Genetic and molecular studies on the accumulation of bioactive compounds in Cannabis sativa L.

Cannabis is a predominantly dioecious and phenotypically diverse, monotypic genus consisting of a single species, the Cannabis sativa. Its cultivation started in Eurasia, several thousand years ago, where the selective pressure that has led it to be cultivated around the globe today began. As a dioecious plant, C. sativa is an obligate outbreeder, and as such every individual plant is genetically unique, and producing offspring with desired traits can be challenging. This genetic variation can be both a blessing and a curse for breeders, because it provides a rich genetic pool from which to select for desired traits, but also it can be difficult to achieve consistency and stability in breeding programs. The current C. sativa constraints, such as its high level of heterozygosity, reside in the limited genetic improvement and poor conservation of genetic resources, resulting from prohibition against the plant as a source of one of the most widespread illicit drugs. Unlike major crops, C. sativa has suffered from a lack of progress in the knowledge of its physiology and has not benefit from advances in breeding technologies. In recent years, there has been growing interest in the potential of C. sativa for both medicinal and industrial purposes. Its legalization in many countries has led to increased research and investment in the cultivation, processing, and marketing of hemp and medical Cannabis products. As a result, the plant is gaining recognition as a valuable crop with significant economic, environmental, and health benefits. The plasticity and wide genetic variability represent the intrinsic functionality of this plant and emphasise the agricultural value of the species. The long stem fibres have been used for millennia as the predominant source of fibre in the textile and paper industry. The seeds are suitable for human and animal consumption and contain a balanced ratio of omega-6/omega-3 essential polyunsaturated fatty acids. In addition to traditional uses, one of the main applications today is Cannabis as a source of countless bioactive compounds. Indeed, C. sativa female flowers are rich in bioactive compounds belonging to the secondary metabolism with remarkable phytochemical potential. These compounds are mostly classified into three classes: phytocannabinoids, terpenoids and phenolics. Phytocannabinoids are the most studied compounds, mainly due to their wide range of pharmaceutical effects in humans, with more than 150 constituents identified, of which THCA, CBDA, CBGA and CBCA are the most representative. Over the last five years, significant advances in C. sativa genetic research have led to a deeper understanding of the plant's genetic background and its potential applications. With the increasing legalization and commercialization, genetic research has become a critical tool for developing new strains with desired traits and improving the quality and consistency of C. sativa products. One major breakthrough in C. sativa genetic research has been the sequencing of the genome, providing valuable insights into the plant's complex genetics and the biochemical pathways that produce its various phytocannabinoids and terpenes. Other recent studies have focused on identifying the genetic markers associated with specific traits, such as high THCA or CBDA content, flowering time, disease resistance, and yield. Advances in new breeding techniques, such as CRISPR-Cas9, have also opened up new possibilities for editing genome for a desired trait. In addition, genetic research has also shed light on the evolutionary history of the plant and its relationship with other members of the Cannabaceae family, helping to clarify the taxonomic classification of different genotypes. Overall, the recent years have seen significant progress in C. sativa genetic research, providing new insights into the plant's biology and potential applications for human health and industry. There is also a growing interest in the study of the plant's genetics and molecular biology. By identifying the genes and pathways involved in phytocannabinoids biosynthesis, as well as the regulatory mechanisms that control their expression, new strategies can be developed for manipulating their production, with important applications in medicine, industry, and agriculture. In this Doctoral Thesis, C. sativa genetic was studied with the aim of better understanding the route leading to phytocannabinoids synthesis. Both industrial and medicinal varieties were used, tapping into the biodiversity available within a germplasm collection maintained at CREA- Research Centre for Cereal and Industrial Crops in Bologna and Rovigo sites, where it was also possible to cultivate high-THC strains in outdoor and indoor facilities authorized with decree n. SP/041 on 13rd March 2017 according to art. 26 of the D.P.R. 309/90). In the first chapter an investigation of the genetic and transcriptional variability of cannabinoid synthases is illustrated, performed on a set of Italian and French C. sativa genotypes with diverse chemotypes. The work focused also on the role of the cannabichromenic acid synthase (CBCAS), for a long time neglected, and considered a gene peculiar to fibre genotypes, considered a mutated, non-functional copy of THCAS for their extremely high sequence similarity. The results showed that CBCAS is present and expressed in both drug-type and fiber-type genotypes, and several genetic variants of CBCAS genes were identified, which may contribute to differences in phytocannabinoid production between different chemotypes. In the second chapter the transcription analyses of cannabinoid pathway genes, during early vegetative stages of plant development are reported. Besides cannabinoid synthases, also other genes responsible for early reaction in the synthesis of these terpeno-phenolic compounds were analysed in order to deepen knowledge for defining the main determinants of this metabolism in the early stages of plant life. Cannabicromenic acid was found the first cannabinoid accumulated in the seedlings, shortly after emergence, and hypotheses are given on the regulation of its synthesis in planta. Moreover, in this chapter is also reported the transcription of cannabinoid synthase genes during seed germination and in long-storage seeds, uncovering a new and important level of gene regulation during seed germination and providing an estimate of the importance of this metabolism for the plant. The third chapter focused on the analysis of a cultivated population of the FINOLA variety, obtained from the certified commercial seed. As with many industrial varieties, FINOLA has a chemotype III and derives from years of breeding, however it still accumulates little amount of THC (residual THC) on inflorescence which can exceed the legal limit and cause seizure and losses to growers. The results show how the B1080/B1192 molecular marker was effective at identifying hemp plants with functional THCA synthase and total THC content above the legal limit. Biochemical analyses also demonstrated a 100% association between the chemotype predicted by molecular markers and the actual chemotype. This study suggests that molecular markers could be used as effective tools for hemp growers and breeders, helping to ensure compliance with regulations and avoid legal issues related to hemp cultivation. The possibility of combining the micropropagation with the maintenance and renewal of mother plants, deputed to the national production of medical cannabis was evaluated and reported in chapter four. Overall, the work provided a valuable protocol for the micropropagation of C. sativa involving the use of cytokinin and gibberellin-based media for the induction and proliferation of shoots, followed by rooting on a separate rooting medium. The results also demonstrated the successful acclimatization and transfer of in vitro-derived plants to ex-vitro conditions and the potential for using micropropagation as a tool for the genetic improvement and development of new C. sativa plants but also for the preservation of genetic diversity