Requirements For Plant Transformation

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A. Overview

In general, plant transformation systems are based on the introduction of DNA into totipotent plant cells, followed by the regeneration of such cells into whole fertile plants. Two essential requirements for plant transformation are therefore an efficient method for introducing DNA into plant cells and the availability of cells or tissues that can easily and reproducibly regenerate whole plants. DNA can be introduced into isolated cells or protoplasts, ex-planted tissues, callus, or cell suspension cultures. However, the process is characteristically inefficient and only a proportion of cells in a target population will be transformed. These cells must be induced to proliferate at the expense of nontransformed cells, and this can be achieved by introducing a selectable marker gene and regenerating plants under the appropriate selective regime. Efficient DNA delivery, competence for regeneration, and a suitable selection system are therefore prerequisites for most plant transformation systems, although there has been recent development in the application of in planta transformation strategies, which circumvent the requirement for extensive tissue culture (discussed later). Other criteria that define an efficient transformation system are listed in Table 1.

B. DNA Transfer Methods

DNA transfer to plants was first attempted in the 1960s, although the lack of selectable markers and molecular tools to confirm transgene integration and expression made the outcome of such experiments unclear (5). A breakthrough came in the late 1970s with the elucidation of the mechanism of crown gall formation by Agrobacterium tumefaciens (6). The discovery that virulent strains of A. tumefaciens carried a large plasmid that conferred the ability to induce crown galls and that part of the plasmid (the T-DNA) was transferred to the plant genome of crown gall cells provided a natural gene transfer mechanism that could be exploited for plant transformation (7). Tobacco plants carrying recombinant T-DNA sequences were first generated in 1981, although the foreign genes were driven by their own promoters and

Table 1 Criteria for an Efficient Plant Transformation System

Essential prerequisites for plant transformation Efficient method of DNA transfer

Availability of cells/tissues competent for regeneration (not required for in planta transformation strategies) Suitable selection system Other criteria that define an efficient transformation system Reproducibly high transformation efficiency (number of transgenic plants recovered as a proportion of cells/explants originally transformed) Minimal culture time, to avoid somaclonal variation and sterility. High-frequency recovery of phenotypically normal, fertile transgenic plants

Technically simple procedure

Economical procedure

Versatile (applicable to many species)

Genotype-independent (applicable to all cultivars and varieties, including elite genotypes)

were not expressed in plant cells (8). The first transgenic tobacco plants expressing recombinant genes in integrated T-DNA sequences were reported in 1983 (9). The technique of Agrobacterium-mediated transformation has been developed and refined since then to become a widely used strategy for gene transfer to plants.

Although it is convenient and versatile, a major limitation of Agro-bacterium-mediated transformation is its restricted host range, which until relatively recently excluded most monocotyledonous plants (3). The development of strategies to extend the range of plants susceptible to Agrobacterium infection is discussed in the following. A number of alternative plant transformation methods were developed to facilitate gene transfer to these recalcitrant species. These methods can be grouped under the term "direct DNA transfer" and include the transformation of protoplasts using polyethylene glycol (PEG) or electroporation, microinjection, the use of silicon carbide whiskers, and particle bombardment. So far, only direct DNA transfer to protoplasts and particle bombardment have gained widespread use (2). The development and application of Agrobacterium-mediated transformation, particle bombardment, protoplast transformation, and other transformation techniques is discussed in more detail below.

C. Cell and Tissue Culture and Plant Regeneration

Small explants of living plant tissue can be maintained on a simple nutrient medium. Transformation may be carried out on tissues dissected from seeds, leaves, stems, roots or buds because under the appropriate conditions these can be induced to dedifferentiate and proliferate to produce undifferentiated callus cultures. Different hormone treatments induce callus to form shoots and roots, allowing the regeneration of whole plants. Alternatively, the callus can be maintained and subcultured indefinitely or can be broken up in liquid medium to provide a cell suspension culture, which can yield individual cells and protoplasts. Depending on the transformation method, tissue explants, callus, dispersed cells, or protoplasts can be used as transformation targets (Fig. 1).

After transformation, cells are allowed to proliferate on selective medium to increase the amount of callus and kill nontransformed cells. Transgenic plants can then be regenerated by two methods: somatic embryogenesis or organogenesis. Somatic embryogenesis involves the formation of embryogenic callus direct from somatic tissues. This recapitulates the entire developmental pathway, including the embryonic stage. Organogenesis involves the direct growth of shoots from the callus of transformed tissues or in some cases direct growth from transformed explants without a callus stage. The shoots can be transferred to rooting medium and regenerated into plants or grafted onto seedling rootstock and propagated. In some species, only one regenerative process is possible under the conditions used for transformation. For example, transgenic rice and maize plants are generated predominantly by somatic embryogenesis, and transgenic cassava plants can be generated only by the organogenesis of shoots. In other species, such as banana and soybean, both processes are possible and the method of choice depends on the starting material and culture conditions and which process produces transgenic plants the most rapidly and with the greatest efficiency.

An alternative to these processes is the development of transgenic plants by true embryogenesis from transformed seeds, zygotes, or gametes that undergo diploidization. Such techniques do not require extensive tissue culture and are discussed along with in planta transformation strategies later.

D. Selectable Marker Genes

Most foreign genes introduced into plants do not confer a phenotype that can be conveniently used for selective propagation of transformed cells. For this reason, a selectable marker gene is introduced at the same time as the nonselectable foreign DNA. This confers upon transformed cells the ability to survive in the presence of a particular chemical, the selective agent, that is toxic to nontransformed cells (10,11). In direct DNA transfer methods, the selectable marker and nonselected transgene(s) may be linked on the same cointegrate vector or may be introduced on separate vectors (cotrans-formation). Both strategies are suitable because exogenous DNA, whether

Zygotic ombryogenosis

Organogenesis or

Somatic embryogenesls

Zygotic ombryogenosis

dp C2> Protoplasts

Figure 1 Strategies for the transformation of higher plants. The boxes show the various different targets for transformation and how they are obtained from whole plants (black arrows). The methods used to transform each of these targets are represented by the square brackets. The gray arrows show the routes used to obtain whole transgenic plants.

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