In vitro Fertilisation

Whereas animal and lower plant IVF-systems can easily make use of naturally free-living gametes, sperm, egg and central cells of angiosperms presuppose their isolation, because the embryo sac is generally deeply embedded in the ovule, and the sperm cells are enclosed in pollen grains or tubes. Micromanipulation techniques and skills are prerequisites for the isolation, fusion and culture of single cells. These methods were developed originally for experiments with somatic cells [19-22]. They were adapted and improved for investigations with gametic cells [23-25]. By use of these methods, experimental access to single gametes, fertilisation and postfertilisation events under continuous microscopic observation with defined conditions are possible, for example, isolation, selection and fusion of pairs of gametic protoplasts. Also, this allows to design detailed experiments to follow precisely timed early events of zygote, embryo and endosperm formation after gamete fusion.

Isolated gametes (Fig. 1 a and b) are protoplasts and therefore can be fused by techniques that have proved to be successful in the fusion of somatic protoplasts. These are electrofusion and the fusion methods using polyethylene glycol or calcium to induce cell fusion. Sperm and egg cell fusion are electrically induced in maize [17, 23, 25-27] and in wheat [28]. Using maize, the same method was applied to central cell fertilisation [29, 30]. Calcium mediated cell fusion of sperm and egg cells [31-33] and of sperm and central cells [29] were also performed in maize. Possibly attributed to the large differences between the cell sizes isolated sperm cells fuse fast, generally in less than one second with egg and central cells [25].

Fig. 1. Isolated female gametic cells and two-celled embryo derived from in vitro fertilised egg cell from maize. (a) egg cell. Bar = 33 ^m. (b) central cell. Bar = 105 ^m. (c) two-celled embryo, 43 h after in vitro fertilisation. Bar = 38 ^m.

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