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.