Abstract
BackgroundThe chromosome preparation is a crucial step for obtaining satisfactory results in molecular cytogenetic researches. The preparation of plant chromosomes for molecular cytogenetic purposes remains a challenge for some species. In contrast to human chromosome preparation, the processes occurring during plant chromosome preparation and causing chromosome spreading are still poorly understood.ResultsWe studied the dynamics of plant chromosome spreading after dropping cell suspension on slides. We showed that steam stimulates cytoplasm hydrolysis and rapid chromosome spreading and that chromosomes stretch during this chromosome spreading. Based on these observations, we developed a novel method, named “SteamDrop”, for the preparation of well-spread mitotic and pachytene chromosomes and successfully used it for 28 plant species with large and small chromosomes. We applied cell suspensions in ethanol instead of the commonly used ethanol/acetic acid fixative. Mitotic and meiotic chromosomes prepared via “SteamDrop” were used in fluorescent in situ hybridization (FISH) experiments with repetitive and unique DNA probes. Long storage of cell suspensions in ethanol did not impair the quality of chromosome preparations.ConclusionThe SteamDrop procedure provides a robust and routine method for high quality plant chromosome preparations. The method can be applied for metaphase as well as pachytene chromosome preparation in wide range of species. The chromosomes prepared by SteamDrop are well suitable for repetitive and unique DNA visualization.
Highlights
The chromosome preparation is a crucial step for obtaining satisfactory results in molecular cytogenetic researches
Several reports were dedicated to elucidating the chromosome spreading dynamic for improving human chromosome preparations [1,2,3,4,5,6,7,8], while equivalent studies are largely lacking for plants
Dynamics of chromosome spreading under steam The experiment has been done using Allium cepa, a species with large chromosomes, and Humulus japonicus, a species with small chromosomes
Summary
The chromosome preparation is a crucial step for obtaining satisfactory results in molecular cytogenetic researches. The preparation of plant chromosomes for molecular cytogenetic purposes remains a challenge for some species. Because the high diversity of species possessing small or large chromosomes, low or high chromosome number and different the human chromosome spreading dynamics have been done [3,5,7]. The method was successfully used for FISH on mitotic metaphase and pachytene chromosomes of maize [20,21,26] and on mitotic metaphase chromosomes of soybean [27,28] Application of this method for species with large sized chromosomes remains problematic because of a low number of non-overlapped metaphases [29]
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