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Summary Rice tungro disease (RTD) is a serious constraint in rice production across tropical Asia. RTD is caused by the interaction between Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. RTSV resistance found in traditional cultivars has contributed to a reduction in the incidence of RTD in the field. Natural RTSV resistance is a recessive trait controlled by the translation initiation factor 4 gamma gene (eIF4G). The Y1059V1060V1061 residues of eIF4G are known to be associated with the reactions to RTSV. To develop new sources of resistance to RTD, mutations in eIF4G were generated using the CRISPR/Cas9 system in the RTSV‐susceptible variety IR64, widely grown across tropical Asia. The mutation rates ranged from 36.0% to 86.6%, depending on the target site, and the mutations were successfully transmitted to the next generations. Among various mutated eIF4G alleles examined, only those resulting in in‐frame mutations in SVLFPNLAGKS residues (mainly NL), adjacent to the YVV residues, conferred resistance. Furthermore, our data suggest that eIF4G is essential for normal development, as alleles resulting in truncated eIF4G could not be maintained in homozygous state. The final products with RTSV resistance and enhanced yield under glasshouse conditions were found to no longer contain the Cas9 sequence. Hence, the RTSV‐resistant plants with the novel eIF4G alleles represent a valuable material to develop more diverse RTSV‐resistant varieties.

Zinc-finger nucleases (ZFNs) have proved to be successful tools for targeted genome manipulation in several organisms. Their main property is the induction of double-strand breaks (DSBs) at specific sites, which are further repaired through homologous recombination (HR) or non-homologous end joining (NHEJ). However, for the appropriate integration of genes at specific chromosomal locations, proper sites for gene integration need to be identified. These regions, hereby named safe harbor loci, must be localized in non-coding regions and possess high gene expression. In the present study, three different ZFN constructs (pZFN1, pZFN2, pZFN3), harboring β-glucuronidase (GUS) as a reporter gene, were used to identify safe harbor loci on rice chromosomes. The constructs were delivered into IR64 rice by using an improved Agrobacterium-mediated transformation protocol, based on the use of immature embryos. Gene expression was measured by histochemical GUS activity and the flanking regions were determined through thermal-asymmetric interlaced polymerase chain reaction (TAIL PCR). Following sequencing, 28 regions were identified as putative sites for safe integration, but only one was localized in a non-coding region and also possessed high GUS expression. These findings have significant applicability to create crops with new and valuable traits, since the site can be subsequently used to stably introduce one or more genes in a targeted manner.

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 development of rice genotypes with micronutrient-dense grains and disease resistance is one of the major priorities in rice improvement programs. We conducted Genome-wide association studies (GWAS) using a Multi-parent Advanced Generation Inter-Cross (MAGIC) Plus population to identify QTLs and SNP markers that could potentially be integrated in biofortification and disease resistance breeding. We evaluated 144 MAGIC Plus lines for agronomic and biofortification traits over two locations for two seasons, while disease resistance was screened for one season in the screen house. X-ray fluorescence technology was used to measure grain Fe and Zn concentrations. Genotyping was carried out by genotype by sequencing and a total of 14,242 SNP markers were used in the association analysis. We used Mixed linear model (MLM) with kinship and detected 57 significant genomic regions with a -log10 ( -value) ≥ 3.0. The and were consistently identified in all the four environments, ten QTLs , , , , and were detected in two environments, while two major loci and were identified for Bacterial Leaf Blight (BLB) resistance. The associated SNP markers were found to co-locate with known major genes and QTLs such as for days to flowering, for plant height, and for grain length. Similarly, and genes were identified for BLB resistance and , and genes were identified for Blast resistance. A number of metal homeostasis genes , , , , and were co-located with QTLs for Fe and Zn. The marker-trait relationships from Bayesian network analysis showed consistency with the results of GWAS. A number of promising candidate genes reported in our study can be further validated. We identified several QTLs/genes pyramided lines with high grain Zn and acceptable yield potential, which are a good resource for further evaluation to release as varieties as well as for use in breeding programs.

Zinc (Zn) is one of the most essential micronutrients required for the growth and development of human beings. More than one billion people, particularly children and pregnant women suffer from Zn deficiency related health problems in Asia. Rice is the major staple food for Asians, but the presently grown popular high yielding rice varieties are poor supplier of Zn in their polished form. Breeding rice varieties with high grain Zn has been suggested to be a sustainable, targeted, food-based and cost effective approach in alleviating Zn deficiency. The physiological, genetic and molecular mechanisms of Zn homeostasis have been well studied, but these mechanisms need to be characterized from a biofortification perspective and should be well integrated with the breeding processes. There is a significant variation for grain Zn in rice germplasm and efforts are being directed at exploiting this variation through breeding to develop high Zn rice varieties. Several QTLs and gene specific markers have been identified for grain Zn and there is a great potential to use them in Marker-Assisted Breeding. A thorough characterization of genotype and environmental interactions is essential to identify key environmental factors influencing grain Zn. Agronomic biofortification has shown inconsistent results, but a combination of genetic and agronomic biofortification strategies may be more effective. Significant progress has been made in developing high Zn rice lines for release in target countries. A holistic breeding approach involving high Zn trait development, high Zn product development, product testing and release, including bioefficacy and bioavailability studies is essential for successful Zn biofortification.

Biofortification of rice (Oryza sativa L.) using a transgenic approach to increase the amount of iron in the grain is proposed as a low-cost, reliable, and sustainable solution to help developing countries combat anemia. In this study, we generated and evaluated a large number of rice or soybean ferritin over-accumulators in rice mega-variety IR64, including marker-free events, by introducing soybean or rice ferritin genes into the endosperm for product development. Accumulation of the protein was confirmed by ELISA, in situ immunological detection, and Western blotting. As much as a 37- and 19-fold increase in the expression of ferritin gene in single and co-transformed plants, respectively, and a 3.4-fold increase in Fe content in the grain over the IR64 wild type was achieved using this approach. Agronomic characteristics of a total of 1,860 progenies from 58 IR64 single independent transgenic events and 768 progenies from 27 marker-free transgenic events were evaluated and most trait characteristics did not show a penalty. Grain quality evaluation of high-Fe IR64 transgenic events showed quality similar to that of the wild-type IR64. To understand the effect of transgenes on iron homeostasis, transcript analysis was conducted on a subset of genes involved in iron uptake and loading. Gene expression of the exogenous ferritin gene in grain correlates with protein accumulation and iron concentration. The expression of NAS2 and NAS3 metal transporters increased during the grain milky stage.