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Fusion with Fluorescent Proteins for Subcellular Localization of Enzymes Involved in Plant Alkaloid Biosynthesis
Time of Update: 2021-01-19
The fusion with fluorescent proteins (FP) may now be used as a quite effective and reliable tool to investigate this question.
This chapter describes the application of this methodology to an enzyme involved in indole alkaloid biosynthesis, with general considerations on the development of the approach.
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Computational Methods for Comparative Analysis of Plant Small RNAs
Time of Update: 2021-01-19
Small RNAs play an important role in plant development, stress responses, and epigenetic regulation, primarily through their role in transcriptional and post-transcriptional silencing of specific target genes and loci.
A database containing upward of hundreds of millions of plant small RNA sequences is being created for comparative analyses.
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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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mRNA Detection by Whole Mount In Situ Hybridization (WISH) or Sectioned Tissue In Situ Hybridization (SISH) in Arabidopsis
Time of Update: 2021-01-19
Gene expression can be analyzed at high spatial resolution via RNA in situ detection methods.
For many tissues and species, these will be performed on sections of embedded and fixed plant material.
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Analysis of Gene Expression in Transgenic Plants
Time of Update: 2021-01-19
The analysis of RNA frequently provides a vital link between changes in enzyme levels and/or metabolites and the phenotype.
Common methods of transcript analysis by Northern and dot-blot hybridizations using nonradioactive probing methods are described.
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In Vitro Propagation of Hydrangea spp.
Time of Update: 2021-01-19
For in vitro culture, Murashige and Skoog medium supplemented with BA (0.25 mg/L) and sucrose (30 g/L) was used.
Following shoot proliferation, the in vitro rooting frequency was 100% on a medium containing NAA 0.5 mg/L.
After 21 days, fully acclimatized and well-established plants were obtained, suitable for commercialization.
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Grafting
Time of Update: 2021-01-19
The technique of grafting has been widely used in other species, but inArabidopsis , its small size makes the process rather more complicated.
However, there are now several well-established grafting procedures available, which we described here, and their use has already contributed greatly to understanding of such processes as shoot branching control, flowering, and disease resistance.
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Gossypol
Time of Update: 2021-01-19
The general symptoms of gossypol toxicity are constipation, depressed appetite, loss of weight, and death, which usually results from circulatory failure.
Although acute toxicity is low, ingestion of small amounts over a prolonged period can be lethal.
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Engineering the Plastid Genome of Nicotiana sylvestris, a Diploid Model Species for Plastid Genetics
Time of Update: 2021-01-19
A uniform population of engineered ptDNA is obtained by selection for marker genes encoded in the vectors.
We report here for the first time plastid transformation inNicotiana sylvestris , a diploid ornamental species.
We demonstrate that the protocols and vectors developed for plastid transformation inN.
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Preparation of DNA from Arabidopsis
Time of Update: 2021-01-19
Successful extraction ofDNA is an essential and time consuming step in many plant molecular biology procedures.
The classical approaches of plant DNA isolation are often designed to produce large amounts of DNA of high molecular weight with sufficient purity.
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Phenotyping of Abiotic Responses and Hormone Treatments in Arabidopsis
Time of Update: 2021-01-19
To be able to assign biological functions to these genes, the mutants need to be evaluated with a broad spectrum of assays to uncover conditional phenotypes.
Here we provide an overview on how to evaluate plants in their development and their response to abiotic factors such as light, hormones, and different stressors.
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PCR Cloning of Coat Protein Genes
Time of Update: 2021-01-19
Amplification between the two primers is achieved by reiterative cycles of template denaturation, primer annealing, and primer extension by a heat-stable DNA polymerase enzyme, which is able to withstand the repeated high temperatures required for DNA denaturation.
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Growth and Storage of Agrobacterium
Time of Update: 2021-01-19
Working with contaminated or genetically changed cultures, or facing the loss of a strain are annoyances that often may be easily prevented.
A reliable preservation method should fulfill the following criteria.
Second, the cultures should be genetically stable during storage.
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Procedures for Plant Rhabdovirus Purification, Polyribosome Isolation, and Replicase Extraction
Time of Update: 2021-01-19
Many of these viruses are persistently transmitted to their mammalian and plant hosts by insect or arthropod vectors in which they are able to multiply.
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Eucalyptus
Time of Update: 2021-01-19
UsingEucalyptus camaldulensis as a model system, we describe here a basicAgrobacterium -mediated genetic transformation protocol through organogenesis for the production of transgenic plants.
Modifications of this protocol to use in mature tissues derived from elite trees and otherEucalyptus species are also described.
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Instrumentation
Time of Update: 2021-01-19
The techniques of electroporation and electrofusion require that cells be subjected to brief pulses of electric fields of the appropriate amplitude, duration, and wave form.
In this chapter, the term electro cell manipulation (ECM) shall describe both techniques.
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Effect of Acetosyringone on Growth and Oncogenic Potential of Agrobacterium tumefaciens
Time of Update: 2021-01-19
Observation of this effect ofvir gene induction depends on the use of particular experimental conditions.
Other parts of the chapter then describe how the mutants that are generated in the presence of acetosyringone are recognized and characterized.
In addition, acetosyringone enhances the ability ofA.
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Production of Transgenic Crops by the Floral-Dip Method
Time of Update: 2021-01-19
Indeed, this simple system, without requiring any knowledge of plant tissue culture, has been a breakthrough in the production of the first transgenic radish plants.
Although the radish is closely related toArabidopsis thaliana , it appears the two plants have different mechanisms of T- DNA transfer using floral dip.
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Use of Molecular Methods for the Detection of Fungal Spores
Time of Update: 2021-01-19
Traditional methods for the isolation and identification of fungal spores can be time-consuming and laborious.
DNA -based methods for fungal detection can be used to detect the spores of plant-pathogenic fungi.
Air borne spores can be collected and identified byPCR allowing identification of the species.
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Soybean (Glycine max) Transformation Using Immature Cotyledon Explants
Time of Update: 2021-01-18
The immature cotyledon method includes direct induction of transgenic somatic embryos from the explant plated on selective media after cocultivation, followed by maturation and regeneration of individual somatic embryos into whole plants.
