The protoplast yield was also dependent on donor material, genotype, and incubation time (Chabaneet al., 2007). The efficiency of protoplast isolation depended on the environmental conditions, particularly the composition of the enzyme solution. The treatment of cellulaseand pectinasegave higher number of protoplasts than that of macerozymeR-10. Regarding the donor material, the best response was obtained with 3% cellulase, 0.5% pectinaseand 0.5% manitol. The protoplast isolation involved the treatment of H.tuberosusleaves with pectinaseto separate the cells, followed by cellulaseto remove their wall. The protocol presented here has established a highly efficient protoplast-based transient system for GFPgene functional expression. GFPhas been successfully used as a visual screenablemarker in H. tuberosus. The protoplast-GFPtransformation system could be useful for transforming varieties with genes of interest