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Characterization of Rice Genes Using a Heterologous Full-Length cDNA Expression System

As a result of the progress in sequencing technology, many plant genomes have now been determined. Functional genomics is required to clarify gene function in many of these species. To identify useful genes easily and quickly, we have developed a FOX (full-length cDNA overexpressor) hunting s ...

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Visual Selection in Rice: A Strategy for the Efficient Identification of Transgenic Calli Accumulating Transgene Products

Fluorescent proteins such as green fluorescent protein (GFP) allow direct visualization of transformed cells without the need for exogenous substrates. Furthermore, visual selection using GFP is a powerful tool that can be used to isolate transformed cells without antibiotic or he ...

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Selection of Transgenic Rice Plants Using a Herbicide Tolerant Form of the Acetolactate Synthase Gene

Acetolactate synthase (ALS) is an enzyme in the biosynthetic pathway for branched-chain amino acids, and bispyribac-sodium (BS), a pyrimidinyl carboxy herbicide, is a well-known inhibitor of ALS activity. However, it appears that a mutated form of rice ALS confers resistance to BS. We succe ...

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A High-Efficiency Agrobacterium-Mediated Transformation System of Rice (Oryza sativa L.)

Agrobacterium-mediated transformation of rice has been routinely performed according to the protocol reported by Hiei et al. (Plant J. 6:271–282, 1994). However, several elite japonica and many indica varieties cannot be efficiently transformed by Agrobacterium system. Also a large ...

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Genetic Transformation of the Model Green Alga Chlamydomonas reinhardtii

Over the past three decades, the single-celled green alga Chlamydomonas reinhardtii has become an invaluable model organism in plant biology and an attractive production host in biotechnology. The genetic transformation of Chlamydomonas is relatively simple and efficient, but a ...

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Use of Northern Blotting for Specific Detection of Small RNA Molecules in Transgenic Plants

Small RNAs (20–24 nucleotides long and nonprotein coding) have been increasingly investigated. They are responsible for phenomena described as RNA interference (RNAi), cosuppression, gene silencing, or quelling. Major classes of small RNAs include microRNAs (miRNAs) and small i ...

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Organophosphorus Hydrolase: A Multifaceted Plant Genetic Marker Which Is Selectable, Scorable, and Quantifiable in Whole Seed

Organophosphorus hydrolase (OPH, EC 3.1.8.1) provides a novel function as an alternative genetic marker system for use in many types of plant transformations. OPH is a high-capacity hydrolase with multiple organophosphorus substrates, many of which are neurotoxins and thus used exten ...

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Employment of Cytokinin Vectors for Marker-Free and Backbone-Free Transformation

Marker-free methods of plant transformation sacrifice the advantages of a selectable marker during regeneration or add work after regeneration to remove the marker. On the positive side, there is no stably integrated marker gene in the plant genome to present regulatory hurdles or poten ...

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Targeting DNA to a Previously Integrated Transgenic Locus Using Zinc Finger Nucleases

Targeting exogenously supplied DNA to a predetermined location within a plant genome provides a powerful tool for basic studies of plant gene function and opens up some intriguing possibilities for crop improvement. The induction of double-strand DNA breaks at specific genomic loci via ...

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Double-Strand Break-Induced Targeted Mutagenesis in Plants

Double-strand breaks are very potent inducers of DNA recombination. There is no recombination between DNA molecules unless one or two DNA strands are broken. It has become feasible to introduce double-strand breaks at specific chromosomal loci by using dedicated, redesigned endonuc ...

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Combinatorial Genetic Transformation of Cereals and the Creation of Metabolic Libraries for the Carotenoid Pathway

Combinatorial nuclear transformation is used to generate populations of transgenic plants containing random selections from a collection of input transgenes. This is a useful approach because it provides the means to test different combinations of genes without the need for separ ...

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Production of a His-Tagged Canecystatin in Transgenic Sugarcane

Transgenic plants have been widely used as expression systems of recombinant proteins in recent years because it can be an efficient alternative for the large-scale production of proteins. This is an area with great potential but is still not much explored. Indeed, this system can bring a breakt ...

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Plastid Transformation as an Expression Tool for Plant-Derived Biopharmaceuticals

The production of biopharmaceuticals in plants is currently one of the most attractive approaches to modern medicine. Several efficient plant-based expression systems have been developed so far. Among them, plastid transformation has attracted biotechnologists because the p ...

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Use of a Callus-Specific Selection System to Develop Transgenic Rice Seed Accumulating a High Level of Recombinant Protein

A mutated rice acetolactate synthase (mALS) gene expressed under the control of the rice callus-specific promoter (CSP) (CSP:mALS) becomes a useful selectable marker for producing transgenic rice seed with higher accumulation of recombinant protein. When amounts of transgene pro ...

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How to Grow Transgenic Arabidopsis in the Field

Arabidopsis is naturally adapted to habitats in which both biotic variables (e.g., light, wind, and humidity) and abiotic variables (e.g., competition, herbivory, and pathogen densities) strongly fluctuate. Hence, conditions in controlled growth chambers (in which Arabidopsis is t ...

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Use of Rolling-Circle Amplification for Large-Scale Yeast Two-Hybrid Analyses

Detection of protein–protein interactions on a large-scale has become a major focus of functional genomics after the completion of genome sequencing. The information generated from these studies not only assembles proteins into signaling networks, but also reveals potential fun ...

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Yeast Two-Hybrid Approaches to Dissecting the Plant Defense Response

We describe a reliable GAL4-based yeast two-hybrid system for identifying and isolating clones encoding proteins interacting with a protein of interest. This two-hybrid system gives extremely low background and few false-positive clones, making it ideal for library screening pur ...

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Identification of Components in Disease-Resistance Signaling in Arabidopsis by Map-Based Cloning

With the whole genome sequence and thousands of defined polymorphisms between ecotypes available, it has become much easier to clone a gene by position (map-based cloning) in Arabidopsis. Recent development of DNA-isolation methods in plants also dramatically facilitated large-s ...

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Agrobacterium-Mediated Transformation to Create an Insertion Library in Magnaporthe grisea

Magnaporthe grisea is the causal agent of rice blast disease and represents a model organism for the study of fungal plant-pathogen interactions. Pathogenicity is a complex phenotype, which is carefully orchestrated by the fungus and begins with recognition and infection of the host pla ...

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Use of Nipponbare BAC Clones for Physical Mapping of an R Gene Locus in Rice

Major advances in rice genomics during the last few years have made positional cloning in rice much more efficient. Nipponbare is a model rice genotype being sequenced by the International Rice Genome Sequencing Project Consortium. Here, we describe an efficient procedure of the constru ...

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