Polyploidization for the Genetic Improvement of Cannabis sativa

Abstract

Cannabis sativa L. is a diploid species, cultivated throughout the ages as a source of fiber, food, and secondary metabolites with therapeutic and recreational properties. Polyploidization is considered as a valuable tool in the genetic improvement of crop plants. Although this method has been used in hemp-type Cannabis, it has never been applied to drug-type strains. Here, we describe the development of tetraploid drug-type Cannabis lines and test whether this transformation alters yield or the profile of important secondary metabolites: Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD), or terpenes. The mitotic spindle inhibitor oryzalin was used to induce polyploids in a THC/CBD balanced drug-type strain of Cannabis sativa . Cultured axillary bud explants were exposed to a range of oryzalin concentrations for 24 h. Flow cytometry was used to assess the ploidy of regenerated shoots. Treatment with 20–40 μM oryzalin produced the highest number of tetraploids. Tetraploid clones were assessed for changes in morphology and chemical profile compared to diploid control plants. Tetraploid fan leaves were larger, with stomata about 30% larger and about half as dense compared to diploids. Trichome density was increased by about 40% on tetraploid sugar leaves, coupled with significant changes in the terpene profile and a 9% increase in CBD that was significant in buds. No significant increase in yield of dried bud or THC content was observed. This research lays important groundwork for the breeding and development of new Cannabis strains with diverse chemical profiles, of benefit to medical and recreational users.

Development of a Direct in vitro Plant Regeneration Protocol From Cannabis sativa L. Seedling Explants: Developmental Morphology of Shoot Regeneration and Ploidy Level of Regenerated Plants

“…In our case, spontaneous rooting of in vitro regenerants represents an added advantage of our regeneration protocol, as a separate auxin containing medium is not required for root induction. In any case, although it was necessary to promote rooting of more in vitro regenerated plants, there are several published protocols concerning in vitro rooting of C. sativa shootlets with an efficiency not below 80% ( Lata et al, 2009 , 2010 , 2016a , b ; Wang et al, 2009 ; Chaohua et al, 2016 ; Parsons et al, 2019 ).…”

“…In the case of C. sativa , results found in the literature are contradictory. While Clarke (1981) found that polyploidization increased THC levels while decreased CBD and cannabinol (CBN) content, and that triploids proved to be inferior to both diploids and tetraploids in terms of cannabinoid production, Parsons et al (2019) got a similar chemical profile between tetraploids and diploids, although notable increases in CBD and sesquiterpenes were associated with tetraploids. In relation to mixoploids and their cannabinoid content, Mansouri and Bagheri (2017) demonstrated that polyploidization increased significantly the content of THC in mixoploid plants compared with tetraploid and diploid plants, and that tetraploid plants had lower amounts of this substance in comparison with diploids, suggesting that mixoploids could be useful to produce THC for commercial use.…”

“…Finally, as it is known that root and shoot development depends on cytokinin:auxin ratio and that high levels of cytokinin supports shoot formation ( Skoog and Miller, 1957 ; Su et al, 2011 ), we tested the effect of media with a cytokinin concentration 50-fold higher than the auxin level, together with different adenine and phenylurea derivatives like BAP, 6-benzylaminopurine riboside (BAP RIB ), TDZ, forchlorfenuron (4-CPPU) and ZEA RIB plus NAA, an auxin commonly employed in protocols for in vitro regeneration of shoots ( Plawuszewski et al, 2006 ; Wielgus et al, 2008 ; Lata et al, 2010 ; Chaohua et al, 2016 ) and in vitro rooting of C. sativa ( Lusarkiewicz-Jarzina et al, 2005 ; Wang et al, 2009 ; Movahedi et al, 2016a ; Parsons et al, 2019 ). The different hormonal combinations present in the different shoot induction media evaluated in this work, are detailed in Table 1 .…”

“…Recent research has reported many cannabinoid pharmacodynamic and pharmacokinetic properties, expanding the potential use of cannabinoids in medical therapies ( Urits et al, 2019 ), and promoting the development of cannabis improved varieties with specific biochemical profiles. In this respect, in vitro culture is a useful tool that has been employed to complement cannabis conventional breeding through large-scale micropropagation of selected elite clones ( Lata et al, 2017 ), development of polyploid varieties with enhanced levels of secondary metabolites ( Mansouri and Bagheri, 2017 ; Parsons et al, 2019 ) or genetic transformation of non-regenerating tissues ( Feeney and Punja, 2003 , 2015 , 2017 ; Wahby et al, 2013 , 2017 ). However, there is still a lack of an in vitro regeneration protocol efficient in the broad range of genetically diverse materials in the species.…”

Effects of In Vitro Polyploidization on Agronomic Characteristics and Fruit Carotenoid Content; Implications for Banana Genetic Improvement

“…Polyploidization has been advocated as a tool for genetic improvement in several crops with the aim of creating new polyploid varieties or increasing fertility of interspecific hybrids (Dhooghe et al, 2011; Sattlser et al, 2016; Denaeghel et al, 2018; Parsons et al, 2019). Polyploid induction has been proposed as an alternative breeding approach for bananas.…”