Effects of buffer system pH and tissue storage on starch gel electrophoresis of allozymes in three tropical tree species

Abstract

The effects of 16 different electrophoresis buffer pHs, 4 tissue storage conditions and 5 storage times on starch gel electrophoresis of 18 enzymes were determined to design a genetic variation sampling strategy for an isozyme study of 3 tropical tree species, Racosperma auriculi-form, R mangium, and Terminalia superba. The pH of the buffer systems had a significant effect on the number of putative gene loci and alleles resolved, and the staining intensity of the 18 enzymes assayed. For Racosperma species, 2 buffer systems B-7 (Tris- citrate gel, pH 9.0: lithium hydroxide-borate electrode, pH 8.5) and H7 (histidine-EDTA gel, pH 7.6: Tris-citrate electrode, pH 7.7) gave the highest average performance in resolving power. All buffer systems yielded poor results for Terminalia. Freezing of Racosperma embryos for up to 2 months did not seriously affect enzyme activity. However, freezing cotyledon tissue of Terminalia increased enzyme activity over a 2-month period. In general, frozen tissues either with or without extraction buffer, were consistently better than frozen tissues with extraction buffer and DMSO. Three classes of enzymes were defined, based on their stability under the standardized storage conditions in vivo. Using the best buffer systems (B-7 and H7) and tissue storage conditions (T0 or T1), 42, 43, and 32 zones of activity were resolved for R auriculiforme, R mangium, and T superba, respectively. Genetic inference of enzyme variants was made for 31 and 32 putative gene loci in R auriculiforme and R mangium, respectively. Mean number of putative alleles per locus was 3. 0 for R auriculiforme and 2.4 for R mangium.