Dataset on nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9

This dataset in brief is related to the research letter entitled "Nicotine-free, nontransgenic tobacco (Nicotiana tabacuml.) edited by CRISPR-Cas9" [1]. Cured tobacco products with a significantly reduced nicotine content helps people to overcome their nicotine addiction. Here we summarize additional data and method descriptions of the generation process of a nicotine-free, nontransgenic tobacco plant. This included the cloning, transformation and regeneration of transgenic tobacco plants, followed by the analysis of the nicotine content and genomic modifications caused by CRISPR-Cas9 mediated gene editing. Subsequently, nicotine-free plants were screened for loss of T-DNA cassette, i.e. nontransgenity. Finally, a metabolic footprint was recorded by 1H NMR analysis

Current Perspectives on Biotechnological Cannabinoid Production in Plants.

The plant Cannabis sativa contains a number of psychoactive chemical compounds, the cannabinoids, which possess a significant pharmaceutical potential. Recently, the usage of Cannabis for medicinal purposes was legalized in many countries. Thus, the study on the influence of different cannabinoids in combination with other Cannabis-derived compounds with respect to the treatment of various diseases becomes increasingly important. Besides the production of distinct cannabinoids in a heterologous host, like tobacco or yeast, transgenic Cannabis plants would be a suitable alternative to modify and therefore optimize the cannabinoid profile. This perspective highlights the current efforts on Cannabis cell culture systems, in vitro propagation, and transformation of the plant and reveals the resulting opportunities concerning biotechnological production of cannabinoids. Furthermore, alternative platform organisms for the heterologous production of cannabinoids, like tobacco, are considered and evaluated

Virus-induced gene silencing (VIGS) in Cannabis sativa L.

Background:The raised demand of cannabis as a medicinal plant in recent years led to an increased interest in understanding the biosynthetic routes of cannabis metabolites. Since there is no established protocol to generate stable gene knockouts in cannabis, the use of a virus-induced gene silencing (VIGS) method, resulting in a gene knockdown, to study gene functions is desirable.Results:For this, a computational approach was employed to analyze the Cannabis sativa L. transcriptomic and genomic resources. Reporter genes expected to give rise to easily scorable phenotypes upon silencing, i.e. the phy-toene desaturase (PDS) and magnesium chelatase subunit I (ChlI), were identified in C. sativa. Subsequently, the targets of specific small interfering RNAs (siRNAs) and silencing fragments were predicted and tested in a post-transcriptional gene silencing (PTGS) approach. Here we show for the first time a gene knockdown in C. sativa using the Cotton leaf crumple virus (CLCrV) in a silencing vector system. Plants transiently transformed with the Agrobacterium tumefaciensstrain AGL1, carrying the VIGS-vectors, showed the desired phenotypes, spotted bleaching of the leaves. The success-ful knockdown of the genes was additionally validated by quantitative PCR resulting in reduced expression of tran-scripts from 70 to 73% for ChlI and PDS, respectively. This is accompanied with the reduction of the chlorophyll a and carotenoid content, respectively. In summary, the data clearly demonstrate the potential for functional gene studies in cannabis using the CLCrV-based vector system.Conclusions:The applied VIGS-method can be used for reverse genetic studies in C. sativa to identify unknown gene functions. This will gain deeper inside into unknown biosynthetic routes and will help to close the gap between avail-able genomic data and biochemical information of this important medicinal plant