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.