Bioengineered ‘golden’ indica rice cultivars with β-carotene metabolism in the endosperm with hygromycin and mannose selection systems

Vitamin-A deficiency (VAD) is a major malnutrition problem in South Asia, where indica rice is the staple food. Indica-type rice varieties feed more than 2 billion people. Hence, we introduced a combination of transgenes using the biolistic system of transformation enabling biosynthesis of provitamin A in the endosperm of several indica rice cultivars adapted to diverse ecosystems of different countries. The rice seed-specific glutelin promoter (Gt-1 P) was used to drive the expression of phytoene synthase (psy), while lycopene β-cyclase (lcy) and phytoene desaturase (crtI), fused to the transit peptide sequence of the pea-Rubisco small subunit, were driven by the constitutive cauliflower mosaic virus promoter (CaMV35S P). Transgenic plants were recovered through selection with either CaMV35S P driven hph (hygromycin phosphotransferase) gene or cestrum yellow leaf curling virus promoter (CMP) driven pmi (phophomannose isomerase) gene. Molecular and biochemical analyses demonstrated stable integration and expression of the transgenes. The yellow colour of the polished rice grain evidenced the carotenoid accumulation in the endosperm. The colour intensity correlated with the estimated carotenoid content by spectrophotometric and HPLC analysis. Carotenoid level in cooked polished seeds was comparable (with minor loss of xanthophylls) to that in non-cooked seeds of the same transgenic line. The variable segregation pattern in T1 selfing generation indicated single to multiple loci insertion of the transgenes in the genome. This is the first report of using nonantibiotic pmi driven by a novel promoter in generating transgenic indica rice for possible future use in human nutrition

Master Greeley -- "See here, this is the way to do it."

Greeley, editor of the Tribune and a war supporter, wanted immediate action against the Rebels. General Scott, fearing that his armies were not ready or trained enough, vascillated, causing cartoons such as this, lampooning both Greeley and Scott. The Manassas disaster that followed for the Union disgraced Scott and the administration, but Greeley held that it was a setback that could be swallowed

The screening of rice germplasm, including those transgenic rice lines which accumulate β-carotene in their polished seeds, for their carotenoid profile

This article does not have an abstract

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

Rhizobial lipochitooligosaccharide nodulation factors activate expression of the legume early nodulin gene ENOD12 in rice

Lipochitooligosaccharide nodulation factors (Nod factors) produced by rhizobia are a major host range determinant. These factors play a pivotal role in the molecular signal exchange, infection and induction of symbiotic developmental responses in legumes leading to the formation of a nodule in which rhizobia carry out N2 fixation. Determining whether rice (Oryza sativa) can respond to Nod factors could lead to strategies that would make rice amenable to develop a nitrogen-fixing endosymbiotic association with rhizobia. We introduced into rice the promoter of the infection-related geneMtENOD12 (from Medicago truncatula) fused to the β-glucuronidase (GUS) reporter gene to serve as a molecular marker to aid in the detection of Nod factor signal perception by rice cells. Treatment of the transgenic rice roots with Nod factors (10–6–10–9m) under nitrogen-limiting conditions induced MtENOD12-GUS expression in cortical parenchyma, endodermis and pericycle. In contrast, chitooligosaccharide backbone alone failed to elicit such a response in the root tissues. These findings demonstrate that rice roots perceive Nod factors and that these lipochitooligosaccharides, but not simple chitin oligomers, act as signal molecules in activating MtENOD12 in cortical parenchyma as in legumes. Exogenous application ofN-naphthaleneacetic acid mimicked the Nod factor-elicited tissue-specific expression of MtENOD12 in roots while cytokinins inhibited it, thus evidencing that Nod factors, auxin and cytokinins probably act on similar signaling elements responsible for the regulation of MtENOD12 activation in rice. Taken together, these results suggest that at least a portion of the signal transduction machinery important for legume nodulation is likely to exist in rice

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

Enhancement of submergence tolerance in transgenic ice overproducing pyruvate decarboxylase

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 T0 transgenic lines showed higher PDC activities and ethanol production compared to the untransformed control. Consequently, ethanol production of tillers of T0 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

The End of the Constitutional Beginning

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