reported for approaches to engineer biofortification by using Agrobacterium-mediated integration into the plant genome. For example, in rice, more than 1,600 transgenic T 0 lines were required to generate two low-T-DNA-copy lines with nutritionally meaningful iron and zinc elevation13. It is therefore possible that even low-level transgenic expression of mutated IRT1 may be effective for driving substantial uptake of iron and zinc from the growth medium into plant cells. The loss of iron and zinc during cassava processing occurred at levels higher than expected (Fig. 3), but, crucially, the loss rates were equivalent in transgenic and nontransgenic plants, and the available iron and zinc in processed foods remained meaningful in terms of nutritional value (Fig. 4). Factors such as the levels of vitamin C and organic acids present in the diet can increase mineral bioaccessibility and improve mineral absorption in the digestive tract29. Cassava is not a recognized source of organic acids30, whereas vitamin C present in fresh storage roots is degraded up to 99% by commonly used processing techniques8. The nutritional value of the elevated iron and zinc present in biofortified cassava foodstuffs can be improved by the consumption of other foods in the diet that contain these absorption promoters. Therefore future studies should evaluate the effects of dietary vitamin C levels on mineral micronutrient bioavailability in biofortified cassava foodstuffs.

The United Nations Sustainable Development Goals call for an end to global hunger and decreases in all forms of malnutrition by the year 2030 (ref. 31). Nutritional security for the global population could be improved through biofortification of staple food crops such as cassava. We report that iron-enriched and iron- and zinc-enriched cassava storage roots can be grown in the field without decreases in yield. Our biofortified plant lines, or indeed other staple dicot crops such as sweet potato and potato, may be exploited as exemplars to improve the nutritional quality of cassava cultivars grown in different regions.