Genome editing in cereal crops: an overview.

Genome-editing technologies offer unprecedented opportunities for crop improvement with superior precision and speed. This review presents an analysis of the current state of genome editing in the major cereal crops- rice, maize, wheat and barley. Genome editing has been used to achieve important agronomic and quality traits in cereals. These include adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Not all traits can be achieved through genome editing, and several technical and regulatory challenges need to be overcome for the technology to realize its full potential. Genome editing, however, has already revolutionized cereal crop improvement and is poised to shape future agricultural practices in conjunction with other breeding innovations.

A Case Study from the Overexpression of OsTZF5, Encoding a CCCH Tandem Zinc Finger Protein, in Rice Plants Across Nineteen Yield Trials.

BACKGROUND: Development of transgenic rice overexpressing transcription factors involved in drought response has been previously reported to confer drought tolerance and therefore represents a means of crop improvement. We transformed lowland rice IR64 with OsTZF5, encoding a CCCH-tandem zinc finger protein, under the control of the rice LIP9 stress-inducible promoter and compared the drought response of transgenic lines and nulls to IR64 in successive screenhouse paddy and field trials up to the T6 generation. RESULTS: Compared to the well-watered conditions, the level of drought stress across experiments varied from a minimum of - 25 to - 75 kPa at a soil depth of 30 cm which reduced biomass by 30-55% and grain yield by 1-92%, presenting a range of drought severities. OsTZF5 transgenic lines showed high yield advantage under drought over IR64 in early generations, which was related to shorter time to flowering, lower shoot biomass and higher harvest index. However, the increases in values for yield and related traits in the transgenics became smaller over successive generations despite continued detection of drought-induced transgene expression as conferred by the LIP9 promoter. The decreased advantage of the transgenics over generations tended to coincide with increased levels of homozygosity. Background cleaning of the transgenic lines as well as introgression of the transgene into an IR64 line containing major-effect drought yield QTLs, which were evaluated starting at the BC3F1 and BC2F3 generation, respectively, did not result in consistently increased yield under drought as compared to the respective checks. CONCLUSIONS: Although we cannot conclusively explain the genetic factors behind the loss of yield advantage of the transgenics under drought across generations, our results help in distinguishing among potential drought tolerance mechanisms related to effectiveness of the transgenics, since early flowering and harvest index most closely reflected the levels of yield advantage in the transgenics across generations while reduced biomass did not.

Coordinated activation of cellulose and repression of lignin biosynthesis pathways in rice.

Cellulose from plant biomass is the largest renewable energy resource of carbon fixed from the atmosphere, which can be converted into fermentable sugars for production into ethanol. However, the cellulose present as lignocellulosic biomass is embedded in a hemicellulose and lignin matrix from which it needs to be extracted for efficient processing. Here, we show that expression of an Arabidopsis (Arabidopsis thaliana) transcription factor, SHINE (SHN), in rice (Oryza sativa), a model for the grasses, causes a 34% increase in cellulose and a 45% reduction in lignin content. The rice AtSHN lines also exhibit an altered lignin composition correlated with improved digestibility, with no compromise in plant strength and performance. Using a detailed systems-level analysis of global gene expression in rice, we reveal the SHN regulatory network coordinating down-regulation of lignin biosynthesis and up-regulation of cellulose and other cell wall biosynthesis pathway genes. The results thus support the development of nonfood crops and crop wastes with increased cellulose and low lignin with good agronomic performance that could improve the economic viability of lignocellulosic crop utilization for biofuels.

Molecular characterization and safety assessment of biofortified provitamin A rice.

Part of the studies involved in safety assessment of genetically engineered crops includes characterizing the organization, integrity, and stability of the inserted DNA and evaluating the potential allergenicity and toxicity of newly-expressed proteins. Molecular characterization of the introduced DNA in provitamin A biofortified rice event GR2E confirmed insertion of a single copy of the transfer-DNA in the genome and its inheritance as a single locus. Nucleotide sequencing of the inserted DNA confirmed it was introduced without modifications. The phytoene synthase, and carotene desaturase proteins did not display sequence similarity with allergens or toxins. Both proteins were rapidly digested in simulated gastric fluid and their enzymatic activity was inhibited upon heat treatment. Acute oral toxicity testing of the protein in mice demonstrated lack of adverse effects. These evidences substantiated the lack of any identifiable hazards for both proteins and in combination with other existing comparative analyses provided assurance that food derived from this rice is safe. This conclusion is in line with those of the regulatory agencies of US Food and Drug Administration, Health Canada and Food Standard Australia and New Zealand.

Promoter deletion analysis reveals root-specific expression of the alkenal reductase gene (OsAER1) in Oryza sativa.

Root-specific promoters are useful in plant genetic engineering, primarily to improve water and nutrient absorption. The aim of this study was to clone and characterise the promoter of the Oryza sativa L. alkenal reductase (OsAER1) gene encoding 2-alkenal reductase, an NADPH-dependent oxidoreductase. Expression analysis using quantitative real-time PCR confirmed the root-specific expression of the OsAER1 gene. Subsequently, a 3082-bp fragment of the OsAER1 promoter was isolated from a local Indonesian rice cultivar, Awan Kuning. Sequencing and further nucleotide sequence analysis of the 3082-bp promoter fragment (PA-5) revealed the presence of at least 10 root-specific cis-regulatory elements putatively responsible for OsAER1 root-specific expression. Using the 3082-bp promoter fragment to drive the expression of the GUS reporter transgene confirmed that the OsAER1 promoter is root-specific. Further, the analysis indicated that OsAER1 promoter activity was absent in leaves, petioles and shoots during sprouting, vegetative, booting and generative stages of rice development. In contrast, the promoter activity was present in anthers and aleurone layers of immature seeds 7-20 days after anthesis. Moreover, there was no promoter activity observed in the aleurone layers of mature seeds. The OsAER1 promoter activity is induced by Al-toxicity, NaCl and submergence stresses, indicating the OsAER1 promoter activity is induced by those stresses. Exogenous treatments of transgenic plants carrying the PA-5 promoter construct with abscisic acid and indoleacetic acid also induced expression of the GUS reporter transgene, indicating the role of plant growth regulators in controlling OsAER1 promoter activity. Promoter deletion analysis was conducted to identify the cis-acting elements of the promoter responsible for controlling root-specific expression. The GUS reporter gene was fused with various deletion fragments of the OsAER1 promoter and the resulting constructs were transformed in rice plants to generate transgenic plants. The results of this analysis indicated that cis-acting elements controlling root-specific expression are located between -1562 to -1026bp of the OsAER1 CDS. Here we discusses the results of the conducted analyses, the possible role of OsAER1 in rice growth and development, possible contributions and the potential usage of these findings in future plant research.

Biofortified indica rice attains iron and zinc nutrition dietary targets in the field.

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.

A trehalose-6-phosphate phosphatase enhances anaerobic germination tolerance in rice.

Global socioeconomic developments create strong incentives for farmers to shift from transplanted to direct-seeded rice (DSR) as a means of intensification and economization(1). Rice production must increase to ensure food security(2) and the bulk of this increase will have to be achieved through intensification of cultivation, because expansion of cultivated areas is reaching sustainable limits(3). Anaerobic germination tolerance, which enables uniform germination and seedling establishment under submergence(4), is a key trait for the development of tropical DSR varieties(5,6). Here, we identify a trehalose-6-phosphate phosphatase gene, OsTPP7, as the genetic determinant in qAG-9-2, a major quantitative trait locus (QTL) for anaerobic germination tolerance(7). OsTPP7 is involved in trehalose-6-phosphate (T6P) metabolism, central to an energy sensor that determines anabolism or catabolism depending on local sucrose availability(8,9). OsTPP7 activity may increase sink strength in proliferating heterotrophic tissues by indicating low sugar availability through increased T6P turnover, thus enhancing starch mobilization to drive growth kinetics of the germinating embryo and elongating coleoptile, which consequently enhances anaerobic germination tolerance.

Meta-analysis of quantitative trait loci for grain yield and component traits under reproductive-stage drought stress in an upland rice population.

A recombinant inbred population developed from a cross between high-yielding lowland rice (Oryza sativa L.) subspecies indica cv. IR64 and upland tropical rice subspecies japonica cv. Cabacu was used to identify quantitative trait loci (QTLs) for grain yield (GY) and component traits under reproductive-stage drought stress. One hundred fifty-four lines were grown in field trials in Indonesia under aerobic conditions by giving surface irrigation to field capacity every 4 days. Water stress was imposed for a period of 15 days during pre-flowering by withholding irrigation at 65 days after seeding. Leaf rolling was scored at the end of the stress period and eight agronomic traits were evaluated after recovery. The population was also evaluated for root pulling force, and a total of 201 single nucleotide polymorphism markers were used to construct the molecular genetic linkage map and QTL mapping. A QTL for GY under drought stress was identified in a region close to the sd1 locus on chromosome 1. QTL meta-analysis across diverse populations showed that this QTL was conserved across genetic backgrounds and co-localized with QTLs for leaf rolling and osmotic adjustment (OA). A QTL for percent seed set and grains per panicle under drought stress was identified on chromosome 8 in the same region as a QTL for OA previously identified in three different populations.

Identification of "safe harbor" loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair.

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 ß-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.

Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene.

Freshwater is a limited and dwindling global resource; therefore, efficient water use is required for food crops that have high water demands, such as rice, or for the production of sustainable energy biomass. We show here that expression of the Arabidopsis HARDY (HRD) gene in rice improves water use efficiency, the ratio of biomass produced to the water used, by enhancing photosynthetic assimilation and reducing transpiration. These drought-tolerant, low-water-consuming rice plants exhibit increased shoot biomass under well irrigated conditions and an adaptive increase in root biomass under drought stress. The HRD gene, an AP2/ERF-like transcription factor, identified by a gain-of-function Arabidopsis mutant hrd-D having roots with enhanced strength, branching, and cortical cells, exhibits drought resistance and salt tolerance, accompanied by an enhancement in the expression of abiotic stress associated genes. HRD overexpression in Arabidopsis produces thicker leaves with more chloroplast-bearing mesophyll cells, and in rice, there is an increase in leaf biomass and bundle sheath cells that probably contributes to the enhanced photosynthesis assimilation and efficiency. The results exemplify application of a gene identified from the model plant Arabidopsis for the improvement of water use efficiency coincident with drought resistance in the crop plant rice.