Modeling and optimizing in vitro seed germination of industrial hemp (Cannabis sativa L.)

Abstract

In vitro seed germination of cannabis as the first physiological stage in the plant life cycle is not only important for studying factors affecting cultivation conditions but also crucial for obtaining juvenile tissue as a potential explant for different in vitro procedures. On the other hand, in vitro seed germination is a multi-variable biological process that can be influenced by genetic (genotype) and physical factors (medium composition and environmental conditions). Therefore, a powerful mathematical methodology such as artificial neural networks (ANNs) is well suited to analyze the data and optimize the conditions this complex system. The current study was aimed to evaluate the effect of different types and concentrations of carbohydrate sources (sucrose and glucose) as well as different strengths of DKW (Driver and Kuniyaki Walnut) and mMS (Murashige and Skoog Medium, Van der Salm modification) media on seed germination indices as well as morphological features of in vitro-grown cannabis seedlings by using three ANNs including multilayer perceptron (MLP), radial basis function (RBF), and generalized regression neural network (GRNN). The GRNN model displayed higher predictive accuracy (r2>0.70) in both training and testing sets for all germination indices and morphological traits in comparison to RBF or MLP. Moreover, non-dominated sorting genetic algorithm-II (NSGA-II) was subjected to the GRNN to find the optimal type and level of media and carbohydrate source for obtaining the best seed germination indices (germination rate and mean germination time). According to the optimization process, 0.43 strength mMS medium supplemented with 2.3 % sucrose would result in the best outcomes. This result showed that a moderate level of salts existing in culture media (0.43 strength of mMS medium) supplemented with a moderate level of sucrose (2.3 %) can improve in vitro seed germination of hemp. The results of a validation experiment revealed that there was a negligible difference between the experimental data and the optimized result. Therefore, GRNN-NSGA-II provided an accurate prediction of seed germination and can likely be employed to optimize different factors involved in in vitro culture of this multi-purpose crop.