Optimizing immersion time and frequency in a twin-bottle temporary immersion system for mass production of Zingiber officinale Roscoe

Ginger (Zingiber officinale Roscoe) is a highly valued medicinal and culinary crop. However, conventional propagation using rhizomes is limited. This study aimed to optimize immersion conditions for the large-scale micropropagation of Z. officinale utilizing a twin-bottle temporary immersion system (TIS), and to evaluate its efficiency in comparison to other culture systems across various concentrations of chlormequat chloride (CCC). The immersion conditions significantly affected shoot multiplication and overall growth parameters. The optimal protocol identified was 6 times/day for 5 min, which resulted in the highest shoot number (4.4 shoots/clump), shoot height (3.5 cm), and fresh weight (4.3 g/clump). Crucially, this treatment achieved the highest ex vitro survival rate of 88.9% following acclimatization. Conversely, excessively long or frequent immersion periods resulted in reduced overall growth and development and diminished plantlet quality. Comparative analysis demonstrated that TIS significantly increased fresh weight, shoot height, and leaf length more than other systems, thereby achieving enhanced mass production, particularly when CCC was absent or at low concentrations. Plantlets regenerated from TIS consistently showed improved acclimatization and high survival rates (up to 80%) under greenhouse conditions. The optimized TIS protocol established in this research offers an efficient, scalable, and cost-effective approach for the mass propagation of high-quality Z. officinale planting material, supporting sustainable commercial cultivation.