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

Esmaeil Nezami-Alanagh,Pedro Pablo Gallego,Ghasem-Ali Garoosi,Mariana Landín

doi:10.1038/s41598-019-46155-2

Esmaeil Nezami-Alanagh, Pedro Pablo Gallego + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-46155-2

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Abstract

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.

Highlights

During the in vitro culture of plants is frequent that physiological disorders appear such as shoot-tip necrosis (STN), callus formation at the base of shoots (BC), hyperhydricity, shoot fasciation (SF), epinasty, leaf necrosis (LN) or leaf color (LC), which reduce the yield and the quality of the production
Several physiological disorders were detected after the micropropagation of pistachio as STN & Leaf necrosis (LN) (Fig. 1A), LC (Fig. 1B), basal callus (BC) (Fig. 1C), SF (Fig. 1D), hyperhydricity (Fig. 1E) and epinasty (Fig. 1F)
As it can be observed in Fig. 2: (i) all treatments produce physiological abnormalities to some degree, including pistachio optimized medium (POM), MS, WPM and DKW media used as controls; (ii) some abnormalities such as STN, LN, LC or BC are frequently, while others are rare such as SF, hyperhydricity or epinasty; (iii) treatments based on MS media caused higher STN, LN, LC and epinasty than www.nature.com/scientificreports those based in POM media, but contrary, POM based treatments caused higher BC and hyperhydricity compared to MS medium; (iv) some treatments based on MS (6, 9, 19, 22 and 23) caused the maximum disorder for STN, LN and LC in both genotypes

Summary

Introduction

During the in vitro culture of plants is frequent that physiological disorders appear such as shoot-tip necrosis (STN), callus formation at the base of shoots (BC), hyperhydricity, shoot fasciation (SF), epinasty, leaf necrosis (LN) or leaf color (LC), which reduce the yield and the quality of the production. Leaf edge necrosis is frequently reported during in vitro culture of variety of species[6,7,8]. The lack of certain nutrients in the culture media lead variations in leaf color (LC) from green to red in micropropagated shoots of different species[6,7,8,9,11]. The formation of basal callus (BC) is important in the commercial shoot micro-propagation of diverse species, since its appearance probably slows down or even inhibits the absorption of nutrients by the shoots, especially alongside the callus senesces[12]. BC has been attributed to the accumulation of calcium in that area of the plant, which can lead to deficiencies of Ca2+ in the upper parts of the shoots[13]

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