Blue and supplemental far-red light were observed to affect in vitro shoot growth with Cannabis sativa (‘BaOx’ and ‘Cherry 1’) in RV750 vessels. A modified “hedging” and fed-batch system for multiple harvests using Oasis^® foam and 120 mL DKW medium was used. Fifteen nodal and/or apical tips were planted and placed into PAR light treatments providing various red to blue ratios (polychromatic white 9:1 RB and dichromatic 2–15: 1 RB, with and without 5% far-red light). Treatments had similar light intensities (190–240 µmol · m⁻² · s⁻¹ PPFD) for a 16 h photoperiod. Shoot tips were harvested in vitro on five successive two-week cycles, with 15 mL of DKW media supplemented to each vessel following harvest. Shoot numbers, length, and fresh and dry mass were recorded at each cycle harvest. Five randomly selected shoot tips per vessel were rooted ex vitro on greenhouse mist bench for 16 days. Over multiple cycles, 5% far-red increased shoot numbers and length in both genotypes tested, regardless of polychromatic or dichromatic source. Shoots harvested per vessel increased from 15 to 28 in three cycles (6 weeks), but increased from 15 to 18 without far-red treatment. Shoot length in far-red-treated plants increased from 19 to 25 mm during cycles 1–3. Plants without far-red treatment were approximately 15 mm during the first three cycles. By cycle 5, both far-red- and non-far-red-treated plants decreased to 10 mm. Dry mass was greatest in cycle 1 for both genotypes (‘Cherry 1’ was 6 mg and ‘BaOx’ was 7 mg) under the highest amount of blue light, but 2 mg under the lowest amount of blue light. Dry mass decreased by 50% in cycle 3, to 4 mg, where it remained for the duration of the experiment. Sixty eight percent of shoots rooted ex vitro on the mist bench, regardless of any prior in vitro treatment.

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.