Resumen de Development of biotechnological tools for the genetic improvement of cannabis sativa l
Cannabis sativa L. (Cannabaceae) is an angiosperm, allogamous and dicotyledonous species that includes short and neutral-day varieties with dioecious specimens (males and females), and monoecious plants. Among its many applications, its industrial and medicinal uses stand out. Despite the fact that cannabis has been used by humans since ancient times and the growing interest that the C. sativa therapeutic properties have aroused in researchers around the world, the psychoactivity of some of its varieties, derived from its ¿9-tetrahydrocannabinol (THC) content, has motivated the prohibition of its cultivation for almost sixty years. The strict control to which cannabis has been subjected has prevented professionals from all over the world from carrying out genetic breeding programs for this species, which has resulted in the absence of uniform varieties.
In this Doctoral Thesis, different biotechnological tools for cannabis genetic improvement have been developed. In the first place, given the lack of reproducibility of some cannabis plant in vitro regeneration protocols and the great influence that the genotype exerts on their effectiveness, plant in vitro regeneration competence of different explants was evaluated. As a result, an hormone-free protocol from C. sativa hypocotyls that presents high regeneration rates (ranging from 32.26% to 71.15%) in all the genotypes evaluated, also presenting a 17.94% of spontaneous rooting rate of regenerants has been developed. At the same time, the polysomatic pattern of different cannabis explants has been studied, and it has been possible to regenerate, from them, a significant percentage of mixoploid specimens (17.65% from cotyledons and 13.33% from hypocotyls) that, as described in the existing literature, could show a greater capacity for cannabinoid synthesis.
On the other hand, given the absence of scientific publications in this regard, and the potential that this technique presents to alleviate the intrinsic variability of this species, the most in-depth study to date on the male floral biology of C. sativa has been developed. Up to 476,903 microspores and pollen grains per male flower, with in vivo microspore viability rates from 53.71 to 70.88% have been found. Furthermore, all stages of development of the microgametophyte have been correlated with an easily measurable floral morphological marker such as the bud length, identifying bud length intervals containing mostly vacuolate microspores and young bi-cellular pollen grains in all the phenotypes evaluated. In this way, and although the starch presence in C. sativa microspores and pollen grains follows a similar pattern to that observed in species recalcitrant to androgenesis, it has been possible to address the induction of microspore embryogenesis in this species, obtaining for the first time microspore-derived multicellular structures after one week long cold-shock bud pretreatment.
Finally, as a prerequisite for the genetic editing of C. sativa by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems, and taking advantage of the in vitro plant regeneration protocol which resulted from this Doctoral Thesis, it has been possible to develop for the first time a protocol for the production of stably transformed cannabis plants, which represents a historical milestone in the genetic improvement of the species. After co-culture with A. tumefaciens and subsequent culture in antibiotic-containing selective regeneration medium, hypocotyls achieved 23.1% and 5.0% of regeneration and transformation rates respectively.
As a whole, the present Doctoral Thesis provides a range of biotechnological tools that will allow the development of a new generation of high-yield cannabis varieties with uniform traits, resistant to multiple biotic and abiotic stresses, and therefore being suitable for both industrial and medicinal use.
