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)].

Quantification of Anti‐Addictive Alkaloids Ibogaine and Voacangine in In Vivo‐ and In Vitro‐Grown Plants of Two Mexican Tabernaemontana Species

“…alba would open the door to genetic engineering of the species, most notably by making it possible to overexpress key enzymes. This approach has been successfully applied to several Apocynaceae species, focusing on important enzymes of the MIA pathway, like strictosidine synthase and tryptophan decarboxylase …”

Agrobacterium‐mediated transformation of Vitis Cv. Monastrell suspension‐cultured cells: Determination of critical parameters

“…SCC from different plant species have been successfully transformed to produce secondary metabolites. For instance, tobacco BY‐2 cell cultures that accumulate interleukin‐10 have been produced or Vinca minor cell cultures have been transformed to produce an increase in the accumulation of the alkaloid vincamine as well as other examples that can be found in the review by Verpoorte et al However, to our knowledge there are not papers reporting the transformation of dedifferentiated Vitis SCC with genes involved in the secondary metabolism, probably because there has not been an efficient transformation protocol until now.…”

Morphogenesis, Genetic Stability, and Secondary Metabolite Production in Untransformed and Transformed Cultures

“…Successful overexpression of tryptophan decarboxylase (TDC) and strictosidine synthase (STR) genes from Catharanthus roseus in rol gene-integrated PVG3 cell suspensions of Vinca minor demonstrated production of 0.003% DW vincamine with overall twofold higher total alkaloid (2.1% DW) accumulation. PVG3 cell line showed enhanced growth, total alkaloid content (~2.7% DW), vincamine content (0.005% DW), and expression of TDC and STR genes (more than seven-to eightfold) in the 5 L stirred tank bioreactor [434].…”