Cannabis sativa L. is a plant of significant interest due to its high content of biologically active compounds, durable fibers, and bioeconomic potential. Despite recent progress in protoplast isolation, complete plant regeneration from cannabis protoplasts remains unachieved, highlighting gaps in protoplast-to-plant systems. This study reports the second successful establishment and partial regeneration of cannabis protoplast cultures, and investigates their molecular dynamics, marking a significant step forward. We demonstrated that the age of donor material is critical for the protoplast isolation, with the optimal source being 1-2-week-old leaves from in vitro-grown seedlings. Cultivation in a modified medium developed for Arabidopsis thaliana supported initial cell divisions and microcallus formation. Transcriptomic analyses of cell proliferation and stress response markers indicate that the cultured protoplasts were viable, re-entered the cell cycle, and exhibited oxidative and abiotic stress resilience. These findings enhance our understanding of cannabis cell biology and lay the groundwork for a protoplast-based regeneration system, paving the way for advanced applications in biotechnology.

• Keywords
• Cannabis culture, cell proliferation, gene expression, in vitro, microcallus formation, oxidative stress, protoplast isolation, stress response,
• Publication type
• Journal Article MeSH

Growing evidence underscores the role of nutrients and fertigation systems in soilless production, influencing medicinal cannabis biomass and secondary metabolite content. This study delves into the impact of enhanced nutrient regimes on the 'ionome' and its ramifications for biomass and cannabinoid production in medicinal cannabis, comparing two distinct fertigation systems: recirculation and drain-to-waste. Notably, we assess the optimal harvest time for maximizing profitability. In comparing the experimental variant with elevated levels of phosphorus (P), potassium (K), and iron (Fe) in the nutrient solution to the control variant, we observe distinct patterns in element composition across stems, leaves, and flowers, with significant differences between fertigation systems. Total nitrogen content was determined through the Kjeldahl method. Flame atomic absorption spectrometry (FAAS) and inductively coupled plasma optical emission spectrometry (ICP-OES) were employed for elemental analysis. Cannabinoid identification and quantification used high-performance liquid chromatography with a diode-array detector (HPLC/DAD). Followed statistical analyses included ANOVA and Tukey's HSD test. Although the augmented nutrient regimen does not substantially increase plant biomass, interesting differences emerge between the two fertigation systems. The recirculation fertigation system proves more profitable during the recommended harvest period. Nonetheless, the altered nutrient regime does not yield statistically significant differences in final inflorescence harvest mass or cannabinoid concentrations in medicinal cannabis. The choice of fertigation system influences the quantity and quality of harvested inflorescence. To optimize the balance between the dry biomass yield of flowers and cannabinoid concentration, primarily total THC yield (sum of tetrahydrocannabinolic acid, Δ9-tetrahydrocannabinol, and Δ8-tetrahydrocannabinol), we propose the 11th week of cultivation as the suitable harvest time for the recirculation system. Importantly, the recirculation system consistently outperformed the drain-to-waste system, especially after the ninth week, resulting in significantly higher total THC yields. Enriched nutrition, when compared with control, increased THC yield up to 50.7%, with a remarkable 182% surge in the recirculation system when compared with the drain-to-waste system.

• Keywords
• Cannabis sativa L., THC, cannabinoids, fertigation, indoor-cultivation, soilless-cultivation,
• Publication type
• Journal Article MeSH

The in vitro shoot propagation of Cannabis sativa L. is an emerging research area for large-scale plant material production. However, how in vitro conditions influence the genetic stability of maintained material, as well as whether changes in the concentration and composition of secondary metabolites can be expected are aspects that need to be better understood. These features are essential for the standardised production of medicinal cannabis. This work aimed to find out whether the presence of the auxin antagonist α-(2-oxo-2-phenylethyl)-1H-indole-3-acetic acid (PEO-IAA) in the culture media influenced the relative gene expression (RGE) of the genes of interest (OAC, CBCA, CBDA, THCA) and the concentrations of studied cannabinoids (CBCA, CBDA, CBC, ∆9-THCA, and ∆9-THC). Two C. sativa cultivars, 'USO-31' and 'Tatanka Pure CBD', were cultivated by in vitro conditions with PEO-IAA presence and then analysed. The RT-qPCR results indicated that even though some changes in the RGE profiles could be observed, no differences were statistically significant compared with the control variant. The results of the phytochemical analyses demonstrate that although there were some differences from the control variant, only the cultivar 'Tatanka Pure CBD' showed a statistically significant increase (at a statistical significance level α = 0.05) in the concentration of the cannabinoid CBDA. In conclusion, it would appear that using PEO-IAA in the culture medium is a suitable approach to improve in vitro cannabis multiplication.

• Keywords
• CBC, CBD, Cannabis sativa, PEO-IAA, THC, auxin antagonist, cannabinoids, in vitro, qRT-PCR, shoot propagation,
• Publication type
• Journal Article MeSH