Background: Vanilla planifolia is an important Orchid com. cultivated for the prodn. of natural vanilla flavor. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass prodn. of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, the authors have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochem. and mol. mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus. Results: For embryogenic callus induction, seeds obtained from 7-mo-old green pods of V. planifolia were inoculated on MS basal medium (BM) contg. TDZ (0.5mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been obsd. The primary embryogenic calli have been formed after transferring on BM contg. IAA (0.5mg l-1) and TDZ (0.5mg l-1). These calli were maintained by subculturing on BM contg. IAA (0.5mg l-1) and TDZ (0.3mg l-1) during 6 mo and formed embryogenic/organogenic calli. Histol. anal. showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By assocg. proteomics and metabolomics analyses, the biochem. and mol. markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) anal. revealed that 15 protein spots are significantly expressed (P \textless 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metab. and photosynthetic activity. In accordance with proteomic anal., metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compds. related with sugar mobilization and nitrogen metab. NMR anal. techniques also allowed the identification of some secondary metabolites such as phenolic compds. whose accumulation was enhanced during shoot differentiation. Conclusion: The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metab. principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compds. synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degrdn. These biochem. mechanisms assocd. with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metab. Indeed, for the first time in vanilla tissue culture, phenolic compds. such as glucoside A and glucoside B were identified. The degrdn. of these compds. in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compd. of vanillin. [on SciFinder(R)]