Integrative approaches to cannabis sativa: genetic classification, transcriptomic analysis, and hormonal modulation

<p dir="ltr">Cannabis sativa (Cannabis) is a versatile medicinal plant, used for millennia with broad applications in the production of textiles, seed oil, and most notably as a recreational intoxicant. A paradigm shift has ushered in an era of contemporary usage that is focusing on the legitimate medicinal potential of the secondary metabolites produced in the flowers of female plants. As traditional medicine makes way for more modern, rigorous clinical applications, the necessary scientific exploration is well underway to dissect the genetic blueprint and metabolic systems of this unusual plant and to establish the efficacy of Cannabis as a treatment option for a number of medical conditions. Overall, the studies encompassed within this thesis were undertaken to address industry issues highlighted by a small number of companies that supported the work described herein. These companies identified areas of interest for their future plant research as part of their larger Cannabis cultivation and processing business models. To that end, the first published Chapter of this study focused specifically on the secondary metabolites grouped together as cannabinoids, their production in the glandular trichomes, their actions on the mammalian endogenous cannabinoid receptors, and the availability of genetic resources for Cannabis research. Using this foundation, the published study encapsulated in Chapter 2 employed a reduced representation shotgun sequencing approach of 87 Cannabis plants from 10 different varieties that included 6 high Δ-9-tetrahydrocannabinol (THC) and 4 high cannabidiol (CBD) producing cultivars to differentiate varieties beyond the widely used, but non-standardised and laborious, metabolomics method. A set of single nucleotide polymorphisms (SNPs) were curated that were capable of grouping plants into varieties. However, some varieties previously considered by Cannabis propagators to be distinct, were unable to be separated at the highest resolution achievable with this approach, likely indicating their recent common pedigree. A subset of 172 SNPs was then generated that were able to group varieties with high confidence and found to be highly associated with the long terminal repeat class of retrotransposable elements. The study in Chapter 3, describes sequencing the transcriptome of the glandular trichomes that produce medically significant cannabinoids and established the transcript profile over a developmental time course of 5-weeks post-floral induction. Differentially expressed transcripts exhibited a clear transition at week 7, indicating a switch from growth and metabolite production to senescence. Leveraging the reduced representation dataset (Chapter 2), the study then examined the relationship between a cohort of SNPs and a set of highly differentially expressed transcripts prior and post transition at week 7, as well as transcripts involved in cannabinoid biosynthesis, with the majority of SNPs falling in non-coding regions of transcripts. Moreover, several differentially expressed transcripts were assembled that were not assigned function and as a result represent a set of potentially ‘novel’ transcripts that warrant further detailed investigation. Finally, the assembly of differentially expressed transcripts relating to hormone biosynthesis indicated the production and action of phytohormones within Cannabis trichomes. With this information, and the reported positive impact of hormones in other cropping plant species, the research described in Chapter 4 sought to determine the outcome of exogenous application of the hormones, jasmonic acid, gibberellic acid and cytokinin to developing lowers. Weekly application for 7 weeks reduced the measured trichome head width in plants subjected to each of the hormone treatments by the final week, while the density of trichomes remained unchanged. The application of gibberellic acid caused a significant reduction in the production of all measured cannabinoids, however the impact of jasmonic acid and cytokinin on cannabinoid concentration was less well defined. The research surrounding Cannabis and its applications continues to accrue, and the constituent parts of this thesis add to the global body of developing work. Directed by industry needs, a novel cheap and effective sequencing method for genetic discrimination of varieties was demonstrated in Cannabis. The transcript profile of the glandular trichomes has previously been assembled and superficially examined, and this was extended by assessing the RNA pool from this specialised cell type across a time course encompassing a critical developmental phase in a commercially relevant variety. Finally, the application of hormones has been established in other plant species as an effective modulator of a variety of biological processes although there are few examples of their exogenous application in Cannabis. This approach was thus applied to an industry grown Cannabis variety to determine the effects upon morphology and chemotype, outcomes that may be harnessed to increase yield and/or the concentration of minor cannabinoids.</p>