Efficiency of Microspore Culture for Doubled Haploid Production in the Breeding Project "Czech Winter Rape"

A selected set of twenty inter-line F1 hybrids of Brassica napus L. was evaluated for their capacity to produce microspore-derived doubled haploid (DH) plants subsequently utilized in the breeding project "Czech Winter Rape". All tested genotypes were able to produce microspore-derived embryos based on the standardized protocol previously adopted for the Czech winter rape varieties/breeding lines; nevertheless, significant differences were found in the frequency of microspore-derived embryo production as related to particular genotypes. However, the fertile DH regenerants were obtained only in 12 hybrid lines of this set. The other 8 lines failed to produce fertile DH plants for several reasons, mainly due to altered morphology of microspore embryos connected with limited germination/conversion potential and due to unsuccessful colchicine treatment resulting in haploid or mixoploid (chimeric) regenerants exhibiting partial or full sterility. In general, colchicine treatment induced diploid (doubled haploid), polyploid (triploid, tetraploid) and mixoploid (containing both euploid and aneuploid nuclei) plants. The fully aneuploid plants were recorded only sporadically. Some modifications of culture protocol (removal of embryo cotyledons before transfer to a germination medium; production of more plantlets from a single embryo by rooting multiple shoots; repeated colchicine treatment of unsuccessfully doubled plants in a later phase of growth) could enhance the final yield of DH fertile regenerants in our experiments. In total, 590 microspore-derived regenerants were produced in a series of experiments, 236 (40%) of them represented fully fertile DH plants. The R2 seeds (progeny of DH R1-generation) of the most promising hybrid-lines were included in the tests of agronomic performance in field conditions.