Optimisation of Plant Growth Regulator Mediated In Vitro Shoot Culture of Solanum nigrum by Managing Endophytic Bacterial Infection

Abstract

Micropropagation of field-collected medicinal plants is frequently defeated by endophytic bacteria that occupy internal tissue, survive surface disinfection and emerge only after the culture appears to have been established. This study optimised plant growth regulator (PGR) mediated in vitro shoot culture of Solanum nigrum L. while simultaneously managing the endophytic bacterial infection that limited it. Nodal explants were surface-sterilised with bavistin (250 µg mL⁻¹) at four immersion durations and cultured on Gamborg B5 medium supplemented with 6-benzylaminopurine (6-BAP) and indole-3-butyric acid (IBA) in three combinations against a hormone-free control. Ten minutes in bavistin gave the most workable balance between asepsis (88%) and explant survival (59%); longer exposure raised asepsis to 97% on tissue of which only 4% survived. All PGR treatments produced significantly higher regeneration than the control (χ² = 27.94, df = 3, p < 0.001), with 6-BAP 0.5 mg L⁻¹ inducing leaves (68.75%), 6-BAP 0.5 + IBA 1.0 mg L⁻¹ inducing roots (81.25%; mean 12 roots per explant, maximum length 5.2 cm) and 6-BAP 2.0 + IBA 2.0 mg L⁻¹ inducing callus whose texture depended on explant origin (friable from nodal segments, 75%; soft and chunky from in vitro-derived roots, 41.70%). The PGR treatments did not differ from one another in response frequency (p ≥ 0.685), indicating that the auxin-to-cytokinin ratio governs the morphogenic pathway rather than the probability of response. Liquid medium supported no growth. Bacterial clouding appeared at the explant–medium interface only after 25 days, confirming an endogenous rather than an environmental origin; the isolate was a motile, cluster-forming, short Gram-negative rod. Agar-well diffusion across six antibiotic classes showed greatest susceptibility to levofloxacin (38 mm at 100 mg L⁻¹; 30 mm at 50 mg L⁻¹), followed by amikacin and cefotaxime sodium. In culture (n = 100 per treatment), however, the ranking inverted: levofloxacin at 50 mg L⁻¹ gave the highest yield of viable, colony-free shoots (49% against 2% untreated; χ² = 70.38, df = 4, p < 0.001) at a phytotoxicity index of 3.9%, whereas levofloxacin at 100 mg L⁻¹ cleared more cultures (66%) but weakened 43.9% of those it cleared (p < 0.001). Amikacin under-performed cefotaxime despite a larger inhibition zone. The study concludes that antibiotic selection for tissue culture must be based on usable yield rather than susceptibility ranking, and that endophyte indexing should precede rather than follow PGR optimisation in field-collected medicinal species.