title: Over-expression of Catharanthus roseus tryptophan decarboxylase and strictosidine synthase in rol gene integrated transgenic cell suspensions of Vinca minor.
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)].
authors.affiliation: 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, priyankaurbest@gmail.com.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Verma
authors.forename: Abhishek
authors.initials: A
authors.lastname: Sharma
authors.forename: Shamshad Ahmad
authors.initials: SA
authors.lastname: Khan
authors.forename: Karuna
authors.initials: K
authors.lastname: Shanker
authors.forename: Ajay K
authors.initials: AK
authors.lastname: Mathur
mesh_terms: D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000378:metabolism*; D019759:Carbon-Nitrogen Lyases / Q000378:metabolism*; D030008:Catharanthus / Q000378:metabolism*; D059828:Plant Cells / Q000378:metabolism*; D031053:Vinca / Q000378:metabolism*
chemical_list: D001142:Aromatic-L-Amino-Acid Decarboxylases; D019759:Carbon-Nitrogen Lyases; C022684:strictosidine synthetase
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