Springer protocols feed by Plant Sciences 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Synchronization of Green Algae by Light and Dark Regimes for Cell Cycle and Cell Division Studies
A synchronous population of cells is one of the prerequisites for studying cell cycle processes such as DNA replication, nuclear and cellular division. Green algae dividing by multiple fission represent a unique single cell system enabling the preparation of highly synchronous cultures by application of a light–dark regime similar to what they experience in nature. This chapter provides detailed protocols for synchronization of different algal species by alternating light–dark cycles; all critical points are discussed extensively. Moreover, detailed information on basic analysis of cell cycle progression in suc...
Source: Springer protocols feed by Plant Sciences - December 14, 2015 Category: Biology Source Type: news
Antibody-Mediated Pathogen Resistance in Plants
We describe the selection from a phage display library of avian scFv antibodies that recognize cell surface proteins on fungi from the genus Fusarium, and the construction of scFv–AFP fusion protein constructs followed by their transient expression in tobacco (Nicotiana spp.) plants and stable expression in Arabidopsis thaliana plants. Using these techniques, the antibody fusion with the most promising in vitro activity can be used to generate transgenic plants that are resistant to pathogens such as Fusarium oxysporum f. sp. matthiolae. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
A Standardized Lepidopteran Bioassay to Investigate the Bioactivity of Insecticidal Proteins Produced in Transgenic Crops
Insecticidal bioassays are the only reliable method to investigate the biological activity of an insecticidal protein and therefore provide an essential toolkit for the characterization and potency determination of these proteins. Here we present a standardized method for a lepidopteran larval bioassay, which is optimized to specifically estimate activity of insecticidal proteins produced in transgenic plants. The treatment can be either applied to the surface of the artificial diet, or blended into the diet. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 4-3-2
This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready® soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples usin...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Analysis of Recombinant Proteins in Transgenic Rice Seeds: Identity, Localization, Tolerance to Digestion, and Plant Stress Response
Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purificati...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Molecular Analyses of Transgenic Plants
One of the major challenges in plant molecular biology is to generate transgenic plants that express transgenes stably over generations. Here, we describe some routine methods to study transgene locus structure and to analyze transgene expression in plants: Southern hybridization using DIG chemiluminescent technology for characterization of transgenic locus, SYBR Green-based real-time RT-PCR to measure transgene transcript level, and protein immunoblot analysis to evaluate accumulation and stability of transgenic protein product in the target tissue. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector
Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE® tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapa...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Continuous Flow Separation of Hydrophobin Fusion Proteins from Plant Cell Culture Extract
Fusion to fungal hydrophobins has proven to be a useful tool to enhance accumulation and recovery of recombinant proteins in plants. Aqueous two-phase separation (ATPS) is an attractive system to capture hydrophobin fusion proteins from plant extracts. The process can simultaneously purify and concentrate target protein with minimal background. ATPS avoids the use of chromatographic column steps, can be carried out in a short time frame, and is amenable to industrial-scale protein purification. A drawback of performing ATPS in large volumes is the lengthy time required for phase separation; however, this can be avoided by ...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Liquid-Liquid Phase Separation of Oil Bodies from Seeds
Fundamentally, oil bodies are discrete storage organelles found in oilseeds, comprising a hydrophobic triacylglycerol core surrounded by a half-unit phospholipid membrane and an outer shell of specialized proteins known as oleosins. Oil bodies possess a number of attributes that were exploited by SemBioSys Genetics to isolate highly enriched fractions of oil bodies through liquid-liquid phase separation for a number of commercial applications. The current chapter provides a general guide for the isolation of oil bodies from Arabidopsis and/or safflower seed, from which protocols can be refined for different oilseed sources...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Plant Cell-Based Recombinant Antibody Manufacturing with a 2 L Orbitally Shaken Disposable Bioreactor
Tobacco BY-2 cells are an attractive platform for the manufacture of a variety of biopharmaceutical proteins, including antibodies. Here, we describe the scaled-up cultivation of human IgG–secreting BY-2 cells in a 200 L orbitally shaken disposable bioreactor, resulting in cell growth and recombinant protein yields that are proportionately comparable with those obtained from cultivations in 500 mL shake flasks. Furthermore, we present an efficient downstream process for antibody recovery from the viscous spent culture medium using expanded bed adsorption (EBA) chromatography. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Temporary Immersion Bioreactors for the Contained Production of Recombinant Proteins in Transplastomic Plants
Despite the largely maternal inheritance of plastid genomes, the risk of transgene dissemination from transplastomic plants can limit the scope for field cultivation. There is a need for a cost-effective, scalable process to grow large quantities of transplastomic plant biomass for biosynthesis of biopharmaceuticals and other high-value heterologous proteins. Temporary immersion culture is a means of achieving this under fully contained conditions. This method describes the organogenesis of transplastomic Nicotiana tabacum callus in RITA® temporary immersion bioreactors to produce rootless leafy biomass, and subsequent...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Total Soluble Protein Extraction for Improved Proteomic Analysis of Transgenic Rice Plant Roots
With the advent of high-throughput platforms, proteomics has become a powerful tool to search for plant gene products of agronomic relevance. Protein extractions using multistep protocols have been shown to be effective to achieve better proteome profiles than simple, single-step extractions. These protocols are generally efficient for above ground tissues such as leaves. However, each step leads to loss of some amount of proteins. Additionally, compounds such as proteases in the plant tissues lead to protein degradation. While protease inhibitor cocktails are available, these alone do not seem to suffice when roots are in...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants
We previously described a procedure for the use of plant protease inhibitors as “companion” accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265–273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along—and co-migrating—with the protein of interest in host plant cells. An alternative, single transgene scheme is then ...
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Transient Expression of Mammalian Genes in N. benthamiana to Modulate N-Glycosylation
Nicotiana benthamiana has shown great success as a platform for the production of recombinant proteins. Here, we describe methods to transiently express high levels of recombinant proteins and simultaneously modulate their glycosylation pattern toward human-like structures. The method aims to generate recombinant proteins with a targeted largely homogeneous glycosylation profile for structure–function studies. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
Efficient, Antibiotic Marker-Free Transformation of a Dicot and a Monocot Crop with Glutamate 1-Semialdehyde Aminotransferase Selectable Marker Genes
Antibiotic-free, efficient in vitro selection in plant genetic engineering can improve risk perception and speed up pre-market scrutiny of genetically modified crops. We provide a protocol for genetic transformation of two important crops, durum wheat and alfalfa, using a bacterial and a plant-derived selectable marker gene encoding mutated, gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA) enzymes. These methods can potentially be applied, with minor adaptations, to many other monocot and dicot crop plants. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 1, 2015 Category: Biology Source Type: news
