Abstract
The establishment of an efficient in vitro propagation system for the conservation of the Mediterranean Mandragora autumnalis is highly desirable due to its scarcity, besides its potential medicinal and pharmacological properties. In a separate unpublished study, this species has proved to be resistant to laboratory plant regeneration from vegetative tissue cultures; therefore, an alternative decoated seed (i.e., endosperm enclosed the zygotic embryo) germination approach was conducted in this study. Pre-cold treatment of M. autumnalis seeds, removal of seed coats, and exogenous application of gibberellic acid (GA3) promoted in vitro seed germination and seedling emergence. In two separate experiments, approximately 10-27% of the germinated decoated seeds developed healthy seedlings within two weeks, compared to the non-germinated intact seeds of the potting soil controls. After 72 days, the highest rates of healthy seedlings development (67.4 and 69.4%) achieved in the in vitro decoated seed cultures supplemented with 60 and 100 mg/L GA3, respectively, compared to only 25% seedlings emergence rate of the in vitro cultures devoid of GA3, and 44.2% of the soil controls. The in vitro developed plants were healthy, survived transplantation conditions, and, significantly, grew faster, formed on average more than the double number of true leaves and shoot fresh weight (p ≤ 0.05), 90% more fresh weight of root system (p ≤ 0.05), and ultimately more than the double gross fresh weight (p ≤ 0.05) than that of the in vivo developed plants of the soil controls. Such in vitro seed germination approaches would be favorable due to the higher capacity of uniform seedling establishment year-round under lab-controlled conditions, facilitating proliferation and conservation of rare and threatened species, and providing fresh and axenic plant materials required for downstream studies such as those associated with leaf-derived protoplasts and genetic transformations.