Different electroporation conditions were evaluated, toward the goal of transformation
of
Coffea arabica
cv. Catimor. The tissues assayed were: embryogenic calli, leaf sections from
in vitro plants, and somatic embryos in globular and torpedo stage obtained
from cell suspensions. The effect of 1 or 24-hour pretreatment with an enzymatic
solution (2% cellulase, 1% macerozyme) and electric field strength (375, 625,
875 V/cm) was evaluated. In all the experiments the tissues were incubated
in ASP buffer (potassium aspartate) during three hours, and then one hour with
plasmid DNA (pCambia3201, containing
gus
and
bar
genes) at room temperature. The electroporation was performed at a capacitance
of 900
μ
F. The effect of the parameters evaluated was determined by the transient expression
of the
gus
gene. The optimal conditions for electroporation were one hour of enzymatic
pretreatment of torpedo shape embryos, electroporation at 375 V and 900
μ
F. The culture of electroporated tissues in liquid media with 8 mg/l benzyladenine
conducted to maximal regeneration through secondary somatic embryogenesis.
The secondary somatic embryos were formed directly in the hypocotyl surface
of the electroporated torpedo shape primary somatic embryos, the production
of secondary somatic embryos was significantly greater than the production
of primary embryos, therefore, this is an excellent method to propagate the
products of genetic transformation. The secondary somatic embryos regenerated
from electroporated torpedo shape somatic embryos were positive for
gus
expression, and also in the PCR analysis for the genes
gus
and
bar
.
