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.

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Summary 1.Peatland soils are estimated to store a third of all terrestrial carbon stocks and are very sensitive to climate change. In these systems, one group of soil mesofauna, enchytraeid worms (Annelida, Oligochaeta), represent up to 70% of total soil fauna biomass and previous studies have highlighted their potential use as 'biological indicators' for functionally important changes in the C cycle. 2. To examine the link between temperature, enchytraeids and carbon fluxes we performed a microcosm experiment in which we assessed the influence of temperature on enchytraeid populations and soil CO 2 and DOC release from a Galician peatland soil over 90 days. Additionally, to unravel the potential underlying mechanisms responsible for DOC production, we also tested the effects of increasing temperatures and enchytraeid activities on the presence of organic chelating metals (iron and aluminium) and hydrogen ions (i.e. acidity) in the soil solution. 3. Enchytraeid population numbers and biomass increased over time at both temperature treatments (14 and 19 ° C), with the greatest increase produced at the highest temperature (to over five and seven times higher initial values, respectively, by day 88). Results also showed that, under warmer conditions, enchytraeid activities increased both CO 2 fluxes and DOC release by twofold ( Q 10 values of 3·9 and 3·6, respectively). 4. The combined effect of temperature and enchytraeids promoted the breakdown of organic substances and consequently, more DOC and iron were leached. An important decline in H + release was observed when enchytraeids were present and possibly eliminating one of the critical mechanisms restricting DOC release. These leachate pH values were also responsible for aluminium immobilisation and hence for its insignificant role in the export of DOC from the peat soil. 5. We conclude that temperature change alone does not explain all the observed increases in soil respiration and DOC production but rather soil invertebrate responses to warming are crucial in controlling C fluxes in peatland soils. This is the result of temperature induced changes of enchytraeid populations and activities which have the potential of speeding up the decomposition of organic matter and altering soil aeration, metal mobilization and acidity of the soil solution, with important implications for the global carbon cycle. 6. There is an urgent need for incorporating the response of soil biology in climate change modelling to make better predictions of future changes in terrestrial carbon pools.

Main conclusion Shoot tip necrosis is a physiological condition that negatively impacts the growth and development of in vitro plant shoot cultures across a wide range of species. Abstract Shoot tip necrosis is a physiological condition and disorder that can arise in plantlets or shoots in vitro that results in death of the shoot tip. This condition, which can spread basipetally and affect the emergence of axillary shoots from buds lower down the stem, is due to the cessation of apical dominance. STN can occur at both shoot multiplication and rooting stages. One of the most common factors that cause STN is nutrient deficiency or imbalance. Moreover, the presence or absence of plant growth regulators (auxins or cytokinins) at specific developmental stages may impact STN. The cytokinin to auxin ratio within an in vitro plant can be modified by varying the concentration of cytokinins used in the culture medium. The supply of nutrients to in vitro shoots or plantlets might also affect their hormonal balance, thus modifying the occurrence of STN. High relative humidity within culture vessels and hyperhydricity are associated with STN. An adequate supply of calcium as the divalent cation (Ca2+) can hinder STN by inhibiting the accumulation of phenolic compounds and thus programmed cell death. Moreover, the level of Ca2+ affects auxin transport and ethylene production, and higher ethylene production, which can occur as a result of high relative humidity in or poor ventilation of the in vitro culture vessel, induces STN. High relative humidity can decrease the mobility of Ca2+ within a plant, resulting in Ca2+ deficiency and STN. STN of in vitro shoots or plantlets can be halted or reversed by altering the basal medium, mainly the concentration of Ca2+, adjusting the levels of auxins or cytokinins, or modifying culture conditions. This review examines the literature related to STN, seeks to discover the associated factors and relations between them, proposes practical solutions, and attempts to better understand the mechanism(s) underlying this condition in vitro.