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.

In vitro plant tissue cultures face various unfavorable conditions, such as mechanical damage, osmotic shock, and phytohormone imbalance, which can be detrimental to culture viability, growth efficiency, and genetic stability. Recent studies have revealed a presence of diverse endophytic bacteria, suggesting that engineering of the endophytic microbiome of in vitro plant tissues has the potential to improve their acclimatization and growth. Therefore, the aim of this study was to identify cultivated tobacco (Nicotiana tabacum L.) endophytic bacteria isolates that are capable of promoting the biomass accumulation of in vitro tobacco shoots. Forty-five endophytic bacteria isolates were obtained from greenhouse-grown tobacco plant leaves and were assigned to seven Bacillus spp. and one Pseudomonas sp. based on 16S rRNA or genome sequence data. To evaluate the bacterial effect on in vitro plant growth, tobacco shoots were inoculated with 22 isolates selected from distinct taxonomic groups. Four isolates of Bacillus cereus group species B. toyonensis, B. wiedmannii and B. mycoides promoted shoot growth by 11%–21%. Furthermore, a contrasting effect on shoot growth was found among several isolates of the same species, suggesting the presence of strain-specific interaction with the plant host. Comparative analysis of genome assemblies was performed on the two closely related B. toyonensis isolates with contrasting plant growth-modulating properties. This revealed distinct structures of the genomic regions, including a putative enzyme cluster involved in the biosynthesis of linear azol(in)e-containing peptides and polysaccharides. However, the function of these clusters and their significance in plant-promoting activity remains elusive, and the observed contrasting effects on shoot growth are more likely to result from genomic sequence variations leading to differences in metabolic or gene expression activity. The Bacillus spp. isolates with shoot-growth-promoting properties have a potential application in improving the growth of plant tissue cultures in vitro.

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Investigation of cellular subfraction proteomes allows the study of specific changes induced by environmental changes, stress and other conditions. Chloroplasts participate in a huge number of complex biochemical processes in plant cells by retrograde signalling, as well as by sensing and responding to cellular dysfunction. Changes in environmental conditions in a controlled way are easily achieved in in vitro model systems. However, growing plants in vitro makes it difficult to obtain sufficient material for chloroplast isolation. Therefore, the chloroplast isolation method needs to be optimised for achieving sufficient yield from a small amount of sample. We used three species of Rosaceae family that are of high agricultural interest for breeding programs in Lithuania. The method used for chloroplast isolation from Arabidopsis thaliana was optimized for Malus domestica, M. platycarpa and Prunus avium. Homogenisation of 3 g of in vitro plant material in sorbitol-based isolation medium with a laboratory blender yielded a sufficient amount of chloroplasts for proteomic analysis. The purity of the fraction was highly increased by additional step of centrifugation at 200× g. The purity of chloroplasts was evaluated visually by microscopy, by immunoblotting with specific antibodies, as well as by using marker proteins and quantitative mass spectrometry. Although microscopy showed negligible amounts of cellular debris in all of the preparations, immunoblotting allowed detection of the presence of cytosolic marker in some of the preparations. Mass spectrometric analysis of marker proteins confirmed the presence of modest amount of non-chloroplast proteins. In conclusion, the presented method for chloroplast isolation for the Rosacea plants in vitro gives sufficient yield and purity for subcellular proteomic studies.

To investigate orchard plant response to cold stress, homologs of thale cress (Arabidopsis thaliana (L.) Heynh.) COR47 (cold responsive) gene in different plants-herbal and woody-were isolated, and the expression of this gene during the cold acclimation process was examined. Conservative COR47 DNA fragments typical of dicotyledonous plants were established and specific primers (18-20 nucleotide length) were created. Two strawberry (Fragaria ananassa Duch.) cultivars (cold-resistant 'Melody' and cold sensitive 'Holiday'), two sweet cherry (Prunus avium L.) cultivars (cold-sensitive 'Kordija' and cold resistant 'Jurgita'), two sour cherry (Prunus cerasus L.) cultivars (cold-sensitive 'Erdi Jubileum' and cold-resistant 'Molodiozhnaja') and the M323 hybrid of sour cherry with sweet cherry were used in the study. Our results show that the COR47 gene homolog transcription is no less than 30 days of cold acclimation at low temperatures. No less than 24-30 days of cold acclimation are necessary for plant hardening. Similar mRNA variations were established in strawberry and cherry plants, implying the similarity of genes involved in the cold acclimation process in orchard plants.

Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro shoot culture that persists after tissue transfer to a medium without antibiotic. The effect is associated with an increase in oxidative stress injury in plant tissues. In this study, we assessed changes of reactive oxygen species accumulation, protein expression, and oxidative protein modification response associated with enduring timentin treatment-induced growth suppression in tobacco (Nicotiana tabacum L.) in vitro shoot culture. The study revealed a gradual 1.7 and 1.9-fold increase in superoxide (O2•−) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O2•− accumulation pattern was different. For PA shoots, the increase in O2•− concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O2•− concentration compared to TC during the period following the first week of cultivation. Although no protein expression differences were detectable between the TC and PA shoots by two-dimensional electrophoresis, the increase in O2•− concentration in PA shoots was associated with a 1.5-fold increase in protein carbonyl modification content after one week of cultivation, and protein carbonylation analysis revealed differential modification of 26 proteoforms involved in the biological processes of photosynthesis and glycolysis. The results imply that the timentin treatment-induced oxidative stress might be implicated in nontranslational cellular redox balance regulation, accelerates the development of senescence of the shoot culture, and contributes to the shoot growth-suppressing effect of antibiotic treatment.

Temperature stress is one of the most common external factors that plants have to adapt to. Accordingly, plants have developed several adaptation mechanisms to deal with temperature stress. Chloroplasts are one of the organelles that are responsible for the sensing of the temperature signal and triggering a response. Here, chloroplasts are purified from low temperature (4° C), control (22° C) and high temperature (30° C) grown Malus x domestica microshoots. The purity of the chloroplast fractions is evaluated by marker proteins, as well as by using in silico subcellular localization predictions. The proteins are digested using filter‐aided sample processing and analyzed using nano‐LC MS/MS. 733 proteins are observed corresponding to published Malus x domestica gene models and 16 chloroplast genome ‐encoded proteins in the chloroplast preparates. In ANOVA, 56 proteins are found to be significantly differentially abundant (p < 0.01) between chloroplasts isolated from plants grown in different conditions. The differentially abundant proteins are involved in protein digestion, cytoskeleton structure, cellular redox state and photosynthesis, or have protective functions. Additionally, a putative chloroplastic aquaporin is observed. Data are available via ProteomeXchange with identifier PXD014212.