In recent years, the use of Cannabis in the adult population has dramatically increased due to legalization of Cannabis in parts of the United States and the use of Cannabis or Cannabis extracts as palliative medication. One of the main barriers hindering the use of Cannabis flowers as medicinal agents is the lack of uniformity in terms of the content of active ingredients. Moreover, Cannabis in nature is highly pollinated, which is the reason for extremely inhomogeneous plant material, hence obtaining new hybrid seeds with consistent genetic makeup is almost an unreachable goal. Furthermore, to stabilize the active ingredients’ concentration in Cannabis plants is even more challenging since it is affected by various factors: plant genetics, growing and storage conditions, the state of maturity at harvest, etc. This is the major reason for avoiding producing Cannabis seeds worldwide and using vegetative propagated plants instead. All these reasons together cause difficulty in the repeatability of the patient’s dosage. Thus, in order to promote Cannabis flowers as validated medicine, the ability to quantify the exact percentage of active ingredients in the plant is required.
The active ingredients of cannabis plants are the cannabinoids, a class of diverse chemical compounds that are concentrated in specialized glandular structures called trichomes. The primary cannabinoid is phytocannabinoid tetrahydrocannabinol (THC) while cannabidiol (CBD), another cannabinoid, is the primary analgesic compound of Cannabis. In addition, there are at least 85 additional plant cannabinoids having varied physiological effects. . Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC) are commonly used approaches for quantification of plant cannabinoids. Although those approaches can be used to type Cannabis strains according to THC and CBD quantities, they require processed plants’ material and are time consuming. Moreover, they can only be implemented on mature flowers, which requires growing a diversity of all plants before selection can be made. Therefore, in order to promote cannabis as medicine there is a need to act on two parallel platforms; stabilization of the active ingredients in the cannabis plant while producing the ability to quantify the percentage of active ingredients in each flower. One possible approach for quantifying active ingredients in whole (unprocessed) plants or production of Cannabis seeds and breeding acceleration process might be achieved by NIR technology. NIR spectrometry supported by image analysis and machine learning can be used to detect and quantify cannabinoids in whole plants or plant material by remote sensing. Recently, my laboratory has revealed the ability to accurately analyse THC levels in Cannabis flowers by NIR spectroscopy (Fig.1). NIR spectrometers/cameras record the absorbance/reflectance spectrum of samples irradiated with light at wavelengths between 700 nm and 2500 nm. NIR radiation is highly penetrative and can be applied to a sample without any preparation/destruction. Although the resulting absorbance or reflectance spectra is not highly discriminative, it can be used to quantify active ingredients and other agricultural features such as germination rate, male/female plants, disease resistance etc., by using NIR calibration models. This new system technology based on NIR spectroscopy will be able to determine cannabinoids content in Cannabis flowers as well as genetic features in Cannabis seeds, and most notably the active ingredients ratio. By this novel technology, we will be one-step ahead towards implementation of consistent cannabis flowers for medical use. Moreover, we will achieve a shorter and much cheaper breeding process, which can cause a dramatic change in the entire plant breeding industry.
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Waste management is a huge concern for the Cannabisgrowers' community. After the female Cannabis flowers are harvested the growers are left to dispose of all the vegetative parts. Legal growers must document every stage of plant management that includes plant material that is being discarded. Thus, Cannabis disposal is subjected to strict waste regulations. Furthermore, traditionally the majority of crop residues are used as animal feed but the strict regulations on Cannabis residues are preventing this use.
One way to manage this crop waste is the incorporation of crop amendments into the soil (green manure). Ploughing plant debris into the soil to generate green manures has been proposed to affect plant health and to control diversity of soil borne and foliar pathogens. My lab expertise in plant defence mechanisms together with our ability to use identified characteristics (e.g. fluorescein diacetate, substrate respiration and biocontrol agent population) that correlates with green manure diseases suppression will advance us towards the calibration of Cannabis waste as green manure.
In our laboratory we also study the accumulation of plant secondary metabolites during plant defence responses. Since it is known that developmental stages and environmental conditions affect the metabolite profiling in Cannabis, we plan to study the suppressive effect of cannabis metabolites throughout the different developmental stages and under different conditions on Botrytis cinerea and other plant pathogens. We will later use those findings to calibrate cannabis waste as suppressive green manure for the control of soil borne pathogens and for metabolic engineering in order to confer resistance against B. cinerea.
Keywords: green manure, plant pathogens, metabolic engineering, secondary metabolites.
Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
Hybrid varieties of Cannabis Sativa, Cannabis Ruderalis and Cannabis Indica are used as drug crops. These crops produce large and dense flowers which are very attractive to Botrytis cinerea - the gray mold disease causal agent. B. cinerea is the most significant pathogen on Cannabis plants and its infection results in enormous economic losses due to yield damages. When Cannabis is grown in-doors under high humidity and in high density of large moisture-retaining female buds, B. cinerea can reach epidemic magnitudes and completely destroy a Cannabis crop within a week. Drug varieties are most susceptible during flowering near harvest time as growers cannot use any chemical fungicides due to consumers' health concerns. Thus, developing new strategies to control this pathogen is essential if we wish to secure Cannabis crops for the health of both consumers and the environment.
In my laboratory, we are focusing on various aspects of the plant's defence responses against the fungal plant-pathogen Botrytis cinerea. To cope with pathogen attack, plants have evolved a wide range of physical, molecular and biochemical defence mechanisms. We target to refine the multi-layer defence responses against B. cinerea by studying the molecular and biochemical interactions of B. cinerea with the plant's defence response.
Our research in the laboratory revealed diverse knowledge on B. cinerea – host interaction and plant induced resistance mechanisms against this pathogen. We also isolated a unique and efficient biocontrol agent that uses a complex mode of actions against B. cinerea including antibiosis and activation of the plant defence response. We aim to use these findings to enhance the defence response of Cannabis plants in order to control B. cinerea and other pathogens infecting this crop.
Genome editing using the CRISPER/Cas9 technology opened new possibilities in research and in biotechnology for many organisms including plants. To date, targeted gene knockout using the CRISPER/Cas9 system has been used in many plant species, with high efficiency. Despite the high potential of the system to manipulate genes and to serve as a tool in breeding programs, there is still no published work on using CRISPER/Cas9 in cannabis. We believe that genome editing can be a powerful tool in generating new cannabis strains having desirable characteristics. To establish the genome editing system in cannabis, we are developing a method for transient transformation in protoplast with a plasmid carrying the Cas9 gene and the guiding molecule. This method allows the Cas9 to induce a mutation without integrating to the genome, generating a non-transgenic cell, with the desirable mutation. Following the transformation, the protoplast are induced to regenerate shoots, resulting in a mutated plant that is unify (non-chimeric) and true-to-type. Calibrating this method to exhibit high efficiency will enable us and other to generate many new plants in short time in a non-transgenic manner. There are many traits that can be manipulated by gene editing. For example, creating plants with agro-technical advantages, or manipulating the cannabinoid biosynthesis pathway.