Transient expression assay for optimization of direct gene transfer into cucumber meristem protoplasts by electroporation

The paper presents a new way of obtaining viable and very homogeneous cucumber protoplasts. Protoplasts from cells formed in the shoot tip meristem culture were isolated from suspension. Plasmid pBI121 was introduced using impulse electric field. Effectiveness of transformation process was determined by the transient expression of ß-glucuronidase (GUS) gene, controlled by promotor 35S. The activity of ß-glucuronidase enzyme as a product of GUS reporter gene was estimated by fluorimetric method (JEFFERSON 1987). Parameters of electroporation process were optimized. The transient expression of GUS gene was measured 24 h after electroporation. The highest effectiveness of transformation process was achieved using three electric impulses at the initial voltage of 250-350 V at 10-sec. intervals as a result of discharging a 140 µF capacitor and 50-70 µg × cm⁻³ plasmid DNA in the presence of 50 µg × cm⁻³ carrier DNA. The system presented is an effective method of exogenic DNA transfer, which is indicated by a high transient expression of the reporter gene. In comparison to Agrobacterium tumefaciens and A. rhizogenes, this alternative method of gene transfer can be used for obtaining transgenic cucumber plants.