Semi-in vivo pollen hydration and pollen tube growth assay
For pollen hydration, freshly opened ms188 pistil was cut and attached upright on agar (1.0%). Then pollen grains (n = 50~80) from a freshly dehiscing anther were transferred onto the stigmatic papilla. The behavior of the pollen was captured under a microscope immediately after pollinations were initiated. Forin vivo pollen tube growth analysis, pollinations were initiated on ms188 stigmas and allowed to proceed for 2 h as described previously (L. Wang et al., 2017). Then stigmas were fixated overnight (60% v/v ethanol, 30% v/v chloroform, 10% v/v acetic acid), incubated in 8 M NaOH for 20 min, and washed in dH2O three times (5 min/each). Samples were dyed in 0.1% decolourized aniline blue (0.1% w/v aniline blue in 0.1 M K3PO4, pH11) for 1 h and images were captured with an Olympus BX51 microscope. Pollen tube growth was calculated as the mean of three biological replicates. All statistical analyses were carried out using Microsoft Excel.