Factors affecting transient gene expression in electroporated black spruce (Picea mariana) and jack pine (Pinus banksiana) protoplasts.
Methods were developed for transient gene expression in protoplasts of black spruce (Picea mariana) and jack pine (Pinus banksiana). Protoplasts were isolated from embryogenic suspension cultures of black spruce and from non-embryogenic suspensions of jack pine. Using electroporation, transient expression of the chloramphenicol acetyltransferase (CAT) gene was assayed and shown to be affected by the cell line used, by voltage, temperature, and by the plasmid concentration and conformation. Increasing the plasmid DNA concentration (0-150μg ml(-1)) resulted in higher levels of transient CAT expression. In jack pine, linearized plasmid gave 2.5 times higher levels of CAT enzyme activity than circular. Optimal voltage varied for each cell line of the two species within the range 200-350 V cm(-1) (960 μF). A heat shock treatment of protoplasts for 5 min at 45 °C resulted in enhanced CAT gene expression for both species.
National Research Council, Plant Biotechnology Institute, 110 Gymnasium Road, S7N OW9, Saskatoon, Saskatchewan, Canada.
T E
TE
Tautorus
F
F
Bekkaoui
M
M
Pilon
R S
RS
Datla
W L
WL
Crosby
L C
LC
Fowke
D I
DI
Dunstan