Efficient Ploidy Determination of Anther-derived Broccoli

Abstract

Broccoli (Brassica oleracea L. Italica Group) breeders routinely use anther or microspore culture to produce doubled-haploid (DH), homozygous lines. In addition to DH (diploid) regenerants, haploid, triploid, tetraploid, octaploid, and aneuploid regenerants may also result from anther culture. Thus, regenerated populations must be screened to identify the diploids, which are the only regenerants likely to set seed and serve as inbred lines. DNA flow cytometry is a useful procedure to determine ploidy of anther-derived regenerants. This study was undertaken to evaluate the effect of plant stage and sampling procedures on ploidy determination by flow cytometry. Anther-derived plants were analyzed at both seedling and mature plant stages. In separate tests, leaves were sampled on a given date, and stability of flow cytometry preparations were evaluated at 1, 2, 4, and 7 days after preparation. In addition, the stability of ploidy readings of excised leaves stored at 4 °C was examined over a 7-day period. In 139 out of 140 comparative assays there was no effect of plant stage on ploidy determination. Flow cytometry preparations stored at 4 °C gave consistent ploidy determinations up to 2 days after they were made, but some instability was observed by 4 and 7 days. Refrigerated leaves were more stable than nuclei preparations, and ploidy determinations did not differ from the first sampling through storage for 7 days. Results indicate that broccoli breeders could make flow cytometry preparations on site and send them offsite for flow cytometry analysis as long as analysis was completed within 1 or 2 days of sample preparation. More consistent results would be obtained by refrigerating leaves and sending them offsite for preparation and analysis at the offsite location.