Generation and selection of events
We generated global maps from updated data on micronutrient deficiency, rice consumption, and poverty distribution2,7,28,29,30 to highlight the strong interconnection of micronutrient deficiency, poverty rate, and rice consumption (see Supplementary Fig. S1 online). An unequivocal overlap between all three of these issues is observed across the maps.
To select a product with the desirable trait of Fe- and Zn-dense rice and robust field performance, we generated and screened 1,689 independent IR64 transgenic events obtained through transformation of seven constructs containing Fe storage and/or chelator genes driven by various promoters (Fig. 1a, Supplementary Table S1 online). The flow of the entire screening process and validation strategy is presented in Fig. 1b.
We prioritized our selection based on the polished grain iron concentration. The highest number of plants showing intense staining when Perls’ Prussian blue was used in T1 grain sections was obtained from three constructs containing OsNAS2 under control of a 35S promoter and/or soybean ferritin under control of a glutelinA2 promoter31 (Supplementary Table S1 online). The transformed plants derived from the construct containing GluA2::SferH-1 in combination with 35S:OsNAS2 (Supplementary Table S1 online, coded as IRS495 or NASFer) gave the highest number of plants with intense staining, higher than the similar construct in which SferH-1 is driven by glutelinB1 promoter (IRS493, Supplementary Table S1 online).
We selected up to 33 events from each construct with the most intense Fe staining (Perls’ Prussian blue) for copy number analysis (Supplementary Table S2 online). A majority of the transgenic events with intense staining contained multiple inserts, but events/lines with single-locus insertion were also identified (Supplementary Table S2 online). To accelerate the selection process, homozygous lines were selected in the segregating T1 generation using a multiplexed PCR assay with three oligonucleotide primers (Supplementary Fig. S2 online) on selected events with high Fe concentration and have a single insert of the three selected constructs (Supplementary Table S2 online). The elemental analysis on homozygous T2 polished seeds using inductively coupled plasma-optical emission spectrometry (ICP-OES) showed a significant 7.5-fold increase in Fe concentration, reaching 15 μg g−1 from the 2 μg g−1 baseline in the non-transformed IR64 control (Fig. 1c). This level of Fe concentration in polished grains was achieved in plants generated using the NASFer construct. Using the single-gene approach of OsNAS2 or SferH-1, the maximum Fe concentration achieved in this study was 8.8 μg g−1 (Supplementary Table S2 online).
Quantitative RT-PCR confirmed enhanced expression of OsNAS2 in roots and leaves of transgenic events (Fig. 2a,b). We also observed enhanced expression of OsNAAT1 (Nicotianamine Aminotransferase 1) and OsDMAS1 (Deoxymugineic Acid Synthase 1) genes in the transgenic events (Supplementary Fig. S3 online). The enhanced expressions of these three genes involved in deoxymugineic acid (DMA) biosynthesis lead to significant increase of NA and DMA concentrations in polished grains of transgenic events by up to thirty two- and thirty three-fold, respectively, compared to WT (Fig. 2c). Accumulation of ferritin in polished grains was detected by immunoblot assay (Fig. 2d).
Field Trials in the Philippines and Colombia
Two high-Fe events with a single insert (NASFer-234 and NASFer-274) and no backbone integration beyond the T-DNA borders (Supplementary Table S2 online) were evaluated in CFTs at IRRI-Philippines and CIAT-Colombia (Supplementary Fig. S4 online). Figure 3a shows that the rice samples were well milled after 2.5 minutes of milling by a Kett Mill laboratory milling machine. The absence of bran in our polished rice was confirmed by microscopy observation (Supplementary Fig. S5 online). We achieved the Fe target of 14.6–15.0 and 13.2–14.7 μg g−1 (a 6-fold increase) for NASFer-274 and NASFer-234, respectively, in both locations (Fig. 3b). Additionally, these two transgenic events accumulated 2.7- to 3.8-fold more Zn when compared with the wild-type IR64 in both locations (Fig. 3c). This simultaneous increase in both Fe and Zn in single grains could have tremendous potential for alleviating both deficiencies.
Agronomic evaluation of the event NASFer-274 showed no yield penalty at both IRRI-Philippines and CIAT-Colombia (Fig. 3d,e), whereas the event NASFer-234 exhibited a lower yield in both locations when compared with IR64 and the null (negative segregant or azygous) (Fig. 3d,e). Grain quality testing on the most promising event (NASFer-274) did not reveal any difference in protein content, amylose content, gel consistency, seed size, and chalkiness (Supplementary Table S3 online).
Elemental maps revealed significant increases of Fe and Zn localization in NASFer-274 endosperm
Synchrotron X-ray fluorescence microscopy (XFM) was used to map the localization of several metal cations transported by NA (Fe, Zn, Cu) as well as elements indicative of phytic acid and protein (P and S, respectively) in transversal sections of null and lead event NASFer-274 whole grain (Fig. 4a). The elemental maps corresponded well with the ICP-OES elemental results (Supplementary Table S4 online) and demonstrated that the grain of event NASFer-274 had higher localization of Fe in aleurone, sub-aleurone and outer endosperm layers relative to null grain. The Zn maps indicated less accumulation in the aleurone and much higher Zn localization in the sub-aleurone and outer endosperm layers of NASFer-274 grain. The localization pattern of P did not differ between NASFer-274 and null grain, indicating no difference in phytate distribution between the two grain types. The S elemental maps, by contrast, showed slightly higher endosperm localization in NASFer-274 grain and, in conjunction with the ICP-OES elemental results, suggest that protein may be slightly increased in NASFer-274 endosperm. The Cu elemental maps also indicated slightly higher Cu localization in NASFer-274 endosperm.
Enhanced Fe in the events is bioavailable
In vitro measurement of bioavailability in the new Fe-enhanced crops is important to predict level of Fe absorption in human. In vitro digestion/Caco-2 cell culture assays using T4 polished grains showed an increase in Fe availability for both of our lead events; this increased bioavailability was more pronounced in the presence of ascorbic acid (Fig. 4b and Supplementary Table S5 online). Ascorbic acid was reported as the most efficient enhancer of the absorption of non-heme Fe32.
Increased NA concentrations in the grain did not enhance grain heavy metal accumulation
Our data showed that cadmium (Cd), arsenic (As), and lead (Pb) concentrations in polished grains harvested from two CFTs were below detection limits by ICP-OES (Supplementary Table S4 online), indicating that increased NA concentrations in the grain did not enhance grain heavy metal accumulation. To further test the extent of possible Cd accumulation, high-Fe/-Zn rice lines were planted in pots containing Cd-contaminated soil from two different locations (0.104 and 0.246 μg g−1 available Cd, respectively). A more sensitive ICP-MS (mass-spectrometry) analysis of harvested polished grains showed no significant difference between transgenic plants and controls, and all transgenic plants had Cd concentrations of <0.05 μg g−1 (Fig. 4c).
Characterization of integration sites of two lead events
DNA blot analysis using a single-cutter endonuclease within the T-DNA indicated single-copy insertions in our two lead events (Fig. 5a). Nevertheless, PCR-based assay analysis and sequencing indicated integration of two T-DNA copies oriented as an inverted repeat in both events (Fig. 5b,c, Supplementary Fig. S2 online, Supplementary Fig. S6 online, Supplementary Fig. S7 online). In agreement with previous studies33, the inverted T-DNA repeat of the two selected events did not trigger transgene silencing as shown by transcript expression and immunoblot analyses (Fig. 2a,b,d).
Trait stability in different genotype backgrounds
The two selected events (274 and 234) were crossed with farmers’ popular varieties from the Philippines (NSic Rc222), Indonesia (Ciherang), and Bangladesh (BR29). The ICP-OES Fe elemental measurement, carried out in the F2 segregant grains, to gain an indication of trait stability in different genotypes showed concentrations ranging from 6 to 11 μg g−1 for Fe (Fig. 6a) and from 33 to 45 μg g−1 for Zn (Fig. 6b). This slight reduction, from 15 μg g−1 in homozygous material to 11 μg g−1, is expected since the F2 bulk polished grains were segregated grains derived from heterozygous parents. In addition, the yield per plant of the crosses with event NASFer-274 increased compared to the IR64 control, and was even higher in the event NASFer-234 (Fig. 6c) in this F1 population; therefore, the grain mineral concentration was most likely diluted because of the higher yield. Actual yield data should be obtained under field conditions on advanced backcross generations. We evaluated the Fe-polished seeds to obtain an indication of the trait stability. These data confirm the trend that this trait in general remains stable in different genotype backgrounds, even though there is some variation.
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