Norman Oliva

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.

Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease

Abstract A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T0, T1, and T2 transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants.

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.

Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars.

Genetic transformation has been attempted for management of rice sheath blight disease, caused by Rhizoctonia solani. We introduced a PR-3 rice chitinase gene (RC7), isolated from R. solani-infected rice plants, into indica rice cultivars IR72, IR64, IR68899B, MH63, and Chinsurah Boro II by the biolistic and PEG-mediated transformation system. Inheritance was studied up to the T(2) generation by Southern blot analysis. Western blot analysis of transgenic plants with polyclonal antibody revealed the presence of chitinase protein with a molecular weight of 35 kDa that reacts with chitinase antibody. The transformants synthesized different levels of chitinase proteins constitutively and progeny from the plants containing the chitinase gene showed different levels of enhanced resistance when challenged with the sheath blight pathogen R. solani.

Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems

Abstract A concise T-DNA element was engineered containing the rice class-I chitinase gene expressed under the control of CaMV35S and the hygromycin phosphotransferase gene (hph) as a selectable marker. The binary plasmid vector pNO1 with the T-DNA element containing these genes of interest was mobilized to Agrobacteriumtumefaciens strain LBA4404 to act as an efficient donor of T-DNA in the transformation of three different indica rice cultivars from different ecosystems. Many morphologically normal, fertile transgenic plants from these rice cultivars were generated after Agrobacterium-mediated transformation using 3-week-old scutella calli as initial explants. Stable integration, inheritance and expression of the chimeric chitinase gene were demonstrated by Southern blot and Western blot analysis of the transformants. Bioassay data showed that transgenic plants can restrict the growth of the sheath blight pathogen Rhizoctonia solani. Bioassay results were correlated with the molecular analysis. Although we obtained similar results upon DNA-mediated transformation, this report shows the potential of the cost-effective, simple Agrobacterium system for genetic manipulation of rice cultivars with a pathogenesis-related (PR) gene.

Transgenic fertile japonica rice plants expressing a modified cryIA(b) gene resistant to yellow stem borer

Abstract The japonica rice variety Taipei 309 was cotransformed by particle bombardment of immature embryo-derived embryogenic calli with a modified δ-endotoxin gene cryIA(b) of Bacillus thuringiensis (Bt) under the control of the rice Actin1 promoter, and the hygromycin resistance gene, hph driven by the CaMV35S promoter. Selected transgenic rice plants showed enhanced insecticidal activity against yellow stem borer (Scirpophaga incertulas), with mortality rates reaching up to 100% in a bioassay with cut stems. Introduction and expression of the Actin1 promoter-Bt gene into rice provides japonica rice germplasm resistant to insect attack.

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.

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.

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.