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Analysis of Interactions Between Viral Replicase Proteins and Plant Intracellular Membranes
Time of Update: 2021-01-19
Differential centrifugation techniques are used to produce membrane-enriched fractions, which can then be analyzed for the presence of viral replicase proteins by immunoblotting.
Because many plant viruses replicate in association with the endoplasmic reticulum (ER), we also discuss two techniques to specifically analyze the interaction of viral proteins with these membranes.
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Using Information From Public Arabidopsis Databases to Aid Research
Time of Update: 2021-01-19
Much of the data are stored in public databases, where data are organized, analyzed, and made freely accessible to the research community.
The methods in this chapter describe ways to access and utilizeArabidopsis data and genomic resources found in databases.
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Cloning of Short Interfering RNAs from Virus-Infected Plants
Time of Update: 2021-01-19
In this chapter, we describe a simple method for cloning siRNA from virus-infected plants.
This protocol includes isolation of small RNAs, their ligation to a pair of 5′ and 3′ adapters, RT- PCR /PCR amplification, and subsequent concatamerization before pGEM-T cloning and sequencing.
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Agrobacterium-Mediated Transformation of Soft Fruit Rubus, Ribes, and Fragaria
Time of Update: 2021-01-19
Gene transfer technology is particularly useful for fruit crops where breeders are faced with long generation times because of the relatively long periods of juvenility associated with perennial plan
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In Situ Hybridization to Plant Tissue Sections
Time of Update: 2021-01-19
An important step in the analysis of gene action is to determine the spatial and temporal regulation of gene expression.
The patterns of gene expression can provide clues to the function of genes and facilitate the mapping of qualitative changes in steady state mRNA levels.
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Comparative Development and Impact of Transgenic Papayas in Hawaii, Jamaica, and Venezuela
Time of Update: 2021-01-19
Although the three sets of transgenic papayas showed effective resistance to PRSV, the adoption rate in each country has varied from full utilization in Hawaii to aggressive testing but delay in deregulating of the product in Jamaica to rejection at an early stage in Venezuela.
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Tools for Expressing Foreign Genes in Plants
Time of Update: 2021-01-19
Since the first reports of tobacco transformation experiments in 1983, a number of fundamental processes, such as gene expression, cell metabolism, or plant development, are being studied using gene transfer experiments.
The spectrum of plant species amenable to transformation is continuously widening.
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Preparation of High Molecular Weight gDNA and Bacterial Artificial Chromosome (BAC) Libraries in Plants
Time of Update: 2021-01-19
Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction.
The construction of BAC libraries is complex and challenging for most molecular laboratories.
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Aspects of Experimental Design for Plant Metabolomics Experiments and Guidelines for Growth of Plant Material
Time of Update: 2021-01-19
For this, each step from plant cultivation to data acquisition needs to be evaluated in order to identify the major sources of error and then an appropriate design can be produced, as with any other experimental approach.
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Identification of Components in Disease-Resistance Signaling in Arabidopsis by Map-Based Cloning
Time of Update: 2021-01-19
Not only can these methods be used to isolate genes that are involved in plant innate immunity, they can also be adapted for any forward genetics projects in Arabidopsis.
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Detection and Quantification of DNA Strand Breaks Using the ROPS (Random Oligonucleotide Primed Synthesis) Assay
Time of Update: 2021-01-19
DNA double strand breaks (DSBs) arise from spontaneous DNA damage due to metabolic activities or from direct and indirect damaging effects of stress.
The level of DSBs positively correlates with the activities of homologous and nonhomologous DNA repair pathways, which in turn inversely correlate with methylation levels and chromatin structure.
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Reovirus Isolation and RNA Extraction
Time of Update: 2021-01-19
They possess 10–12 segmented double-stranded RNAs (dsRNAs) as a genome and are transmitted propagatively by leafhoppers or planthoppers.
Rice dwarf virus has 12 genomic segments separated by PAGE.
Genome characterization of other plant reoviruses is reviewed by Uyeda et al.
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Analysis of Bisulfite Sequencing Data from Plant DNA Using CyMATE
Time of Update: 2021-01-19
Several software tools were developed to assist in quantitative evaluation of bisulfite sequencing data from complex methylation patterns occurring in plants.
The software is able to reveal methylation patterns on complementary strands by handling data from hairpin bisulfite sequencing.
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A Technical Platform for PCR-Based SNP Screening in Cereals and Other Crops
Time of Update: 2021-01-19
With the rapid development of sequencing technologies and sequenced genomes, single-nucleotide polymorphisms (SNPs) have become a common genomic tool in the study of biological diversity, genome variation, gene mapping, cloning, and marker-assisted selection.
In this chapter,PCR -based SNP screening is discussed in detail.
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Phenotyping the Development of Leaf Area in Arabidopsis thaliana
Time of Update: 2021-01-19
The protocols described here are for the comparison ofArabidopsis thaliana genotypes, but can be easily adapted to compare leaf expansion under different environmental conditions and in other dicotyledonous plants.
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Applications of Kinetic Modeling to Plant Metabolism
Time of Update: 2021-01-19
Kinetic models encapsulate the available kinetic information of all the enzymes in a pathway, and then calculate the complex behavior that emerges from the interactions between these network components.
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Bioinformatics Analysis of Small RNAs in Plants Using Next Generation Sequencing Technologies
Time of Update: 2021-01-19
Like many other groups, we use these new technologies, especially SBS (Sequence-By-Synthesis), for deep profiling of small RNA molecules in plants.
We have generated numerous SBS small RNA libraries; each can consist of more than three million signatures of more than 33 nucleotides in length.
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The Isolation of Intact Chloroplasts
Time of Update: 2021-01-19
The isolation of intact chloroplasts from plant tissue allows the study of processes carried out in this subcellular compartment.
The latter is important in the case of the plant molecular biologist wishing to study the subcellular localization of a given protein.
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14Seed Mutagenesis of Arabidopsis
Time of Update: 2021-01-19
The focus of this chapter is on the production of mutations inArabidopsis seed with chemical mutagens.
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Medicago truncatula Transformation Using Leaf Explants
Time of Update: 2021-01-19
The choice and use of model legume plants ( Medicago truncatula andLotus japonicus ) for molecular studies has triggered extensive studies devoted to the development of efficientAgrobacterium -mediated transformation protocols for these two plants.
