The activity of DAHPS was measured at 549 nm. Regarding PAL, it was extracted in 1 mL of 200 mM sodium borate buffer and then evaluated by measuring the production of trans-cinnamic acid at 290 nm. For centuries, Cannabis sativa L. has been widely used around the world for various applications . These days, interest has been focused on medicinal and recreational facets, furthering commercial expansion. With Canada recently adopting the more globally appreciated view of cannabis, there exists an ever evolving, multi-billion-dollar industry focused on vegetative propagation . Despite the reliance on clonal propagation, there is a continual need to germinate seeds to select new elite genotypes, perform pheno-hunting, as well as supporting breeding programs. To select new elite genotypes, plants are started from seed .
During the vegetative phase of growth, a cutting is taken and maintained as a vegetative plant while the seedling is grown to maturity. Once the elite genotypes are selected, the cutting is then used as a source to propagate the clonal line. Maintaining the large population of cuttings during the phenotyping exercise represents a significant cost to producers and leaves the cutting derived mother plants exposed to insects and diseases. To address the issues of insect, disease, and viral infections in mother plants, many producers use plant tissue culture to ensure that they are starting with clean material. Forthis process, nodal segments are disinfected and established in culture, a time consuming and relatively expensive endeavor. Once the seedlings are established and multiplied, micropropagated clones can be transferred into the growth facility and cultivated to maturity to identify elite genotypes. After selecting the elite genotypes, the in vitro parent material would be available for clonal propagation. This approach would greatly reduce the amount of space required for selecting new cultivars and provide a ready source of clean planting material once elite genotypes are identified.
However, this approach requires an effective in vitro seed germination protocol with high germination speed and frequency. An efficient in vitro seed germination system would also support downstream biotechnologies in which seedling-derived tissues are preferred .We previously reported the effect of different types and strengths of media in addition to carbohydrate types and levels as primarily important factors contributing to in vitro cannabis seed germination indices and morphological seedling traits . Our results demonstrated that maximum germination percentage was achieved with 0.43 strength mMS medium and 2.3% sucrose . While the germination rate was over 80%, this was after 40 days of culture. Typically, the cannabis seed germinates within several days in the greenhouse/growth room, suggesting that something during the disinfection process was interfering with subsequent germination. To improve our previous protocol, we hypothesize that optimizing the disinfection protocol and seed scarification would increase the speed and frequency of seed germination. As with most aspects of a tissue culture system, in vitro disinfection is a complex and non-linear process that is affected by numerous factors such as disinfectant and contaminant types and levels, media pH, immersion time, temperature, and theirinteractions .
In the disinfection process, the concentration of disinfectants plays a conflicting dual role relating to contamination frequency and seed viability. Higher disinfectant concentrations generally lead to a greater control over contaminants; however, lower seedling viability is often the trade-off. Therefore, it is necessary to optimize the disinfection process. The disinfection process cannot be represented by a simple stepwise algorithm, especially when the datasets are highly imbalanced and noisy . Therefore, artificial intelligence models combined with optimization algorithms such as a genetic algorithm can be employed as an efficient and reliable computational method to inter-pret, forecast, and optimize this complex system . This strategy has been successfully used for modeling and optimizing different tissue culture systems, including in vitro decontamination, shoot proliferation, androgenesis, somatic embryogenesis, secondary metabolite production, and rhizogenesis . Ivashchuk et al. employed multilayer perceptron and radial basis function as two well-known artificial neural networks for modeling and predicting the effect of different disinfectants and immersion times for Bellevalia sarmatica, Echinacea purpurea, and Nigella damascene explant decontamination. They reported that both algorithms were able to accurately model and predict the disinfection process . In another study, Hesami et al. applied a hybrid MLP and non-dominated sorting genetic algorithm-II for the modeling and optimization of disinfectants and immersion times for chrysanthemum leaf segment decontamination. It was reported that MLP-NSGA-II had a high performance to predict and optimize the system .