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United States Department of Agriculture

Agricultural Research Service

Title: Use of DNA Flow Cytometry to Evaluate Ploidy of Broccoli Derived from Anther Culture

Author
item Farnham, Mark

Submitted to: Biotechnology at the 1890 Institutions Proceedings
Publication Type: Proceedings
Publication Acceptance Date: November 8, 1997
Publication Date: N/A

Interpretive Summary: Plant breeders develop inbred plants that are genetically uniform and that produce offspring identical in appearance. Usually, inbreds are made by self-pollinating plants six to eight generations. A newer method, called anther culture, produces broccoli inbreds in one generation, saving years of work. A problem with anther culture is that some plants developed using git are genetically abnormal. Complicating this, abnormal plants cannot be readily distinguished from normal ones. To use anther culture efficiently, breeders need to identify the normal broccoli that are potentially useful, distinguishing them from abnormal ones having no value. In this research, broccoli inbreds were developed by anther culture, and a technology, called flow cytometry, was used to assess the normal or abnormal status of plants. With flow cytometry, cells of anther derived plants are compared to cells from normal check plants to see if they are normal. If they are, plants are kept for further work, and if they are not, plants are deemed abnormal and discarded. The use of flow cytometry in this research demonstrated that the final steps of making an inbred can be two to three times more efficient. Results give public and private breeders information to help them breed improved, high quality broccoli. Such improved broccoli ultimately stimulates grower production and provides a stable supply of high quality broccoli for consumers.

Technical Abstract: The use of anther culture to generate doubled-haploid lines to serve as parents for F1 hybrids is a common practice among broccoli breeders. Anther-derived regenerants from culture do not all exhibit the same ploidy; plants can be haploids, diploids, tetraploids, and in a few cases aneuploids. Effective methods are needed to identify diploids among populations of regenerants and are essential to use anther culture efficiently in a broccoli breeding program. In this study, anther-derived regenerants from hybrid sources were assayed by extracting nuclei from leaves, staining them with propidium iodide, and then by passing stained nuclei suspensions through a flow cytometer. Histograms characteristic of regenerants of a given ploidy were established allowing populations to be characterized for frequency of diploids, tetraploids, haploids, and aneuploids. Most hybrid sources had anther-derived populations with 50-60% %diploids and 30-40% tetraploids. An exception to this was the population derived from 'Marathon' which was 78% diploid and only 15% tetraploid. These results indicate that genotype of the anther source can affect the frequency of a particular ploidy among regenerants derived from culture. In addition, results show that flow cytometry is an effective technology for assessing ploidy of regenerants, allowing for clear identification of diploids that may serve as sources of parental lines.

Last Modified: 4/19/2014
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