Current protocols in plant biology最新文献

Rice (Oryza sativa L.) is the staple food for more than half of the world's population, and has also become an important model monocot. As a result, numerous genetic transformation protocols have been developed to improve and better understand this particular agronomic plant. Here we introduce a convenient transformation method using Agrobacterium. The explants used are embryogenic calli derived from mature seeds, which are easily obtained and can be used all year round. After selection and regeneration, transformants are obtained from resistant calli cultured on the regeneration medium. This protocol has been used to generate transgenic rice (Oryza sativa L.) in as little as 4 months. © 2016 by John Wiley & Sons, Inc.

Chromosome painting (CP) refers to fluorescence in situ hybridization (FISH) of chromosome-specific DNA probes to identify large chromosome regions, chromosome arms, and whole chromosomes. For CP and CCP (comparative chromosome painting) in plants, most often, contigs of chromosome-specific bacterial artificial chromosomes (BAC) from the species of origin or a related species are used as painting probes. CP enables visualization and tracing of particular chromosome regions and/or chromosomes throughout all mitotic and meiotic stages as well as the corresponding interphase chromosome territories. CCP enables identification of large-scale homeologous chromosome regions and chromosomes shared among two or more species. Here, a step-by-step protocol for carrying out CP in Arabidopsis thaliana (Arabidopsis) and CCP in other crucifer taxa based on the use of Arabidopsis chromosome-specific BAC contigs is described. © 2016 by John Wiley & Sons, Inc.

In multi-cellular organisms, gene expression is orchestrated by thousands of transcription factors (TF). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a robust tool to investigate gene expression because this technique profiles in vivo protein-DNA interaction at a genome-wide scale. Eight years after the first ChIP-seq paper, there are limited reports of ChIP-seq experiments in plants, especially for sequence-specific DNA binding TFs. This lag greatly prevents our understanding of transcriptional regulation in an entire kingdom. In order to bridge the technical gap, we describe a ChIP-seq procedure that we have successfully applied to dozens of sequence-specific DNA binding TFs. The basic protocol includes procedures to isolate nuclei, sonicate chromatin, immunoprecipitate TF-DNA complex, and recover ChIP-enriched DNA fragments. The support protocol also describes practices to optimize library preparation by a gel-free DNA size selection. Lastly, examples are given to optimize library amplification using real-time PCR. © 2016 by John Wiley & Sons, Inc.

Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200 to 300 base pair size range. The recombinant viruses are used to infect experimental plants, and wherever the virus invades, the target gene or genes will be silenced. VIGS is thus transient, and in the span of a few weeks, it is possible to design VIGS constructs and then generate loss-of-function phenotypes through RNA silencing of the target genes. In soybean (Glycine max), the Bean pod mottle virus (BPMV) has been engineered to be valuable tool for silencing genes with diverse functions and also for over-expression of foreign genes. This protocol describes a method for designing BPMV constructs and using them to silence or transiently express genes in soybean. © 2016 by John Wiley & Sons, Inc.

Genetic bottlenecks during domestication and modern breeding limited the genetic diversity of soybean (Glycine max (L.) Merr.). Therefore, expanding and diversifying soybean genetic resources is a major priority for the research community. These resources, consisting of natural and induced genetic variants, are valuable tools for improving soybean and furthering soybean biological knowledge. During the twentieth century, researchers gathered a wealth of genetic variation in the forms of landraces, Glycine soja accessions, Glycine tertiary germplasm, and the U.S. Department of Agriculture (USDA) Type and Isoline Collections. During the twenty-first century, soybean researchers have added several new genetic and genomic resources. These include the reference genome sequence, genotype data for the USDA soybean germplasm collection, next-generation mapping populations, new irradiation and transposon-based mutagenesis populations, and designer nuclease platforms for genome engineering. This paper briefly surveys the publicly accessible soybean genetic resources currently available or in development and provides recommendations for developing such genetic resources in the future. © 2016 by John Wiley & Sons, Inc.

Terpenes/terpenoids constitute one of the largest classes of natural products, due to the incredible chemical diversity that can arise from the biochemical transformations of relatively simple prenyl diphosphate starter units. All terpenes/terpenoids comprise a hydrocarbon backbone generated from various length prenyl diphosphates (a polymer chain of prenyl units). Upon ionization (removal) of the diphosphate group, the remaining allylic carbocation intermediates can be coaxed down complex chemical cascades leading to diverse linear and cyclized hydrocarbon backbones that can be further modified with a wide range of functional groups (e.g., alcohols or ketones) and substituent additions (e.g., sugars or fatty acids). Because of this chemical diversity, terpenes/terpenoids have great industrial uses as flavors, fragrances, high grade lubricants, biofuels, agricultural chemicals, and medicines. 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. © 2016 by John Wiley & Sons, Inc.

Transient gene expression in protoplasts provides a powerful tool to study protein expression, protein localization, protein-protein association, and gene expression regulation, etc. There are several methods including electroporation, which have been reported to introduce DNA into protoplasts. However, one of the best methods used is polyethylene glycol (PEG)-mediated transfection. Here, we describe an improved PEG-mediated transformation method including preparation of protoplasts, PEG-mediated transformation, and, by way of example, expression of the AtLYK5 gene (AT2G33580) in protoplasts. The protoplast transient expression system provides unique capabilities to support cell-based experiments involved in plant biochemistry and physiology. © 2016 by John Wiley & Sons, Inc.

Precise genome modification using artificial nucleases is a powerful tool for in-depth understanding of gene functions and for creating new varieties. The CRISPR/Cas system, derived from an adaptive immunity system in bacteria and archaea, can introduce DNA double-strand breaks (DSBs) into pre-selected genomic loci and lead to loss of gene function due to error-prone non-homologous end joining (NHEJ). RNA-guided nucleases have been widely used in several eukaryotic organisms. In this article, we provide a detailed protocol for designing and constructing gRNA targets, detecting nuclease activity in transient protoplast assays, and identifying mutations in transgenic plants (including rice, wheat and maize). Targeted mutations in T0 plants can be generated in 14 to 18 weeks. © 2016 by John Wiley & Sons, Inc.

A simple, robust, inexpensive, high-throughput method for isolating genomic DNA from maize (Zea mays) leaf tissues is described. The DNA obtained using this extraction protocol is suitable for polymerase chain reaction (PCR) genotyping, which can be employed for the identification of alleles in diverse genetic and breeding approaches, such as marker-assisted selection, genetic fine mapping, and mutant introgression. This method utilizes 96-well plates for the collection of leaf tissue and the subsequent isolation of genomic DNA. The DNA isolation step is performed inexpensively within 3 hr and uses a urea-based extraction buffer that does not require an organic extraction step. Yields of genomic DNA are sufficient to perform ∼25 PCR-genotyping reactions per sample. These qualities, coupled with the protocol being robust and easy for inexperienced users to master, make this method ideal for new researchers. © 2016 by John Wiley & Sons, Inc.

In Arabidopsis thaliana, various attempts have been made to create artificial chromosomes as a new tool for cytological and genetic analyses. However, most of the efforts have been unsuccessful until recently. Most eukaryotic chromosomes are linear, and therefore the Arabidopsis artificial chromosomes have also been designed to be linear and to carry the telomere structure at both ends. In contrast, circular artificial chromosomes were successfully created by the Cre/LoxP system combined with Ac/Ds transposon system, on the basis of the discovery that ring minichromosomes are relatively stable and transmissible to the next generations in A. thaliana. Because ring minichromosomes ∼1 to 6 Mb in size have been generated, in this article, the protocol for inducing large chromosomal rearrangements resulting in ring chromosome formation is described. © 2016 by John Wiley & Sons, Inc.