Overcoming serious incompatibilities resulting from intergeneric hybridisation between Diplotaxis tenuifolia and Brassica oleracea to develop CMS wall-rocket.

Alloplasmic male-sterile breeding lines of Diplotaxis tenuifolia were developed by intergeneric hybridisation between cytoplasmic male-sterile cauliflower (Brassica oleracea L.) and perennial wall-rocket. Amphidiploid filial generation (F1) plants (2n = 2x = 20, CD) were obtained by manual crosses and ovary culture. The phenotype of the hybrids was mostly similar to that of the wall-rocket parent. The plants were male-sterile, but absolutely no seed set was achieved after pollination with D. tenuifolia. Amphitetraploid F1 hybrid plants (4n = 4x = 40, CCDD) obtained by colchicine treatment were backcrossed seven times with pollen of the Diplotaxis parent to develop alloplasmic diploid (2n = 2x = 22, DD) D. tenuifolia lines. The reduction of Brassica chromosomes was recorded by flow cytometry and chromosome counting. Ovary culture was essential up to the Backcross generation 4 (BC4 generation) because of absent or disturbed seed development. From the BC5 generation onward, no further embryo rescue was necessary. The developed D. tenuifolia lines are completely stable with regard to their cytoplasmic male sterility (CMS) status. Fertile or partially fertile pollen was observed neither in the F1 generation nor in any of the subsequent backcross generations (BC1 to BC7). Male sterility was attributed to both premeiotic (sporophytic) and postmeiotic (pollen-abortive) effects, similar to those observed in the CMS Brassica oleracea parent. Regarding morphological traits and growth dynamics, only variations in the dimensions and dissection of leaves between different cross progenies were observed. The plant phenotype, vigour, and yield potential of the CMS lines (BC7) are comparable to those of the D. tenuifolia parental line. The female fertility has been only moderately restored; however, the proportion of normally shaped seeds per pod is still lower than that of the Diplotaxis control. A significant number of the developed seeds showed disturbed and shrunken embryogenic tissue, which could be visualised and measured in more detail by 3D X-ray CT. The reasons for these developmental abnormalities need to be investigated in further studies. No further incompatibilities were observed between the used CMS-inducing cybrid cytoplasm and the D. tenuifolia genome. Therefore, the used CMS-inducing cybrid cytoplasm has been proven to be a vital source of CMS for D. tenuifolia, and the developed lines are applicable for hybrid breeding programs.