Researchers at CRAG, in collaboration with the John Innes Centre (UK) and the company Front Range Biosciences (USA), have developed TILLCANN, the first publicly available mutant resource for Cannabis sativa. This platform represents a significant breakthrough in cannabis research, offering unprecedented opportunities to enhance genetic diversity and enable trait discovery for both medicinal and industrial applications.

Cannabis sativa is a highly versatile crop valued for its fibre, oil-rich seeds, and phytochemicals used in medicinal and psychoactive products. However, stringent regulations and selective breeding focused on a narrow set of traits have severely reduced its genetic diversity, hindering efforts to develop improved varieties.

In this work, published in the Molecular Horticulture journal, the research team led by Jason Argyris and Amparo Monfort, IRTA researchers at CRAG, has developed an efficient mutagenesis and mutation detection protocol tailored to cannabis. The study establishes a high-throughput screening platform using the well-known TILLING (Targeting Induced Local Lesions in Genomes) approach. Despite being widely applied in many crop species, this method had not been systematically implemented in cannabis until now.

The TILLCANN platform: A novel resource for cannabis breeding
Using ethyl methanesulfonate (EMS) as a mutagen, the researchers generated 1,633 M2 cannabis families, forming the basis of the TILLCANN platform. To demonstrate the utility of the resource, they conducted TILLING-by-Sequencing (TbyS) on 512 M2 families, focusing on genes with key agronomic and biochemical functions.

Their screening identified mutations in several genes, including those involved in cannabinoid biosynthesis, trichome development, and leaf morphology. Notably, they discovered that heterozygous mutations in the cannabis homologue of the transcription factor TCP4 led to significant changes in leaflet number and shape—traits with potential impact on plant architecture and productivity. They also identified functional mutations in CsOLS1, a gene involved in cannabinoid synthesis, which may be useful for increasing or reducing THC content depending on the intended use (e.g., medicinal cannabis vs. industrial hemp).

© Centre for Research in Agricultural Genomics

A step forward for global cannabis innovation
The TILLCANN platform provides a unique tool for forward and reverse genetic studies in cannabis. Researchers now have a high-probability resource for identifying mutations in genes of interest, which can accelerate functional genomics research and the development of improved cultivars incorporating novel genetic variability.

Potential traits that could be explored using TILLCANN include: understanding flowering time control, enhancing disease resistance against pathogens, improving seed yield and nutritional quality for food applications, or optimizing fibre quality for industrial uses.

CRAG is one of the few centres in Spain that has the permit from the Spanish Agency of Medicines and Medical Devices (AEMPS) to grow cannabis plants for scientific research, an emerging field in the EU. This work was carried out in collaboration with Dr. Cristobal Uauy’s group at the John Innes Centre—world leaders in TILLING technology—and with Front Range Biosciences, a US biotechnology company specializing in cannabis genetics, with whom CRAG and IRTA have collaborated since 2019.

“The novel genetic variability unlocked through the TILLCANN platform offers powerful opportunities for gene discovery and breeding innovation in both medicinal and industrial cannabis”, said Drs. Monfort and Argyris. “We believe this resource can serve as a springboard for trait improvement efforts that have long been hampered by limited genetic diversity in this important crop”.

Source: Centre for Research in Agricultural Genomics