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Profiling Primary Metabolites of Tomato Fruit with Gas Chromatography/Mass Spectrometry

Metabolite profiling is a rapidly expanding technology which aims to quantify the entire metabolome of biological samples. Gas Chromatography Mass Spectrometry (GC-MS) is one of the most widely used analytical tools for profiling highly complex mixtures of primary metabolites, su ...

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Solid Phase Micro-Extraction GCMS Analysis of Natural Volatile Components in Melon and Rice

The natural fragrance compounds produced by plants play key roles in the long-term fitness and survival of these plants as well as being of direct/indirect benefit to man. Almost all plant fragrances, either pleasant or unpleasant, comprise many different compounds, from different chemi ...

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The Use of Genomics and Metabolomics Methods to Quantify Fungal Endosymbionts and Alkaloids in Grasses

The association of plants with endosymbiotic micro-organisms poses a particular challenge to metabolomics studies. The presence of endosymbionts can alter metabolic profiles of plant tissues by introducing non-plant metabolites such as fungal specific alkaloids, and by meta ...

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ICP-MS and LC-ICP-MS for Analysis of Trace Element Content and Speciation in Cereal Grains

Trace elements are unevenly distributed and speciated throughout the cereal grain. The germ and the outer layers of the grain have the highest concentrations of trace elements. A large fraction of the trace elements is therefore lost during the milling process. The bioavailability of the re ...

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Combined NMR and Flow Injection ESI-MS for Brassicaceae Metabolomics

High-throughput screening of large collections of plants, whether in the context of gene function analysis, quality trait selection, or metabolic engineering requires robust and rapid methodologies that provide maximum information with minimum sample pre-fractionation. H ...

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Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Plant Metabolite Profiling and Metabolite Identification

Mass spectrometry (MS) is usually the technique of choice for metabolomic studies where the volume of sample material is too limited for applications employing nuclear magnetic resonance (NMR) spectroscopy. With the advent of ultra-high accuracy mass spectrometers such as the Orbit ...

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High Precision Measurement and Fragmentation Analysis for Metabolite Identification

The degree of precision in measuring accurate masses in LC MS/MS-based metabolomics experiments is a determinant in the successful identification of the metabolites present in the original extract. Using the methods described here, complex broccoli extracts containing hundreds ...

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Tissue Preparation Using Arabidopsis

The ability to track changes in the levels of many metabolites in plants has great utility in a number of biological contexts. A metabolomics experiment usually requires the comparison of different varieties in either a functional genomics context or in response to perturbation by an exter ...

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Precautions for Harvest, Sampling, Storage, and Transport of Crop Plant Metabolomics Samples

Plant metabolomics is increasingly a routine option for plant biologists and food scientists. Here, we suggest some precautions for preparation and handling of samples issued from crop plants, in order to ensure sample representativeness and quality before their biochemical anal ...

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Separating the Inseparable: The Metabolomic Analysis of PlantPathogen Interactions

Plant–microbe interactions—whether pathogenic or symbiotic—exert major influences on plant physiology and productivity. Analysis of such interactions represents a particular challenge to metabolomic approaches due to the intimate association between the interact ...

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Aspects of Experimental Design for Plant Metabolomics Experiments and Guidelines for Growth of Plant Material

Experiments involve the deliberate variation of one or more factors in order to provoke responses, the identification of which then provides the first step towards functional knowledge. Because environmental, biological, and/or technical noise is unavoidable, biological expe ...

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Practical Applications of Metabolomics in Plant Biology

The technologies being developed for the large-scale, essentially unbiased analysis of the small molecules present in organic extracts made from plant materials are greatly changing our way of thinking about what is possible in plant biology. A range of different separation and detect ...

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Marker-Free Gene Targeting by Recombinase-Mediated Cassette Exchange

Repeated gene targeting by recombinase-mediated cassette exchange (RMCE) is an efficient tool for the study of gene function and regulation because of the high predictability and repeatability of gene expression. We have developed the site-directed integration (SDI) vector syst ...

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A One-Time Inducible Transposon to Create Knockout Mutants in Rice

Use of a transposon is an efficient tagging tool for exploring the function of the gene it inserts into or is adjacent to. A few modifications have been applied to the native Ac transposon to allow it to transpose efficiently or spontaneously and stop quickly thereafter. Furthermore, locating the t ...

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Exploiting MultiSite Gateway and pENFRUIT Plasmid Collection for Fruit Genetic Engineering

MultiSite Gateway cloning techniques based on homologous recombination facilitate the combinatorial assembly of basic genetic pieces (i.e., promoters, CDS, and terminators) into gene expression or gene silencing cassettes. pENFRUIT is a collection of MultiSite Triple Gatew ...

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A Developmentally Regulated Cre-lox System to Generate Marker-Free Transgenic Brassica napus Plants

In this chapter, a strategy for engineering marker-free Brassica napus plants is described. It is based on the Cre-lox site-specific recombination system and includes three essential steps. At first, the binary vector pLH-nap-lx-cre-35S-bar-lx-vst has been designed. In this vector, the c ...

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High Frequency of Single-Copy T-DNA Transformants Produced After Floral Dip in CRE-Expressing Arabidopsis Plants

Transgenic plants that harbor a single copy of the introduced transgene are preferable to those with multiple transgene copies because multiple T-DNA copies correlate with expression variability and susceptibility to silencing. Especially after the commonly used floral-dip A ...

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Expression of Artificial MicroRNAs in Physcomitrella patens

MicroRNAs (miRNAs) are ∼21-nt-long small RNAs transcribed from endogenous MIR genes which form precursor RNAs with a characteristic hairpin structure. MiRNAs control the expression of cognate target genes by binding to reverse complementary sequences resulting in cleavage or tr ...

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Efficient Modification of Floral Traits by Heavy-Ion Beam Irradiation on Transgenic Torenia

While heavy-ion beam irradiation is becoming popular technology for mutation breeding in Japan, the combination with genetic manipulation makes it more convenient to create greater variation in plant phenotypes. We have succeeded in producing over 200 varieties of transgenic tor ...

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A Protocol for Transformation of Torenia

This chapter describes an Agrobacterium tumefaciens-mediated transformation protocol for torenia, a plant that has several useful characteristics and is primarily used for ornamental and experimental purposes. Leaf segments of torenia were co-cultured with A. tumefaciens ...

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