Shoot tip necrosis of in vitro plant cultures: a reappraisal of possible causes and solutions.

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. 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.

Computer-based tools provide new insight into the key factors that cause physiological disorders of pistachio rootstocks cultured in vitro.

During the in vitro culture of plants some physiological disorders caused major problems that have been associated with culture media composition. The objective of this study was to better understand the abnormal physiological response of two pistachio rootstocks to changes in culture media ingredients. On this purpose, two computer-based tools were employed: design of experiment (DOE) and neurofuzzy logic. DOE was employed to generate a five-dimensional IV-design spaces allowing to reduce the number of treatments from 6,250 to 61. The second one, an artificial intelligence (AI) tool, neurofuzzy logic, was used to understand the cause-effect relationships between the factors studied (25) and seven physiological disorders including shoot-tip necrosis (STN), leaf necrosis (LN), leaf color (LC), basal callus (BC) formation, shoot fasciation (SF), hyperhydricity and epinasty, typically described during pistachio in vitro culture. Four out of the seven disorders were successfully modeled, being significantly affected by a limited number of factors. STN and BC were significantly affected by the concentration of EDTA-. However, while a low concentration of EDTA- reduces the STN, promotes BC. LN and LC were strongly alleviated by high amounts of thiamine-HCl. Undoubtedly, the results demonstrate the importance of recording and using data related to physiological disorders along with growth parameters when developing suitable culture media for plant tissues. The computer-based tools have been useful to: i) well sample experimental design; ii) reduce the final number of treatments and the experimental work; iii) identify the key factors affecting each disorder; iv) get insight about the causes that promote the appearance of physiological disorders. Our findings demonstrate that the recently AI designed POM media, although not optimal, is the most suitable (favouring growth and limiting physiological abnormalities) media for in vitro culture of pistachio compared to those media, currently used.

Combining DOE With Neurofuzzy Logic for Healthy Mineral Nutrition of Pistachio Rootstocks in vitro Culture.

The aim of this study was to determine the effects of Murashige and Skoog (MS) salts on optimal growth of two pistachio rootstocks, P. vera cv. "Ghazvini" and "UCB1" using design of experiments (DOE) and artificial intelligence (AI) tools. MS medium with 14 macro-and micro-elements was used as base point and its concentration varied from 0 to 5 × MS concentrations. Design of experiments (DOE) software was used to generate a five-dimensional design space by categorizing MS salts into five independent factors (NH4NO3, KNO3, mesos, micros and iron), reducing the experimental design space from 3,125 to just 29 treatments. Typical plant growth parameters such as shoot quality (SQ), proliferation rate (PR), shoot length (SL), and some physiological disorders including shoot-tip necrosis (STN) and callus formation at the base of explants (BC) were evaluated for each treatment. The results were successfully modeled using neurofuzzy logic software. The model delivered new insights, by different sets of "IF-THEN" rules, pinpointing the key role of some ion interactions ( SO 4 2 - × Cl-, K+ × SO 4 2 - × EDTA-, and Fe2+ × Cu2+ × NO 3 - ) for SQ, PR, and SL, whilst physiological disorders (STN and BC) were governed mainly by independent ions as Fe2+ and EDTA-, respectively. In our opinion, the methodology and results obtained in this study is extremely useful to understand the effect of mineral nutrients on pistachio in vitro culture, through discovering new complex interactions among macro-and micro-elements which can be implemented to design new media of plant tissue culture and improve healthy plant micropropagation for any plant species.