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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.

Background: In vitro cultivation and cryopreservation techniques are essential tools for genetic diversity conservation and pathogen-free plant propagation of horticultural crops. The optimisation of cryopreservation protocols typically focuses on minimising the negative effects of pretreatment with cryoprotectors (CPs), cryogenic freezing (CF) treatment, and recovery procedures on explants. However, the impact of in vitro and CF techniques on plant-associated microbiota remains poorly understood, and their potential to improve plant adaptation after cryopreservation is underexplored. The aim of the present study was to investigate in vitro shoot culture and cryopreservation-induced changes in the endophytic bacterial diversity of two sweet cherry cultivars and to assess the potential of an inoculum of bacterial isolates to improve the growth of shoot culture after CF., Results: Cultivars 'Sunburst' and 'Mindaugė' showed different responses to cold hardening preconditioning as well as different survival and regrowth rates after cryopreservation. Metataxonomic analysis revealed variation in the abundance and taxonomic composition of bacteria assigned to 35 families in samples of field-grown tree leaves, dormant buds, and in vitro shoot culture before and after CF treatment. Bacillaceae and Enterobacteriaceae bacteria were predominant in the leaf samples of both cultivars. For 'Sunburst', Pseudomonadaceae and Sphingomonadaceae bacteria were dominant in dormant buds and in vitro shoots, respectively, while Burkholderiaceae was largely predominant in the shoots following CF treatment. Conversely, 'Mindaugė' tissues exhibited more consistent colonisation by Bacillaceae and Enterobacteriaceae across the experimental groups, except for in vitro shoots where Mycobacteriaceae prevailed. A pure bacterial isolate inoculum was applied to the 'Mindaugė' shoot culture to counter the CF treatment-induced suppression of shoot growth (~ 40%). Cocultivation with Brevibacterium sp. S1-2, Bacillus cereus S1-3, or B. toyonensis Nt18 increased the shoot leaf area from 48 to 75%., Conclusions: This study revealed that endophytic bacterial diversity is significantly reduced under in vitro conditions, often leading to a genotype-specific increase in the abundance and dominance of bacteria attributed to a single bacterial family. Moreover, shoot cocultivation with endophytic bacterial isolates has potential for improving the recovery of in vitro shoots after cryopreservation., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

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 (O 2 •- ) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O 2 •- accumulation pattern was different. For PA shoots, the increase in O 2 •- concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O 2 •- 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 O 2 •- 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.

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.