Factors controlling variability in measurements of the transient gene expression in electroporated protoplasts of wheat callus.
Factors controlling variability in enzyme transient expression assays have been investigated in electroporated protoplasts isolated from wheat embryogenic and nonembryogenic calli. The level of variation was reduced to a minimum through the optimization of the beta-glucuronidase measurements in the pellet and the supernatant of the homogenized protoplasts, by expressing the data on the basis of the number of protoplasts found to be viable immediately before the assay and on the amount of protein in the pellet and supernatant. Protoplast separation on the basis of size was also useful in eliminating some of the variation resulting from the heterogeneity of the callus used. Efforts to partially synchronize the callus tissue by auxin starvation have not resulted in a significant decrease of this variation. Our results indicate that the level of variation in enzyme transient activity in protoplasts resulting from calli can be reduced by implementation of the experimental techniques presented here.
Zaghmout
O M
OM
Department of Agronomy, Horticulture, and Entomology, Texas Tech University, Lubbock 79409.
Wang
W C
WC
D002463:Cell Membrane Permeability; D046508:Culture Techniques; D004560:Electricity; D015870:Gene Expression; D005966:Glucuronidase / Q000378:metabolism; D010957:Plasmids; D014162:Transfection; D014908:Triticum / Q000235:genetics*
D005966:Glucuronidase