Abstract
Medical cannabis (Cannabis sativa L.) is quickly becoming a central agricultural crop as its production has continued to increase globally. The recent release of the cannabis reference genomes provides key genetic information for the functional analysis of cannabis genes. Currently, however, the established tools for in vivo gene functional analysis in cannabis are very limited. In this study, we investigated the use of the tobacco rattle virus (TRV) as a possible tool for virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE). Using leaf photobleaching as a visual marker of PHYTOENE DESATURASE (PDS) silencing, we found that VIGS was largely restricted to the agro-infiltrated leaves. However, when agro-infiltration was performed under vacuum, VIGS increased dramatically, which resulted in intense PDS silencing and an increased photobleaching phenotype. The suitability of TRV as a vector for virus-aided gene expression (VAGE) was demonstrated by an analysis of DsRed fluorescence protein. Interestingly, a DsRed signal was also observed in glandular trichomes in TRV2-DsRed-infected plants, which suggests the possibility of trichome-related gene function analysis. These results indicate that TRV, despite its limited spread, is an attractive vector for rapid reverse-genetics screens and for the analysis of gene function in cannabis.
Highlights
Cannabis sativa L. is an important medical and industrial crop; its use continues to expand as its consumption for both medicinal and recreational use increases [1]
It is of great importance to identify and characterize the genetic factors regulating the attributes that affect the value of the cannabis crop, such as quality and quantity of cannabinoids, sex determination, resistance to biotic and abiotic stresses, and the mechanism responsible for flowering
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Summary
Cannabis sativa L. (cannabis) is an important medical and industrial crop; its use continues to expand as its consumption for both medicinal and recreational use increases [1]. As a very efficient alternative for stable transformation in other crops, virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE) have been used for gene functional analyses and for extensive reverse genetics screens in a variety of plants, including petunias, tomatoes, Arabidopsis, and strawberries [26,27,28,29,30,31,32]. These wellestablished approaches involve the viral inoculation of target plants using a viral vector, cloned in a binary vector, to induce gene silencing or, alternatively, gene expression. VIGS is an attractive approach for the functional analysis of genomics in cannabis
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