Enduring Effect of Antibiotic Timentin Treatment on Tobacco In Vitro Shoot Growth and Microbiome Diversity

Abstract

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial communities. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation; however, the effect of antibiotic treatment on the diversity of indigenous microbial populations and the consequences on the performance of tissue culture is not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco (Nicotiana tabacum L.) shoots in vitro as well as the composition of the plant-associated microbiome. The study revealed that shoot cultivation on a medium supplemented with 250 mg L−1 timentin resulted in 29 ± 4% reduced biomass accumulation and a 1.2–1.6-fold higher level of oxidative stress injury compared to the control samples. Moreover, the growth properties of shoots were only partially restored after transfer to a medium without the antibiotic. Microbiome analysis of the shoot samples using multivariable region-based 16S rRNA gene sequencing revealed a diverse microbial community in the control tobacco shoots, including 59 bacterial families; however, it was largely dominated by Mycobacteriaceae. Antibiotic treatment resulted in a decline in microbial diversity (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. These results imply that the diversity of the plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture.

Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture

“…•− accumulation in tobacco in vitro shoot tissues could be related to processes of tissue culture senescence and oxidative stress response to timentin treatment, both of which might contribute to an increase in oxidative lipid injury observed previously [35]. Proteomics analysis was used to study potential links between the molecular mechanisms of these two processes.…”

“…To study the enduring growth suppression effect in tobacco in vitro shoot culture observed after the timentin treatment as described previously [35], the shoots were cultured on a medium supplemented with timentin at a sub-cytotoxic concentration that was previously shown to be effective for Agrobacterium elimination after tobacco genetic transformation [33]. The post-antibiotic treatment (PA) experimental group was prepared by cultivating the timentin-treated shoots on medium without antibiotic under the same conditions as the treatment control (TC) shoots for at least one cultivation passage before the analysis.…”

“…The accumulation of ROS was assayed at five time points during the tobacco shoot propagation cycle that were selected based on previous observations of the stress-induced oxidative lipid injury variation during the TC and PA shoot growth and culture senescence [35] (Figure 1; Supplementary Materials Table S1). During the first week of cultivation, the O 2…”

“…The relatively low cytotoxic effect of direct exposure to antibiotic timentin on in vitro plant tissues has been described [30,33,34]. However, our previous studies revealed that treatment of tobacco in vitro culture with the antibiotic at sub-cytotoxic levels resulted in a shoot growth suppressing effect that persists after transfer to medium without antibiotic and is associated with increased levels of oxidative lipid peroxidation injury [35,36]. Therefore, the antibiotic-induced enduring growth suppressing effect could have a negative impact on tobacco plant transformation efficiency, plant regeneration, propagation of the tissue culture or subsequent plant rooting and acclimation.…”

“…The generation of ROS and oxidative damage could lead to the accelerated senescence of plant tissues [58]. Considering elevated levels of oxidative lipid injury enduring for at least several propagation cycles in tobacco in vitro shoots as a consequence of timentin treatment [35], it could be presumed that the oxidative stress-mediated acceleration of senescence could be an important factor contributing to the growth-suppressive effect observed in the timentin-treated tobacco shoot culture. Therefore, in the current study, we assessed the accumulation of ROS (O 2…”