Microspores are attractive targets for transformation of maize through DNA microinjection since they may allow, through the development of embryogenic structures, the regeneration of transgenic plants without chimerism. Unfortunately, microinjection into unicellular microspores is technically difficult because of the presence of the exine. However, in later stages of development, this difficulty may be overcome. In this regard, our objective was to determine the most suitable stages of early androgenic development for microinjection using lucifer yellow as a marker dye. Experiments have shown that the fluorescent dye is able to spread inside the whole embryogenic structure (about 4–10 days old), only in those cases where internal cell walls are still absent or incompletely formed, as assessed by cytological observations. At later stages, when the septation is completed and the embryo is cellularized, diffusion of the dye is no longer possible. These results demonstrate that multinuclear structures with the broken exine and a cytoplasm not yet partitioned, may be conveniently used for direct transformation via microinjection.
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Gaillard et al. (1992) studied this question.