Modified Media for Repeated In Vitro Cutting Cycles of Cannabis sativa Without the Use of Cytokinin

In vitro hedging; combined with the fed-batch liquid media process is an innovative system that generates multiple sterile plants without the use of exogenous cytokinin. This combined process was demonstrated with Cannabis sativa ('Cherry1', 'BaOx', 'T1', 'Peach') grown in vessels of three different physical states-stationary agar (A); stationary Oasis^® infused with liquid (OILs); and agitated Oasis^® infused with liquid (OILa). Vessels were pre-selected as control or supplemented; where supplement vessels received 15 mL DKW liquid media each cycle harvest. The number of shoot tips harvested; shoot length; and dry shoot mass from repeated cutting cycles was recorded. In a single harvest; 'BaOx' and 'Cherry 1' produced one shoot per plant from the original 15 planted on all treatments. 'Peach' and 'T1' produced less shoots on average; but the most in OIL treatments. All shoots harvested were longer in OIL compared to A; regardless of genotype. Over multiple cycles; 'Peach' and 'T1' were unable to reliably produce shoots on a repeated schedule and were, therefore, eliminated from the experiment. By cycle 3; maximum number of plants were produced; regardless of supplementation ('Cherry 1'; 30; 'BaOx'; 22). Shoot length was above 10 mm (planting standard) for both genotypes until after the third cycle (10 weeks) where number and quality decreased (nodes and internodes easily discerned). By the end of the experiment; the only shoots that remained productive for over 16 weeks and multiple repeated harvest cycles were those in OIL treatments with supplements.