Sterilization protocol for bud break in Eucalyptus clone-413: Overcoming challenges in tissue culture

Abstract

Effective sterilization is crucial for successful plant tissue culture, ensuring high survivability and minimal microbial contamination. This study evaluated the efficacy of ten sterilization protocols (T1-T10) involving combinations of 70% ethanol, Tween 20, Bavistin, mercuric chloride (HgCl2), sodium hypochlorite (NaClO), and antibiotics (ampicillin or streptomycin). Each treatment's impact on explant survivability and contamination rates was analyzed. The results revealed significant differences in efficacy among protocols. T8, incorporating 70% ethanol, Tween 20 (10-15 minutes), 1% Bavistin (3-5 minutes), 0.1% NaClO (12-15 minutes), and ampicillin, achieved the highest mean survivability (92%) and lowest contamination (8%). Similarly, T7, which utilized a shorter exposure to Tween 20, showed high survivability (84%) and low contamination (16%). Sodium hypochlorite-based treatments supplemented with ampicillin consistently outperformed HgCl2-based treatments. T3, the best HgCl2-based protocol, achieved 84% survivability and 16% contamination. However, increasing HgCl2 concentration to 0.15% (T4 and T5) reduced survivability (56-58%) and increased contamination (42-44%). Protocols without antibiotics demonstrated suboptimal results, as evidenced by T1 and T6, which had lower survivability rates (60-70%) and higher contamination (30-40%). These findings highlight the advantages of sodium hypochlorite and antibiotic combinations in improving sterilization efficacy. This study provides a framework for optimizing sterilization strategies, promoting sustainable and effective practices in plant tissue culture, and advancing biotechnological innovations.