CsCBDAS2-Driven Enhancement of Cannabinoid Biosynthetic Genes Using a High-Efficiency Transient Transformation System in Cannabis sativa 'Cheungsam'

Abstract

Cannabis sativa produces pharmacologically valuable cannabinoids. In this study, we developed and optimized a transient transformation system using Cannabis sativa 'Cheungsam' to facilitate gene functional analysis. Various experimental conditions, including plant developmental stages, light conditions, Agrobacterium strains, tissue types, and physical treatments such as sonication and vacuum infiltration, were systematically evaluated using GUS histochemical staining and qPCR analysis. Among these, 7-day-old seedlings cultured under dark conditions and transformed with the GV3101 strain exhibited high transformation efficiency. Leaf tissue showed a higher GUS staining proportion and GUS staining area compared to hypocotyl and cotyledon tissues. The application of a combination of sonication and vacuum infiltration techniques resulted in the most intense GUS expression. Using the optimized protocol, we introduced a recombinant vector carrying CsCBDAS2, a key gene in cannabidiol (CBD) biosynthesis. qPCR analysis revealed that CsCBDAS2 overexpression led to significant upregulation of multiple upstream CBD biosynthetic genes (CsOAC, CsGOT, CsPT1, CsPT4, CsCBDAS1, and CsCBDAS2) and the transcription factor (TF) CsWRKY20, suggesting coordinated co-expression and potential involvement of a transcriptional feedback loop. These results demonstrate the effectiveness of our transient transformation system and provide insights into the regulatory mechanisms of cannabinoid biosynthesis in cannabis.

Keywords: Cannabis sativa Cheungsam; GUS; agroinfiltration; cannabinoid; qPCR; transient transformation.

Figure 1

Cannabinoid biosynthesis pathway.

Figure 2

Histochemical GUS staining in Cannabis sativa ‘Cheungsam’ tissues transformed with different Agrobacterium tumefaciens strains. The blue-stained tissue indicates the presence of GUS activity. (a–f) Representative images showing GUS expression patterns in transformed tissues. (a,b) GV3101/pDS-GUS; (c,d) LBA4404/pDS-GUS; (e,f) EHA105/pDS-GUS; (g) Percentage of GUS-stained seedlings transformed with different Agrobacterium strains; (h) Quantification of GUS-stained area in tissues using ImageJ (version 1.54g). Data represent the means of three replicates, and error bars indicate SEM. Means with the same letters are not significantly different (Tukey’s test, p < 0.05).

Figure 3

Effects of sonication and vacuum infiltration treatment on GUS expression in transformed plant tissues (leaf). (a–h) Representative images of histochemical GUS staining in different treatment groups. (a,b) untreated control; (c,d) sonication-only; (e,f) vacuum-infiltration only; (g,h) combination of sonication and vacuum infiltration. (a,c,e,g) leaf tissue, (b,d,f,h) cotyledon tissue. (i) Percentage of GUS-stained seedlings under various treatment conditions. (j) Relative GUS gene expression analyzed by qPCR. (k) Quantification of GUS-stained area under various treatments using ImageJ. Data represent the means of three replicates, and error bars indicate SEM. Means with the same letters are not significantly different (Tukey’s test, p < 0.05).

Figure 4

Comparison of the expression levels of CBD biosynthesis-related genes (a–f) in Cannabis sativa ‘Cheungsam’ plants overexpressing CsCBDAS2 under dark conditions. Data represent the means of three replicates, and error bars indicate SEM. Statistical significance was determined using Student’s t-test, with *** p < 0.001.

Figure 5

Comparison of the expression levels of transcription factor genes (a,b) in Cannabis sativa ‘Cheungsam’ plants overexpressing CsCBDAS2 under dark conditions. Data represent the means of three replicates, and error bars indicate SEM. Statistical significance was determined using Student’s t-test, with ns (not significant), *** p < 0.001.

Figure 6

Schematic representation of the Gateway cloning system.

Figure 7

Schematic of the T-DNA structure of the binary vectors used for transient gene expression. (a) pGWB502; (b) GUS gene; and (c) CsCBDAS2 gene.

Figure 8

Vacuum applied to seedlings submerged in Agrobacterium cell suspension.

Figure 9

Explants that were transferred to MS medium after co-cultivation. (a) 16 h/8 h (light/dark) condition; (b) dark condition.