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Ocimum species are widely recognized for their aromatic and therapeutic properties and frequently used in the pharmaceutical, cosmetic and food industries. However, large-scale cultivation is often restricted by seasonal dependence, genetic variability, and limited propagation capacity. This study aims to establish efficient in vitro culture protocols and also to assess their transformation efficiency using diverse Rhizobium rhizogenes strains in four key Ocimum species- O. basilicum, O. sanctum, O. gratissimum and O. kilimandscharicum.In the present study, the protocol for in-vitro culture establishment for all the selected Ocimum species was carried out using two different explants sources- seeds and nodal segments. Among these, O. gratissimum exhibited the highest seed germination rate (90%) and nodal establishment compared to the rest of the species. Moreover, leaf-derived callus was also induced using different combinations and concentrations of auxins 2,4-Dichlorophenoxyacetic acid (2,4-D) or Naphthalene acetic acid (NAA) along with fixed concentration of kinetin (Kin). Notably, O. basilicum and O. kilimandscharicum responded best to 2,4-D with Kin (specifically at 2.0 to 4.0mg/L 2,4-D +0.25 kin, producing friable callus. The friable callus was then transferred to the optimized liquid medium with 3.0 and 4.0mg/L 2,4-D +0.25 kin suitable for suspension culture for O. gratissimum and O. kilimandscharicum. However, the supplementation of NAA along with the fixed concentration of kinetin (Kin) also resulted in the formation of compact callus with adventitious roots. In another objective, hairy root was induced from the leaves of all four species with two different Rhizobium rhizogenes strains-A and B. Strain B was noted to be best for all the species for hairy root emergence and establishment compared to the strain A. This study presents a robust protocol for in vitro propagation and Rhizobium mediated-transformation in these four Ocimum species. These methods provide a plethora for genetic transformation and better secondary metabolite production in Ocimum species for commercial applications.
Keywords: Ocimum, In vitro cultures, callus, hairy root, Rhizobium rhizogenes.
