Development of electrophoresis technique for isoenzymatic analysis of rubber tree (Hevea sp.) clones

This study aimed at optimizing techniques for preparing clonal leaflet extracts and identifying buffers and migration conditions so as to achieve better resolution of electrophoretic patterns in rubber tree clones. Twenty-five enzymatic systems in eleven gel and electrode buffers were tested. Similar electrophoretic profiles were obtained from extracts of clones in different phenological stages, in samples submitted to different centrifugation conditions and in plants from the same clone. Extracts from liophylized leaflets showed the same electrophoretic resolution as nonliophylized leaflets after two days for Adh, Pgi, 6Pgdh, Lap-1, Skd, Acp, Mdh-1, ß-Glu, Pgm and Idh. Got and Est electrophoretic patterns were well resolved only in fresh leaflets extracts. The other enzymes studied did not show a good electrophoretic resolution. Employing different types of substrates, nine regions of esterase activity were observed under the conditions used, although genetic variation has been characterized for only three loci in the clones analysed. Est-7 phenotypes observed using naphtyl-esters were similar to patterns found using L-benzoyl-β-naphtylamide assubstrate, suggesting that this esterase is an endopeptidase. The use of fluorogenic substrates allows the detection of a new electrophoretic Acp variant in Hevea nitida and another βGlu variant in Hevea rigidifolia leaflet extracts.

rubber tree; clones; electrophoresis; isoenzymes