Biology subjects

Jones, A. M. P.

Publications and source records attributed to Jones, A. M. P..

5 recordsLinked to original sources

bioRxiv2024

Floral Reversion based micropropagation of Day-Neutral Cannabis sativa L.

Micropropagation systems have been developed for photoperiod sensitive cultivars of cannabis, but there are currently no published methods for day-neutral genotypes. Most established cannabis micropropagation systems rely on shoot proliferation using modal explants that need them to be maintained in vegetative growth, which is not possible for day-neutral genotypes. Floral reversion, the process by which plants revert to vegetative growth from the generative stage, has been demonstrated as an alternative and potentially more efficient approach to the micropropagation of photoperiodic cannabis. The current study investigated if this process could be adapted to facilitate the micropropagation of day-neutral genotypes and overcome existing barriers. During the process, various factors such as photoperiod and light intensity may influence the efficiency of floral reversion. To evaluate this approach in day-neutral cultivars, various photoperiods were compared to assess the impact on reversion rate and subsequent growth. Excised florets obtained from in vitro day-neutral C. sativa cv. "Blue Auto Mazar x auto Blueberry" seedlings were subjected to five photoperiods including 12.0, 16.0, 18.0, 20.0, and 24.0 hours of light per day for four weeks. Reversion rates and time, shoot length, shoot number, and node number were measured. Statistical analyses revealed significant differences (p-values < 0.05) in terms of reversion time among photoperiod treatments. The highest reversion rate happened under the 16.0 hr photoperiod with 72% success. The highest shoot lengths were observed under 20.0 hr of photoperiods with a mean of 10.1 mm and the lowest shoot length belonged to 12.0 hr of photoperiods with a mean of 5.6 mm, respectively. Reverted shoots developed vegetatively for some time before initiating new flowers. However, during this period the shoots were successfully rooted in vitro and then acclimated to the growth chamber where they completed their life cycle, including seed production. This process was also successfully achieved from a 2-year old culture of C. sativa cv. "Blue Auto Mazar", demonstrating that it is feasible to use this approach for long term culture maintenance and multiplication. This study demonstrates that floral reversion can successfully be used to clonally propagate day-neutral cannabis plants and serves as a basis for developing large-scale clonal propagation and breeding strategies.

plant biology↗

bioRxiv2023

Comparative Restriction Enzyme Analysis of Methylation (CREAM) Reveals Methylome Variability Within a Clonal In Vitro Cannabis Population

The primary focus of medicinal cannabis research is to ensure the stability of cannabis lines for consistent administration of chemically consistent products to patients. In recent years, tissue culture has emerged as a valuable technique for genetic preservation and rapid production of cannabis clones. However, there is concern that the physical and chemical conditions of the growing media can induce somaclonal variation, potentially impacting the viability and uniformity of clones. To address this concern, we developed Comparative Restriction Enzyme Analysis of Methylation (CREAM), a novel method to assess DNA methylation patterns and used it to assess a population of 78 cannabis clones maintained in tissue culture. Through bioinformatics analysis of the methylome, we successfully detected 2,272 polymorphic methylated regions among the clones. Remarkably, our results demonstrated that DNA methylation patterns were preserved across subcultures within the clonal population, allowing us to distinguish between two subsets of clonal lines used in this study. These findings significantly contribute to our understanding of the epigenetic variability within clonal lines in medicinal cannabis produced through tissue culture techniques. This knowledge is crucial for understanding the effects of tissue culture on DNA methylation and ensuring the consistency and reliability of medicinal cannabis products with therapeutic properties. Additionally, the CREAM method is a fast and affordable technology to get a first glimpse at methylation in a biological system. It offers a valuable tool for studying epigenetic variation in other plant species, thereby facilitating broader applications in plant biotechnology and crop improvement.

plant biology↗

bioRxiv2021

Comparative analysis of machine learning and evolutionary optimization algorithms for precision tissue culture of Cannabis sativa: Prediction and validation of in vitro shoot growth and development based on the optimization of light and carbohydrate sources

Micropropagation techniques offer opportunity to proliferate, maintain, and study dynamic plant responses in highly controlled environments without confounding external influences, forming the basis for many biotechnological applications. With medicinal and recreational interests for Cannabis sativa L. growing, research related to the optimization of in vitro practices is needed to improve current methods while boosting our understanding of the underlying physiological processes. Unfortunately, due to the exorbitantly large array of factors influencing tissue culture, existing approaches to optimize in vitro methods are tedious and time-consuming. Therefore, there is great potential to use new computational methodologies for analysing data to develop improved protocols more efficiently. Here, we first tested the effects of light qualities using assorted combinations of Red, Blue, Far Red, and White spanning 0-100 mol/m2/s in combination with sucrose concentrations ranging from 1-6 % (w/v), totaling 66 treatments, on in vitro shoot growth, root development, number of nodes, shoot emergence, and canopy surface area. Collected data were then assessed using multilayer perceptron (MLP), generalized regression neural network (GRNN), and adaptive neuro-fuzzy inference system (ANFIS) to model and predict in vitro Cannabis growth and development. Based on the results, GRNN had better performance than MLP or ANFIS and was consequently selected to link different optimization algorithms (genetic algorithm, biogeography-based optimization, interior search algorithm, and symbiotic organisms search) for prediction of optimal light levels (quality/intensity) and sucrose concentration for various applications. Predictions of in vitro conditions to refine growth responses were subsequently tested in a validation experiment and data showed no significant differences between predicted optimized values and observed data. Thus, this study demonstrates the potential of machine learning and optimization algorithms to predict the most favourable light combinations and sucrose levels to elicit specific developmental responses. Based on these, recommendations of light and carbohydrate levels to promote specific developmental outcomes for in vitro Cannabis are suggested. Ultimately, this work showcases the importance of light quality and carbohydrate supply in directing plant development as well as the power of machine learning approaches to investigate complex interactions in plant tissue culture.

plant biology↗

bioRxiv2021

Accumulation of somatic mutations leads to genetic mosaicism in Cannabis

Cannabis is typically propagated using stem cuttings taken from mother plants to produce genetically uniform propagules. However, producers anecdotally report that clonal lines deteriorate over time and eventually produce clones with less vigour and lower cannabinoid levels than the original mother plant. While the cause of this deterioration has not been investigated, one potential contributor is the accumulation of somatic mutations within the plant. To test this, we used deep sequencing of whole genomes (>50x) to compare the variability within an individual Cannabis sativa cv. "Honey Banana" plant sampled at the bottom, middle and top. We called over 6 million sequence variants based on a reference genome and found that the top had the most by a sizable amount. Comparing the variants among the samples uncovered that nearly 600K (34%) were unique to the top while the bottom only contained 148K (12%) and middle with 77K (9%) unique variants. Bioinformatics tools were used to identify mutations in critical cannabinoid/terpene biosynthesis pathways. While none were identified as high impact, four genes contained more than double the average level of nucleotide diversity ({pi}) in or near the gene. Two genes code for essential enzymes required for the cannabinoid pathway while the other two are in the terpene pathways, demonstrating that mutations were accumulating within these pathways and could influence their function. Overall, a measurable number of intra-plant genetic diversity was discovered that could impact long-term genetic fidelity of clonal lines and potentially contribute to the observed decline in vigour and cannabinoid content.

plant biology↗

bioRxiv2020

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study

Cannabis sativa is relatively recalcitrant to regeneration from somatic tissues, but several reports have been published demonstrating a response. Most reports show low levels of regeneration from somatic tissues, but a landmark publication by Lata et al. in 2010 reported regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single, high-THC genotype. Despite the importance regeneration plays in plant biotechnology this protocol has not been used in subsequent papers in the decade since it was published, raising the concern that it is not reproducible. Many researchers are looking to build research programmes in this growing field, and it is important that the reproducibility and robustness of single-genotype C. sativa regeneration protocols undergo multi-lab validations to ensure they are reproducible across the species. Replication studies in this burgeoning field will help research groups avoid lost time and resources which arise from pursuing protocols that are not reproducible. Here we test the replicability of this protocol across 10 drug-type C. sativa genotypes. This protocol successfully induced callus in all 10 genotypes. Callus size and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive genotype. However, the most successful shoot induction medium developed in the 2010 paper failed to induce regeneration in any of the cultivars tested, resulting in the eventual necrosis of the calli. Based on this replication study, it is evident that the existing regeneration protocol is not robust and could not be replicated in any of the 10 genotypes tested.View Full Text

plant biology↗