Dynamics of Endophytic Microbiome in Tobacco During Dedifferentiation, Regeneration, and Antibiotic Treatment
Plants do not live in isolation; they host a wide range of bacterial endophytes within their tissues that contribute to growth, nutrient uptake, hormone balance, disease resistance, and stress tolerance. Despite growing interest in plant-associated microbiomes, how developmental changes and laboratory manipulations alter these microbial communities remains poorly understood. This study examines the endophytic bacterial communities of tobacco (Nicotiana tabacum) across several developmental stages and experimental conditions, with particular focus on how dedifferentiation, tissue culture, regeneration, and antibiotic exposure shift microbial composition and diversity. Plant material was collected at distinct stages including callus, suspension cultures, and regenerated tissues, under both control and antibiotic-treated conditions. Sample categories included control suspensions (SC), antibiotic-treated suspensions (ST), callus (Cl), control regenerated roots (RR) and shoots (RS), and regenerated tissues from treated suspensions (RRT and RST). Genomic DNA was extracted from plant tissues under sterile conditions, and the 16S rRNA gene variable regions were amplified for microbial profiling. Paired-end sequencing was carried out on a next-generation sequencing platform, and the resulting reads were processed to generate OTUs with taxonomic assignments based on the SILVA reference database. Initial analysis revealed clear differences in bacterial community composition across sample types. Proteobacteria were notably enriched in certain groups, pointing to selective pressures tied to developmental stage and treatment. Antibiotic exposure produced comparatively modest additional changes relative to the pronounced microbial filtering observed during dedifferentiation, indicating that tissue culture itself acts as a primary bottleneck in shaping the endophytic microbiome. This study provides a comprehensive dataset on plant-associated microbiome dynamics during developmental transitions and experimental perturbations. Sequence data deposited in the Sequence Read Archive (SRA) will support future comparative and functional studies in plant-microbe interactions, microbial ecology, and tissue culture research, with broader relevance to improving plant health and understanding microbiome resilience.