Figure 5.

Alexander viability staining demonstrates that cngc16 pollen are hypersensitive to elevated CaCl₂ concentrations. Pollen grains were harvested into a water suspension from plants grown under normal conditions. Aliquots were modified as indicated and incubated for 3 h at 20°C in parallel. Incubations were done in solutions corresponding to water only, standard liquid in vitro germination medium, pH 7.5 (GM), or Tris-MES buffer (pH 7.5). Solutions were amended as indicated with Ca²⁺ using CaCl₂. Alexander staining was done after 3 h by pelleting pollen and resuspending pellets in 1 mL of Alexander stain for 30 min. Within the relatively short post hydration time frame assayed, wild-type (wt) controls for each solution showed less than 0.5% pollen grain germination. Viability counts were done with a digital camera mounted on a Leica DM IRE2 microscope. Values represent means ± sd of three independent experiments, each with approximately 50 pollen grains. Student’s t test was done to compare the pollen viability of cngc16 mutants to wild-type plants incubated at the same condition. *Student’s t test significant at P < 0.05. **Student’s t test significant at P < 0.01.