Chapter 6 Cell Cycle Synchronization, Chromosome Isolation, and Flow-Sorting in Plants

Abstract

Publisher Summary This chapter discusses the cell-cycle synchronization, chromosome isolation, and flow sorting in plants. Plant cell populations synchronously traversing the cell division cycle or accumulated within a specific phase of the cycle represent a valuable model for studying a wide range of biological questions. Techniques for cell synchronization, chromosome sorting, and the production of chromosome-specific libraries were initially developed in mammalian species. The delay in developing equivalent techniques for the flow cytometric analysis and sorting of plant chromosomes was mainly due to difficulties in obtaining chromosome suspensions that contained intact and well-dispersed chromosomes and that were free of contaminating cellular debris. The procedure for chromosome isolation involves four fundamental steps: (1) induction of cell-cycle synchrony, (2) accumulation of cells in metaphase, (3) isolation of chromosomes from the cells, and (4) flow sorting of chromosomes. For the isolation of chromosome suspensions, problems posed by the use of plant tissues include (1) difficulties in obtaining a high level of mitotic synchrony, (2) the variable degree of chromosome stickiness and clumping observed after treatment with mitotic drugs, (3) a tendency for metaphase-blocked chromosomes to split into single chromatids, (4) the presence of the rigid plant cell wall which, in comparison to mammalian cells, increases debris and inhibits the release of intact chromosomes during homogenization.