In Vitro Pollen Germination and Pollen Tube Length Measurement

Pollen from single flowers were collected in a reaction tube and frozen in liquid nitrogen. For in vitro germination, pollen grains from 5 to 10 flowers were mixed and incubated for the indicated times at 26°C in 1 mL of standard germination medium (Schmülling et al., 1993) containing 10% Suc. In order to study the effect of sugars on pollen germination, the standard concentration of Suc in the medium was substituted by different concentrations of Glu, Suc, Pal, and Mhl as indicated. An aliquot of 50 µL was counted for the determination of pollen germination efficiency. To determine the effect of different sugars on pollen tube length, an aliquot of 50 µL was analyzed by light microscopy (100× magnification). Photographs were taken of nine randomly selected microscope fields with more than 50 pollen grains. Pollen tube length was determined for an average number of 100 pollen tubes per individual experiment. To assess the inhibitory effect of miglitol on pollen germination and tube growth, 20 mm miglitol was added to germination medium with or without 1% Suc. Photographs of three randomly selected microscope fields from three independent aliquots with more than 100 pollen were determined per individual experiment.