Optimizing the surface sterilization protocol for in vitro culture of ginger (Zingiber officinale Rosc.)

Abstract

Ginger is a widely cultivated tropical plant renowned for its culinary, medicinal, and economic significance. Rhizome buds of ginger are commonly used as explants in micropropagation, a technique employed to rapidly produce disease-free planting material. However, these buds are often heavily contaminated with soil-borne microbes, making surface sterilization a critical step in the successful establishment of in vitro culture establishment. This study aimed to develop an effective surface sterilization protocol that minimizes contamination while maintaining high explant viability. The rhizome buds measuring 0.5–1.0 cm in length were subjected to a multi step surface sterilization process. Initial washing was carried out using a detergent (Teepol) and a surfactant (Tween 20), followed by treatment with 30% Thiram fungicide for 30 minutes. Subsequently, the buds were immersed in 70% ethanol for 2-3 minutes and exposed to 30% sodium hypochlorite (Clorox) under two-time regimes. In the first treatment, Clorox was applied for 10 minutes, replaced with a fresh solution and applied for another 10 minutes using 102 buds. In the second treatment, the same procedure was followed with 15-minute exposures each time using 92 buds. Each Clorox treatment was followed by 2–3 rinses with sterile distilled water. After final trimming, the buds were re-dipped in 70% ethanol for 5–10 seconds and immediately washed with sterile distilled water. The experiment was conducted using a Completely Randomized Design. Results indicated that the 30% Clorox treatment for 15 minutes, applied twice, was the most effective surface sterilization protocolachieving the highest survival rate (67.39%) and the lowest contamination rate (30.43%) while minimizing plant mortality. These findings highlight the importance of extended, repeated, time-regulated sterilization steps over a single prolonged exposure for effective surface sterilization without compromising explant viability.