Qihua Cai

Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology


Flow cytometric analysis and flow sorting have great potential in facilitating the mapping and cloning of agriculturally important genes. The objective of this research was to optimize procedures for increasing metaphase index in root-tip meristem, isolating mitotic metaphase chromosomes and analyzing and sorting isolated chromosomes with flow cytometry. Three spindle toxins (i.e. trifluralin, amiprophos-methyl and oryzalin) at various concentrations were compared for their effects on metaphase chromosome accumulation. The effects of different types of buffer (mainly magnesium sulfate, hexylene glycol, polyamine buffer and their variations and/or combinations) on the quality of wheat chromosome suspension were studied. Chromosome suspensions were physically prepared from several aneuploid lines of the wheat cultivar Chinese Spring. Metaphase indices around 70% were achieved in synchronized root tips with the treatment of 20 μM APM for 6 hours and 10-20 μM oryzalin for 5 hours. Chromosome suspensions prepared in magnesium sulfate buffer with 1 % hexylene glycol and polyamine (0.2 mM sperrnine and 0.5 mM spermidine) had the highest frequency of intact chromosomes, the least fluorescence debris and preserved chromosome structure for at least 7 days. Flow karyotyping of wheat chromosomes was established through univariate analysis. The FL3-H histogram of the wheat cultivar, Chinese Spring, showed three major chromosome peaks. Each discrete peak represented chromosome types with similar DNA content. Unique peaks corresponding to the short arms of 4D and ID wheat chromosomes were observed in the FL3-H histogram of ditelosomic lines CSDt4DS and CSDt1DS. Over 2000 chromosome arms of 4DS and I DS were sorted, respectively. C-banding analysis of the sorted chromosome particles indicated that the purity of sorted chromosomes was 91.1 % and 95.4% for 4DS and 1DS, respectively.

Library of Congress Subject Headings

Wheat -- Genetics Plant chromosomes Flow cytometry



Number of Pages



South Dakota State University