Plant Regeneration from Haploid Stem Peel Protoplasts of Brassica napus L.

Abstract

Summary Protoplasts were isolated from stem peels of 55 microspore-derived haploid plants of B. napus and cultured in liquid VN medium alone or in VN medium on top of an agarose layer containing 1/2 strength VN medium. At day five 0 to 70% of the protoplasts had divided, depending on the haploid line from which they were isolated. After 7-10 days in culture the cells in VN alone were embedded in a modified MS medium containing 0.625 mg·1 -1 BA, 0.625 mg· 1 -1 Kinetin, 0.625· mg·1 -1 2iP, 0.625· mg·1 -1 zeatin 0.5 mg·1 -1 NAA, 50 g·1 -1 mannitol, 6 mg· 1 -1 agarose, pH 5.7. The cells in liquid medium or embedded in agarose were exposed to light gradually. Two weeks after adding agarose to the liquid VN cultures, the agarose containing protoplast-derived microcolonies was spread on 2N medium which was similar to the embedding medium but contained 200 mg·1 -1 casein hydrolysate, 15 g·1 -1 sucrose and 4 g·1 -1 agarose to induce callus proliferation. Microcalli from the liquid VN cultures on the agarose layer were plated on the same medium. After 3-4 weeks on callus proliferation medium the calli were transferred to 3 regeneration media where 20-30% of the calli from the 4 haploid lines tested developed plantlets within 3-4 weeks.