Abstract:Protoplasts were released in large quantities from mature pollen of Brassica napus L. and B. campestris var. purpurea for the first time, with a yield up to 66.7% and 70.4% respectively. Most of the pollen protoplasts were viable as tested by fluorochromatic reaction with fluorescein diacetate. The success of isolation of pollen protoplasts in these two Brassica species relied on a technique modified from the previous method developed for several monocotyledonous flowers. The procedure included two steps: First, the pollen was hydrated in 1 mol/L of sucrose solution at 28–30℃ for ca. 9h. During this process, the exine of most pollen dehisced and was detached from the pollen grains which were then covered by intine alone. Second, the hydrated pollen was transferred into an enzyme solution containing 1% cellulase, 1% pectinase, I mol/L mannitol, 0.5% potassium dextran sulphate and Ks medium salts. After 4–6 of enzymatic maceration, the intine was degradated resulting in the release of protoplasts. Factors affecting the two steps have been investigated.