High Frequency Plant Regeneration from Leaf Derived Callus of High Δ9-Tetrahydrocannabinol Yielding Cannabis sativa L.

An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC-FID), high THC yielding elite female clone of a drug-type Cannabis variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 mu M) of indole- 3-acetic acid (IAA), indole- 3-butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 mu M of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 mu M NAA plus 1.0 mu M TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 mu M TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 - MS) supplemented with 2.5 mu M IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95% in the indoor growroom.