植物学技术

Molecular techniques have created the opportunity for great advances in plant mutation genetics and the science of mutation breeding. The powerful targeted induced local lesions in genomes (TILLING) technique has introduced the possibility of reverse genetics—the ability to sc ...

The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to u ...

Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simpl ...

The construction of full-length cDNA libraries allows researchers to study gene expression and protein interactions and undertake gene discovery. Recent improvements allow the construction of high-quality cDNA libraries, with small amounts of mRNA. In parallel, these improve ...

Cereal grains, as storage tissues of the plant, contain high amounts of starch. Purification of RNA from plant tissue especially from seed tissue can be challenging due to this high starch content. Starch coprecipitates with RNA in the presence of isopropanol or ethanol and can interfere with the ...

Ribonucleic acid (RNA) extraction is the necessary first step in many protocols, primarily to investigate genes and gene expression. RNA comes in a variety of forms: total RNA, ribosomal RNA, messenger RNA (mRNA), and small interfering RNA (siRNA) to name a few. In some instances, total RNA is all that is ...

Deoxyribonucleic acid (DNA) extracted from endosperm can be effectively used for rapid genotyping using seed tissue, to evaluate seed quality from packaged grains and to determine the purity of milled grains. Methods outlined here are optimal procedures to isolate DNA from endosperm t ...

Advances in sequencing technologies have aided the discovery of millions of genome-wide DNA polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertion–deletions (InDels) which are an invaluable resource for marker-assisted breeding. Presently availa ...

With the rapid development of sequencing technologies and sequenced genomes, single-nucleotide polymorphisms (SNPs) have become a common genomic tool in the study of biological diversity, genome variation, gene mapping, cloning, and marker-assisted selection. In this chapter, ...

Agrobacterium tumefaciens-mediated transformation is one of the most efficient and simple gene delivery systems for genetic improvement and biology studies in maize. This system has become more widely used by both public and private laboratories. However, transformation effic ...

Agrobacterium is a common soil bacterium with natural capacity for trans-kingdom transfer of genetic information by transferring its T-DNA into the eukaryotic genome. In agricultural plant biotechnology, combination of non-phytopathogenic strain of Agrobacterium tumef ...

Agrobacterium-mediated transformation of barley is a valuable tool for determining gene function by over-expression of a gene of interest or by RNAi-based gene silencing. The method is based on the inoculation of immature embryos with Agrobacterium and uses a hygromycin resistance ge ...

The quality of extracted DNA is crucial for several applications in molecular biology. If the DNA is to be used for next-generation sequencing (NGS), then microgram quantities of good-quality DNA is required. In addition, the DNA must substantially be of high molecular weight so that it can be used f ...

The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is ...

Particle bombardment transformation describes the acceleration of high-velocity microparticles coated with exotic genes through the plant-protective cell walls, in order for the introduced genes to be integrated into the host genome. This technique has proven to be an effective a ...

Since its first invention in the late 1980s the particle gun has evolved from a basic gunpowder driven machine firing tungsten particles to one more refined which uses helium gas as the propellant to launch alternative heavy metal particles such as gold and silver. The simple principle is that DNA- ...

The transfer and immobilization of biological macromolecules onto solid nitrocellulose or nylon (polyvinylidene difluoride (PVDF)) membranes subsequently followed by specific detection is referred to as blotting. DNA blots are called Southerns after the inventor of the tec ...

The significance of small RNAs, being an important part of the cellular machinery, is undeniable. Several techniques have been employed for detection of specific small RNAs. Among these techniques northern hybridization is the most popular due to its universal application to RNAs of vari ...

This chapter describes a detailed protocol for genomic Southern blot analysis which can be used to detect transgene or endogenous gene sequences in cereal genomes. The protocol follows a standard approach that has been shown to generate high-quality results: size fractionation of geno ...

To date a number of cereal genomes are fully sequenced and more are near completion. The information within these genomes will be of most use to scientists when every gene has been functionally characterized leading to the complete annotation of these genomes. This chapter describes how funct ...