Plant DNA flow cytometry and estimation of nuclear genome size.

Plant Genome Size Estimation by Flow Cytometry: Inter-laboratory Comparison

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4′,6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

A new model for grass functional genomics is described based on Brachypodium distachyon , which in the evolution of the Pooideae diverged just prior to the clade of “core pooid” genera that contain the majority of important temperate cereals and forage grasses. Diploid ecotypes of B . distachyon (2 n = 10) have five easily distinguishable chromosomes that display high levels of chiasma formation at meiosis. The B . distachyon nuclear genome was indistinguishable in size from that of Arabidopsis, making it the simplest genome described in grasses to date. B . distachyon is a self-fertile, inbreeding annual with a life cycle of less than 4 months. These features, coupled with its small size (approximately 20 cm at maturity), lack of seed-head shatter, and undemanding growth requirements should make it amenable to high-throughput genetics and mutant screens. Immature embryos exhibited a high capacity for plant regeneration via somatic embryogenesis. Regenerated plants display very low levels of albinism and have normal fertility. A simple transformation system has been developed based on microprojectile bombardment of embryogenic callus and hygromycin selection. Selected B . distachyon ecotypes were resistant to all tested cereal-adapted Blumeria graminis species and cereal brown rusts ( Puccinia reconditia ). In contrast, different ecotypes displayed resistance or disease symptoms following challenge with the rice blast pathogen ( Magnaporthe grisea ) and wheat/barley yellow stripe rusts ( Puccinia striformis ). Despite its small stature, B . distachyon has large seeds that should prove useful for studies on grain filling. Such biological characteristics represent important traits for study in temperate cereals.

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Brachypodium distachyon. A new model system for functional genomics in grasses.

Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species.

Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.

S. Lcretti

Papers

Analysis of Nuclear DNA content in plant cells by Flow cytometry