A Cytospin Technique for Spreading Plant Metaphases Suitable for Immunofluorescence Studies

Abstract

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.

The inter‐specific hybrid Silene latifolia×S. viscosa reveals early events of sex chromosome evolution

“…Cell cycle synchronization and metaphase accumulation were performed as described in Lengerova et al (2004). Slides were prepared (via isolation of metaphase protoplasts) according to Hladilova et al (1998), and the preparations were pre‐treated as described in Lengerova et al (2004). Total genomic DNA from both S. latifolia and S. viscosa was used for genomic in situ hybridization, using either green labeling (with SpectrumGreen direct‐labeled dUTP and Nick Translation Kit both from Vysis, Abbott Park, IL, USA) or red labeling (Fluorolink Cy3‐dUTP from Amersham Pharmacia Biotech (Little Chalfont, UK) in combination with Roche Nick Translation Mix).…”

An accumulation of tandem DNA repeats on the Y chromosome in Silene latifolia during early stages of sex chromosome evolution

“…Mitotic slides were prepared according to Hladilova et al (1998). Briefly, seedlings were synchronized with aphidicolin (30 μM, Sigma, for 16 h) and metaphases accumulated with oryzalin (15 μM, Sigma, for 4 h).…”

Multicolor FISH mapping of the dioecious model plant, Silene latifolia

“…After 16 h in the presence of this inhibitor, the seedlings were extensively washed with water (5 h) and then exposed to 15 mM oryzalin (Sigma) (for 5 h) to accumulate the dividing cells in metaphase. During the last hour of oryzalin treatment, root tips of the seedlings were cut off, and metaphase protoplasts were isolated as described in Hladilova et al (1998). Slides were prepared by dropping fixed protoplast suspensions onto slides.…”