Journal
PROTOPLASMA
Volume 252, Issue 1, Pages 373-381Publisher
SPRINGER WIEN
DOI: 10.1007/s00709-014-0685-1
Keywords
Bioreactor; Cell suspensions; Strictosidine synthase; Transgenic; Tryptophan decarboxylase; Vinca minor
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Funding
- DST-FAST TRACK [SERC/LS-261/2012]
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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 beta-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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