Plant transformation by microinjection techniques

Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking.