Hypocotyl protoplast culture in Brassica napus L

Protoplasts were isolated from the hypocotyls of two oilseed rape (Brassica napus L.) cultivars, Yunbei 2 and Ningyou 7. Light treatment during seed germination could apparently increase their cell division frequency. The purified protoplasts were cultured with three different methods. The method called "agarose island" was found superior to two others ("thin liquid layer" and "agarose solid layer"). The protoplasts cultured in the "agarose island" divided more rapidly and developed into calli more frequently. Shoot regeneration occurred soon after the transfer of protoplast-derived calli onto the differentiation medium. Individual shoots were rooted on the rooting medium. Whole plants were transplanted into pots, and grew well in the phytotron. The "agarose island" method might be suitable for genetic transformation of protoplasts because the liquid medium surrounding the "island" could be conveniently replaced without strong disturbing the microenvironment of protoplasts plated.