Development of a petal protoplast transfection system for Sinningia speciosa

Abstract

Premise Transient gene expression systems are powerful tools for studying gene interactions in plant species without available or stable genetic transformation protocols. We optimized a petal protoplast transformation protocol for Sinningia speciosa, a model plant, to study the development of floral symmetry. 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. Conclusions The transient transfection system presented herein can be effectively used to study gene‐regulatory interactions in Gesneriaceae species.

Isolation, Purification, and Application of Protoplasts and Transient Expression Systems in Plants

“…The choice of materials for isolating plant cell protoplasts plays a pivotal role in determining both the yield and viability of the resulting protoplasts [4]. Commonly employed materials for protoplast isolation include young leaves [5], petals [6], roots [7], callus [8], and suspension cultures [9]. And it has been shown that vigorously growing and younger tissues used as isolation materials tend to yield protoplasts with higher yields and vitality [10].…”