Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Protoplasts isolated from the economically important grasses do not readily divide in culture. This is especially true of Zea mays L. from which only one cell line, B73, has been established from a primary protoplast culture. The cell line is not morphogenetic and has cytogenetic abnormalities. The early physiological events of cell wall regeneration and DNA synthesis in protoplast cultures of Zea mays L. (cultivar Seneca 60), the B73 maize cell line and Hyoscyamus muticus L. were studied to compare the non-regenerating primary protoplast culture of Zea mays with two regenerating protoplast populations, B73 and H. muticus.;Cell wall regeneration and DNA synthesis in the cultured protoplasts were analyzed with fluorescent dyes on a fluorescence activated cell sorter, the FACS II. Cell walls were stained with Calcofluor White. DNA content was measured by the binding of Hoechst 33258, a quantitative fluorochrome for DNA.;The fluorescence activated cell sorter (FACS II) operates by passing stained cells in a flow stream past an argon-krypton laser beam and measuring the relative size of each cell given by the low angle, forward light scatter and the amount of fluorescence of each cell. Approximately 2.5 x 10('4) cells per second can be analyzed, and the data from 10('5) cells were collected.;Survival studies on the three cultures showed protoplast death in the early days of culture. Analysis of DNA showed that all three cultures had protoplasts in G1 and G2 of the cell cycle and that the loss of protoplasts preferentially occurred among G2 protoplasts. All cultures, after day 3 showed an increase of G2 protoplasts indicating that DNA synthesis was occurring in all populations. Se60 protoplasts showed a third peak of DNA content that occurred at less than the G1 peak and, presumably, represented degenerating cells. Hyoscyamus and B73 protoplasts did not show this peak.;Hyoscyamus and B73 protoplasts both showed cell wall fluorescence and both had subpopulations having varying rates of cell wall synthesis. Se60 showed a very small peak of increased synthesis on day 4 of culture but this population was lost by day 6.



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