Figure 6

Analysis of changes in cpDNA content during development using fluorescence microscopy and flow cytometry. (A-D) The frequency of relative fluorescence values of DAPI-stained, glutaraldehyde-fixed chloroplasts isolated from four different tissues at two stages of growth measured by fluorescence microscopy. Flow cytometric analysis (E) and PPoD (F) of the same four samples shown in (A-D) using SYTO 42 (chloroplasts were not fixed). D12 L1,2: first two leaves of 12-day-old seedlings. D12 Cotyledons: cotyledons of 12-day-old seedlings. D23 L5: fifth leaf of 23-day-old plant. D23 L1: first leaf of 23-day-old plant. The means (arrows) ± standard error are 6.14 ± 0.76 (A), 4.39 ± 0.81 (B), 2.62 ± 0.2 (C), 2.14 ± 0.18 (D). The numbers of chloroplasts with no detectable DNA (and the number of chloroplasts assayed) by DAPI-staining are 0(37), 0(36), 0(62), 4(66) for the samples shown in (A-D), respectively. The means of the SYTO 42 fluorescence profiles shown in (E) are 5835 (D12 L1,2), 4128 (D12 Cotyledons), 2344 (D23 L5), and 2042 (D23 L1). At least 3000 chloroplasts were analyzed. Mean SYTO 42 fluorescence values were 7607, 6007, 3115, 2431 and 8471, 5440, 3523, 2549 for the same tissues obtained from the ecotypes Nossen and Estland, respectively (profiles not shown).