Scientific Paper

An Investigation into the Development of Chloroplast Isolation and “In-Organelle” Digestion Procedures for the Routine Production of Chloroplast DNA Fingerprints of Eucalyptus species

Published in: South African Forestry Journal
Volume 164, issue 1, 1993 , pages: 1–7
DOI: 10.1080/00382167.1993.9629371
Author(s): M. CoulsonUN/FRD Photosynthetic Nitrogen Metabolism Research Unit, Department of Biology, South Africa, B.I. HuckettUN/FRD Photosynthetic Nitrogen Metabolism Research Unit, Department of Biology, South Africa, M.P. WattUN/FRD Photosynthetic Nitrogen Metabolism Research Unit, Department of Biology, South Africa

Abstract

The technique of “in-organelle” DNA digestion was assessed with regard to its applicability to the production of eucalypt chloroplast DNA (cpDNA) restriction fragments. Because of the waxy cuticle, high incidence of thickened tissue and presence of phenolic compounds in eucalypt leaves, it was necessary to devise an effective protocol for the isolation of high yields of purified chloroplasts. Eucalyptus grandis S/N M6 was selected as the model for this purpose. “In organelle” digestion of the resultant E. grandis chloroplasts (450–600 μg chlorophyll) with Bgl II, followed by electrophoretic separation of fragments, produced a well resolved profile clearly distinguishable from that of an outgroup, Spinacea oleracea. However, the described chloroplast isolation procedure was found to be non-optimal when applied to E. grandis TAG 5, E. grandis TAG 14, E. nitens and E. macarthurii (yields ranged from 0,0 to 5,3 μ g chlorophyll/g fresh mass). On account of the requirement that the chloroplast extraction protocol be optimised for each lineage, it is concluded that cpDNA analysis of eucalypts via the route of “in-organelle” digestion is impractical for incorporation into a diagnostic routine.

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