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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.

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The financial support for this research work from the Department of Biotechnology, Ministry of Science and Technology, Government of India is gratefully acknowledged by the authors (Sanction Order No. BT/PR6829/GBD/27/489/2012). The authors thank Dr. Nandita Madhavan for providing access to semi-preparative HPLC facility at IIT Madras (Currently: Associate Professor, Indian Institute of Technology Bombay, Mumbai, India). The authors wish to thank Dr. Suresh Baburaj [Survey Officer, Central Council for Research in Homoeopathy (CCRH)], for providing the Viola odorata plant material required for the study. The authors thank Prof. P. Balaram (Molecular Biophysics Unit, Indian Institute of Science Bangalore, India) for his valuable suggestions for deciphering the sequence of the cyclotide(s) identified in this study. The authors also thank Dr. V. Sabareesh (Advanced Centre for Bio Separation Technology, Vellore Institute of Technology University, Tamil Nadu, India) for initial discussion on de novo sequencing of peptides.

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Correspondence to Smita Srivastava.

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Supplementary Fig. S1. De novo peptide sequencing of vodo I66 using collision-induced dissociation (CID) based MS/MS spectra of (a) 3643.61 Da; (b) 3428.48 Da; (c) 919.47 Da and (d) 1258.51 Da. The b and y ions are indicated in blue and red font respectively. The peptide sequence deduced from each MS/MS data is indicated in green font in the respective panel. (TIFF 353 KB). (TIF 352 KB)


Supplementary Fig. S2. De novo peptide sequencing of vodo I98 using collision-induced dissociation (CID) based MS/MS spectra of (a) 3522.54 Da; (b) 2729.15 Da; (c) 1433.62 Da and (d) 1589.70 Da.The b and y ions are indicated in blue and red font respectively. The peptide sequence deduced from each MS/MS data is indicated in green font in the respective panel, where ‘X’ indicates I/L (TIFF 334 KB). (TIF 333 KB)

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Supplementary material 4 (DOCX 12 KB)

Supplementary material 5 (DOCX 12 KB)

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Narayani, M., Chadha, A. & Srivastava, S. Callus and cell suspension culture of Viola odorata as in vitro production platforms of known and novel cyclotides. Plant Cell Tiss Organ Cult 130, 289–299 (2017).

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  • Viola odorata
  • Cyclotides
  • Callus
  • Cell suspension culture
  • Liquid chromatography
  • Fourier transform mass spectrometry