Tropane alkaloids are a class of secondary metabolites occurring in several solanaceous and Erythroxylum plant species. The alkaloid family includes several important compounds that have both positive and negative effects on the mammalian and insect nervous systems, and for commercial purposes, are plant derived. Leaf waxes and polyphenols are major bearers that inhibit protoplast isolation from leaves of alkaloid bearing taxons of Erythroxylum which are useful for electrochemical fusion to produce transgenic plants. In this study several digestion enzymes were used to overcome these barriers to obtain ideal protoplasts for electrochemical fusion. The most efficient was a combination of Cellulase (2%), Hemicellulase (1%) and Macerase (1%) in a 0.7 M mannitol and 0.01 M MES osmoticum (pH 5.8). Young leaves were harvested from four tropane bearing Erythroxylum taxons and thoroughly washed in a 10% bleach solution, rinsed 3 × in de-ionized water, cut into 1 mm strips and placed into petri dishes for digestion at 24 ̊C. The enzymes and osmoticum above were used for the digestion. Leaves during digestion were agitated for 3 hr at 45 rpm and thereafter, digestion continued for 5 hr. After digestion, the osmoticum and free protoplasts were filtered, to remove debris, centrifuged and separated by sucrose density gradient. Digestion yielded a cell density of ≈8.0 × 105. For electroporation, volumes of 20 µl of protoplasts were placed in BTX electroporation chambers and ‘pearl chained’ for 20 sec at 20 V, and fused at 120 V for 10 µs. Within the microscopic field of view (200×), an average of 15 protoplasts could be viewed with 5 to 7 successful fusions. There were ≈ 6000 protoplasts per slide or 2,400 fusions and the rate of fusion was 40%. Vector incorporation, transgenic growth and development and metabolites are currently being monitored for herbicide activity and useful medicinal products.

Key words: Erythroxylum , electrofusion, enzymes, protoplasts