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Over-expression of Catharanthus roseus tryptophan decarboxylase and strictosidine synthase in rol gene integrated transgenic cell suspensions of Vinca minor

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Abstract

Tryptophan decarboxylase (TDC) and strictosidine synthase (STR) genes from Catharanthus roseus have been successfully over-expressed in the rol gene integrated cell suspensions of V. minor. Thirty seconds SAAT (sonication-assisted Agrobacterium transformation) treatment of plant cell suspension with LBA1119 having construct (<hpt-<Tdc2-<Str-gus>) generated three stable TDC + STR over-expressing cell lines—PVG1, PVG2, and PVG3. The transgenes were confirmed by β-glucuronidase GUS histochemical assay and PCR amplification of rol genes/GUS gene. All the three cell suspension lines were found to be slow growing. In comparison to the control cell suspensions (GI = 241.0 ± 5.8), PVG3 cell line registered a growth index (GI) of 208.0 ± 10.0 followed by PVG1 (GI = 140.0 ± 14.2) and PVG2 (GI = 85.0 ± 9.6). The PVG3 cell line was also up-scaled in the 5-l stirred tank bioreactor with GI of 745.6 ± 35.3 under optimized parameters. Only PVG3 line registered a twofold increase in total alkaloid content (2.1 ± 0.1 % dry wt.) and showed vincamine presence (0.003 ± 0.001 % dry wt.) which was further enhanced at the bioreactor level (2.7 ± 0.3 and 0.005 ± 0.001 % dry wt., respectively). Real-time (RT) qPCR analysis of PVG3 showed more than sevenfold to eightfold increase in TDC and STR expression [relative quantity value (RQ) = 7.6 ± 0.8 (TDC); RQ = 8.5 ± 0.9 (STR)].

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Acknowledgments

The work presented here has been supported by DST-FAST TRACK SERC/LS-261/2012. PV is highly thankful to Johan Memelink (Leiden University) for providing the Agrobacterium tumefaciens strain LBA1119 with construct (<hpt-<Tdc2-<Str-gus>). Help rendered by Jyotsana Priya during HPLC analysis has been highly acknowledged.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Plant Biotechnology, Central Institute of Medicinal and Aromatic Plants (CIMAP), Council of Scientific and Industrial Research, PO-CIMAP, Kukrail Picnic Spot Road, Lucknow, 226015, India

    Priyanka Verma, Abhishek Sharma, Shamshad Ahmad Khan & Ajay K. Mathur

  2. Department of Analytical Chemistry, Central Institute of Medicinal and Aromatic Plants (CIMAP), Council of Scientific and Industrial Research, PO-CIMAP, Lucknow, 226015, India

    Karuna Shanker

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  1. Priyanka Verma
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  2. Abhishek Sharma
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  3. Shamshad Ahmad Khan
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  4. Karuna Shanker
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  5. Ajay K. Mathur
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Correspondence to Priyanka Verma.

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Verma, P., Sharma, A., Khan, S.A. et al. Over-expression of Catharanthus roseus tryptophan decarboxylase and strictosidine synthase in rol gene integrated transgenic cell suspensions of Vinca minor . Protoplasma 252, 373–381 (2015). https://doi.org/10.1007/s00709-014-0685-1

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