In tissue culture, efficient nutrient availability and effective control of callus contamination are crucial for successful plantlet regeneration. This study was aimed to enhance callogenesis, callus regeneration, control callus contamination, and substitute iron (Fe) source with FeO-NPs in Murashige and Skoog (MS) media. Nanogreen iron oxide (FeO-NPs) were synthesized and well characterized with sizes ranging from 2 to 7.5 nm. FeO-NPs as a supplement in MS media at 15 ppm, significantly controlled callus contamination by (80%). Results indicated that FeCl3-based FeO-NPs induced fast callus induction (72%) and regeneration (43%), in contrast FeSO4-based FeO-NPs resulted in increased callus weight (516%), diameter (300%), number of shoots (200%), and roots (114%). Modified media with FeO-NPs as the Fe source induced fast callogenesis and regeneration compared to normal MS media. FeO-NPs, when applied foliar spray, increased Plant fresh biomass by 133% and spike weight by 350%. Plant height increased by 54% and 33%, the number of spikes by 50% and 265%, and Chlorophyll content by 51% and 34% in IRRI-6 and Kissan Basmati, respectively. Additionally, APX (Ascorbate peroxidase), SOD (Superoxide dismutase), POD (peroxidase), and CAT (catalase) increased in IRRI-6 by 27%, 29%, 283%, 62%, while in Kissan Basmati, APX increased by 70%, SOD decreased by 28%, and POD and CAT increased by 89% and 98%, respectively. Finally, FeO-NPs effectively substituted Fe source in MS media, shorten the plant life cycle, and increase chlorophyll content as well as APX, SOD, POD, and CAT activities. This protocol is applicable for tissue culture in other cereal crops as well.
