Author
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Johnson, Emanuel |
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Submitted to: Meeting Abstract
Publication Type: Abstract Only Publication Acceptance Date: 9/4/2000 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: 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 inhibit protoplast isolation from leaves of Erythroxylum which are useful for electrochemical fusion to produce transgenic plants. In this study several digestion enzymes were used to obtain ideal protoplast population for electrochemical fusion. The most efficient was a combination of Cellulase, Hemicelulase and Macerase in a 0.7 M mannitol and 0.01 M MES osmoticum (pH 5.8). Young leaves were harvested from Erythroxylum spps., and washed in a 10% bleach solution, rinsed in de-ionized water, cut into strips and placed into petri dishes for digestion at 24 deg C. Enzymes and osmoticum above were used for the digestion. Leaves during digestion were agitated for 3 hr at 45 rpm. Digestion was continued for 15 hr. After digestion, the osmoticum and free protoplasts were filtered, to remove debris, centrifuged and separated by sucrose density gradient. Digestion yielded approx. 800,000 protoplast. For electroporation, volumes of 20 ul of protoplasts were placed in BTX (micro slide) electroporation chambers, #pearl chained# for 20 sec at 20 V, and fused at 120 V for 10 us. The microscopic field (200#) showed an average of 15 protoplasts with 5 to 7 successful fusions. There were approx. 6,000 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. |
