Current protocols in plant biology最新文献

In order to study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique does not require separation of cells by enzymatic digestion of any kind, does not require cell-specific reporter genes, and allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capture of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2018 by John Wiley & Sons, Inc.

Constitutive and dynamic protein-protein interactions are fundamental to all aspects of cellular processes. Compared to other techniques measuring protein-protein interactions in plants, the luciferase complementation assay has a number of advantages: it detects plant protein-protein interactions in real time, requires little hands-on manipulation of samples, is highly quantitative, has extremely low background, and can be easily scaled up for high-throughput interactome studies. Here, we describe a protocol that includes two alternate data collection methods to quantify luminescence results based on Agrobacterium-mediated transient luciferase expression in Nicotiana benthamiana. One data collection method employs a charge-coupled device imaging system that allows the interactions to be presented as images, and the other employs a luminometer, which enables the assay to be conducted in a 96-well plate. Technical parameters related to frequently encountered problems and common errors, presented here, are important for performing this assay successfully. © 2018 by John Wiley & Sons, Inc.

The following method enables the rapid production of transgenic potato plants and microtubers for gene validation and expression, or promoter studies. The method is highly efficient, with reproducible transformation efficiencies of at least 50% to 60% with potato cv. Desiree, and can produce transgenic microtubers within 6 months of initiation of the experiment. Microtubers are produced in the absence of hormones, giving an in vitro gene testing system broadly analogous to the natural state. © 2018 by John Wiley & Sons, Inc.

Among targeted proteomic techniques, AQUA-MRM is considered as one of the most reliable for accurate protein quantitation. This method displays high sensitivity, specificity, and reproducibility compared to many common biochemical techniques by coupling the use of unique, heavy-labeled peptide standards and triple-quadrupole mass spectrometry. However, there are several important steps that are required for successful development and validation of a robust AQUA-MRM assay. The following protocol outlines and details the key steps necessary for plant sample preparation as well as AQUA-MRM development and validation, specifically for absolute quantitation of plant proteins in vivo. © 2018 by John Wiley & Sons, Inc.

Activation of pattern-triggered plant immunity requires recognition of microbe-derived molecular patterns (MAMPs) by plant-encoded pattern recognition receptors (PRRs). Many plant PRRs are found in selected plant genera only. Transfer of single PRRs or of cassettes expressing several PRRs (PRR stacking) across plant genus boundaries offers the potential to boost disease resistance by improving pathogen recognition features in economically important crop plants. The success of such an approach is most dependent on the availability of a large number of plant PRRs. Here, an efficient method for the identification of novel PRRs in the model plant Arabidopsis thaliana (hereafter, Arabidopsis for simplicity) is described. This method takes advantage of natural variation in microbial pattern sensitivity among hundreds of Arabidopsis accessions currently available. Identification of pattern-sensitive as well as pattern-insensitive accessions facilitates next-generation sequencing (NGS)–assisted mapping of PRRs. This approach is potentially applicable to the identification of PRRs that recognize patterns of any chemical nature. © 2017 by John Wiley & Sons, Inc.

Oilseed rape (Brassica napus) is a commercially important member of the Brassicacea family. It is grown for its edible and industrial oils as well as for animal feed. Genetic transformation technology has been used to study gene function and produce oilseed rape with improved agronomic characteristics. This protocol describes a method for the Agrobacterium tumefaciens–mediated transformation of oilseed rape cotyledonary petioles. The method is reproducible and has been used to transform both spring and winter cultivars. Modifications have been made to the rooting stage, which have reduced the vitrification of shoots. This has not only increased the number of phenotypically normal shoots but has also resulted in an increase in transformation efficiency, concomitant with a dramatic reduction in the number of escapes regenerated. Transformation frequencies typically range from 5% to 10%, with an average of 12% using doubled haploid model varieties, but a maximum efficiency of 20% has been achieved. © 2017 by John Wiley & Sons, Inc.

Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

Plant recognition of invading organisms occurs through identification of foreign molecules associated with attackers and of self-derived, damage-associated molecules. Perception of these molecules activates signaling processes including dynamic changes in ion balance, production of second messengers such as reactive oxygen species and nitric oxide, increased levels of plant hormones, and map kinase cascade activation. Together these signaling events stimulate transcriptional changes to initiate plant defense responses. Among the earliest detectable signaling events is a rapid increase in apoplastic pH, i.e., extracellular alkalinization. Here, an assay for quantification of this alkalinization response using suspension-cultured cell lines for Arabidopsis, potato, and maize is described. This assay is an inexpensive, fast, simple, and reproducible method to quantify defense signaling output, providing a powerful tool for evaluating early plant responses to elicitors and pathogens. Results from the alkalinization assay are comparable to other more costly and time-consuming methods for assessing defense signaling, such as measurement of the oxidative burst, calcium influx, and marker gene expression. This bioassay is a quantitative and robust method for evaluation of plant defense output. © 2017 by John Wiley & Sons, Inc.

Non-destructive methods to quantify the root system architecture of a plant grown in soil are essential to aid our understanding of the factors that impact plant root development in natural environments. With environmental change threatening our ability to sustain agricultural productivity for an expanding global population, the application of these methods has never before seen such an increase in demand. X-ray computed tomography (CT) based phenotyping techniques permit the spatio-temporal quantification of roots, helping to identify novel adaptive root architectural responses to abiotic and biotic factors. This protocol reports an integrated workflow from column preparation and plant growth to image and quantification of the root system using novel open source software applications, RooTrak and RooTh. © 2017 by John Wiley & Sons, Inc.