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.

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 shoot culture and cryopreservation (CP) are techniques essential for the ex situ preservation of genetic resources and the production of plant propagation material of clonally propagated horticultural crops. Changes in plant-associated microbiota diversity and composition induced by in vitro cultivation and CP treatment could have a negative effect on the growth and ex vitro adaptation of the in vitro propagated shoots. Therefore, the aim of the present study was to assess changes in endophytic bacteria diversity in domestic apple tissues induced by in vitro cultivation and CP treatment and to investigate the potential of the bacterial inoculum to improve the rooting and ex vitro acclimatisation of the propagated shoots. Metataxonomic analysis revealed a variation in the endophytic bacteria diversity and taxonomic composition between the field-grown tree dormant bud and the in vitro propagated or CP-treated shoot samples of apple cv. Gala. Whereas Sphingobacteriaceae, Sphingomonadaceae, Pseudomonadaceae, and Beijerinckiaceae families were the most prevalent families in the bud samples, Enterobacteriaceae, Bacillaceae, and Lactobacillaceae were dominant in the in vitro shoots. The bacterial inoculum effect on rooting and ex vitro acclimatisation was assessed using four isolates selected by screening the endophytic isolate collection. Bacillus sp. L3.4, B. toyonensis Nt18, or a combined inoculum resulted in a 21%, 36%, and 59% increase in cumulative root length and a 41%, 46%, and 35% increase in the biomass accumulation of ex vitro acclimatised plantlets, respectively. Root zone microbiota functional diversity analysis implied that growth stimulation was not related to improved nutrient uptake but could involve a pathogen-suppressing effect. The results demonstrate that the application of plant growth-promoting bacteria can potentially improve the performance of the in vitro propagated germplasm.

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.

Plants inhabiting environments with suboptimal growth conditions often have a more pronounced capacity to attract and sustain microbial communities that improve nutrient absorption and expand abiotic stress tolerance. Rhodiola rosea L. is a succulent plant of the Crassulaceae family adapted to survive in sandy or rocky soils or dry tundra. The aim of the present study was to investigate the diversity and plant growth-stimulating potential of R. rosea endophytic microbiota. Metataxonomic analysis of the bacterial diversity in the rhizome of R. rosea revealed 108 families. Among these, three families were found exclusively in the core microbiome of 1-year-old plants, while nine families were unique to the core microbiome of mature plants grown in the field for more than 4 years. Seventy-three endophytic bacteria isolates were obtained from the rhizome of R. rosea plants and were assigned into 14 distinct bacterial genera of Firmicutes (26%) or Proteobacteria (74%) phyla. Screening for functional genes related to the nitrogen cycle, phosphorus mineralisation or dissolution, and traits associated with nitrogen fixation (56% of isolates), siderophore production (40%), inorganic phosphorus solubilisation (30%), and production of indole-related compounds (51%) led to the classification of the isolates into 16 distinct clusters. Co-cultivation of 45 selected isolates with germinating Arabidopsis seedlings revealed 18 and 5 isolates that resulted in more than a 20% increase in root or shoot growth, respectively. The study results established the complexity of the succulent R. rosea endophytic microbiome and identified isolates for potential plant growth-stimulating applications.

Surface immobilization and characterization of the functional activity of fibronectin (Fn) type-III domains are reported. The domains FnIII9-10 or FnIII10 containing the RGD loop and PHSRN synergy site were recombinantly produced and covalently bound to chemically activated PEG methacrylate (MA) hydrogel coatings by microcontact printing. Such fabricated biochip surfaces were 6 mm in diameter and consisted of 190 µm wide protein stripes separated by 200 µm spacing. They were analyzed by imaging null ellipsometry, atomic force microscopy and fluorescence microscopy. Also, the coatings were tested in human foreskin fibroblast and HeLa cultures for at least 96 h, thus evaluating their suitability for controlled cell adhesion and proliferation. However, while HeLa cultures were equally well responsive to the FnIII9-10, FnIII10 and Fn surfaces, the fibroblasts displayed lower cell and lower focal adhesion areas, as well as lower proliferation rates on the Fn fragment surfaces as compared to Fn. Nevertheless, full functional activity of the fibroblasts was confirmed by immunostaining of Fn produced by the cells adherent on the biochip surfaces. The observed interaction differences that were either cell type or surface composition-dependent demonstrate the potential use of specifically engineered Fn and other ECM protein-derived domains in biochip architectures.