In vitro direct regeneration and Agrobacterium tumefaciens mediated in planta transformation of Ocimum sanctum L.

Ocimum sanctum is a multipurpose herb with highly significant medicinal properties. An in vitro direct regeneration protocol for propagation of a valuable medicinal plant Ocimum sanctum , using petiole explants has been successfully developed. The protocol employed regeneration of shoots directly, without any intervening callus using Murashige and Skoog (MS) medium fortified with 3 mg L -1 BAP + 1 mg L -1 NAA. The maximum regeneration frequency of 98% with 9.6 shoots per explants was achieved. Agrobacterium tumefaciens mediated genetic transformation (ATMT) protocol (transient and stable) was established using LBA4404 strain harboring pBI121 with uid-A reporter gene and neomycin phosphotransferase ( npt-II ) as selection marker. The putative transformants were screened on MS with 50 mg L -1 kanamycin and subsequently rooted on the half-strength MS medium. The confirmation was done via polymerase chain reaction (PCR) using npt-II and gus-A gene-specific primers. The maximum stable transformation frequency 70% ± 0.35. Thus, it is apparent that the established in vitro direct regeneration and ATMT method was suitable for integrating novel genes and modulating the metabolic flux for obtaining desired agronomic trait in planta .