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Mol Cytogenet. 2014 Mar 6;7(1):21. doi: 10.1186/1755-8166-7-21.

An easy "SteamDrop" method for high quality plant chromosome preparation.

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1
Department of Genetics and Biotechnology, Russian State Agrarian University-MTAA, Timiryazevskay str, 49, 127550 Moscow, Russia. ludmila.khrustaleva19@gmail.com.

Abstract

BACKGROUND:

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. In contrast to human chromosome preparation, the processes occurring during plant chromosome preparation and causing chromosome spreading are still poorly understood.

RESULTS:

We 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.

CONCLUSION:

The 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.

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