The use of an automated cell tracking system to identify specific cell types competent for regeneration and transformation

Abstract

Combining stepper-motors for microscope stage movement with a specially designed software program has led to the establishment of an efficient cell tracking system. Cell immobilization, fixed reference points for calibration of target cell positions, and a video recording system complete the cell finder system. Specific cells can be identified (either beforehand or in retrospect), their locations fixed, and subsequent development of the individual cells monitored daily using computer-assisted relocation. In this way, the specific cell type capable of sustained division and regeneration has recently been identified within a low efficiency protoplast system of a recalcitrant species, sugarbeet. These totipotent cells originated from stomatal guard cells. Isolation and purification procedures were then optimized in a directed way to yield millions of guard cell protoplasts (GCPs). Using polyethylene glycol (PEG)-mediated gene transfer and glucuronidase (GUS) activity for transient expression studies proved that GCPs were amenable to transformation. Gene transfer efficiency was high, as was the number of stably transformed plants that can be produced. At present, the optimized procedure yields 600 transgenic individuals per person per year. This number allows for the selection of the best plants with regard to copy number, DNA insert size, gene expression, and field performance. Prospects for future application of the cell finder system will be discussed.