An efficient and reliable shoot organogenesis protocol was developed from leaf explants taken from microshoots of Ocimum tenuiflorum L. Cultures were established using nodal explants taken from field grown mature plant on MS medium supplemented with 6-benzyladenine (BA; 2.5 μM). The effect of different cytokinins namely BA, kinetin (KIN) and thidiazuron (TDZ) in combination with 0.5 μM α-naphthalene acetic acid (NAA) was examined on shoot proliferation and elongation. Maximum number of shoots per culture vessel (38) was recorded on MS medium supplemented with 5.0 μM BA, whereas the maximum number of elongated shoots per culture vessel (15) along with maximum shoot length (4.80 cm) was observed on medium supplemented with 2.5 μM BA. Shoot organogenesis was attempted from the segments of fully expanded leafs and 34.45% explants regenerated shoots on MS medium supplemented with 5.0 µM each of BA and NAA. Incorporation of cefotaxime (300 mg/l) was found to be beneficial for shoot organogenesis and further increased shoot regeneration frequency to 36% explants. On the other hand, addition of carbenicillin into the medium was found to inhibit shoot organogenesis. For root induction in microshoots, among various auxins used, IBA was found to be the best. A pulse treatment of 50.0 μM IBA for 36 h improved rooting of microshoots and a maximum of 89% microshoots rooted. Rooted microshoots were successfully acclimatized under polyhouse conditions with a survival rate of 80%. Later acclimatized plants were established in their natural conditions. Random Amplified polymorphic DNA and Inter Simple Sequence Repeat markers established a clonal fidelity of micropropagated plants with that of mother plant.
The authors are thankful to Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala and TIFAC-CORE, Thapar Institute of Engineering and Technology, Patiala for providing necessary facilities required to carry out the work.