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Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae

Featured protocol,  Authors: Yun-Long Zhao
Yun-Long ZhaoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
For correspondence: zyl714@126.com
Bio-protocol author page: a4092
Tao Zhang
Tao Zhang Affiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4093
 and Hui-Shan Guo
Hui-Shan GuoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a2721
date: 2/20/2017, 46 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2133.

Brief version appeared in PLoS Pathog, Jul 2016
Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et al., 2011; Yadeta and Thomma, 2013). Our recent work has shown that V. dahliae develops hyphopodium as an infection structure to breach plant root cell wall (Zhao et al., 2016). Here, we provide a detailed protocol to analyze the penetration ability and the pathogenicity of V. dahliae as well as recover fungal hyphae from infected cotton stems developed from our previous studies (Zhang et al., 2016a and 2016b; Zhao et al., 2016). Cellophane membrane has been used in inducing appressorium development of foliar pathogens but not root pathogens (Bourett and Howard, 1990). We adopted the method of using the cellophane membrane to induce and assess the development of hyphopodium. Hopefully, it will greatly promote the research of molecular events involved in recognition of the host that regulate infectious development. This protocol is also helpful to identify the key component controlling the pathogenicity of V. dahliae and widen our understanding of the mechanism of plant-microbe interaction.

Rice Root Organic Acid Efflux Measurement by Using Ion Chromatography

Featured protocol,  Authors: Chun-quan Zhu
Chun-quan ZhuAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4131
Xiao-fang Zhu
Xiao-fang Zhu Affiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4132
 and Ren-fang Shen
Ren-fang ShenAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
For correspondence: rfshen@issas.ac.cn
Bio-protocol author page: a4133
date: 2/20/2017, 52 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2141.

Brief version appeared in Plant Physiol, Jun 2016
Organic acids secreted from plant roots play important roles in various biological processes including nutrient acquisition, metal detoxification, and pathogen attraction. The secretion of organic acids may be affected by various conditions such as plant growth stage, nutrient deficiency, and abiotic stress. For example, when white lupin (Lupinus albus L.) is exposed to phosphorus (P)-deficient conditions, the secretion of citrate acid from its proteoid roots significantly increases (Neumann et al., 1999). This protocol describes a method for the collection and measurement of the efflux of organic acids (oxalate, malate, and citrate) from the roots of rice cultivar Nipponbare (‘Nip’) under different nitrogen forms (NH4+ and NO3-), together with different P supply (+P and -P) conditions.

Establishment of New Split-root System by Grafting

Featured protocol,  Authors: Xiangqiang Kong
Xiangqiang KongAffiliation: Cotton Research Center, Shandong Key Lab for Cotton Culture and Physiology, Shandong Academy of Agricultural Sciences, Jinan, China
Bio-protocol author page: a4098
Zhen Luo
Zhen Luo Affiliation: Cotton Research Center, Shandong Key Lab for Cotton Culture and Physiology, Shandong Academy of Agricultural Sciences, Jinan, China
Bio-protocol author page: a4099
 and Hezhong Dong
Hezhong DongAffiliation: Cotton Research Center, Shandong Key Lab for Cotton Culture and Physiology, Shandong Academy of Agricultural Sciences, Jinan, China
For correspondence: donghz@saas.ac.cn
Bio-protocol author page: a4100
date: 2/20/2017, 24 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2136.

Brief version appeared in J Exp Bot, Mar 2012
A new split-root system was used to simulate non-uniform salt, drought or nutrient deficiency stress in the root zone, in which the root system was divided into two or more equal portions. Here, we established a split-root system by grafting of cotton seedlings. In contrast to the conventional split-root, the main roots of the new system remained intact, which provided a better system for studying cotton response to unequal treatment in the root zone. The new system was suitable for plant growth in nutrient solution and the two root systems can fully be immerged in the nutrient solution.

Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere

Featured protocol,  Authors: Carlos A. Ruiz-Pérez
Carlos A. Ruiz-PérezAffiliation 1: School of Biological Sciences, Georgia Institute of Technology, Ford Environmental Sciences & Technology Building, Atlanta, USA
Affiliation 2: Molecular Genetics, Corporación Corpogen, Bogotá, Colombia
For correspondence: cruizperez3@gatech.edu
Bio-protocol author page: a4126
 and María Mercedes Zambrano
María Mercedes ZambranoAffiliation: Molecular Genetics, Corporación Corpogen, Bogotá, Colombia
Bio-protocol author page: a4127
date: 2/20/2017, 30 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2142.

Brief version appeared in Appl Environ Microbiol, Jan 2016
The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of microbial diversity and they can be important for plant health. The characterization of microbial communities in diverse plants, such as Espeletia plants that are endemic to the Paramo ecosystem in the Andes Mountains, can shed light regarding possible interactions among microorganisms and microbial functional properties. Obtaining DNA from plant endophytic microbial communities involves various steps to ensure that samples are free of contamination from microorganisms present on the plant surface (epiphytes). Plant leaves are first surface sterilized, cut into pieces, homogenized using glass beads, and then used for DNA extraction using a commercially available kit. DNA samples are then quantified and analyzed using Qubit® 2.0 for use in PCR amplification of 16S rRNA genes.

Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters

Featured protocol,  Authors: Tao Zhang*
Tao ZhangAffiliation: National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a4181
Yangbin Gao*
Yangbin GaoAffiliation: Section of Cell and Developmental Biology, University of California, San Diego, USA
Bio-protocol author page: a4182
Rongchen Wang
Rongchen WangAffiliation: National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a4183
 and Yunde Zhao
Yunde ZhaoAffiliation 1: National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China
Affiliation 2: Section of Cell and Developmental Biology, University of California, San Diego, USA
For correspondence: yundezhao@ucsd.edu
Bio-protocol author page: a4184
 (*contributed equally to this work) date: 2/20/2017, 31 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2148.

Brief version appeared in J Integr Plant Biol, Apr 2014
CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct tissue/cell specific gene editing.

Chromatographic Separation of the Codonocarpine Type Alkaloids from the Root Bark of Capparis decidua

Featured protocol,  Authors: Yvonne Forster
Yvonne ForsterAffiliation: Department of Chemistry, University of Zurich, Zurich, Switzerland
Bio-protocol author page: a4136
Abdul Ghaffar
Abdul GhaffarAffiliation: Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
Bio-protocol author page: a4135
 and Stefan Bienz
Stefan BienzAffiliation: Department of Chemistry, University of Zurich, Zurich, Switzerland
For correspondence: stefan.bienz@chem.uzh.ch
Bio-protocol author page: a4137
date: 2/20/2017, 41 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2144.

Brief version appeared in Phytochemistry, Aug 2016
Various parts of the caper tree Capparis decidua have found application in traditional medicine. The isolation and structural elucidation of the codonocarpine type alkaloids contained in the root bark, however, is not trivial and has probably led to misinterpretation in the past. This protocol describes the extraction and chromatographic separation of the four major alkaloids of the root bark of Capparis decidua. The delivered samples of cadabicine, codonocarpine, isocodonocarpine and capparidisinine were suitable for their unambiguous structural elucidation by NMR, MS and MS/MS.

Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells

Featured protocol,  Authors: Vincent Bayle
Vincent BayleAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
Bio-protocol author page: a4180
Matthieu Pierre Platre
Matthieu Pierre PlatreAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
Bio-protocol author page: a4102
 and Yvon Jaillais
Yvon JaillaisAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
For correspondence: yvon.jaillais@ens-lyon.fr
Bio-protocol author page: a4103
date: 2/20/2017, 36 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2145.

Brief version appeared in Nat Plants, Jun 2016
In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility between laboratories. In this protocol, we describe procedures for automatic counting of the number of intracellular compartments in Arabidopsis root cells, which can be used for example to study endocytosis or secretory trafficking pathways and to compare membrane organization between different genotypes or treatments. While developed for Arabidopsis roots, this method can be used on other tissues, cell types and plant species.

Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies

Featured protocol,  Authors: Shengping Li
Shengping LiAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4023
Yanhe Zhao
Yanhe ZhaoAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4024
 and Yunkun Wu
Yunkun WuAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
For correspondence: wuyk@fjirsm.ac.cn
Bio-protocol author page: a4025
date: 2/5/2017, 121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2122.

Brief version appeared in Plant Cell, May 2015
GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally containing a conserved GRAS domain, which is about 350-450 resides and can be subdivided into five distinct motifs with their name derived from the most prominent amino acids: LRI (leucine-rich region I), VHIID, LRII (leucine-rich region II), PFYRE and SAW and mainly function in the interaction between GRAS proteins and their partners (Sun et al., 2012).By phylogenetic analysis, the GRAS family can be divided into more than ten subfamilies, of which SCL4/7 is one important subgroup and functions in response to environmental stresses. Here we describe a detailed protocol forthe expression and purification of the GRAS domain of Os-SCL7, a SCL4/7 member in rice, which enable us to crystallize it and determine its structure.

Miniature External Sapflow Gauges and the Heat Ratio Method for Quantifying Plant Water Loss

Featured protocol,  Author: Robert Paul Skelton
Robert Paul SkeltonAffiliation: University of California Berkeley, Department of Integrative Biology, 4003 Valley Life Sciences Building, Berkeley, CA, USA
For correspondence: skelrob@gmail.com
Bio-protocol author page: a4055
date: 2/5/2017, 130 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2121.

Brief version appeared in Funct Plant Biol, Oct 2013
External sapflow sensors are a useful tool in plant ecology and physiology for monitoring water movement within small stems or other small plant organs. These gauges make use of heat as a tracer of water movement through the stem and can be applied in both a laboratory and a field setting to generate data of relatively high temporal resolution. Typical outputs of these data include monitoring plant water use on a diurnal time scale or over a season (e.g., in response to increasing water deficit during drought) to gain insight into plant physiological strategies. This protocol describes how to construct the gauges, how best to install them and some expected data outputs.

Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae

Authors: Yun-Long Zhao
Yun-Long ZhaoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
For correspondence: zyl714@126.com
Bio-protocol author page: a4092
Tao Zhang
Tao Zhang Affiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4093
 and Hui-Shan Guo
Hui-Shan GuoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a2721
date: 2/20/2017, 46 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2133.

[Abstract] Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et ...

Rice Root Organic Acid Efflux Measurement by Using Ion Chromatography

Authors: Chun-quan Zhu
Chun-quan ZhuAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4131
Xiao-fang Zhu
Xiao-fang Zhu Affiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4132
 and Ren-fang Shen
Ren-fang ShenAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
For correspondence: rfshen@issas.ac.cn
Bio-protocol author page: a4133
date: 2/20/2017, 52 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2141.

[Abstract] Organic acids secreted from plant roots play important roles in various biological processes including nutrient acquisition, metal detoxification, and pathogen attraction. The secretion of organic acids may be affected by various conditions such as plant growth stage, nutrient deficiency, and abiotic ...

Establishment of New Split-root System by Grafting

Authors: Xiangqiang Kong
Xiangqiang KongAffiliation: Cotton Research Center, Shandong Key Lab for Cotton Culture and Physiology, Shandong Academy of Agricultural Sciences, Jinan, China
Bio-protocol author page: a4098
Zhen Luo
Zhen Luo Affiliation: Cotton Research Center, Shandong Key Lab for Cotton Culture and Physiology, Shandong Academy of Agricultural Sciences, Jinan, China
Bio-protocol author page: a4099
 and Hezhong Dong
Hezhong DongAffiliation: Cotton Research Center, Shandong Key Lab for Cotton Culture and Physiology, Shandong Academy of Agricultural Sciences, Jinan, China
For correspondence: donghz@saas.ac.cn
Bio-protocol author page: a4100
date: 2/20/2017, 24 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2136.

[Abstract] A new split-root system was used to simulate non-uniform salt, drought or nutrient deficiency stress in the root zone, in which the root system was divided into two or more equal portions. Here, we established a split-root system by grafting of cotton seedlings. In contrast to the conventional split-root, ...

Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere

Authors: Carlos A. Ruiz-Pérez
Carlos A. Ruiz-PérezAffiliation 1: School of Biological Sciences, Georgia Institute of Technology, Ford Environmental Sciences & Technology Building, Atlanta, USA
Affiliation 2: Molecular Genetics, Corporación Corpogen, Bogotá, Colombia
For correspondence: cruizperez3@gatech.edu
Bio-protocol author page: a4126
 and María Mercedes Zambrano
María Mercedes ZambranoAffiliation: Molecular Genetics, Corporación Corpogen, Bogotá, Colombia
Bio-protocol author page: a4127
date: 2/20/2017, 30 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2142.

[Abstract] The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of ...

Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters

Authors: Tao Zhang*
Tao ZhangAffiliation: National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a4181
Yangbin Gao*
Yangbin GaoAffiliation: Section of Cell and Developmental Biology, University of California, San Diego, USA
Bio-protocol author page: a4182
Rongchen Wang
Rongchen WangAffiliation: National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a4183
 and Yunde Zhao
Yunde ZhaoAffiliation 1: National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China
Affiliation 2: Section of Cell and Developmental Biology, University of California, San Diego, USA
For correspondence: yundezhao@ucsd.edu
Bio-protocol author page: a4184
 (*contributed equally to this work) date: 2/20/2017, 31 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2148.

[Abstract] CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method ...

Chromatographic Separation of the Codonocarpine Type Alkaloids from the Root Bark of Capparis decidua

Authors: Yvonne Forster
Yvonne ForsterAffiliation: Department of Chemistry, University of Zurich, Zurich, Switzerland
Bio-protocol author page: a4136
Abdul Ghaffar
Abdul GhaffarAffiliation: Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
Bio-protocol author page: a4135
 and Stefan Bienz
Stefan BienzAffiliation: Department of Chemistry, University of Zurich, Zurich, Switzerland
For correspondence: stefan.bienz@chem.uzh.ch
Bio-protocol author page: a4137
date: 2/20/2017, 41 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2144.

[Abstract] Various parts of the caper tree Capparis decidua have found application in traditional medicine. The isolation and structural elucidation of the codonocarpine type alkaloids contained in the root bark, however, is not trivial and has probably led to misinterpretation in the past. This protocol describes ...

Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells

Authors: Vincent Bayle
Vincent BayleAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
Bio-protocol author page: a4180
Matthieu Pierre Platre
Matthieu Pierre PlatreAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
Bio-protocol author page: a4102
 and Yvon Jaillais
Yvon JaillaisAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
For correspondence: yvon.jaillais@ens-lyon.fr
Bio-protocol author page: a4103
date: 2/20/2017, 36 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2145.

[Abstract] In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility ...

Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies

Authors: Shengping Li
Shengping LiAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4023
Yanhe Zhao
Yanhe ZhaoAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4024
 and Yunkun Wu
Yunkun WuAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
For correspondence: wuyk@fjirsm.ac.cn
Bio-protocol author page: a4025
date: 2/5/2017, 121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2122.

[Abstract] GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally ...

Miniature External Sapflow Gauges and the Heat Ratio Method for Quantifying Plant Water Loss

Author: Robert Paul Skelton
Robert Paul SkeltonAffiliation: University of California Berkeley, Department of Integrative Biology, 4003 Valley Life Sciences Building, Berkeley, CA, USA
For correspondence: skelrob@gmail.com
Bio-protocol author page: a4055
date: 2/5/2017, 130 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2121.

[Abstract] External sapflow sensors are a useful tool in plant ecology and physiology for monitoring water movement within small stems or other small plant organs. These gauges make use of heat as a tracer of water movement through the stem and can be applied in both a laboratory and a field setting to generate ...

Nitrate Assay for Plant Tissues

Authors: Lufei Zhao
Lufei ZhaoAffiliation: State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, China
For correspondence: lufeizh@163.com
Bio-protocol author page: a3770
 and Yong Wang
Yong WangAffiliation: State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, China
Bio-protocol author page: a3771
date: 1/20/2017, 413 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2029.

[Abstract] Nitrogen is an essential macronutrient for plant growth and nitrate content in plants can reflect the nitrogen supply of soil. Here, we provide the salicylic acid method to evaluate the nitrate content in plant tissues. The method is reliable and stable, thus it can be a good choice for measurement ...
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Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves Updates
The author made some updates (highlighted in blue) to the protocol on 09/19/2016.

Authors: Arsalan Daudi
Arsalan DaudiAffiliation 1: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Affiliation 2: Department of Plant Pathology, University of California, Davis, CA, USA
For correspondence: aadaudi@ucdavis.edu
Bio-protocol author page: a107
 and Jose A. O’Brien
Jose A. O’BrienAffiliation: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Bio-protocol author page: a108
date: 9/20/2012, 31197 views, 15 Q&A
DOI: https://doi.org/10.21769/BioProtoc.263.

[Abstract] In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi ...

[Bio101] Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/20/2011, 30176 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.95.

[Abstract] Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, ...

[Bio101] Pollen Fertility/viability Assay Using FDA Staining

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 5/20/2011, 15913 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.75.

[Abstract] Pollen grains can be fertile or sterile by nature. This method stains pollen grains for an enzyme as the vital indicator of membrane integrity. Only fertile grains fluoresce under microscopic examination....

[Bio101] Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/5/2011, 15173 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.93.

[Abstract] Promoter-driven GUS (beta-glucuronidase) activity is the most commonly used technique for tissue-specific expression patterns in Arabidopsis. In this procedure, GUS enzyme converts 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) to a blue product. The staining is very sensitive. Processed samples can ...

[Bio101] Arabidopsis Pollen Tube Aniline Blue Staining

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Scienc, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 6/20/2011, 14881 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.88.

[Abstract] The aim of this experiment is to track pollen tube growth in vivo in the female tissues after pollination. This can be used to phenotype pollen germination, tube growth and guidance, and reception....

[Bio101] Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well)

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/5/2011, 14165 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.90.

[Abstract] This is a simple protocol for isolating genomic DNA from fresh plant tissues. DNA from this experiment can be used for all kinds of genetics studies, including genotyping and mapping. This protocol uses Edward’s extraction buffer to isolate DNA....

In vitro Protein Ubiquitination Assays

Authors: Qingzhen Zhao
Qingzhen ZhaoAffiliation: College of Life Science, Liaocheng University, Liaocheng, China
Bio-protocol author page: a887
 and Qi Xie
Qi XieAffiliation: State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology Chinese Academy of Sciences, Beijing, China
For correspondence: qxie@genetics.ac.cn
Bio-protocol author page: a888
date: 10/5/2013, 13797 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.928.

[Abstract] Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity ...

[Bio101] Arabidopsis Growing Protocol – A General Guide

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 9/5/2011, 12804 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.126.

[Abstract] Arabidopsis as the model organism for higher plants is widely studied among plant biology labs around the world. However, taking care of this tiny plant may not be trivial. Here is a general guide used for the Heven Sze lab at the University of Maryland, College Park. A lot of efforts have been taken ...

Seed Germination and Viability Test in Tetrazolium (TZ) Assay

Authors: Pooja Verma
Pooja VermaAffiliation: NIPGR, National Institute of Plant Genome Research, New Delhi, India
Bio-protocol author page: a799
 and Manoj Majee
Manoj MajeeAffiliation: NIPGR, National Institute of Plant Genome Research, New Delhi, India
For correspondence: manojmajee@nipgr.ac.in
Bio-protocol author page: a800
date: 9/5/2013, 11978 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.884.

[Abstract] Tetrazolium (TZ) assay is the fast evaluation for seed viability and alternative quick method for seed’s germinability (Porter et al., 1947; Wharton, 1955). All respiring tissues are capable of converting a colourless compound, TZ (2,3,5 triphenyl tetrazolium chloride) to a carmine red coloured water-insoluble ...

[Bio101] A Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 5/20/2011, 10942 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.70.

[Abstract] This method can be used to free and separate the mesophyll cells from Arabidopsis leaves. The protoplasts that are generated in this way can be used for transient expression for protein activity and subcellular localization assays....
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