Other Direct Transformation Methods

Although the three transformation methods just discussed provide a route for the transformation of most plant species, there is value in the continuing exploration of novel transformation techniques. In some cases, it is possible to learn lessons from animal cell technology, where a number of alternative transformation systems have been established. Microinjection of DNA into animal eggs and zygotes, for example, is a routine procedure for generating transgenic animals that is applicable to all animal species. Microinjecting DNA into plant cells, while laborious and technically challenging, is advantageous in that it is the only current strategy available to study transformation events at a single-cell level. The injection of DNA into plant zygotes is being explored as a method for regenerating whole transgenic plants; however, it is currently highly inefficient. For example, isolated maize zygotes microinjected with DNA mimic embryonic development but abort at an early stage (55). Recently, the same technique has been applied to barley protoplasts, resulting in the recovery of numerous embryo-like structures but only two transgenic plants (56).

In 1986, Kurata et al. (57) devised a novel transformation strategy for animal cells in which DNA was taken up from solution after piercing the cell membrane with a finely focused laser beam. Although applicable to only a small number of cells, this technique achieved stable transformation efficiencies of 0.5%. A similar technique for plant transformation is available (58), although stable transformation has not been achieved.

Electroporation is used for the transformation of plant protoplasts but has been adapted for the transformation of walled plant cells in tissues. For example, maize explants have been transformed by electroporation either following partial enzymatic removal of the cell wall (59) or without treatment (60). This technique has also been successfully applied to rice and sugarcane explants (61,62). Pollen has also been transformed using electroporation (63).

Finally, a novel approach for the transformation of maize cells is to mix cells with silicon carbide whiskers. These penetrate the cells, allowing DNA uptake presumably through transient pores. This method is cheap, reproducible, and very simple to perform. Although initially used only for maize transformation (64), the technique has now been applied to other cereals (3,65).

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