JOHNSON, EMANUEL L. AND STEPHEN D. EMCHE.* USDA ARS Weed Science Laboratory PSI, Bldg 001 Rm 329 , Beltsville, MD 20705-2350 USA. - Transgenic Plants: A source for medicinal plants.
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