THE USE OF AN AUTOMATED CELL TRACKING SYSTEM TO IDENTIFY SPECIFIC CELL-TYPES COMPETENT FOR REGENERATION AND TRANSFORMATION

Combining stepper-motors far microscope stage movement with a speciall y designed software program has led to the establishment of an efficie nt cell tracking system. Cell immobilization, fixed reference points f or calibration of target cell positions, and a video recording system complete the cell finder system. Specific cells can be identified (eit her beforehand or in retrospect), their locations fixed, and subsequen t development of the individual cells monitored daily using computer-a ssisted relocation. In this way, the specific cell type capable of sus tained 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. Isolati on and purification procedures were then optimized in a directed way t o yield millions of guard cell protoplasts (GCPs). Using polyethylene glycol (PEG)-mediated gene transfer and glucuronidase (GUS) activity f or transient expression studies proved that GCPs were amenable to tran sformation. Gene transfer efficiency was high, as was the number of st ably transformed plants that can be produced. At present, the optimize d procedure yields 600 transgenic individuals per person per year. Thi s number allows for the selection of the best plants with regard to co py number, DNA insert size, gene expression, and field performance. Pr ospects for future application of the cell finder system will be discu ssed.