Establishment of optimized in vitro disinfection protocol of Pistacia vera L. explants mediated a computational approach: multilayer perceptron-multi-objective genetic algorithm.

BACKGROUND: Contamination-free culture is a prerequisite for the success of in vitro - based plant biotechnology. Aseptic initiation is an extremely strenuous stride, particularly in woody species. Meanwhile, over-sterilization is potentially detrimental to plant tissue. The recent rise of machine learning algorithms in plant tissue culture proposes an advanced interpretive tool for the combinational effect of influential factors for such in vitro - based steps. RESULTS: A multilayer perceptron (MLP) model of artificial neural network (ANN) was implemented with four inputs, three sterilizing chemicals at various concentrations and the immersion time, and two outputs, disinfection efficiency (DE) and negative disinfection effect (NDE), intending to assess twenty-seven disinfection procedures of Pistacia vera L. seeds. Mercury chloride (HgCl2; 0.05-0.2%; 5-15 min) appears the most effective with 100% DE, then hydrogen peroxide (H2O2; 5.25-12.25%; 10-30 min) with 66-100% DE, followed by 27-77% DE for sodium hypochlorite (NaOCl; 0.54-1.26% w/v; 10-30 min). Concurrently, NDE was detected, including chlorosis, hard embryo germination, embryo deformation, and browning tissue, namely, a low repercussion with NaOCl (0-14%), a moderate impact with H2O2 (6-46%), and pronounced damage with HgCl2 (22-100%). Developed ANN showed R values of 0.9658, 0.9653, 0.8937, and 0.9454 for training, validation, testing, and all sets, respectively, which revealed the uprightness of the model. Subsequently, the model was linked to multi-objective genetic algorithm (MOGA) which proposed an optimized combination of 0.56% NaOCl, 12.23% H2O2, and 0.068% HgCl2 for 5.022 min. The validation assay reflects the high utility and accuracy of the model with maximum DE (100%) and lower phytotoxicity (7.1%). CONCLUSION: In one more case, machine learning algorithms emphasized their ability to resolve commonly encountered problems. The current successful implementation of MLP-MOGA inspires its application for more complicated plant tissue culture processes.

Somatic embryogenesis from seeds in a broad range of Vitis vinifera L. varieties: rescue of true-to-type virus-free plants.

BACKGROUND: Somatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L. However, low frequencies of plant embryo conversion are commonly found. In a previous work we obtained from cut-seeds of a grapevine infected with the Grapevine leafroll associated viruses 1 and 3 (GLRaV-1 and GLRaV-3), high rates of direct regeneration, embryo plant conversion and sanitation. The aim of this study is to evaluate the usefulness of this procedure for regeneration of other grapevine varieties which include some infected with one to three common grapevine viruses (GLRaV-3, Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV)). As grapevine is highly heterozygous, it was necessary to select from among the virus-free plants those that regenerated from mother tissues around the embryo, (true-to-type). RESULTS: Somatic embryogenesis and plant regeneration were achieved in a first experiment, using cut-seeds from the 14 grapevine varieties Airén, Cabernet Franc, Cabernet Sauvignon, Mencía, Merlot, Monastrell, Petit Verdot, Pinot Blanc (infected by GFLV and GFkV), Pinot Gris, Pinot Meunier, Pinot Noir, Syrah, Tempranillo (infected by GFLV), and Verdil. All regenerated plants were confirmed to be free of GFkV whereas at least 68% sanitation was obtained for GFLV. The SSR profiles of the virus-free plants showed, in both varieties, around 10% regeneration from mother tissue (the same genetic make-up as the mother plant). In a second experiment, this procedure was used to sanitize the varieties Cabernet Franc, Godello, Merlot and Valencí Blanc infected by GLRaV-3, GFkV and/or GFLV. CONCLUSIONS: Cut-seeds can be used as explants for embryogenesis induction and plant conversion in a broad range of grapevine varieties. The high regeneration rates obtained with this procedure facilitate the posterior selection of true-to-type virus-free plants. A sanitation rate of 100% was obtained for GFkV as this virus is not seed-transmitted. However, the presence of GLRaV-3 and GFLV in some of the regenerated plants showed that both viruses are seed-transmitted. The regeneration of true-to-type virus-free plants from all infected varieties indicates that this methodology may represent an alternative procedure for virus cleaning in grapevine.

Hydrotime analysis to explore the effect of H2O2-priming in the relationship between water potential (Ψ) and germination rate of Capsicum annuum L. seed under NaCl- and PEG-induced stress.

Hydrogen peroxide (H2O2) priming was proved to be effective to boost salt and drought stress tolerance in Capsicum annuum L. Hither, hydrotime (HT) approach, a population-based threshold model-germination dependence on water availability, was introduced to account for the effects of reduced water potential on H2O2-primed seeds in progress toward germination. Pre-optimized H2O2 concentrations (1 and 10 mM for 24 h at 25 °C) were applied. Subsequently, seeds were germinated over a range of water potential (Ψ) (0 to -1.2 MPa) induced by sodium chloride (NaCl) and polyethylene glycol (PEG-6000) at 25 °C. We suggest that H2O2-priming affects differently the three derived items of HT model; induced lower hydrotime constant (θH) thus, revealed more rapid germination particularly evident with PEG, shifted the threshold or base water potential for 50% germination (Ψb(50)) towards a more negative value in NaCl culture, reflected a better salinity tolerance, though, the opposite effect was recorded with PEG, and reduced the standard deviation (σΨb), proved a better uniformity of the germination process for both cultures. Thus, H2O2-priming increased GRg (1/tg) at all Ψ>Ψb(g) which is due to both a lower Ψb(50) and a smaller θH in the primed seeds for NaCl culture and primarily owing to reducing θH with no positive effect on Ψb(50) for PEG culture. A normalized time-scale, for comparing responses, was introduced and confirmed the aforementioned impact. Therefore, the crosstalk between H2O2 molecule and different cell pathways generates a constructive response in accordance with imposed stress.