Current Protocols in Plant Biology Virtual Issue

The Editors of Current Protocols in Plant Biology are pleased to bring you this special protocol-based virtual issue. This collection highlights the most downloaded protocols from CPPB in the past two years, in the areas of DNA and RNA extraction, protein extraction and quantification, protein-protein interactions, genetics and genomics, transformation, and genotyping.

Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation

In order to study the transcriptome of individual plant cells at specific points in time, protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery were developed. 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.

Highly Efficient Agrobacterium‐Mediated Transformation of Potato (Solanum tuberosum) and Production of Transgenic Microtubers

This 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 cultivar Desiree, and can produce transgenic microtubers within 6 months of initiation of the experiment.

A Guide to Genome‐Wide Association Mapping in Plants

An overview of the design, analysis, and interpretation of GWAS. Strategies for prioritizing GWA candidates for further statistical and empirical validation are highlighted. While focused on plants, the material covered is also applicable to other systems.

Generation of Soybean (Glycine max) Transient Transgenic Roots

This protocol describes a method to generate transgenic soybean roots in as little as 3 weeks. While using the traditional Agrobacterium tumefaciens method, stable transgenic plants take 6 to 12 months to create, depending on species.

Absolute Quantitation of Plant Proteins

This protocol outlines and details the key steps necessary for plant sample preparation as well as development and validation of the mass spectrometry-based method known as AQUA‐MRM. The focus is specifically for absolute quantitation of plant proteins in vivo.

Luciferase Complementation Assay for Protein‐Protein Interactions in Plants

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.

Using Xenopus laevis Oocytes to Functionally Characterize Plant Transporter

Isolation of Xenopus laevis oocytes and preparation of the capped sense RNA (cRNA) of the maize boron importer TASSEL‐LESS1 (TLS1) were described. The cRNA was microinjected into the isolated oocytes, and functionally assess the boron import capabilities of TLS1 in an oocyte swelling assay. These protocols can be easily adapted to study other plant and non‐plant transporters with putative import function.

Genotyping‐by‐Sequencing

Genotyping‐by‐sequencing refers to a suite of related methods that obtain genotype data from samples by using restriction enzyme digestion followed by high‐throughput sequencing. This protocol contains the steps necessary to go from purified DNA to Illumina‐ready libraries. It also covers the considerations that go into planning a genotyping‐by‐sequencing experiment.

Extraction and Analysis of Terpenes/Terpenoids

The protocols presented here focus on the extraction of terpenes/terpenoids from various plant sources and have been divided into extraction methods for terpenes/terpenoids with various levels of chemical decoration—from relatively small, nonpolar, volatile hydrocarbons to substantially large molecules with greater physical complexity due to chemical modifications.