Transient gene expression in secondary somatic embryos from coffee tissues electroporated with the genes gus and bar
Da Silva,Rafael Fernandez
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 μF. 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 μF. 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.