Kurniawan Rudi Trijatmiko

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.

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.

NAL1 allele from a rice landrace greatly increases yield in modern indica cultivars

Significance This work reports discovery of a unique gene important for rice agriculture. A significant yield enhancement in rice modern cultivar was achieved by identification of a gene, SPIKELET NUMBER (SPIKE) in Indonesian rice landrace. The SPIKE increased grain yield of an indica cultivar IR64, which is widely grown in the tropics, over four seasons at the field level and improved plant architecture without changing grain quality or growth period, which are important for regional adaptability. These results indicate finding of SPIKE will be extremely valuable for contributing to increase grain production of indica rice cultivars. Increasing crop production is essential for securing the future food supply in developing countries in Asia and Africa as economies and populations grow. However, although the Green Revolution led to increased grain production in the 1960s, no major advances have been made in increasing yield potential in rice since then. In this study, we identified a gene, SPIKELET NUMBER (SPIKE), from a tropical japonica rice landrace that enhances the grain productivity of indica cultivars through pleiotropic effects on plant architecture. Map-based cloning revealed that SPIKE was identical to NARROW LEAF1 (NAL1), which has been reported to control vein pattern in leaf. Phenotypic analyses of a near-isogenic line of a popular indica cultivar, IR64, and overexpressor lines revealed increases in spikelet number, leaf size, root system, and the number of vascular bundles, indicating the enhancement of source size and translocation capacity as well as sink size. The near-isogenic line achieved 13–36% yield increase without any negative effect on grain appearance. Expression analysis revealed that the gene was expressed in all cell types: panicles, leaves, roots, and culms supporting the pleiotropic effects on plant architecture. Furthermore, SPIKE increased grain yield by 18% in the recently released indica cultivar IRRI146, and increased spikelet number in the genetic background of other popular indica cultivars. The use of SPIKE in rice breeding could contribute to food security in indica-growing regions such as South and Southeast Asia.

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 economization1. Rice production must increase to ensure food security2 and the bulk of this increase will have to be achieved through intensification of cultivation, because expansion of cultivated areas is reaching sustainable limits3. Anaerobic germination tolerance, which enables uniform germination and seedling establishment under submergence4, is a key trait for the development of tropical DSR varieties5,6. 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 tolerance7. OsTPP7 is involved in trehalose-6-phosphate (T6P) metabolism, central to an energy sensor that determines anabolism or catabolism depending on local sucrose availability8,9. 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.

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.

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 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.

Mapping quantitative trait loci conferring blast resistance in upland indica rice (Oryza sativa L.)

A genetic analysis of blast resistance in upland rice variety is very crucial. In this study, we performed a linkage mapping of quantitative trait loci (QTLs) for blast resistance using an advanced backcross population from a cross between Way Rarem (susceptible indica variety) and Oryzica Llanos 5 (durable resistant indica variety). A transgressive segregation was observed in the advanced backcross population of Way Rarem//Oryzica Llanos 5. A total of 16 QTLs have been identified along chromosomes 1, 3, 5, 6, 7, 8, 9, and 11 against eight blast pathogen isolates. Each QTL accounted from 11.31 to 45.11% of the variation in blast resistance. Most QTLs showed race specificity, demonstrating the small effect of such QTLs. Unexpectedly, several superior blast resistance alleles were contributed by Way Rarem, the susceptible-recurrent parent. Among eight candidate defense response genes detected in several loci, a single gene (oxalate oxidase) present on chromosome 3 was found to be associated with blast resistance in upland indica rice. Ultimately, these advanced backcross lines with resistance to blast tagged by markers might be useful for pyramiding blast resistance alleles in upland rice.

Molecular Analyses of Transgenic Plants.

One of the major challenges in plant molecular biology is to generate transgenic plants that express transgenes stably over generations. Here, we describe some routine methods to study transgene locus structure and to analyze transgene expression in plants: Southern hybridization using DIG chemiluminescent technology for characterization of transgenic locus, SYBR Green-based real-time RT-PCR to measure transgene transcript level, and protein immunoblot analysis to evaluate accumulation and stability of transgenic protein product in the target tissue.

Evaluasi Sifat Daya Tembus Akar dan Identifikasi Mutan Stabil pada Populasi Penanda Aktivasi

Abstract Drought stress is one of the important limiting factors in increasing rice production in Indonesia. Development of rice varieties with increased tolerance to drought is needed to meet the rice production challenge. Some transgenic Nipponbare rice lines that carry activation tag have been generated from the previous study. The purpose of this study was to evaluate the root penetration ability and the stability of Ds element in the T1 generation of activation tag rice lines. Materials used in the study were T1 seeds of 47 transgenic lines, drought tolerant check varieties (Cabacu and IRAT112), and sensitive check varieties (IR64 and wild type Nipponbare). T1 seeds were prescreened by germination in Basta herbicide solution to eliminate T1 individuals that did not carry activation tag element. Root penetration ability was evaluated using wax-petrolatum layers as a proxy for compacted soil layers. The presence of bar gene and the absence of hpt gene as detected by PCR were used to identify T1 stable mutants. Out of 47 transgenic lines tested, 38 lines showed better root penetration ability than non transformed Nipponbare. PCR analysis identified four stable mutants, namely M-Nip-12.12, M-Nip-19.8, M-Nip-19.9, and M-Nip- 20.13. One stable mutant, M-Nip-20.13, showed better root penetration ability than tolerant check varieties. This mutant is a good candidate for isolation of drought tolerance gene. Abstrak Cekaman kekeringan merupakan salah satu faktor pembatas penting dalam peningkatan produksi padi di Indonesia. Perakitan varietas unggul padi toleran kekeringan diperlukan untuk menjawab tantangan tersebut. Beberapa galur padi Nipponbare transgenik yang membawa penanda aktivasi telah dihasilkan pada penelitian sebelumnya. Tujuan penelitian ini adalah mengevaluasi daya tembus akar dan stabilitas elemen Ds pada galur-galur penanda aktivasi generasi T1. Bahan yang digunakan dalam penelitian ini adalah benih T1 47 galur transgenik, varietas cek toleran kekeringan (Cabacu dan IRAT112), dan varietas cek peka (IR64 dan Nipponbare tipe liar). Benih T1 ditapis terlebih dahulu dengan perkecambahan pada larutan herbisida Basta untuk mengeliminasi individu yang tidak membawa elemen penanda aktivasi. Daya tembus akar dievaluasi menggunakan lapisan lilin sebagai tiruan lapisan tanah yang padat dan keras (hardpans). Keberadaan gen bar dan ketiadaan gen hpt yang dideteksi dengan PCR digunakan untuk mengidentifikasi mutan-mutan stabil. Dari 47 galur yang diuji, 38 galur menunjukkan daya tembus akar yang lebih baik daripada Nipponbare non transforman. Analisis PCR mengidentifikasi empat mutan stabil, yaitu M-Nip-12.12, M-Nip-19.8, M-Nip-19.9, dan M-Nip-20.13. Satu mutan stabil, M-Nip-20.13, menunjukkan daya tembus akar yang lebih baik daripada varietas cek toleran. Mutan ini menjadi kandidat yang baik untuk isolasi gen toleran kekeringan.

Deteksi Gen HptII dan Keragaan Agronomis pada Populasi BC1F1 Tanaman Padi Transgenik

Rice varieties tolerant to drought stress are needed to stabilize rice production under drought stress condition. We developed transgenic rice cv. Nipponbare carrying hptII gene that might also contain OsDREB1A gene. OsDREB1A gene responsible to drought tolerance trait need to be transferred into cultivated rice in order to obtain new local rice variety tolerant to drought stress. The aims of this research were to detect the presence of hptII gene in the F1 and BC1F1 transgenic rice and to observe the agronomic performace of those populations and their plant physiology. F1 population was developed by crossing transgenic Nipponbare, as donor parent, with Batutegi, Code, Ciherang, and Konawe genotypes, as recipient parents. BC1F1 population was developed by backcrossing F1 transgenic line with recipient parents, respectively. The presence of hptII gene was analyzed by PCR using a pair of primers for hptII. The observation of agronomic performance was carried out in the green house, meanwhile the observation of stomata was done using microscope. The result of PCR analysis showed that BC1F1 Batutegi trans, BC1F1 Code trans, BC1F1 Konawe trans1, BC1F1 Konawe trans3, dan BC1F1 Konawe trans4 were detected carrying the hptII gene. Agronomic data showed that BC1F1 transgenic rice lines yielded panicles, filled grains, and total grains higher than those of recipent parents. Comparing to the recipient parents, BC1F1 Konawe trans1 and BC1F1 Konawe trans3 had less stomata on the lower side of the leaf, but had more stomata on the upper side of the leaf.