Optimized tissue culture protocol for high-frequency multiplication and genetic fidelity evaluation in ginger (Zingiber officinale Roscoe)

Abstract

Ginger (Zingiber officinale Roscoe) is a widely valued spice and medicinal plant, primarily propagated vegetatively through rhizomes. However, conventional propagation methods are inefficient and prone to soil-borne pathogens. The present study reports an optimized in vitro micropropagation protocol for large scale and disease-free production of ginger using rhizome bud as explants. Axenic cultures were successfully established using a surface sterilization protocol comprising 70% ethanol (30 sec), 1000 ppm Cefotaxime (15 min), 1000 ppm Streptomycin (15 min), Kasugamycin (5 mL L-1) for 15 min, 2500 ppm Bavistin (20 min) and 0.1% HgCl2 (6 min), resulting in 60% contamination free cultures. For sprout induction, MS basal medium supplemented with 1.0 mg L-1 BAP and 0.5 mg L-1 NAA led to 100% bud break, with 3.00 sprouts and 2.00 leaves per explant. During shoot multiplication, the average highest number of shoots (5.81) was observed on MS medium containing 5.0 mg L-1 BAP and 0.5 mg L-1 NAA, while the average best multiplication rate (3.15), leaf number (13.55), and mean shoot length (2.98 cm) were recorded on medium with 3.0 mg L-1 BAP and 0.1 mg L-1 NAA. in vitro rooting was most effective in ½ MS medium with 2.0 mg L-1 IAA, with 100% rooting in most treatments, and maximum root number (7.80) recorded in these media, whereas highest root length was observed in MS medium containing 2.0 mg L-1 IBA. Ex vitro hardening with a mixture of Cocopeat: Vermiculite (1:1) resulted in 90% survival rate. Genetic fidelity of 55 in vitro regenerated ginger plants was assessed using eight ISSR primers, of which six produced 100% monomorphic banding patterns, indicating complete genetic uniformity among all samples. This optimized protocol offers a reproducible, scalable system for commercial propagation and conservation of genetically stable ginger plants.