Development of a Directin vitroPlant Regeneration Protocol FromCannabis sativaL. Seedling Explants: Developmental Morphology of Shoot Regeneration and Ploidy Level of Regenerated Plants

In vitroshoot regeneration can efficiently contribute to the improvement of recalcitrantCannabis sativaL. We aimed at developing a highly efficient protocol forin vitrodirect regeneration ofC. sativaplants from different explants (cotyledon, hypocotyl, and true leaf) from seedlings of monoeciousC. sativashort-day varieties Ferimon, Felina32, Fedora17, and USO31, together with dioecious neutral-day variety Finola. Ten regeneration media, including already published protocols, and self-designed combinations of plant growth regulators were tested. The developmental morphology since germination of seeds to the development of rooted plantlets was followed. Additionally, the ploidy level of explants andin vitroregenerants was analyzed. We concluded that hypocotyl is the best explant forin vitrodirect regeneration ofC. sativaplants with 49.45% of responding explants, while cotyledon and true leaf had a poor response with, respectively, 4.70 and 0.42% of explants developing plantlets. In terms of shoot regeneration, we found significant differences among the culture media evaluated and the varieties studied. Overall, the best regeneration media were ZEA(RIB)2.0 (mg/L) and ZEA(RIB)1.0 (mg/L) + NAA 0.02 (mg/L) with 66.67% of responding hypocotyls. Amazingly, hypocotyls cultured in medium without plant growth regulators showed an excellent response (61.54% of responding hypocotyls) and spontaneous rooting of regenerants (17.94%).In vitroregenerated plants were acclimatized just 6 weeks after culture initiation. The developmental morphology study suggests that regenerated shoots originate from pericycle cells adjacent to xylem poles. Polysomaty was detected in hypocotyls and cotyledons of all varieties studied, and diploid (>80%) and mixoploid (with diploid and tetraploid cells) plants were regenerated. Our protocol allows a high shoot organogenesis efficiency in differentC. sativavarieties. The fact that a significant percentage of plants are mixoploid may provide an alternative way to develop polyploids inC. sativa. Our results show that directin vitroregeneration may make a significant contribution to the development of improvedC. sativamaterials for medical applications.