Lina Torrizo

Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene

In this report, we show that the expression of the soybean ferritin gene, driven by the endosperm-specific glutelin promoter, leads to higher iron and zinc levels in transgenic indica rice grains. Brown rice is rarely consumed, and polishing of the rice grain brings considerable loss of micronutrients by removing its outer layers. No data until now have shown that after commercial milling the micronutrient concentration remains higher than that of the control. In our experiment, expression of the soybean ferritin gene under the control of the glutelin promoter in rice has proven to be effective in enhancing grain nutritional levels, not only in brown grains but also in polished grains. Besides determining the iron levels in transgenic rice grains, we also checked for zinc concentration, and it was found to be higher in transgenic seeds than in the control. Moreover, we introduced this gene in an elite indica rice line that has highly desirable agronomic and field-performance traits. Prussian blue staining reaction clearly revealed the presence of iron in the endosperm cells of transgenic rice grains, and immunolocalization revealed the presence of the expression gene in the endosperm of the transgenic material. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest

Abstract The truncated chimeric Bt gene, cryIA(b) of Bacillus thuringiensis, driven by two constitutive promoters, 35S from CaMV and Actin-1 from rice, and two tissue-specific promoters, pith tissue and pepcarboxylase (PEPC) for green tissue from maize, was introduced into several varieties of rice (indica and japonica) by microprojectile bombardment and protoplast systems. A total of 1800 putative transgenic Bt rice plants could be produced. Southern analysis revealed that more than 100 independently transformed plants could be confirmed for integration of the cryIA(b) gene. High levels of CryIA(b) proteins were obtained in the green tissue (leaves and stem) of many plants using the PEPC promoter. There was little difference in Bt protein level in leaves and stems from transgenic plants with the 35 S or Actin-1 promoter. Out of 800 Southern-positive plants that were bioassayed, 81 transgenic plants showed 100% mortality of insect larvae of the yellow stem borer (Scirpophaga incertulas). The transgene, cryIA(b), driven by different promoters showed a wide range of expression (low to high) of Bt proteins stably inherited in a number of rice varieties with enhanced yellow stem borer resistance. This first report of transgenic indica Bt rice plants with the PEPC or pith promoter either alone or in combination should provide a better strategy for providing rice plants with protection against insect pest resistance, minimizing the expression of the CryIA(b) protein in seeds and other tissues.

Enhancement of submergence tolerance in transgenic rice overproducing pyruvate decarboxylase.

Summary Transgenic rice ( Oryza sativa L.) lines were produced through transformation with rice pdc1 gene coding for pyruvate decarboxylase (PDC), one of the enzymes involved in alcohol fermentation. The over-expression of PDC was used not only to assess the role of alcohol fermentation but also to produce lines with enhanced metabolic capacity under anaerobiosis conferring submergence tolerance to these lines. Tillers of confirmed T 0 transgenic lines showed higher PDC activities and ethanol production compared to the untransformed control. Consequently, ethanol production of tillers of T 0 transgenic plants was positively correlated with survival after submergence. This is the first known report of transformation of an economically-important crop resulting in increased submergence 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.

Large-scale production and evaluation of marker-free indica rice IR64 expressing phytoferritin genes

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.

Golden rice: introgression, breeding, and field evaluation

Considerable progress has been made on the genetic engineering of rice for improved nutritional content involving micronutrients and carotenoid content. Golden Rice, developed by genetic engineering (Agrobacterium and biolistic transformation) was used in rice breeding for the transfer of high-nutritional value to the local rice cultivars. Simultaneously, commercial Asian indica rice cultivars were also developed with expression of high-carotenoid levels. The lines were developed based on POSITECH (PMI) selection system or made marker free by segregating out the marker gene from the gene of interest. Anther culture was used to develop the homozygous stable lines, which could be of much use in further introgress-breeding and in farmer’s field. Enhanced carotenoids levels (up to T3 generation) were observed in a number of lines compared to the T0-T1 seeds which could be due to transgeneration effect of growing under greenhouse versus field conditions. However, a few introgressed lines showed less carotenoid levels than the original lines used in the breeding process. Agronomic performance of introgressed lines, non-transgenic controls, and transgenic golden rice (IR64 and BR29) developed at IRRI showed acceptable and comparable data under identical limited field conditions (screenhouse data). Syngenta generated a new Golden Rice (US cultivar) containing high level of carotenoids grown in the field at Louisiana, USA is expected to be available to the public domain. Incorporation of genes for carotenogenesis in seeds by transgenesis or by introgression did not change any significant agronomic characteristics in rice plants. The ongoing and future study of bioavailability, quality, larger field testing and freedom to operate will ensure the benefit of Golden Rice to the people who need them most.

Agrobacterium-mediated transformation: rice transformation.

Agrobacterium is a common soil bacterium with natural capacity for trans-kingdom transfer of genetic information by transferring its T-DNA into the eukaryotic genome. In agricultural plant biotechnology, combination of non-phytopathogenic strain of Agrobacterium tumefaciens with modified T-DNA and vir-genes in a binary vector system is the most widely utilized system for genetic improvement in diverse plant species and for gene function validation. Here we have described a highly efficient A. tumefaciens-mediated transformation system for indica and japonica rice cultivars based on an immature embryo system.