Submitted to: HortScience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 25, 1997
Publication Date: N/A
Interpretive Summary: The old and time-consuming method for making inbred broccoli varieties is to self pollinate plants for several generations. A faster method is to use anther or pollen culture which can result in inbreds in a single generation. A problem with anther culture is that many broccoli lines developed using it are genetically abnormal and sterile. Genetically abnormal plants cannot be readily distinguished from genetically normal ones. A new technology, flow cytometry, can distinguish genetically normal and abnormal plants. With flow cytometry, cells from leaves of anther- derived plants are compared to cells from normal control plants. Unfortunately, most broccoli breeders do not have flow cytometry and do not know how to use the technology. This study describes precise cytometry methods for identifying normal, anther-derived plants. It shows that it does not matter if plants are old or young when sampled since results will be the same. It also shows that breeders without cytometry equipment can refrigerate leaves, store them at cold temperatures, and send them to a laboratory that has the necessary equipment and expertise to analyze samples. This research provides public and private broccoli breeders information to help them breed improved, high quality varieties. These better varieties will enhance production and provide a reliable supply of quality broccoli for consumers.
Technical Abstract: Broccoli (Brassica oleracea L.) breeders use anther culture to produce doubled-haploid (diploid) lines. During culture, polyploidization occurs and diploids can result. However, polyploidization may not occur at all, or may involve a tripling, quadrupling, or octupling of the genome. Thus, regenerants must be screened to identify diploids, the only regenerants that will serve as potential inbred lines. DNA flow cytometry can be used to determine ploidy of anther-derived plants. In this study, leaf age and sampling procedures were evaluated for their effects on ploidy determination by flow cytometry. Regenerants were analyzed at seedling and mature plant stages. In addition, leaves were sampled on a given date and stability of preparations was evaluated at 1, 2, 4, and 7 days after preparation. Also, stability of ploidy readings of leaves stored at 4 C was examined over 7 days. 139 of 140 comparative assays at two stages resulted in the same ploidy determination. Flow cytometry preparations stored at 4 gave consistent determinations for up to two days, but some instability was evident after that. Refrigerated leaves were more stable than refrigerated preparations and ploidy did not differ from the first sampling through 7 days of storage. Results show that broccoli breeders can get consistent and accurate ploidy determinations with flow cytometry by refrigerating leaves and sending them offsite for preparation and analysis.