Current protocols in plant biology最新文献

Parallel DNA assembly methods allow multiple fragments of DNA to be compiled in a desired order in a single reaction. Several methods enable the efficient one-step assembly of multiple DNA parts into a suitable plasmid acceptor at high efficiency. Type IIS-mediated assembly offers the specific advantage of a one-step reaction that does not require proprietary reagents or the amplification and purification of linear DNA fragments. Instead, multiple plasmids housing standardized DNA parts of interest are combined in an enzyme cocktail. To make these standard parts, DNA sequences with defined functions are assigned specific sequence features. This allows parts to be interoperable and reusable. The availability of collections of DNA parts and molecular toolkits that allow the facile assembly of multigene binary constructs and the establishment of standards for the creation of new parts means Type IIS-mediated assembly has become a powerful technology for modern plant molecular biologists. © 2016 by John Wiley & Sons, Inc.

This protocol describes one method for generating transgenic soybean (Glycine max) using particle bombardment of embryogenic cultures. Embryogenic cultures consist of proliferating masses of somatic embryos and are initially obtained from the cotyledons of immature seeds. Embryogenic cultures are bombarded with small DNA-coated particles and the cells that receive, incorporate and express the DNA are selected, based on a co-delivered antibiotic resistance gene. For recovery of whole plants from the antibiotic resistant embryogenic tissues, the tissues are first placed on a medium conducive to embryo development. Mature embryos are then dried for a short period and placed on a germination medium. Plantlets are then gradually exposed to ambient light and humidity conditions, prior to transfer to a greenhouse. Operators need to be constantly monitoring the protocol and observant of all outcomes. These procedures also are mostly descriptive and need to be precisely followed to be successful. © 2016 by John Wiley & Sons, Inc.

Chemical mutagenesis provides an inexpensive and straightforward way to generate a high density of novel nucleotide diversity in the genomes of plants and animals. Mutagenesis therefore can be used for functional genomic studies and also for plant breeding. The most commonly used chemical mutagen in plants is ethyl methanesulfonate (EMS). EMS has been shown to induce primarily single base point mutations. Hundreds to thousands of heritable mutations can be induced in a single plant line. A relatively small number of plants, therefore, are needed to produce populations harboring deleterious alleles in most genes. EMS mutagenized plant populations can be screened phenotypically (forward-genetics), or mutations in genes can be identified in advance of phenotypic characterization (reverse-genetics). Reverse-genetics using chemically induced mutations is known as Targeting Induced Local Lesions IN Genomes (TILLING). This unit gives information on EMS treatment of seed and vegetative propagules. © 2016 by John Wiley & Sons, Inc.

Epigenetic modification of DNA through methylation is known to be involved in multiple biological processes such as gene suppression. However, the exact mechanism of how DNA methylations play their part is yet unclear. In mammals, CpG islands (CGI) have been studied extensively for their involvement in cancer. Whereas in plants, despite the fact that there are not only CpG but also CHG and CHH contexts of methylation, an efficient and easy-to-use pipeline to decipher these phenomena is still to be developed. Both ZED-align and BisuKit are user-friendly apps deployed on CyVerse infrastructure where users can use their bisulfite sequence files to run multiple command line-based packages with minimal intervention. © 2016 by John Wiley & Sons, Inc.

Minichromosomes have been generated in maize using telomere-mediated truncation. Telomere DNA, because of its repetitive nature, can be difficult to manipulate. The protocols in this unit describe two methods for generating the telomere DNA required for the initiation of telomere-mediated truncation. The resulting DNA can then be used with truncation cassettes for introduction into maize via transformation. © 2016 by John Wiley & Sons, Inc.

High-quality preparations of chromosomes are useful for many purposes. To prepare metaphase chromosome spreads in maize, root tips are harvested and treated with nitrous oxide to stop cell division at metaphase before being fixed in acetic acid. This process allows a high number of condensed chromosome spreads to be obtained at the end of the procedure. To prepare chromosome spreads from various stages of meiosis, anthers are first fixed before being examined for developmental stage. Cells are digested with a mixture of enzymes before the chromosomes are dropped onto glass sides and fixed under UV light. © 2016 by John Wiley & Sons, Inc.

A wealth of maize mutants is now available with known sites of transposon insertions in over 45% of maize genes. Materials can be obtained free of charge from the UniformMu public resource through MaizeGDB.org or directly from the Maize Genetics COOP Stock Center. Specific mutants can be sought online based on gene-sequences of interest. A key feature of the UniformMu resource is the uniformity of wild-type controls, which facilitates characterization of mutant phenotypes. Methods developed for construction (transposon mutagenesis), analysis and utilization of the resource are described here. These include the high-throughput Mu-seq genotyping protocol that enables both forward and reverse approaches for linking genotypes-to-phenotypes. © 2016 by John Wiley & Sons, Inc.

Rice (Oryza sativa) is the most important consumed staple food for a large and diverse population worldwide. Since databases of genomic sequences became available, functional genomics and genetic manipulations have been widely practiced in rice research communities. Insertional mutants are the most common genetic materials utilized to analyze gene function. To mutagenize rice genomes, we exploited the transpositional activity of an Activator/Dissociation (Ac/Ds) system in rice. To mobilize Ds in rice genomes, a maize Ac cDNA was expressed under the CaMV35S promoter, and a gene trap Ds was utilized to detect expression of host genes via the reporter gene GUS. Conventional transposon-mediated gene-tagging systems rely on genetic crossing and selection markers. Furthermore, the activities of transposases have to be monitored. By taking advantage of the fact that Ds becomes highly active during tissue culture, a plant regeneration system employing tissue culture was employed to generate a large Ds transposant population in rice. This system overcomes the requirement for markers and the monitoring of Ac activity. In the regenerated populations, more than 70% of the plant lines contained independent Ds insertions and 12% expressed GUS at seedling stages. This protocol describes the method for producing a Ds-mediated insertional population via tissue culture regeneration systems. © 2016 by John Wiley & Sons, Inc.

Fluorescence In Situ Hybridization (FISH) is the annealing of fluorescent DNA probes to their complementary sequences on prepared chromosomes and subsequent visualization with a fluorescent microscope. In maize, FISH is useful for distinguishing each of the ten chromosomes in different accessions (karyotyping), roughly mapping single genes, transposable elements, transgene insertions, and identifying various chromosomal alterations. FISH can also be used to distinguish chromosomes between different Zea species in interspecific hybrids by use of retroelement painting. © 2016 by John Wiley & Sons, Inc.

The measurement of enzyme activities represents an important step towards the understanding of biological networks. Continuous or discontinuous assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, two protocols for phosphoenolpyruvate carboxylase activity (EC 4.1.1.31) in plant extracts are given. For this, an extraction protocol is also described. Then, an optimization protocol for enzyme assays using enzymatic, chemical, or biological standards is proposed. This protocol evaluates in one run the optimal extract dilution, the recovery of a standard, and the technical error in a given matrix. The interest of using biological standard in routine measurements is highlighted. © 2016 by John Wiley & Sons, Inc.