Callus and cell suspension culture of Viola odorata as in vitro production platforms of known and novel cyclotides

Cyclotides are unique plant cyclic-peptides that can serve as agrochemicals, pharmaceutical scaffolds for drug delivery, and therapeutic agents. Currently, cyclotides are obtained only via direct extraction from limited plants. Hence, they serve as valuable candidates for synthesis via plant cell bioprocesses. In this study, callus lines (47 in total) were successfully induced from the leaf and petiole explants of the Indian medicinal plant, V. odorata, on a solidified woody plant medium (WPM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (4.5 mg/l). Two fast growing callus lines, VOP-4 and VOL-44, were selected for the development of cell suspension cultures having a doubling time of 8 and 6 days, respectively. Further, known (15) and novel (9) cyclotides were identified for the first time in the callus and cell suspension cultures of V. odorata, using liquid chromatography and Fourier transform mass spectrometry. The cyclotides were identified based on their monoisotopic mass (2.5-4 kDa), hydrophobic nature, disulfide bonds, circular structure and amino acid sequence. Some of the cyclotides identified in the study (vodo I96, vodo I97, vodo I98) were exclusively produced in callus/cell suspension cultures and not in the parent plant. The study revealed that besides germplasm conservation, plant cell bioprocessing of V. odorata could be a potential alternative for in vitro production of known and novel cyclotides.