MEDIA AND PROTOCOLS FOR THE ISOLATION OF INDEPENDENT MUTANTS AND TRANSFORMANTS FROM NICOTIANA-PLUMBAGINIFOLIA PROTOPLASTS

Developing protoplasts of N. plumbaginifolia can be spatially separate d at an early age by embedding them in agarose. If 0.2% bovine serum a lbumin (BSA) is added to the medium, embedding substantially improves plating efficiencies of these protoplasts over unshaken liquid culture . This procedure allows regeneration of thousands of independent mutan t or transformant cell lines with minimal chance of chimeras or multip le regenerants from one transformation event. Reproducible plating eff iciencies of 8-20% were achieved under these conditions. A medium that increased the stability of isolated protoplasts also increased the ab solute transformation efficiency obtained with polyethylene glycol (PE G)-mediated transformation by over 40%. Thus, the protoplast culture a nd transformation system described here constitutes a very efficient s ystem for the generation of large numbers of independently transformed callus lines and is suitable for large scale insertion mutagenesis ex periments based on transformation of haploid protoplasts.