Crop improvement through genetic engineering: development of transformation technologies and production of stress tolerant transgenic crops

With the increase of environmental degradation, the crop improvement of stress tolerance becomes more important. Little success, however, has been achieved through traditional breeding method because of the limited availability of germplasm and the complexity of the genetic control of stress tolerance traits. Genetic engineering allows gene transfer between unrelated species. It greatly widens the genetic resource and is a good alternative for the improvement of stress tolerance. The goal of this study is to produce stress-tolerant maize and soybean to adapt to environmental stresses. Genetic transformation is used as a tool to achieve the goal. Since transformation technologies in maize and soybean are not robust in the respects of transformation efficiency and the quality of resulted products, the study also was focused on the development and optimization of maize and soybean transformation technologies.;Two available soybean transformation protocols were explored. Factors affecting the transformation efficiency of Agrobacterium-mediated cotyledonary node protocol were studied and optimized. Efforts also were made to repeat the controversial transformation protocol-soybean pollen-tube pathway transformation protocol because of the great interest with the tissue-culture free feature of the protocol. The result indicated that soybean pollen-tube pathway transformation is not reproducible. This study established a new Agrobacterium-mediated maize transformation protocol using a standard binary vector system. The resulting transgenic maize plants then were evaluated. Results showed that transformants generated from this new method have better qualities compared with those obtained from particle bombardment transformation.;This study showed that the expression of a tobacco mitogen-activated protein kinase kinase kinase gene, Nicotiana protein kinase 1 (NPK1) gene improved the freezing and drought tolerance in maize. This is the first report that freezing and drought traits were achieved in major crop maize through genetic engineering approach. Transgenic maize was identified to have elevated levels of several stress related gene expression, including DREB1, EREBP, EREBR1, GST and small HSP, indicating that the active NPK1 has induced the oxidative signaling pathway as expected and, therefore, protected maize plants from stress damage. NPK1 transgenic soybean also was produced. However, no advantage in drought tolerance was detected in these transgenic soybeans

Identification of a novel iron regulated basic helix-loop-helix protein involved in Fe homeostasis in Oryza sativa

<p>Abstract</p> <p>Background</p> <p>Iron (Fe) is the most limiting micronutrient element for crop production in alkaline soils. A number of transcription factors involved in regulating Fe uptake from soil and transport in plants have been identified. Analysis of transcriptome data from <it>Oryza sativa </it>grown under limiting Fe conditions reveals that transcript abundances of several genes encoding transcription factors are altered by Fe availability. These transcription factors are putative regulators of Fe deficiency responses.</p> <p>Results</p> <p>Transcript abundance of one nuclear located basic helix-loop-helix family transcription factor, <it>OsIRO3</it>, is up-regulated from 25- to 90-fold under Fe deficiency in both root and shoot respectively. The expression of <it>OsIRO3 </it>is specifically induced by Fe deficiency, and not by other micronutrient deficiencies. Transgenic rice plants over-expressing <it>OsIRO3 </it>were hypersensitive to Fe deficiency, indicating that the Fe deficiency response was compromised. Furthermore, the Fe concentration in shoots of transgenic rice plants over-expressing <it>OsIRO3 </it>was less than that in wild-type plants. Analysis of the transcript abundances of genes normally induced by Fe deficiency in <it>OsIRO3 </it>over-expressing plants indicated their induction was markedly suppressed.</p> <p>Conclusion</p> <p>A novel Fe regulated bHLH transcription factor (OsIRO3) that plays an important role for Fe homeostasis in rice was identified. The inhibitory effect of <it>OsIRO3 </it>over-expression on Fe deficiency response gene expression combined with hypersensitivity of <it>OsIRO3 </it>over-expression lines to low Fe suggest that OsIRO3 is a negative regulator of the Fe deficiency response in rice.</p

Editorial: improving the nutritional content and quality of crops: promises, achievements, and future challenges

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Editorial: improving the nutritional content and quality of plants: promises, achievements, and future challenges, volume II

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Comparative analysis and integrative classification of NCI60 cell lines and primary tumors using gene expression profiling data

BACKGROUND: NCI60 cell lines are derived from cancers of 9 tissue origins and have been invaluable in vitro models for cancer research and anti-cancer drug screen. Although extensive studies have been carried out to assess the molecular features of NCI60 cell lines related to cancer and their sensitivities to more than 100,000 chemical compounds, it remains unclear if and how well these cell lines represent or model their tumor tissues of origin. Identification and confirmation of correct origins of NCI60 cell lines are critical to their usage as model systems and to translate in vitro studies into clinical potentials. Here we report a direct comparison between NCI60 cell lines and primary tumors by analyzing global gene expression profiles. RESULTS: Comparative analysis suggested that 51 of 59 cell lines we analyzed represent their presumed tumors of origin. Taking advantage of available clinical information of primary tumor samples used to generate gene expression profiling data, we further classified those cell lines with the correct origins into different subtypes of cancer or different stages in cancer development. For example, 6 of 7 non-small cell lung cancer cell lines were classified as lung adenocarcinomas and all of them were classified into late stages in tumor progression. CONCLUSION: Taken together, we developed and applied a novel approach for systematic comparative analysis and integrative classification of NCI60 cell lines and primary tumors. Our results could provide guidance to the selection of appropriate cell lines for cancer research and pharmaceutical compound screenings. Moreover, this gene expression profile based approach can be generally applied to evaluate experimental model systems such as cell lines and animal models for human diseases

Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize

We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R1 kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R1 kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R3 seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R3 seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens

Nicotianamine, a Novel Enhancer of Rice Iron Bioavailability to Humans

Background: Polished rice is a staple food for over 50 % of the world’s population, but contains little bioavailable iron (Fe) to meet human needs. Thus, biofortifying the rice grain with novel promoters or enhancers of Fe utilization would be one of the most effective strategies to prevent the high prevalence of Fe deficiency and iron deficiency anemia in the developing world. Methodology/Principal Findings: We transformed an elite rice line cultivated in Southern China with the rice nicotianamine synthase gene (OsNAS1) fused to a rice glutelin promoter. Endosperm overexpression of OsNAS1 resulted in a significant increase in nicotianamine (NA) concentrations in both unpolished and polished grain. Bioavailability of Fe from the high NA grain, as measured by ferritin synthesis in an in vitro Caco-2 cell model that simulates the human digestive system, was twice as much as that of the control line. When added at 1:1 molar ratio to ferrous Fe in the cell system, NA was twice as effective when compared to ascorbic acid (one of the most potent known enhancers of Fe bioavailability) in promoting more ferritin synthesis. Conclusions: Our data demonstrated that NA is a novel and effective promoter of iron utilization. Biofortifying polished rice with this compound has great potential in combating global human iron deficiency in people dependent on rice for thei

Agrobacterium tumefaciens-Mediated Transformation of Maize Embryos Using a Standard Binary Vector System

We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciensstandard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L−1 l-cysteine. Inclusion of l-cysteine in cocultivation medium lead to an improvement in transient β-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar andgustransgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciensstandard binary vector system

Transparency in planning, warranting and interpreting research

409 PSI DMRs in the DCL3a RNAi line, and 88 PSI DMRs in the DCL3a RNAi line, associated with significant changes in nearby gene expressio