In Vitro Pollen Germination Assay

To calculate the in vitro germination ratio, the viable pollen grains were germinated in solid and liquid pollen germination media. The liquid pollen germination medium consists of 20% sucrose, 10% polyethylene glycol 4000 (PEG4000), 3 mM $\mathrm{Ca}{\left({\mathrm{NO}}_3\right)}_2$ , 40 mg/L ${\mathrm{H}}_3{\mathrm{BO}}_3$ , and 10 mg/L thiamine (Vitamin B1) [33 (link)]. To make a solid germination medium, we added 1% agarose to the liquid medium. When the rice flowers reached anthesis, the fully mature pollen grains were collected in germination media immediately. Afterward, pollens on the germination media were incubated at 28 °C for about 30 min. We kept the humidity to prevent the germination medium from drying and observed the pollens using a BX61 microscope (Olympus, Tokyo, Japan). The pollen germination state and tube length were measured using Image J software [58 (link)]. More than 100 pollens were analyzed daily for a week. Ruthenium red and Calcofluor white were used for pectin, intine staining. All histochemical staining was incubated at room temperature for 15 min [31 (link),33 (link)]. The stained pollen grains were observed using the Olympus BX61 microscope.