Unveiling innovations for enhancing in vitro propagation of Vanilla phaeantha Rchb.f. through the use of double-phase technique and temporary immersion bioreactor systems.

The study aimed to introduce in vitro propagation techniques for Vanilla phaeantha , focusing on the effectiveness of the double-phase technique and temporary immersion bioreactor systems for clonal multiplication. Optimal conditions for plantlets acclimatization were also sought. Endophytic bacteria occurring in the culture were isolated, analyzed, and controlled. Explants obtained from greenhouse mother plants underwent a decontamination process using sodium ampicillin at different concentrations (0, 250, and 500 mg L−1) for up to five subcultures. Bacteria present during establishment phase were isolated and analyzed via 16 S rRNA sequencing. The study also encompassed an evaluation of clonal multiplication strategies, which included assessing the influence of different consistencies of culture media (liquid, double-phase, and semisolid), and the use of three temporary immersion bioreactor models (RITA®, RALM® and TIB®). The resulting plantlets underwent acclimatization in a mixture of commercial substrate and coconut fiber with varying composition and were kept in a growth chamber before being transferred to a greenhouse. It was verified that the use of ampicillin sodium led to a significant reduction in bacterial contamination over five subcultures. Sequencing using the 16 S rRNA gene enabled the identification of some contaminant bacteria, including species of the genera Rhizobium , Herbaspirillum , and Methylobacterium. Clonal multiplication strategies were evaluated, with the double-phase method proving superior in shoot production per explant (7.0 ± 0.4) compared to the semisolid medium (5.3 ± 0.3). Among temporary immersion bioreactor models tested, RALM® was most effective in shoot formation when compared to RITA® and TIB® (8.2 ± 0.3; 4.9 ± 0.2, and 1.2 ± 0.1, respectively). The use of substrates composed of commercial substrate and coconut fiber, in any combination tested, ensuring 100 % survival of vanilla plantlets in the greenhouse. The findings provide a micropropagation protocol for Vanilla phaeantha , offering a diversification avenue for vanilla cultivation. • This study describes innovative techniques for the in vitro propagation of vanilla species. • Endophytic bacteria were isolated, identified, and controlled through the continuous use of antibiotics. • The double-phase system is more efficient than tradicional multiplication in a conventional culture medium. • This study test a new temporary immersion bioreactor model, proving efficient for enhancing in vitro vanilla propagation. [ABSTRACT FROM AUTHOR]