Plant Biology

The generation of transgenic plants can be a lengthy and difficult process. Transient expression assays have been developed as faster and more convenient alternatives for investigating gene function. These assays often take advantage of the ability of Agrobacterium to transfer foreign DNA into plant cells with intact cell walls. Agrobacterium-mediated transformation is, however, inefficient and shows great variability. In the May issue of Cold Spring Harbor Protocols, Andreas Nebenführ and colleagues from the University of Tennessee present FAST Technique for Agrobacterium-Mediated Transient Gene Expression in Seedlings of Arabidopsis and Other Plant Species, a quick, efficient and economical assay for gene function in intact plants. The technique involves cocultivation of young plant seedlings and Agrobacterium in the presence of Silwet-77. The Silwet-77 facilitates transformation, thus replacing a wounding or device-dependent vacuum step. As one of May’s featured articles, it is freely available to subscribers and non-subscribers alike.

Cold Spring Harbor Protocols is hosting the movie figures that accompany the new lab manual, Live Cell Imaging, Second Edition, edited by Robert Goldman, Jason Swedlow and David Spector, . These movies are freely accessible to all, and worth a look if you’re interested in seeing the state of the art in time lapse imaging.

With the recent progress in understanding epigenetic mechanisms, methods for profiling patterns of DNA modification have become important tools for analysis of gene regulation. DNA methylation, in which cytosine is modified to form 5-methylcytosine, is a well-characterized epigenetic modification essential for normal development in plants and mammals. In the December issue of Cold Spring Harbor Protocols, Jon Reinders presents Amplification of Bisulfite-Converted DNA for Genome-Wide DNA Methylation Profiling. This method utilizes the treatment of DNA with sodium bisulfite, which converts unmethylated cytosine to uracil (5-methylcytosine is not converted). This is followed with PCR amplification, where the uracil amplifies as thymine, creating a C-to-T transition. The genome can then be analyzed for these transitions using an array-based platform. Reinders protocol mitigates the major issues with bisulfite conversion (DNA fragmentation and poor reproducibility) and reduces bias during the amplification step. While the protocol is optimized for use in Arabidopsis, it can potentially be adapted for use in other organisms.

Live cell imaging techniques are driving a revolution in biological research. Instead of viewing dead tissues and cells fixed at a particular stage of activity, scientists can now visualize dynamic changes as they happen, permitting a better understanding of complete processes. The revolution has been fueled by the implementation of genetically encoded fluorescent proteins, the subject of the 2008 Nobel Prize in Chemistry.

The diverse array of applications benefiting from fluorescent proteins ranges from markers targeted at organelles and protein fusions designed to monitor intracellular dynamics to reporters of transcriptional regulation and in vivo probes for whole-body imaging and detection of cancer. Fluorescent proteins have enabled the creation of highly specific biosensors to monitor a wide range of intracellular phenomena, including pH and metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. In the December issue of Cold Spring Harbor Protocols, David Piston and colleagues present Fluorescent Protein Tracking and Detection: Fluorescent Protein Structure and Color Variants, a comprehensive overview of the wide variety of fluorescent proteins that are currently available. The article features more than twenty movies of different fluorescent proteins in action and is a great primer for planning imaging experiments. As one of December’s featured articles, it is freely available to subscribers and non-subscribers alike.

In addition, the same authors have also contributed Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells. This article provides some general tips for the practical aspects of using and imaging enhanced green fluorescent protein (EGFP) and newer members of the color palette, as well as quantitative imaging of fluorescent proteins and imaging of several fluorescent proteins at the same time. Finally, an overview is provided for the different types of biosensors that have been derived from flourescent proteins.

Both articles are adapted from the spectacular new manual, Live Cell Imaging: A Laboratory Manual, Second Edition which is due out by month’s end.

CSH Protocols December Cover

CSH Protocols December Cover

We’re getting toward the end of the second volume of our Emerging Model Organisms series in Cold Spring Harbor Protocols, and November’s issue brings us a look at the Hawaiian Bobtail Squid and the genus Dioscorea, or True Yams.

Euprymna scolopes, the Hawaiian Bobtail Squid (our cover model this month, see below) is a cephalopod that’s well-suited for study in the laboratory. E. scolopes is primarily studied in three contexts:
1) as a model for cephalopod development–the embryos and protective chorions are clear, making it amenable for the observations and manipulations common in other studied model systems
2) as a model of animal-bacteria symbioses with the luminous marine bacterium Vibrio fischeri
3) as a system for studying the interaction of tissues with light, as the squid features a specialized light organ.

Heinz Gert de Couet and colleagues supply an overview of the Hawaiian Bobtail Squid as a model system, along with protocols for Preparation of Genomic DNA, Confocal Immunocytochemistry, Whole-Mount In Situ Hybridization (parts 1 and 2), and Culture and Observation.

Dioscorea is a large genus of plants that are monocots but that look like dicots, and are closely related to the phylogenetically derived group containing the grasses. It’s interesting evolutionarily because of the position it occupies, as a link between the eudicots and grasses–groups that contain all the model flowering plant species. The true yam is also important as a food crop. R. Geeta and colleagues provide an overview of the genus, and protocols for husbandry, culturing tissues, management of plantlets, controlled crosses, and DNA extraction.

CSH Protocols November Cover

CSH Protocols November Cover

Volume 2 of our Emerging Model Organisms series rolls on in the October issue of Cold Spring Harbor Protocols. This month brings a look at two emerging models, one all-time classic.

Neelima Sinha and colleagues present “The Mother of Thousands” (Kalanchoë daigremontiana), a plant which has the fascinating ability to regenerate and entire organism from somatic cells. The process of forming a somatic embryo outside of a seed environment provides an attractive model system for studying embryogenesis. Kalanchoë is also used in the study of Crassulacean acid metabolism (CAM), which is an important evolutionary adaptation of the photosynthetic carbon assimilation pathway to arid environments. In addition, natural compounds extracted from tissues of Kalanchoë have potential applicability in treating tumors and inflammatory and allergic diseases, and have been shown to have insecticidal properties. Protocols are provided for fixing and sectioning tissues, in situ hybridization, transformation using agrobacterium, DNA extraction and RNA extraction.

John Werren and colleagues provide The Parasitoid Wasp Nasonia: An Emerging Model System with Haploid Male Genetics. Nasonia is a genus consisting of four interfertile species. They’re particularly useful as a genetic tool for study because females are diploid and develop from fertilized eggs, and males are haploid and develop from unfertilized eggs. This allows geneticists to exploit many of the advantages of haploid genetics in an otherwise complex eukaryotic organism. Protocols are available for field collection, strain maintenance, rearing fly hosts, egg collection, virgin collection and crossing methods, larval RNAi and curing Wolbachia bacterial infections.

As for that “classic” system mentioned above, if you know genetics, then you know Barbara McClintock, and you know that Maize has been a keystone model system for nearly a century. Micheal Scanlon and colleagues have written up Maize (Zea mays): A Model Organism for Basic and Applied Research in Plant Biology, which gives an up-to-date discussion of the state of Maize research.

March’s issue of Cold Spring Harbor Protocols includes a set of three articles detailing common methods for DNA isolation from plants:

Quick Miniprep for Plant DNA Isolation gives a rapid method, good when processing large numbers of samples where high purity of the resulting DNA is not needed.

Dellaporta Miniprep for Plant DNA Isolation is quick and inexpensive and results in a higher quality end product. Because it yields enough DNA to test a large number of markers, it’s recommended in cases when many markers will be tested on the same samples.

Cetyltrimethyl Ammonium Bromide (CTAB) DNA Miniprep for Plant DNA Isolation is useful for isolation of DNA from tissues containing high amounts of polysaccharides. The presence of polysaccharides can inhibit PCR reactions. Under high salt conditions, CTAB binds the polysaccharides and takes them out of solution.

These articles join a previously published protocol for plant DNA purification, from C. Eduardo Vallejos, An Expedient and Versatile Protocol for Extracting High-Quality DNA from Plant Leaves. This method results in DNA that has virtually no protein or phenolic contaminants, is RNA-free, contains high-molecular-weight fragments according to CHEF electrophoresis, and is fully digestible by restriction enzymes.

Our series highlighting new and lesser-known laboratory model organisms continues in February, with two sets of articles detailing the use of moss and choanoflagellates.

The moss Physcomitrella patens has been used in laboratory research for more than 80 years, but the last 15 have seen a resurgence in moss research. P. patens can easily be grown in the lab, and spends most of its life in a haploid state that allows many of the approaches used in yeast and microbes to be applied. Methods for RNAi have been worked out, the genome has been sequenced and assembled, physical and genetic maps are available, and more than 250,000 expressed sequence tags (ESTs) are known. Ralph Quatrano and colleagues have contributed an overview of the use of P. patens as a laboratory organism to February’s issue of Cold Spring Harbor Protocols along with protocols for culture, isolation of protoplasts, somatic hybridization, chemical and UV mutagenesis, transformation via direct DNA uptake, T-DNA mutagenesis, and biolistic delivery systems, and isolation of DNA, RNA and proteins.

Choanoflagellates are a varied group of protozoa that are the closest living relative to the metazoa, and their study is leading to new insights into metazoan ancestry and origins. Barry Leadbeater and colleagues have written up a series of articles highlighting the use of Monosiga brevicollis as a representative species, as it has recently had its genome sequenced, and is readily grown and manipulated in the laboratory (protocols included are generally transferable to most choanoflagellate species). The set of articles includes an overview of choanoflagellates, and protocols for isolation from field samples and culture of choanoflagellates, long-term storage, visualization of actin and beta-tubulin, purification of total RNA, and preps for rapid DNA isolation, high molecular weight DNA isolation and separation of choanoflagellate and bacterial genomic DNA.

These articles on Emerging Model Organisms are being collected in a series of lab manuals, the first of which is currently available here (now on sale 25% off!).

One of our featured articles for February comes from S.P. Dinesh-Kumar and colleagues at Yale University and is an update to a method first published a few years ago in our RNAi manual (now marked way down to a bargain price in both hardcover and paperback). RNAi has become a commonly-used tool for the down-regulation of genes in plants. The most effective means of accomplishing this gene silencing is through the use of viral vectors, with the Tobacco Rattle Virus (TRV) providing the most robust results. Virus-Induced Gene Silencing as a Tool for Delivery of dsRNA into Plants outlines a simple procedure for introducing TRV-based vectors into plants such as Arabidopsis, Nicotiana benthamiana and tomato.

Like all of our featured articles, access to this protocol is free for both subscribers and non-subscribers alike.

The October issue of CSH Protocols presents a new focus on Emerging Model Organisms.

Much of twentieth century biological research has focused on a limited number of model organisms, such as Arabidopsis, C. elegans, mouse, Drosophila, and E. coli. These classical model species, chosen because they are amenable to laboratory research and suitable for studying a range of biological problems, have served to elucidate many biological processes that can be generalized across a wider array of organisms. It is only a slight exaggeration to say that the basic workings of the cell were elucidated mostly from experiments on a few single-celled organisms — primarily E.coli and yeast. Our understanding of animal development was largely based on the genetics of fruit fly and worm and on the manipulation of a handful of amphibians and mouse; most of what we learned about the molecular and developmental biology of plants came from examining Arabidopsis and just a few other species. But biology wasn’t always done this way.
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