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Sperm cells were isolated from corn (Zea mays L.) tricellular pollen gains. They were released using a light osmotic chock, and separated from pollen contaminants (especially starch grains) by a Percoll gradient centrifugation. Isolated sperm cells (3 x 10' per milliliter) show a high viability score (90%) as demonstrated with the fluorochromatic reaction. They appeared as spherical cells which lack cell wall and plastids, and can be considered as haploid protoplasts.In angiosperms, sperm cells are formed either in the pollen grain (tricellular pollen) or in the pollen tube during germination (bicellular pollen). In (FCR+) after exposure to the fluorescein diacetate mixture.Light Microscopy Controls. Throughout the isolation procedure, sperm cells were examined with phase contrast or fluorescent microscopy. Sperm cells were counted using 0.05% ethidium bromide in the isolation medium. Sperm cells could also be observed using phase contrast combined with fluorescence microscopy (6).Yield of Sperm Cells. The number of pollen grains was estimated at 2500 grains/mg. The initial number of sperm cells was represented by the total weight of pollen (in mg) multiplied by 2500 and by 2 (two sperm cells per pollen grain). The

A multidisciplinary study was carried out to analyse the chromosome doubling process during the early stages of in vitro maize microspore embryogenesis. The main stages (microspore derivatives) that were formed in the course of the culture were analysed. Chromosome number was determined from squashed cells, and DNA content was measured by cytometry. In parallel, an ultrastructural analysis of the microspore derivatives demonstrated the occurrence of a nuclear fusion process. It seems likely that nuclear fusion ensures chromosome doubling at early stages of induced microspore embryogenesis. It occurs precisely at the 5/7 day stage in the embryonic domain and probably leads to polyploidy in the endosperm domain of the microspore derivatives. As a conclusion a scheme summarises the results and proposes an interpretation of the sequence of chromosome doubling events during early maize microspore embryogenesis. Understanding of this process will be important for future efforts to increase the percentage of homozygous plants for crop improvement.