Genetic and epigenetic stability in Cannabis sativa mother plants

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Medicinal and recreational Cannabis has become a mult-billion dollar industry in Canada and is rapidly expanding around the world. Due to the variability among seedlings and Health Canada's stringent requirements, Cannabis is propagated using clonal means to produce uniform plants and products. This requires mother plants to be maintained indefinitely as a source of propagation material. A commonly observed phenomenon is that cuttings taken from a mother plan change over time, generally becoming less vigorous with lower levels of cannabinoids. These changes lead to lower productivity and batch to batch variability. The objective of this proposal is to investigate two potential mechanisms for this and develop best practices to prevent or remediate the decline. The first potential mechanism that will be investigated is genetic drift due to the accumulation of somatic mutations. To investigate this, samples from various parts of the plant will be sequenced to quantify the mutation rate. If mutations are found to accumulate, plants will be propagated from different regions of the plant (bottom vs top) over multiple cycles to compare genetic fidelity. This experiment will help to determine the rate of genetic drift in Cannabis and establish best practices to minimize it. The second potential mechanism that will be investigated is epigenetic changes due to loss of juvinility. To test this, cuttings will be taken from the bottom (more juvinile) and top (more mature) portion of the plant over several cycles. These cuttings will be evaluated for morphological juvinility traits. Transcriptome and DNA methylation will be evaluated to determine the cause of any differences. Additionally, plants produced through tissue culture, which is generally known to re-juvinate plants, will be compared to stem cuttings in the same manner. This part of the project will determine if epigenetic changes are contributing to the observed changes and develop approaches to minimize the decline. Overall, this project will help to identify the mechanism underlying and establish approaches to prevent or reverse the decline in plant quality that has been observed.