In Vitro regeneration of bio-immunized banana cv. Grand Naine using a novel double-decker temporary immersion bioreactor

Considering the havoc caused by Fusarium oxysporumf. sp. cubense (TR4) at the global level on banana cultivation, especially in India, there is a high demand for an economical bio-immunized tissue culture technique for the sustainable production of disease-resistant plantlets, wherein bio-immunization is achieved through treatment of tissue culture-derived plants with antifungal secondary metabolites to mitigate the impact of Fusarium wilt. In the current investigation, a novel, user-friendly, and cost-effective Double Decker Temporary Immersion Bioreactor (DDTIB) was designed and optimized for the mass multiplication of bio-immunized Grand Naine plantlets to cater to the huge demand of planting material of banana. An immersion frequency of six hours with three minutes duration of media immersion in DDTIB significantly improved the regeneration frequency (22.32 shoots/clump) as compared to the Semi Solid Tissue Culture System (SSTCS), which yielded only 7.86 shoots/clump. Additionally, after three consecutive multiplication cycles, DDTIB produced significantly higher biomass (1112.24 mg) as compared to SSTCS (996.36 mg). A novel primary hardening technique was also introduced in DDTIB-regenerated bio-immunized plantlets, resulting in a 91.52% survival rate. The genetic fidelity of DDTIB-regenerated plants was confirmed using ISSR markers and further confirmed by metabolomics analysis. The DDTIB developed in the current study would be the most suitable bioreactor for the rapid and large-scale commercial multiplication of bio-immunized Grand Naine plantlets.