Membrane and conditioned media proteins from embryogenic cultures of carrot (Daucus carota L.)

Raymond Mark Slay, Purdue University

Abstract

For the regeneration of whole plants from a single cell or small group of cells in tissue culture, a specific developmental program must be followed. A process in the regeneration of plants is somatic embryogenesis. Carrot cells in culture provide a system that is easily manipulated to induce somatic embryogenesis. Regeneration via somatic embryogenesis for other plant systems may not be as easy and in some cases may not be possible. This variability in regeneration would suggest that development can be regulated or impaired if the program to be followed cannot be completed. To better understand somatic embryogenesis in carrot, I have looked at two aspects of cell growth in culture. First, I have compared membrane proteins from embryo-forming (induced) and nonembryogenic (uninduced) cells of carrot in culture. Membranes were separated by isopycnic centrifugation and correlated to known enzyme marker activities. Enriched membrane factions were collected. Proteins and glycoproteins were analyzed using SDS-PAGE (1-dimensional) or isoelectric-focusing (IEF) followed by SDS-PAGE (2-dimensional) as well affino-blotting of membrane proteins. A plasma membrane surface protein label also was tested in this same comparison. Second, proteins that were secreted or released into the culture medium of embryogenic carrot cells were investigated. Proteins and glycoproteins were precipitated and collected and analyzed using SDS-PAGE and affino-blotting. In addition, growth data were correlated to protein content of the culture medium. Tunicamycin was used to inhibit N-linked protein glycosylation to determine the importance of these glycoproteins in development. The synthetic plant auxin, 2,4-dichlorophenoxyacetic acid also was tested since it promoted nonembryogenic (uninduced) cell growth. It is evident from the results that proteins from both membranes and conditioned medium can be described that are specific in development. Some developmentally specific membrane proteins also may be potentially useful as noninvasive markers of uninduced (72 kd, 46 kd) and induced (16 kd and 15 kd) carrot cells since they are surface exposed. Proteins that appear in culture medium during uninduced or induced cell growth may also hold some promise, but stage specificity is less clear. Some proteins do, however, correlate with development of embryogenic (induced) cells (69 kd and 78 kd) and some with nonembryogenic (uninduced) growth (42, 47, and 54 kd).

Degree

Ph.D.

Advisors

Hodges, Purdue University.

Subject Area

Botany

COinS