Artificial Neural Networks Elucidated the Essential Role of Mineral Nutrients versus Vitamins and Plant Growth Regulators in Achieving Healthy Micropropagated Plants.

The design of an adequate culture medium is an essential step in the micropropagation process of plant species. Adjustment and balance of medium components involve the interaction of several factors, such as mineral nutrients, vitamins, and plant growth regulators (PGRs). This work aimed to shed light on the role of these three components on the plant growth and quality of micropropagated woody plants, using Actinidia arguta as a plant model. Two experiments using a five-dimensional experimental design space were defined using the Design of Experiments (DoE) method, to study the effect of five mineral factors (NH4NO3, KNO3, Mesos, Micros, and Iron) and five vitamins (Myo-inositol, thiamine, nicotinic acid, pyridoxine, and vitamin E). A third experiment, using 20 combinations of two PGRs: BAP (6-benzylaminopurine) and GA3 (gibberellic acid) was performed. Artificial Neural Networks (ANNs) algorithms were used to build models with the whole database to determine the effect of those components on several growth and quality parameters. Neurofuzzy logic allowed us to decipher and generate new knowledge on the hierarchy of some minerals as essential components of the culture media over vitamins and PRGs, suggesting rules about how MS basal media formulation could be modified to assess the quality of micropropagated woody plants.

Use of phage ϕ6 to inactivate Pseudomonas syringae pv. actinidiae in kiwifruit plants: in vitro and ex vivo experiments.

Over the last years, the global production and trade of kiwifruit has been severely impacted by Pseudomonas syringae pv. actinidiae (Psa), a phytopathogen that causes a disease in kiwifruit plants known as bacterial canker. The available treatments for this disease are still scarce, with the most common involving frequently spraying the orchards with disinfectants, copper-based bactericides and/or antibiotics. Moreover, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Phage therapy may be an alternative approach to inactivate Psa. The present study investigated the potential application of the already commercially available bacteriophage (or phage) ϕ6 to control Psa infections. The inactivation of Psa was assessed in vitro, using liquid culture medium, and ex vivo, using artificially contaminated kiwifruit leaves with two biovar 3 (a highly aggressive pathogen) strains (Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10). In the in vitro experiments, the phage ϕ6 was effective against both strains (maximum reduction of 2.2 and 1.9 CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). In the ex vivo tests, the decrease was lower (maximum reduction 1.1 log and 1.8 CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). The results of this study suggest that the commercially available phage ϕ6 can be an effective alternative to control Psa infections in kiwifruit orchards.

Modeling and Optimizing Culture Medium Mineral Composition for in vitro Propagation of Actinidia arguta.

The design of plant tissue culture media remains a complicated task due to the interactions of many factors. The use of computer-based tools is still very scarce, although they have demonstrated great advantages when used in large dataset analysis. In this study, design of experiments (DOE) and three machine learning (ML) algorithms, artificial neural networks (ANNs), fuzzy logic, and genetic algorithms (GA), were combined to decipher the key minerals and predict the optimal combination of salts for hardy kiwi (Actinidia arguta) in vitro micropropagation. A five-factor experimental design of 33 salt treatments was defined using DOE. Later, the effect of the ionic variations generated by these five factors on three morpho-physiological growth responses - shoot number (SN), shoot length (SL), and leaves area (LA) - and on three quality responses - shoots quality (SQ), basal callus (BC), and hyperhydricity (H) - were modeled and analyzed simultaneously. Neurofuzzy logic models demonstrated that just 11 ions (five macronutrients (N, K, P, Mg, and S) and six micronutrients (Cl, Fe, B, Mo, Na, and I)) out of the 18 tested explained the results obtained. The rules "IF - THEN" allow for easy deduction of the concentration range of each ion that causes a positive effect on growth responses and guarantees healthy shoots. Secondly, using a combination of ANNs-GA, a new optimized medium was designed and the desired values for each response parameter were accurately predicted. Finally, the experimental validation of the model showed that the optimized medium significantly promotes SQ and reduces BC and H compared to standard media generally used in plant tissue culture. This study demonstrated the suitability of computer-based tools for improving plant in vitro micropropagation: (i) DOE to design more efficient experiments, saving time and cost; (ii) ANNs combined with fuzzy logic to understand the cause-effect of several factors on the response parameters; and (iii) ANNs-GA to predict new mineral media formulation, which improve growth response, avoiding morpho-physiological abnormalities. The lack of predictability on some response parameters can be due to other key media components, such as vitamins, PGRs, or organic compounds, particularly glycine, which could modulate the effect of the ions and needs further research for confirmation.

Computer-Assisted Recovery of Threatened Plants: Keys for Breaking Seed Dormancy of Eryngium viviparum.

Many endangered plants such as Eryngium viviparum (Apiaceae) present a poor germination rate. This fact could be due to intrinsic and extrinsic seed variability influencing germination and dormancy of seeds. The objective of this study is to better understand the physiological mechanism of seed latency and, through artificial intelligence models, to determine the factors that stimulate germination rates of E. viviparum seeds. This description could be essential to prevent the disappearance of endangered plants. Germination in vitro was carried out under different dormancy breaking and incubation procedures. Percentages of germination, viability and E:S ratio were calculated and seeds were dissected at the end of each assay to describe embryo development. The database obtained was modeled using neurofuzzy logic technology. We have found that the most of Eryngium seeds (62.6%) were non-viable seeds (fully empty or without embryos). Excluding those, we have established the germination conditions to break seed dormancy that allow obtaining a real germination rate of 100%. Advantageously, the best conditions pointed out by neurofuzzy logic model for embryo growth were the combination of 1 mg L-1 GA3 (Gibberellic Acid) and high incubation temperature and for germination the combination of long incubation and short warm stratification periods. Our results suggest that E. viviparum seeds present morphophysiological dormancy, which reduce the rate of germination. The knowledge provided by the neurofuzzy logic model makes possible not just break the physiological component of dormancy, but stimulate the embryo development increasing the rate of germination. Undoubtedly, the strategy developed in this work can be useful to recover other endangered plants by improving their germination rate and uniformity favoring their ex vitro conservation.