Ploidy of Regenerated Broccoli Derived from Microspore Culture Versus Anther Culture

Anther and microspore culture are commonly utilized to produce doubled-haploid (diploid), homozygous lines in broccoli (Brassica oleracea L. Italica Group). It is well-documented that doubled-haploid regenerants are produced by means of polyploidization during anther culture. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement. As a consequence, regenerated populations from anther culture contain diploids, but also haploids, triploids, and tetraploids. Microspore culture represents a simpler and more direct method for producing doubled-haploids. Although a similar mix of ploidy types is likely to be observed among regenerants derived from microspore culture, the actual ploidy levels of such regenerants have not been documented for broccoli. Thus, the objectives of this study were to compare ploidy levels of regenerants developed using both anther and microspore culture in broccoli, and to examine phenotypic variation in ploidy makeup of populations developed from both anther and microspore culture using different F1 hybrids. Broccoli regenerants were derived simultaneously from both anther and microspore cultures using the same four F1 hybrids, including Everest, Patriot, Greenbelt and Major. Ploidy level was determined by flow cytometry. A majority of regenerants derived from both anther and microspore culture, were determined to be diploids or tetraploids. Significant differences in ploidy makeup of populations were observed among hybrid varieties for both culture techniques. Regardless of the culture method used, `Everest' produced a greater percentage of diploids and a lower percentage of tetraploids than `Patriot' did. Haploids were observed more frequently from microspore culture than from anther culture when `Everest' and `Major' served as parents.