Methods and Results
A high yield of petal protoplasts was obtained using a 6-h enzyme digestion in a solution of 1.5% cellulase and 0.4% macerozyme. Modest transfection efficiency (average 41.4%) was achieved. The viability of the transfected protoplasts remained at more than 90%. A fusion of green fluorescent protein and CYCLOIDEA (SsCYC), the Teosinte branched 1/Cincinnata/Proliferating cell factor transcription factor responsible for floral symmetry, was subcellularly localized inside the nuclei of the protoplasts. Transiently overexpressing SsCYC indicates the success of this system, which resulted in the predicted increased (but nonsignificant) expression of its known target RADIALIS (SsRAD1), consistent with gene network expectations.
The transient transfection system presented herein can be effectively used to study gene-regulatory interactions in Gesneriaceae species.