Floral Reversion based micropropagation of Day-Neutral Cannabis sativa L

Micropropagation systems have been developed for photoperiod sensitive cultivars of cannabis, but there are currently no published methods for day-neutral genotypes. Most established cannabis micropropagation systems rely on shoot proliferation using modal explants that need them to be maintained in vegetative growth, which is not possible for day-neutral genotypes. Floral reversion, the process by which plants revert to vegetative growth from the generative stage, has been demonstrated as an alternative and potentially more efficient approach to the micropropagation of photoperiodic cannabis. The current study investigated if this process could be adapted to facilitate the micropropagation of day-neutral genotypes and overcome existing barriers. During the process, various factors such as photoperiod and light intensity may influence the efficiency of floral reversion. To evaluate this approach in day-neutral cultivars, various photoperiods were compared to assess the impact on reversion rate and subsequent growth. Excised florets obtained from in vitro day-neutral C. sativa cv. “Blue Auto Mazar x auto Blueberry” seedlings were subjected to five photoperiods including 12.0, 16.0, 18.0, 20.0, and 24.0 hours of light per day for four weeks. Reversion rates and time, shoot length, shoot number, and node number were measured. Statistical analyses revealed significant differences (p-values < 0.05) in terms of reversion time among photoperiod treatments. The highest reversion rate happened under the 16.0 hr photoperiod with 72% success. The highest shoot lengths were observed under 20.0 hr of photoperiods with a mean of 10.1 mm and the lowest shoot length belonged to 12.0 hr of photoperiods with a mean of 5.6 mm, respectively. Reverted shoots developed vegetatively for some time before initiating new flowers. However, during this period the shoots were successfully rooted in vitro and then acclimated to the growth chamber where they completed their life cycle, including seed production. This process was also successfully achieved from a 2-year old culture of C. sativa cv. “Blue Auto Mazar”, demonstrating that it is feasible to use this approach for long term culture maintenance and multiplication. This study demonstrates that floral reversion can successfully be used to clonally propagate day-neutral cannabis plants and serves as a basis for developing large-scale clonal propagation and breeding strategies.