Establishment and optimization of a hemp (Cannabis sativa L.) agroinfiltration system for gene expression and silencing studies

Abstract

industrial hemp (Cannabis sativa L.) is a high-yielding annual crop primarily grown for fiber, seeds, and oil. Due to the phytochemical composition of hemp, there has been an increased interest in the market for nutraceuticals and dietary supplements for human health. Recent omics analysis has led to the elucidation of hemp candidate genes involved in the syntheses of specialized metabolites. However, a detailed study of these genes has not been undertaken due to the lack of a stable transformation system. We report for the first time an agroinfiltration system in hemp utilizing vacuum infiltration, which is an alternative method to stable transformation. A combination of 0.015% Silwett L-77, 5 mM ascorbic acid, and thirty second sonication followed by a 10-minute vacuum treatment resulted in the highest β-glucuronidase expression in the leaf, male and female flowers, stem, and root tissues. The phytoene desaturase gene was silenced with a transient hairpin RNA expression, resulting in an albino phenotype in the leaves and the male and female flowers. This agroinfiltration system would be useful for overexpression and silencing studies of target genes to regulate the yield of specialized metabolites in hemp.

Agrobacterium-Mediated Genetic Transformation and Cloning of Reference Genes in Suspension Cells of Artemisia Pallens

“…There were no signi cant changes observed in transient transformation e ciency on the addition of Silwet L-77; however, it increased GUS expression in Cannabis sp. (Deguchi et al, 2020). However, the addition of Pluronic F-68 in bacterial solution increased the GUS intensity and activity (Fig.…”

“…Similarly, the addition of Pluronic F-68 surfactants effectively increased the transformation e ciency in Theobroma cacao (Fister et al, 2016) and Cannabis sativus (Deguchi et al, 2020). After transformation, cefotaxime was used in the selection medium to kill adhered Agrobacterium.…”

“…The effect of two chemical additives, namely, Silwett L-77 (0.05%) and Pluronic F-68 (0.05%), on transient transformation was assessed. These chemical additives are well known to reduce surface tension and enhance bacterial entry into plant cells (Deguchi et al, 2020). Transformation e ciency was calculated based on GUS spots visible under a microscope in 200 µl transformed SC.…”

Metabolic Engineering Strategies of Industrial Hemp (Cannabis sativa L.): A Brief Review of the Advances and Challenges

“…Introduction of genes into the hemp genome does not appear to be limiting as for example the creation of “hairy roots;” however, the transition of that cell into a viable plant requires a major reprogramming of plant cell development. Using appropriate surfactant and antioxidant reagents, Agrobacterium infection to hemp explant became no longer a difficult step via vacuum infiltration ( Deguchi et al, 2020 ). Once somatic embryogenesis is achieved by altering the transient expression of morphologic regulator genes, hemp explants would regenerate at a high rate and be available for further genetic modifications with value added traits.…”

“…Nevertheless, strong transient gene expression achieved via agroinfiltration is limited to some model crops, and agroinfiltration protocols for many agronomically important crops have only recently been optimized ( King et al, 2015 ). Most recently, Deguchi et al (2020) optimized the proper concentration of surfactant and antioxidants for Agrobacterium vacuum infiltration and achieved gene overexpression and silencing in hemp trichome. Schachtsiek et al (2019) inoculated Agrobacterium carrying VIGS-vectors to Cannabis mature leaf which led to the reduction of 70% of gene expression in phytoene desaturase and magnesium chelatase subunit I.…”