Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B-campestris)

A simple and reliable method for evaluating the viability of Brassica pollen was established in which the in vitro germination rate of pollen was adopted as the index of the viability of pollen grains. Pollen grains were preincubated in an atmosphere in which the relative humidity (RH) was fixed to 52% or 66% at 20 degrees C for 5 hours. They were cultured for 16 hours at 25 degrees C in a liquid Kwack's medium (1964) supplemented with 20% sucrose, and the pH was adjusted to 8.0. They were then observed under a microscope and the number of germinating and unchanged pollen grains were counted. The germination rate of pollen was improved and stabilized by preincubation and the use of a high pH medium. More than 90% of the freshly harvested pollen grains of Brassica rapa (syn. B. campestris) germinated constantly in these conditions. Undehisced anthers were collected from flowers at anthesis and dehydrated by incubation at 20 degrees C for 16-24 hours in an atmosphere where the RH was fixed to 15% or 32%. They were put into a plastic vial and preserved in a freezer at -20 degrees C. The germination percentage of the preserved pollen was scored at intervals during preservation. The germination rate of the pollen grains preserved at -20 degrees C for 1 year was higher than 50% and the pollen proved to be efficient for seed set. Most of the seeds germinated normally.