Cytometric analysis of growth-regulator-dependent transcription and cell-cycle progression in Petunia protoplast cultures

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Abstract
Acridine orange simultaneously stains DNA and RNA. Using flow cytometry, synthesis of these nucleic acids can be related throughout a culture time-course. This technique has been used with nuclei isolated from Petunia hybrida protoplasts during 48 h of culture. Nuclear RNA content has been evaluated with respect to DNA levels, namely the cell-cycle phase. Nuclear RNA synthesis was not dependent upon exogeneous hormones during the first 18 h of culture, but either auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) or cytokinin (N6-benzyladenine) were necessary for entry into the S phase. Cytokinin alone could stimulate maximal RNA synthesis within each cell-cycle phase up to 24 h. In complete medium, DNA synthesis only began from a phase “G1B” having substantial RNA, although a subnormal amount of RNA (in protoplasts cultivated only with 2,4-D) did not prevent protoplast entry into the S phase. However, both hormones were necessary for highest RNA levels and G2 frequencies after 48 h. As in mammalian cells, the mean RNA level in plant 4C nuclei is double that of 2C nuclei. G2 nuclei are larger than G1 nuclei, but upon activation G1 nuclei in fact diminsh in size. This study aimed to identify restriction points in the cell cycle as affected by growth regulators and the specific synthesis of nucleic acids. For example, the RNA levels induced by N6-benzyladenine, although similar to those in complete medium, were not sufficient to induce mitosis. Conversely, 2,4-D action was probably limited by low nucleotide synthesis in the absence of cytokinin.