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The Peptide Microarray ChloroPhos1.: A Screening Tool for the Identification of Arabidopsis thaliana Chloroplast Protein Kinase Substrates
We present the method and discuss limitations and challenges associated with the determination of phosphorylation activity on peptide substrates in vitro. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news

Phosphopeptide Immuno-Affinity Enrichment to Enhance Detection of Tyrosine Phosphorylation in Plants
We describe a detailed protocol that is based on immuno-affinity enrichment step using an anti-phospho-tyrosine (pTyr)-specific antibody. This single enrichment strategy effectively enriches pTyr-containing peptides from complex total plant cell extracts, which can be measured by LC-MS/MS without further fractionation or enrichment. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news

Hemp (Cannabis sativa L.)
suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the presence of the PMI gene was confirmed using PCR an...
Source: Springer protocols feed by Plant Sciences - January 1, 2015 Category: Biology Source Type: news

Sugarcane (Saccharum Spp. Hybrids)
We describe here Agrobacterium tumefaciens-mediated transformation of sugarcane. Embryogenic callus induced from immature leaf whorls was used as target for transformation with the hypervirulent Agrobacterium strain AGL1 carrying a constitutive nptII expression cassette in vector pPZP200. Selection with 30 mg/L geneticin during the callus phase and 30 mg/L paromomycin during regeneration of shoots and roots effectively suppressed the development of non-transgenic plants. This protocol was successful with a commercially important sugarcane cultivar, CP-88-1762, at a transformation efficiency of two independent transgenic pl...
Source: Springer protocols feed by Plant Sciences - January 1, 2015 Category: Biology Source Type: news

Populus trichocarpa
Nisqually-1 is a clone of black cottonwood that is widely used as a model woody plant. It was the first woody plant to have a full genome sequence and remains today as the model for growth, metabolism, development, and adaptation for all woody dicotyledonous plants. It is one of the best-annotated plant genomes available. It is also currently studied to improve bioenergy feedstocks and to learn about responses to environmental variation that may result from climate change. It is the best characterized woody plant for lignin biosynthesis. In spite of its role as a model woody plant, many important genetic applications have...
Source: Springer protocols feed by Plant Sciences - January 1, 2015 Category: Biology Source Type: news

Poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch)
We describe in this chapter an Agrobacterium tumefaciens-mediated transformation protocol for poinsettia. A detailed description of genetic transformation, antibiotic selection, subsequent regeneration via somatic embryogenesis, and rooting as well as molecular and morphological analyses is included. The methodology described here could facilitate the future engineering of poinsettia for research purpose as well as commercial production of poinsettia plants with improved resistance or novel traits. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - January 1, 2015 Category: Biology Source Type: news

Tall Fescue (Festuca arundinacea Schreb.)
Tall fescue (Festuca arundinacea Schreb.) is the predominant cool-season perennial grass in the United States. It is widely used for both forage and turf purposes. This chapter describes a protocol that allows for the generation of a large number of transgenic tall fescue plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calli induced from caryopsis are used as explants for inoculation with A. tumefaciens. The Agrobacterium strain used is EHA105. Hygromycin phosphotransferase gene (hph) is used as the selectable marker, and hygromycin is used as the selection agent. Calli resistant to hygromycin are ...
Source: Springer protocols feed by Plant Sciences - January 1, 2015 Category: Biology Source Type: news

Setaria viridis
Interest in plant model systems for genetic, biological, and functional genomics studies stems from advantages they provide in terms of fast generation time, small stature, and simple growth requirements. A model species would be especially advantageous for the studies of C4 photosynthetic grasses, which currently present practical challenges. These include long seed-to-seed generation times, and because of their large size at maturity they require large growing areas. One potential model of interest for C4 photosynthetic grasses is Setaria viridis. It has all the desirable aforementioned attributes for a model; however, f...
Source: Springer protocols feed by Plant Sciences - October 14, 2014 Category: Biology Source Type: news

Medicago truncatula Transformation Using Leaf Explants
We describe here the protocol developed for M. truncatula R108-1 (c3). (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - October 14, 2014 Category: Biology Source Type: news

Medicago truncatula Transformation Using Cotyledonary Explants
Medicago truncatula has been developed into a model species for legumes. The M. truncatula genotype Jemalong A17 has been used for EST and genome sequencing. However, this genotype is difficult to regenerate from callus cultures. By using cotyledons as explants for Agrobacterium infection and direct shoot formation, this protocol allows for rapid production of transgenic plants from Jemalong A17 and other genotypes. Transgenic plants can be regenerated and established in the greenhouse in only 3–4 months after Agrobacterium-mediated transformation. Transformation frequency was in the range of 5–12 %. (Source: S...
Source: Springer protocols feed by Plant Sciences - October 14, 2014 Category: Biology Source Type: news

Brachypodium distachyon
The small grass Brachypodium distachyon has attributes that make it an excellent model for the development and improvement of cereal crops and bioenergy feedstocks. To realize the potential of this system, many tools have been developed (e.g., the complete genome sequence, a large collection of natural accessions, a high density genetic map, BAC libraries, EST sequences, microarrays, etc.). In this chapter, we describe a high-efficiency transformation system, an essential tool for a modern model system. Our method utilizes the natural ability of Agrobacterium tumefaciens to transfer a well-defined region of DNA from its tu...
Source: Springer protocols feed by Plant Sciences - October 14, 2014 Category: Biology Source Type: news

Gateway®-Compatible Plant Transformation Vectors
Studies in functional genomics and crop improvement programs often rely on the introduction and expression of transgenes in plants. There are two essential components required for in planta transgene expression, a plasmid vector on which the transgene sequence is carried and a delivery system capable of transferring the vector to the target cells. Agrobacterium-mediated plant transformation and the binary plasmid vector system is the preferred method of transgene delivery. The cloning technologies used for DNA manipulation underpin many of these studies. Increased demand for efficient high-throughput transformation systems...
Source: Springer protocols feed by Plant Sciences - October 14, 2014 Category: Biology Source Type: news

Cloning and Profiling of Small RNAs from Cucumber Mosaic Virus Satellite RNA
RNA silencing is not only a gene regulation mechanism that is conserved in a broad range of eukaryotes but also an adaptive immune response against foreign nucleic acids including viruses in plants. A major feature of RNA silencing is the production of small RNA (sRNA) of 21–24 nucleotides (nt) in length from double-stranded (ds) or hairpin-like (hp) RNA by Dicer-like (DCL) proteins. These sRNAs guide the binding and cleavage of cognate single-stranded (ss) RNA by an RNA silencing complex. Like all plant viruses and subviral agents, replication of viral satellite RNAs (satRNAs) is associated with the accumulation of ...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news

Detection of Plant Viruses in Natural Environments by Using RNA-Seq
Sequencing of RNA by next generation sequencers, RNA-Seq, is revolutionizing virus detection. In addition to the unbiased detection of various viruses from wild plants in natural environments, RNA-Seq also allows for the parallel collection of host plant transcriptome data. Host transcriptome data are highly valuable for studying the responses of hosts to viral infections, as well as viral host manipulation. When detecting viruses using RNA-Seq, it is critical to choose appropriate methods for the removal of rRNA from total RNA. Although viruses with polyadenylated genomes can be detected by RNA-Seq following mRNA purifica...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news

Detection and Analysis of Non-retroviral RNA Virus-Like Elements in Plant, Fungal, and Insect Genomes
Endogenous non-retroviral RNA like sequences (NRVSs) have been discovered in the genome of a wide range of eukaryotes. These are considered as fossil RNA viral elements integrated into host genomes by as-yet-known mechanisms, and in many cases, those fossils are estimated to be millions-of-years-old. It is likely that the number of NRVS records will increase rapidly due to the growing availability of whole-genome sequences for many kinds of eukaryotes. Discovery of the novel NRVSs and understanding of their phylogenetic relationship with modern viral relatives provide important information on deep evolutionary history of R...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news