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
Cytokinins play an important role in plant development. We investigated the possibility that the nopaline Ti plasmid gene (tzs) from Agrobacterium tumefaciens could encode a protein able to participate in plant cytokinin production and lead to alterations in plant phenotype as a result of the expression of endogenous tzs. tzs was placed under the control of a heat‐inducible promoter from the Zea mays hsp70 gene. The expression of this fused gene was examined in transgenic Brassica napus plants. The tzs gene, which encodes the enzyme dimethylallyl transferase, was used as a cytokinin biosynthetic gene. The expression of the tzs gene was monitored by RNA hybridization and analysis of cytokinin content. Overproduction of cytokinin was observed even when the plants had not been heat‐shocked, and the plants displayed a reduced root system, increased height and branching, and delayed flowering. In addition, a significant increase in seed yield was observed in the transgenic plants, accounted for by increased number of seeds per silique and seed weight. The results suggest that increased levels of cytokinins, through the expression of tzs, are correlated with growth rather than with differentiation processes.
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