Abstract
BackgroundCannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type.ResultsHyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise Δ9-tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures.ConclusionHere we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information.
Highlights
Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests
Morphology of glandular trichomes In order to investigate the sample morphology images of glandular Cannabis trichomes are recorded with transmission, single-photon fluorescence and scanning electron microscopy (SEM)
In the work presented we successfully apply a Coherent anti-Stokes Raman scattering (CARS) imaging setup to visualise the main cannabinoids in intact Cannabis glandular trichomes and to distinguish between different cell types
Summary
Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Other cannabinoids like cannabidiol (CBD), terpenes and phenolic compounds show further pharmacological effects, which make this plant a highly interesting pharmaceutical target [6] The biosynthesis of these metabolites typically occurs in specialised plant surface structures, so-called glandular trichomes. The second group are capitate glands that have a large globular head and (massive) stalks They are produced on flowering bracts of female flowers and on anthers of male flowers. These glands are thought to be the primary site of cannabinoid biosynthesis and storage [10, 11].only glandular trichomes were analysed in this study
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