A cytospin technique for spreading plant metaphases suitable for immunofluorescence studies

Recent immunofluorescence techniques enable the localization of various cellular antigens, thus providing a powerful tool for cell and molecular biology research. Serious problems occur, however, when these techniques are applied to plant material. The presence of the cellulose wall can be a barrier to reproducible penetration of antibodies into cells and it often displays a confusing autofluorescence. A novel technique to prepare mitotic chromosome spreads from root tip meristems of germinating seeds is presented. Synchronous mitotic cells arrested in metaphase are converted into protoplasts using pectin and cellulose hydrolytic enzymes, and the purified protoplasts are fixed either in a methanol-acetic acid mixture to study DNA epitopes or in a nonextracting fixative to study chromosomal proteins. The latter fixative contains Triton X-100 to lyse the protoplasts and neutral formaldehyde to fix proteins by cross-linking. The protoplasts are immediately centrifuged onto microscopic slides as commonly done for mammalian cytogenetics. Using commercially available antibodies and both epifluorescence and confocal laser scanning microscopy, we demonstrated that the acid fixed chromosome slides are suitable for detection of DNA (anti-DNA antibody) or incorporated 5-bromodeoxyuridine (anti-BrdU antibody), while the cytospun formaldehyde and Triton X-100 fixed samples are convenient for detecting histones (antihistone antibody, pan). This technique should provide a general tool to study structural and functional domains of plant chromosomes.