植物学技术

The small size of most plant virus genomes and their very limited coding capacities requires that plant viruses are dependent on proteins expressed by the host plant for all stages of their life cycle. Identification of these host proteins is essential if we are to understand in any meaningful way t ...

Direct gene transfer into tobacco leaf protoplasts via electroporation is used in many laboratories for transient-expression studies. Protoplasts in general are a convenient model to study events that occur in planta rapidly. Transient expression provides answers in a matter of da ...

A main objective of electroporation is to transform cells by stable incorporation and expression of foreign genetic material. Incorporation may result in transformation with marker genes, as well as desired traits from a breeder’s point of view (e.g., male sterility, virus resistance, in ...

Plant cell walls present a barrier to DNA uptake not present in mammalian cells or plant protoplasts. Transformation methods that force DNA through this network of cellulose fibers include particle gun technology (ballistics; 1), imbibition of dried seeds (2), and use of silicon carbide “wh ...

Protoplast fusion and subsequent in vitro plant regeneration, leading to somatic hybridization, offer opportunities for transferring entire genomes from one plant into another, regardless of the interspecific crossing barriers. In contrast to techniques for plant transfor ...

The complex nature of plant metabolism often can seriously hinder the functional analysis of many proteins; and this has led some researchers to attempt to express such proteins in less sophisticated organisms. Unfortunately, the obvious choice, Escherichia coli, is less than ideal with ...

Oocytes of the South African clawed toad, Xenopus laevis, have proved a versatile and powerful heterologous expression system for eukaryotic genes (1). Xenopus oocytes were first used to express plant genetic material in 1979 (2), and in the past decade it has become increasingly evident that t ...

In Chapter 28, a method for the isolation of intact chloroplasts was described. The availability of this technique has allowed researchers the opportunity to investigate the mechanisms of chloroplast biogenesis. The chloroplast is a complex organelle, being made up of six subcompartm ...

The isolation of intact chloroplasts from plant tissue allows the study of processes carried out in this subcellular compartment. Such processes may be enzymatic, e.g., photosynthetic oxygen-evolution, or mechanistic, like the import and targeting of precursor proteins synthesi ...

The chloroplast is the product of two genomes with the majority of its polypeptide complement encoded by nuclear DNA and synthesized on cytosolic ribosomes. Proteins destined for the chloroplast must therefore contain targeting information to allow their correct localization. It h ...

During the last years the structure and gene content of the mitochondrial genome of land plants have been subject of intensive molecular investigations. Because of the large size (200–2500 kb), and the complex, multipartite organization, the actual configuration of the mitochondrial g ...

Protein synthesis by isolated plant mitochondria under in vitro conditions is a direct method to study the translational products of this organellar genetic system. Compared to fungal and animal mitochondrial systems, a considerably higher number of proteins is expected to be encoded ...

Direct identification of the product of a transferred gene is clearly an important part of the characterization of a transgenic plant. It is possible to detect proteins using highly sensitive ELISA techniques (Chapter 36, this volume), and to determine their precise histological or subce ...

The immunodetection of proteins bound to a membrane has widespread applications in plant biochemistry and molecular biology, including the identification and semiquantitative determination of foreign proteins expressed in transgenic plants. The approach is usually appl ...

Immunoassays were first developed over 30 yr ago. The radioimmunoassay for insulin described by Yalow and Berson (1) heralded a new era in the use of antibody reagents for the quantification of proteins and peptides. The Nobel Prize-winning research revolutionized analysis by virtue of much ...

The localization of proteins within plant tissues is readily accomplished using the techniques of immunocytochemistry: the identification of a cell-bound antigen in situ by means of a specific antigen-antibody reaction, tagged microscopically by a visible label (1–3).

Electroporation is now a standard method of transfection and cell loading. There is a variety of commercial electroporation equipment, and many published and manufacturer-supplied protocols. Many of these protocols are results of trial and error. These empirical protocols are val ...

Direct DNA transformation is currently the method of choice for obtaining transformed cereals, such as Zea mays, owing to the inherent host limitations of Agrobacterium tumefaciens. This soil bacterium, which naturally acts by transferring its own genes into a host plant, is commonly eng ...

The techniques of electroporation and electrofusion require that cells be subjected to brief pulses of electric fields of the appropriate amplitude, duration, and wave form. In this chapter, the term electro cell manipulation (ECM) shall describe both techniques. ECM is a quite universal ...

The introduction of DNA into plant cells, protoplasts, or intact tissues has been accomplished by a variety of mechanisms, including electroporation, electrofusion, particle bombardment, liposome transfer, the use of bacterial vectors, polyethylene glycol treatment, and oth ...