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 successful isolation, purification, culture, and regeneration of diploid cotyledon-derived protoplasts of Brassica napus L, cv. 'Topas'. Various protoplast isolation media, nutrient media, subculture procedures, and protoplast sources were tested under two culture temperatures. Protoplast viability, cell wall regeneration, and cell division were monitored. Single cotyledon-derived protoplasts formed calli in liquid protoplast medium, and when these were subcultured on solid proliferation medium and solid regeneration medium of appropriate composition, plants regenerated either by shoot formation or embryogenesis. Continuous culture at 32 degrees C instead of 25 degrees C favoured the initiation of cell division and cell proliferation but prevented regeneration, although calli maintained regeneration capacity. Viable haploid protoplasts were isolated from cotyledons of heat-shock-induced, microspore-derived haploid embryos and from young leaves of secondary embryos that were formed on microspore-derived embryos. Cell divisions were triggered in the two types of haploid protoplast cultures, and microcalli were formed at high frequencies. Differences between haploid and diploid protoplast cultures are discussed.