Trends in intellectual property rights protection for medical cannabis and related products.

The purpose of this review is to advance the field of applied cannabis research by providing insights into the patenting of medical cannabis and current intellectual property rights (IPR) data.Medical cannabis (MC) patent and plant breeders' rights (PBR) registrations are filed on industrially applicable aspects of research. Studying the filing data and trends informs researchers of both gaps in current applied knowledge in MC (where patents have not been filed) and prior knowledge (where patents have already been filed).Our focus is on those intellectual property rights (IPR) that are registered and germane to technical innovations in MC and related products. These are patents and PBR and thus exclude trade secrets, copyrights, franchises, or trademarks. Methods used for surveying the defined IPR landscape include searches of publicly available patent and PBR data and classifying the data according to the upstream-midstream-downstream innovation paradigm of the MC industry.The findings suggest that the technical knowledge as expressed by patent filings is growing commensurate to the economic and legislative activity. Specific cannabis patents in agricultural technologies directed at improving yield, efficiency, and quality (known as "agritech") are being filed and granted. These agritech-focused patents represent original novel and applied MC research achievements that address specific problems in cannabis cultivation, such as protection of the cannabis crop, maximizing cannabis yield, harvesting and post-harvesting of cannabis, and new advantageous varieties. Patents on ex planta and in planta cannabis genes expression have been published in recent years while patents on extraction methods for cannabinoids have increased since 2015. Much patent activity is in the downstream category of MC patient-oriented products and delivery systems for a very wide range of medical indications and disease conditions.The emerging importance of access and benefit-sharing treaties and regulations is noted with implications on the industry briefly discussed. Patent data on leading and emerging patentee companies and institutions are shown. We also provide evidence of prior art and freedom to operate.

Characterization of novel pollen-expressed transcripts reveals their potential roles in pollen heat stress response in Arabidopsis thaliana.

KEY MESSAGE: Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response. The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response.

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress.

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H2 DCFDA-staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into 'low-ROS' and 'high-ROS' subpopulations. Pollen germination assays following fluorescence-activated cell sorting revealed that the high-ROS pollen germinated with a frequency that was 35-fold higher than the low-ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose-dependent alterations in DCF-fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry-based approaches to investigate metabolic changes during stress responses in pollen.