Efficient Doubled Haploid Production in Brassica napus via Microspore Colchicine Treatment in vitro and Ploidy Determination by Flow Cytometry

An efficient microspore culture protocol was used to rapidly produce homozygous doubled haploid (DH) progeny of crosses between low erucic canola and high erucic resynthesized rapeseed (Brassica napus L.). Microspores of Canadian cultivars ‘Excel’ and ‘Profit’ as well as three F1 hybrids with the resynthetic line RS239 were treated with colchicine immediately after isolation. Flow cytometry was applied for early identification of DH regenerants. The diploidization rate was subsequently verified by scoring flower morphology . In vitro colchicine treatment had a positive effect on induced diploidization in dependence on the frequency of preliminary spontaneous diploidization which was, however, determined by the genotype. In addition, effects of colchicine treatment on embryoid formation and regeneration have been evaluated. The method presented is feasible for commercial large-scale production of DH lines in oilseed rape since the genotype-specific diploidization can be efficiently balanced by in vitro colchicine treatment. In addition, the use of flow cytometry immediately after in vitro culture allows to efficiently select for doubled haploids thus saving labor and cost and in the laboratory and subsequent greenhouse phase.