http://www.plantmethods.com The latest research articles published by Plant Methods 2010-03-15T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ Background: Microarrays are a powerful tool used for the determination of global RNA expression. There is an increasing requirement to focus on profiling gene expression in tissues where it is difficult to obtain large quantities of material, for example individual tissues within organs such as the root, or individual isolated cells. From such samples, it is difficult to produce the amount of RNA required for labelling and hybridisation in microarray experiments, thus a process of amplification is usually adopted. Despite the increasing use of two-cycle amplification for transcriptomic analyses on the Affymetrix ATH1 array, there has been no report investigating any potential bias in gene representation that may occur as a result. Results: Here we compare transcriptomic data generated using Affymetrix one-cycle (standard labelling protocol), two-cycle (small-sample protocol) and IVT-Express protocols with the Affymetrix ATH1 array using Arabidopsis root samples. Results obtained with each protocol are broadly similar. However, we show that there are 35 probe sets (of a total of 22810) that are misrepresented in the two-cycle data sets. Of these, 33 probe sets were classed as mis-amplified when comparisons of two independent publicly available data sets were undertaken. Conclusions: Given the unreliable nature of the highlighted probes, we caution against using data associated with the corresponding genes in analyses involving transcriptomic data generated with two-cycle amplification protocols. We have shown that the Affymetrix IVT-E labelling protocol produces data with less associated bias than the two-cycle protocol, and as such, would recommend this kit for new experiments that involve small samples. http://www.plantmethods.com/content/6/1/9 Tara Holman Michael Wilson Kim Kenobi Ian Dryden T. Charlie Hodgman Andrew Wood Michael Holdsworth Plant Methods 2010, 6:9 2010-03-15T00:00:00Z doi:10.1186/1746-4811-6-9 Plant Methods 1746-4811 6 9 2010-03-15T00:00:00Z PDF Background: Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed. Results: In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2) gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV) 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis. Conclusions: The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought. http://www.plantmethods.com/content/6/1/8 James Petrie Pushkar Shrestha Qing Liu Maged Mansour Craig Wood Xue-Rong Zhou Peter Nichols Allan Green Surinder Singh Plant Methods 2010, 6:8 2010-03-11T00:00:00Z doi:10.1186/1746-4811-6-8 Plant Methods 1746-4811 6 8 2010-03-11T00:00:00Z PDF Background: In situ hybridization is a general molecular method typically used for the localization of mRNA transcripts in plants. The method provides a valuable tool to unravel the connection between gene expression and anatomy, especially in species such as pines which show large genome size and shortage of sequence information. Results: In the present study, expression of the catalase gene (CAT) related to the scavenging of reactive oxygen species (ROS) and the polyamine metabolism related genes, diamine oxidase (DAO) and arginine decarboxylase (ADC), were localized in developing Scots pine (Pinus sylvestris L.) seeds. In addition to specific signals from target mRNAs, the probes continually hybridized non-specifically in the embryo surrounding region (ESR) of the megagametophyte tissue, in the remnants of the degenerated suspensors as well as in the cells of the nucellar layers, i.e. tissues exposed to cell death processes and extensive nucleic acid fragmentation during Scots pine seed development. Conclusions: In plants, cell death is an integral part of both development and defence, and hence it is a common phenomenon in all stages of the life cycle. Our results suggest that extensive nucleic acid fragmentation during cell death processes can be a considerable source of non-specific signals in traditional in situ mRNA hybridization. Thus, the visualization of potential nucleic acid fragmentation simultaneously with the in situ mRNA hybridization assay may be necessary to ensure the correct interpretation of the signals in the case of non-specific hybridization of probes in plant tissues. http://www.plantmethods.com/content/6/1/7 Jaana Vuosku Suvi Sutela Mira Saaskilahti Johanna Kestila Anne Jokela Tytti Sarjala Hely Haggman Plant Methods 2010, 6:7 2010-02-05T00:00:00Z doi:10.1186/1746-4811-6-7 Plant Methods 1746-4811 6 7 2010-02-05T00:00:00Z XML Background: Samples for plant metabolic fingerprinting are prepared generally by metabolism quenching, grinding of plant material and extraction of metabolites in solvents. Further concentration and derivatisation steps follow in dependence of the sample nature and the available analytical platform. For plant material sampled in the field, several methods are not applicable, such as, e.g., collection in liquid nitrogen. Therefore, a protocol was established for sample pre-treatment, grinding, extraction and storage, which can be used for analysis of field-collected plant material, which is further processed in the laboratory. Ribwort plantain (Plantago lanceolata L., Plantaginaceae) was used as model plant. The quality criteria for method suitability were high reproducibility, extraction efficiency and handling comfort of each subsequent processing step. Results: Highest reproducibility of results was achieved by sampling fresh plant material in a solvent mixture of methanol:dichloromethane (2:1), crushing the tissue with a hand-held disperser and storing the material until further processing. In the laboratory the material was extracted threefold at different pH. The gained extracts were separated with water (2:1:1 methanol:dichloromethane:water) and the aqueous phases used for analysis by LC-MS, because the polar metabolites were in focus. Chromatograms were compared by calculating a value Ξ for similarities. Advantages and disadvantages of different sample pre-treatment methods, use of solvents and solvent mixtures, influence of pH, extraction frequency and duration, and storing temperature are discussed with regard to the quality criteria. Conclusions: The proposed extraction protocol leads to highly reproducible metabolic fingerprints and allows optimal handling of field-collected plant material and further processing in the laboratory, which is demonstrated for an exemplary field data-set. Calculation of Ξ values is a useful tool to judge similarities between chromatograms. http://www.plantmethods.com/content/6/1/6 Tanja Maier Juergen Kuhn Caroline Mueller Plant Methods 2010, 6:6 2010-01-29T00:00:00Z doi:10.1186/1746-4811-6-6 Plant Methods 1746-4811 6 6 2010-01-29T00:00:00Z XML Background: Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here. Results: Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice. Conclusions: In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems. http://www.plantmethods.com/content/6/1/5 Edit Kotogany Denes Dudits Gabor Horvath Ferhan Ayaydin Plant Methods 2010, 6:5 2010-01-28T00:00:00Z doi:10.1186/1746-4811-6-5 Plant Methods 1746-4811 6 5 2010-01-28T00:00:00Z XML Background: In plant transformation, method compliance is critical for success. Transformation methods are complicated and tend to evolve over time. Until the complete method is published, method details are often partially orally transmitted and thus bound to a few people. Their documentation in text files are often a mixture of material and method description with many references to other sources especially to media description. These media are complex and often composed from several commercially available mixtures plus individually prepared stocks. The actual transformation experiment is generally documented in lab books, in which deviations from the methods and results are reported. Additionally, work schedules are planned in diaries. Both paper-based sources lack backup copies and miss unambiguous links to method descriptions and media recipes.DescriptionTo solve the problem, we devised a standard-operation-procedure system based on a Microsoft Access database containing the interlinked modules 'Media', 'Methods' and 'Experiments'. The Media module contains all basic chemicals, stocks and complex media. In this module, complex media are composed from other elements of the Media module, thus mimicking the workflows of media preparation in the lab. The Media module is made attractive to the user by functions that generate file cards and labels. The Methods module describes each method stepwise and links the steps to the media. Copy functions allow cloning of old methods to document method evolution without alteration of the old methods. Activation and inactivation functions in the Media and the Methods module remove outdated entries from active use. The Experiments module links the method to experiment specific information. This module generates a lab-book like user interface and a work schedule, and it contains a simple result section. Conclusion: The system has been evolved and tested over several years in a transformation service unit, where it increased efficiency. Additionally, the system provided rapid access to data for quality control and decision making. The system can be easily modified for the use in other research environments. http://www.plantmethods.com/content/6/1/4 Karin Koehl Juergen Gremmels Plant Methods 2010, 6:4 2010-01-27T00:00:00Z doi:10.1186/1746-4811-6-4 Plant Methods 1746-4811 6 4 2010-01-27T00:00:00Z XML Background: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetics procedure for identifying point mutations in selected gene(s) amplified from a mutagenized population using high-throughput detection platforms such as slab gel electrophoresis, capillary electrophoresis or dHPLC. One essential pre-requisite for TILLING is genomic DNA isolation from a large population for PCR amplification of selected target genes. It also requires multiplexing of genomic DNA isolated from different individuals (pooling) in typically 8-fold pools, for mutation scanning, and to minimize the number of PCR amplifications, which is a strenuous and long-drawn-out work. We describe here a simplified procedure of multiplexing, NEATTILL (Nucleic acid Extraction from Arrayed Tissue for TILLING), which is rapid and equally efficient in assisting mutation detection. Results: The NEATTILL procedure was evaluated for the tomato TILLING platform and was found to be simpler and more efficient than previously available methods. The procedure consisted of pooling tissue samples, instead of nucleic acid, from individual plants in 96-well plates, followed by DNA isolation from the arrayed samples by a novel protocol. The three variants of the NEATTILL procedure (vast, in-depth and intermediate) can be applied across various genomes depending upon the population size of the TILLING platform. The 2-D pooling ensures the precise confirmation of the coordinates of the positive mutant line while scanning complementary plates. Choice of tissue for arraying and nucleic acid isolation is discussed in detail with reference to tomato. Conclusion: NEATTILL is a convenient procedure that can be applied to all organisms, the genomes of which have been mutagenized and are being scanned for multiple alleles of various genes by TILLING for understanding gene-to-phenotype relationships. It is a time-saving, less labour intensive and reasonably cost-effective method. Tissue arraying can cut costs by up to 90% and minimizes the risk of exposing the DNA to nucleases. Before arraying, different tissues should be evaluated for DNA quality, as the case study in tomato showed that cotyledons rather than leaves are better suited for DNA isolation. The protocol described here for nucleic acid isolation can be generally adapted for large-scale projects such as insertional mutagenesis, transgenic confirmation, mapping and fingerprinting which require isolation of DNA from large populations. http://www.plantmethods.com/content/6/1/3 Yellamaraju Sreelakshmi Soni Gupta Reddaiah Bodanapu Vineeta Chauhan Mickey Hanjabam Sherinmol Thomas Vijee Mohan Sulabha Sharma Rajeswari Srinivasan Rameshwar Sharma Plant Methods 2010, 6:3 2010-01-26T00:00:00Z doi:10.1186/1746-4811-6-3 Plant Methods 1746-4811 6 3 2010-01-26T00:00:00Z XML Background: Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells. Results: We used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B. Conclusions: The results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending on the promoter used to drive expression of the RP-FP fusion protein gene, the fluorescent tagged sites can be visualized at high frequency in different cell types. The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle. These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants. http://www.plantmethods.com/content/6/1/2 Antonius Matzke Koichi Watanabe Johannes van der Winden Ulf Naumann Marjori Matzke Plant Methods 2010, 6:2 2010-01-19T00:00:00Z doi:10.1186/1746-4811-6-2 Plant Methods 1746-4811 6 2 2010-01-19T00:00:00Z XML Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research. http://www.plantmethods.com/content/6/1/1 Jian-Feng Li Li Li Jen Sheen Plant Methods 2010, 6:1 2010-01-14T00:00:00Z doi:10.1186/1746-4811-6-1 Plant Methods 1746-4811 6 1 2010-01-14T00:00:00Z XML Background: The β-glucuronidase (GUS) gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has thus far been underestimated. Results: We report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression. Conclusions: In order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented. http://www.plantmethods.com/content/5/1/19 Simone Fior Paolo Gerola Plant Methods 2009, 5:19 2009-12-30T00:00:00Z doi:10.1186/1746-4811-5-19 Plant Methods 1746-4811 5 19 2009-12-30T00:00:00Z XML