Over-Expression of Catharanthus roseus Apoplastic Peroxidase (CrPrx) Gene Associated with Late Downstream Steps of TIAs Pathway at whole plant level.

Abstract Agrobacterium rhizogenes mediated genetic transformation for hairy root production was carried out to explore the possibility of recovering transgenic plants from hairy roots with over expressed limiting pathway Apoplastic peroxidase gene ( CrPrx ). For generating ( CrPrx ) over-expressing hairy roots, the leaves of C. roseus cv. Dhawal culture were infected with A. rhizogenes strain A 4 harboring Ri plasmid and a pBI 121- CrPrx construct. 15 independent roots clones finally established were christened as NV-C1 to NV-C15 clones. Maximum expressions of genes (RQ=8.23 ± 0.23) was observed in root lines NV-C7 followed by NV-C6(RQ=6.44 ± 0.21) and NV-C11(RQ=5.59 ± 0.30). The direct regeneration of shoots from hairy root explants co-cultured with A.rhizogenes resulted in integration of CrPrx gene in the regenerated progeny. Nearly 78.3% root explants formed adventitious shoot buds on to an optimized regeneration medium (1.0 m/l NAA+5.0 mg/l BAP). CrPrx overexpressing tisssues registered 2.00-2.54 higher level of mRNA transcripted strictosidine synthase (STR), tryptophan decarboxylase (TDC), 0.8 and 0.75 fold lesser level of CrPrx 1 in NV-C7 root tissues as compare to their non-transformed controls. HPLC analysis revealed that CrPrx overexpressing NV-C7 roots had 3 folds more ajmalicine and serpentine content over parent NVST roots comparatively for Catharanthine, this enhancement was 2 folds, while shoots regenerated from NV-C7 and NVST showed ajmalicine accumulation elevated by a factor of 2.5 as well as also favored the near doubling of vindoline content which in turn resulted in better vincristine and vinblastine content. NV-C7 root line and its respective shoot progeny is promising tissue in terms of TIA’s productivity at the whole plant level.