In vitro mass propagation of Cannabis sativa L.: A protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants

• An improved micropropagation protocol has been developed for Cannabis sativa L.
• The maximum response for shoot and root formation was achieved on MS + 2 μM meta-topolin.
• A separate medium containing auxin was not required for root induction.
• Micropropagated plants were successfully acclimatized ex-vitro.
• Regenerated plants were highly comparable to that of the mother plant.

The present study describes a simple, efficient and one step regeneration system for rapid shoot proliferation and in vitro rooting of Cannabis sativa nodal explants using meta-topolin (mT), an aromatic natural cytokinin. The best response in terms of explants producing maximum number of shoots with maximum shoot length and percent explants producing shoots was recorded on Murashige and Skoog (MS) medium supplemented with 2 μM mT. Shoots multiplied on the same medium for two sub-cultures were able to induce healthy roots within 4–6 weeks. A separate medium containing auxin was not required for root induction. Regenerated plantlets were successfully acclimatized and hardened off in the climatic controlled grow room with 100% survival rate. Genetic fidelity of in vitro propagated plants was tested using inter simple sequence repeat (ISSR) markers. Our results show that all the ISSR profiles from in vitro propagated plants were monomorphic and comparable to that of the mother plant, thereby confirming the genetic fidelity. Qualitatively and quantitatively, cannabinoid profiles and the content, using gas chromatography-flame ionization detector (GC–FID), in mother plant and in vitro propagated plants were found to be similar to each other. Furthermore, regenerated plants were eco-physiologically and functionally comparable to that of the mother plant. The maximized regeneration protocol using mT is thus effective and safe for large scale production of true to type C. sativa plants.

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