Abstract
Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium x hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-mu F capacitor in a 250-V cm(-1) electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33-36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of > 1,500 mu S cm(-1) allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l(-1) kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l(-1) kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.