COTYLEDON-DERIVED DIPLOID AND HAPLOID PROTOPLAST CULTURE AND DIPLOID PLANT-REGENERATION IN BRASSICA-NAPUS CV TOPAS

The present paper describes a simple and reliable protocol for the suc cessful isolation, purification, culture, and regeneration of diploid cotyledon-derived protoplasts of Brassica napus L, cv. 'Topas'. Variou s protoplast isolation media, nutrient media, subculture procedures, a nd protoplast sources were tested under two culture temperatures. Prot oplast viability, cell wall regeneration, and cell division were monit ored. Single cotyledon-derived protoplasts formed calli in liquid prot oplast medium, and when these were subcultured on solid proliferation medium and solid regeneration medium of appropriate composition, plant s regenerated either by shoot formation or embryogenesis. Continuous c ulture at 32 degrees C instead of 25 degrees C favoured the initiation of cell division and cell proliferation but prevented regeneration, a lthough calli maintained regeneration capacity. Viable haploid protopl asts were isolated from cotyledons of heat-shock-induced, microspore-d erived haploid embryos and from young leaves of secondary embryos that were formed on microspore-derived embryos. Cell divisions were trigge red in the two types of haploid protoplast cultures, and microcalli we re formed at high frequencies. Differences between haploid and diploid protoplast cultures are discussed.