EFFICIENT PLOIDY DETERMINATION OF ANTHER-DERIVED BROCCOLI

Broccoli (Brassica oleracea L. Italica Group) breeders routinely use a nther or microspore culture to produce doubled-haploid (DH), homozygou s lines. In addition to DH (diploid) regenerants, haploid, triploid, t etraploid, octaploid, and aneuploid regenerants may also result from a nther culture. Thus, regenerated populations must be screened to ident ify the diploids, which are the only regenerants likely to set seed an d serve as inbred lines. DNA flow cytometry is a useful procedure to d etermine ploidy of anther-derived regenerants. This study was undertak en to evaluate the effect of plant stage and sampling procedures on pl oidy determination by flow cytometry, Anther-derived plants were analy zed at both seedling and mature plant stages. In separate tests, leave s were sampled on a given date, and stability of flow cytometry prepar ations were evaluated at 1, 2, 4, and 7 days after preparation. In add ition, the stability of ploidy readings of excised leaves stored at 4 degrees C was examined over a 7-day period. In 139 out of 140 comparat ive assays there was no effect of plant stage on ploidy determination, Flow cytometry preparations stored at 4 degrees C gave consistent plo idy determinations up to 2 days after they were made, but some instabi lity was observed by 4 and 7 days. Refrigerated leaves were more stabl e than nuclei preparations, and ploidy determinations did not differ f rom the first sampling through storage for 7 days. Results indicate th at broccoli breeders could make flow cytometry preparations on site an d send them offsite for flow cytometry analysis as long as analysis wa s completed within 1 or 2 days of sample preparation. More consistent results would be obtained by refrigerating leaves and sending them off site for preparation and analysis at the offsite location.