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Broccoli ( Brassica oleracea  L. Italica group) breeders routinely use anther or microspore culture to produce dihaploid (diploid), homozygous lines. During the culture process, polyploidization occurs and diploid regenerants can result. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement. Thus, regenerated populations must be screened to identify the diploids that are the regenerants most likely to set seed and serve as inbred lines. DNA flow cytometry has proven a useful procedure for determining ploidy of anther derived regenerants. This study was undertaken to evaluate the effect of leaf age and sampling procedures on ploidy determination via flow cytometry. Anther-derived plants were analyzed at a four- to five-leaf stage (transplant stage) and at time of heading (mature plant stage). In addition, leaves were sampled on a given date and stability of the flow cytometry preparations was evaluated over 7 days. Lastly, the stability of ploidy readings of leaves stored at 4°C was examined over a 7-day period. In only one case out of 123 comparative assays did leaf age affect ploidy determination. For that exception, a haploid at transplant stage was a diploid at the mature plant stage. Flow cytometry preparations and also leaves stored at 4°C gave consistent ploidy determinations up to four days after preparations were made or tissue was refrigerated, respectively. These results indicate that broccoli breeders can make flow cytometry preparations on site and send them offsite for flow cytometry analysis. Alternatively, leaves could be refrigerated, sent offsite, and then prepared and analyzed at another location.

Utility of DNA Flow Cytometry for Ploidy Determination of Anther-derived Broccoli: Effect of Leaf Age and Sampling Protocols